Hindawi Publishing CorporationArchaeaVolume 2013 Article ID 289236 16 pageshttpdxdoiorg1011552013289236

Research ArticleMolecular Characterization of Copper and Cadmium ResistanceDeterminants in the Biomining Thermoacidophilic ArchaeonSulfolobus metallicus

Alvaro Orell1 Francisco Remonsellez12 Rafaela Arancibia1 and Carlos A Jerez1

1 Laboratory of Molecular Microbiology and Biotechnology Department of Biology and Millennium Institute for Cell Dynamics andBiotechnology Faculty of Sciences University of Chile Santiago Chile

2 Department of Chemical Engineering North Catholic University Antofagasta Chile

Correspondence should be addressed to Carlos A Jerez cjerezuchilecl

Received 5 November 2012 Accepted 4 January 2013

Academic Editor Elisabetta Bini

Copyright copy 2013 Alvaro Orell et al This is an open access article distributed under the Creative Commons Attribution Licensewhich permits unrestricted use distribution and reproduction in any medium provided the original work is properly cited

Sulfolobus metallicus is a thermoacidophilic crenarchaeon used in high-temperature bioleaching processes that is able to growunder stressing conditions such as high concentrations of heavy metals Nevertheless the genetic and biochemical mechanismsresponsible for heavy metal resistance in S metallicus remain uncharacterized Proteomic analysis of S metallicus cells exposed to100mM Cu revealed that 18 out of 30 upregulated proteins are related to the production and conversion of energy amino acidsbiosynthesis and stress responses Ten of these last proteins were also up-regulated in S metallicus treated in the presence of 1mMCd suggesting that at least in part a common general response to these two heavy metals The S metallicus genome containedtwo complete cop gene clusters each encoding a metallochaperone (CopM) a Cu-exporting ATPase (CopA) and a transcriptionalregulator (CopT) Transcriptional expression analysis revealed that copM and copA from each cop gene cluster were cotranscribedand their transcript levels increased when S metallicus was grown either in the presence of Cu or using chalcopyrite (CuFeS

2) as

oxidizable substrate This study shows for the first time the presence of a duplicated version of the cop gene cluster in Archaea andcharacterizes some of the Cu and Cd resistance determinants in a thermophilic archaeon employed for industrial biomining

1 Introduction

Bioleaching is the biological conversion of an insoluble metalcompound into a water soluble form [1 2] Microbe-basedprocesses have clear economic advantages in the extractionof metals from many low-grade deposits [3] and thesemetal-extraction processes are usually more environmen-tally friendly than physical-chemical processes [3ndash5] Someores are refractory to mesophilic leaching and temperaturespreferably as high as 75ndash85∘C are required [6 7] At hightemperatures biomining consortia are dominated by ther-moacidophilic Archaea from the genus Sulfolobus AcidianusandMetallosphaera [8]

Metals play an integral role in the life process of microor-ganisms but at high levels both essential and nonessentialmetals can damage cell membranes alter enzyme specificitydisrupt cellular functions and damage the structure of DNA

[9 10] Acid-leaching solutions are characterized by highmetal concentrations that are toxic to most life and asmight be expected microorganisms that grow in mineral-rich environments are in most cases remarkably tolerant toa wide range of metal ions [3 11] and should possess robustmetal resistance mechanisms [11ndash15]

Despite this only some metal tolerance values have beenreported [6] and the genetic and biochemical mechanismsresponsible for metal resistance in biomining acidophilicArchaea are just beginning to be characterized [16] It istherefore important to further understand the mechanismsused by these microorganisms to adapt to and to resist highconcentrations of heavy metals

Related to archaeal copper (Cu) resistancemechanisms afew metal efflux pumps have been identified from sequencedgenomes of some members of this domain [17] A Cu-resistance (cop) locus has been described in Archaea

2 Archaea

which includes genes encoding a new type of archaealtranscriptional regulator (CopT) a putative metal-bindingchaperone (CopM) and a putative Cu-transporting P-typeATPase (CopA) [10] The same Cu-resistance mechanismwas described in Sulfolobus solfataricus P2 and Ferroplasmaacidarmanus In both microorganisms the putative metalchaperones and the ATPase are cotranscribed and theirtranscriptional levels increase significantly in response to Cuexposure suggesting that the transport system is operatingfor Cu efflux [18 19] Recently it was described that thecopRTA operon from S solfataricus strain 982 (copTMAin S solfataricus P2) is cotranscribed at low levels fromthe copR promoter under all conditions whereas increasedtranscription from the copTA promoter took place in thepresence of Cu excess These authors proposed a model forCu homeostasis in Sulfolobus which relies on Cu efflux andsequestration [20]

In silico studies have further identified a CPx-ATPasewhich most likely mediates the efflux of heavy metalcations in the biomining archaeon Metallosphaera sedula[21] This putative protein has significant identity to a P-type ATPase from S solfataricus (CopA) [19] MoreoverM sedula contains ORFs with significant similarity to bothCopM (Msed0491) andCopT (Msed0492) from S solfataricus[21] Very recently Maezato et al [22] have reported a geneticapproach to investigate the specific relationship betweenmetal resistance and lithoautotrophy during biotransforma-tion of chalcopyrite by M sedula The functional role of itscopRTA operon was demonstrated by cross-species comple-mentation of a Cu-sensitive S solfataricus copRmutant [22]

Cadmium (Cd) is very toxic and probably carcinogenicat low concentrations However the biological effects of thismetal and the mechanism of its toxicity are not yet clearlyunderstood [23ndash26] In some neutrophilic microorganismsCd is taken up via the magnesium or manganese uptakesystems [23] Although the mechanisms in acidophiles havenot been elucidated putative Cd resistance operons in somesequenced genomes from acidophilic microorganisms havebeen identified The species with the highest homologyto the cadA motif were Acidithiobacillus ferrooxidans andThermoplasma spp These high similarities suggest that Cdexport may be a common resistance mechanism amongacidophiles [11] Recently a time-dependent transcriptomicanalysis using microarrays in the radioresistant archaeonThermococcus gammatolerans cells exposed to Cd showedthe induction of genes related to metal homeostasis drugdetoxification reoxidation of cofactors ATP production andDNA repair [27]

One alternative mechanism proposed for metal tolerancein microorganisms is the sequestration of metal cations byinorganic polyphosphates (polyP) [28] and at the same timethe intracellular cations concentration would regulate thehydrolysis of this polymer [29] S metallicus can toleratevery high concentrations of copper and accumulates highamounts of polyP granules [14 30] Furthermore the levels ofintracellular polyP are greatly decreased when this archaeonis either grown in 200mM Cu or shifted to 100mM Cu[14 30] An increase in exopolyphosphatase (PPX) activ-ity and Pi efflux due to the presence of Cu suggests a

metal tolerance mechanismmediated through polyP [14 30]Actual evidence suggests that polyPmay providemechanisticalternatives in tuning microbial fitness for the adaptationunder stressful environmental situations and may be of cru-cial relevance amongst extremophiles The genes involved inpolyP metabolism in Crenarchaeota have been only partiallyelucidated as long as a polyP synthase activity is still to bereported and characterized in this kingdom [15]

Thus far there are no studies on the prospective geneticand biochemical mechanisms that enable S metallicus tothrive in such high concentrations of Cu and other metalsUnderstanding these mechanisms could be particularly use-ful in potential improvement of the bioleaching microorgan-isms which could likely increase the efficiency of bioleachingprocesses in due course Since the genome of S metallicusis not currently available possible genes involved in Curesistance were searched by using a CODEHOP and ldquogenomewalkingrdquo approaches and the transcriptional expression ofthese isolated genes was assessed by real-time RT-PCRFurthermore a proteomic approach was used to identifypossible proteins involved in resistance to Cu and Cd inS metallicus To our knowledge this is the first paper thatshows the occurrence of a duplicated version of the cop genecluster in Archaea and gives insights into the molecular Cuand Cd resistance determinants in a thermophilic archaeonemployed for industrial biomining

2 Materials and Methods

21 Strains and Growth Conditions S metallicus DSM 6482was grown at 65∘C in medium 88 (Deutsche Sammlung vonMikroorganismen und Zellkulturen) containing 005 (wv)elemental sulfur and 002 (wv) yeast extract S solfataricusDSM 1617 was grown at 70∘C in medium 182 (DeutscheSammlung von Mikroorganismen und Zellkulturen) with01 (wv) yeast extract and with 01 (wv) Casamino acidsTo study Cd tolerance of S metallicus and S solfataricusthe microoganisms were grown in their respective mediaexcept that different concentrations of Cd (0005ndash5mM)were present initially as indicated in the correspondingexperiment

For differential expression assays S metallicus cells weregrown in the absence of Cu or Cd to the early stationaryphase and after removing the medium from the cells bycentrifugation they were then shifted to a new mediumcontaining 100ndash200mM CuSO

4(Cu from now on) or 1mM

CdSO4(Cd from now on) during 24 h After this time cells

were treated to obtain protein extracts Growth was moni-tored by measuring unstained cells numbers by means of aPetroff-Hausser chamber under a phase contrast microscope

22 Preparation of Protein Extracts from S metallicus Cellsfrom 800mL of a control culture grown to 108 cellsmL(early stationary phase) or cultures shifted to 100ndash200mMCu or 1mM were harvested by centrifugation at 7700 g for15min The pellets were washed with medium 88 to removethe sulfur Cells were then resuspended in 50mM Tris-HClpH 815 10mM EDTA 100 120583gmL PMSF buffer (20120583L per

Archaea 3

mg wet weight) frozen and sonicated six times for 30 seach time The lysates were centrifuged at 4300 g for 5minto eliminate cellular debris The protein concentration ofsupernatants was determined by the method of Bradford(Coomasie Plus protein assay reagent Pierce) Between 120and 500120583g of proteins from the protein extracts were mixedwith rehydration IEF buffer as described by Hatzimanikatiset al [31] and Choe and Lee [32] with some modificationsincluding urea 8M thiourea 2M CHAPS 2 (wv) Bio-Lyte3ndash10 027 (vv) Bio-Lyte 5ndash8 013 (vv) and bromophenolblue 0001 (wv) followed by incubation at 25∘C for 30minDTT (003 g) and sterile nanopure water were then added tocomplete a final volume of 300 120583L The samples were thenincubated at room temperature for 1 h

23 Isoelectric Focussing (IEF) Each sample (300 120583L) wasloaded onto the pH 3ndash10 (nonlinear) 17 cm IPG strips(Biorad) in the first dimension chamber and was incubatedat room temperature for 1 h Mineral oil (25mL) was thenadded to prevent evaporation of the sample and precipitationof urea and strips were passively rehydrated for 18 h Theisoelectric focusing was performed with the PROTEAN IEF(Biorad) using the following conditions 250V for 15min2000V for 2 h 8000V for 4 h 10000V for 11 h and 50Vfor 4 h reaching a total of 120000Vh

24 SDS-PAGE Prior to the second-dimensional elec-trophoresis strips were equilibrated as described by Hatz-imanikatis et al [31] with some modifications Strips wereincubated for 15min with a solution containing 6M urea156mM DTT 30 (vv) glycerol 2 (wv) SDS and 24mMTris-HCl pH 68 and subsequently for 15 more min in a solu-tion containing 6M urea 135mM iodoacetamide 30 (vv)glycerol 2 (pv) SDS and 24mM Tris-HCl pH 68 Finallystrips were incubated with electrophoresis buffer containing192mM glycine 1 (wv) SDS and 250mM Tris-HCl pH 83until the second-dimensional run SDS-PAGEwas performedusing the PROTEAN II xi cell as described by Laemmli [33]and gels consisted of 115 or 15 (wv) polyacrylamide Stripswere then overlaid onto the second-dimensional gels sealedwith 1 (wv) agarose in electrophoresis buffer containinga trace amount of bromophenol blue Electrophoresis wascarried out at constant 70V for 15 h All experiments wereperformed in triplicate Gels were stained with silver orCoomasie Blue G-250 as described by Shevchenko et al [34]and Giavalisco et al [35] respectively

25 Gels Analysis and Mass Spectrometry Gel images weredigitized by scanning (Epson) and analyzed with the Delta2D software (Decodon) to identify the spots differentiallyexpressed due to the presence of toxic metals An estimateof relative quantitative changes was made on the basis ofthe change in percent volume among silver stained gelsSpots of interest were recovered from Coomasie Blue G-250stained gels manually and were sent to electron spray tandemionizationmass spectrometric analysis (tandemMS-MS ESI-QUAD-TOF) The results obtained were analyzed with Mas-cot algorithm (httpwwwmatrixsciencecomindexhtml)

by using the MSMS Ion search and all genomes availableat databases were used as queries The entire MS analysiswas performed at the Cambridge Center for ProteomicsUniversity of Cambridge UK

26 CODEHOP-PCR CopA and CopM amino acid sequen-ces from S acidocaldarius S tokodaii and S solfataricuswereobtained fromNCBI (httpwwwncbinihgov) After align-ing the sequences by using the CLUSTAL X program toidentify areas of homology consensus-degenerate PCR pri-mers were designed according to the CODEHOP strategy[36 37] using the WWW access at httpblocksfhcrcorgblockscodehophtml Two regions of high sequence sim-ilarity were identified for both CopA and CopM seq-uences respectively (Figure S1 see Supplementry Materi-al available online at httpdxdoiorg1011552013289236)and used to design the consensus-degenerate hybrid oligonu-cleotide primers (Table 1) Consensus-degenerate hybridoligonucleotide primers were designed for the N- and C-terminus of CopA while a pair of degenerate hybrid oligonu-cleotide primers was designed for CopM (Figure S1)

Amplification reactions contained 1x thermophilic DNApolymerase buffer with 2mMMgCl

2 02mMdNTPs 05 120583M

of each primer 5U Taq DNA polymerase 40 ng of S metal-licus genomic DNA as a template and water to a final volumeof 50120583L The thermal cycling conditions were 3min at 95∘Cfollowing 30 cycles of 95∘C for 30 s 50∘C for 30 s and 72∘Cfor 1min 1 final additional cycle at 72∘C for 10min Productsof amplification were applied onto 10 (wv) agarose gelsand main amplification bands were excised purified andTA-cloned into pCR21-TOPO (Invitrogen) vector and finallysequenced

27 GenomeWalking Experiments Genome walking strategywas performed as described by Acevedo et al [38] Thusa double-stranded oligo-cassette AdaptT adapter was con-structed by annealing of the two unphosphorylated primersAdaptF (51015840-CTAGGCCACGCGTCGACTAGTACTA-G-CTT-31015840) and AdaptR (51015840-AGCTAGTACTAGTCGACGCG-TGGCCTAG-31015840) Annealing was performed by heating theprimers (10 120583M) in a boiling water bath for 5min and thenslowly cooling to room temperature

Six different DNA libraries were constructed by meansof digesting 1120583g of S metallicus genomic DNA with the fol-lowing restriction enzymes HindIII BamHI EcoRI EcoRVNcoI and PstI respectively DNA digestion reactions werecarried out using 10U of restriction enzyme 2120583L of thecorresponding enzyme reaction buffer in 20120583L of totalreaction volume The reaction mix was incubated at 16∘Cduring 16 h To complete the 31015840 recessive end of the DNAfragments and to add a 31015840 overhanging adenine 500 ng ofthe digested and purified DNA were incubated with 5U ofTaq DNA polymerase 1 120583L of 10mM dNTPs mix and 5 120583Lof 10x thermophilic DNA polymerase buffer in 50120583L totalvolume at 70∘C for 45min Seven 120583L of thismixture was thenincubatedwith 15 pmol of AdaptT oligo-cassette 1 U T4DNAligase (Promega) and 2120583L of 5x ligase buffer in a total volume

4 Archaea

Table 1 Oligonucleotides

Name Sequence DescriptioncopA cdegF 51015840-gatgtagtaatagtaaaaactggagaaataataccngcngaygg CODEHOP-PCRcopA cdegR 51015840-tcatcagcaaaattagaagaatctccngtngcdat CODEHOP-PCRcopT cdegF1 51015840-ctcaaatagaatataaagtattacaaatgttaaaagargaywsnmg CODEHOP-PCRcopT cdegR1 51015840-ggattaccatttatttcatttccacartartcrca CODEHOP-PCRcopT cdegR2 51015840-cttatcatattcataaatcttccatctattaatttrtarcaytc CODEHOP-PCRcopT degF1 51015840-gartgytayaarctnat DOP-PCRcopT degR1 51015840-atnagyttrtarcayct DOP-PCRcopM degF 51015840-gayccngtntgyggnatgga DOP-PCRcopM degR 51015840-ccnggnttyccntacgg DOP-PCRAdaptF2 51015840-cacgcgtcgactagtactagctt Genome WalkingSP1copA1 31015840 51015840-aaggatgagggggaccttatgg GenomeWalkingSP2copA1 31015840 51015840-ggagataagaaatggggtaaaagag GenomeWalkingSP1copA1 51015840 51015840-tgataccatcatggaacctgtcag GenomeWalkingSP2copA1 51015840 51015840-tcctccacaatcccatccgctg GenomeWalkingSP1copA1 51015840 2 51015840-gattgtagctaagttaacctcggcctcg GenomeWalkingSP2copA1 51015840 2 51015840-cttctcaccctcagtctggttgg GenomeWalkingSP1copA2 31015840 51015840-gaaagaggaatatatgcaagggtaaacgg GenomeWalkingSP2copA2 31015840 51015840-gtgttaatgggagagctggaggg GenomeWalkingSP1copA2 51015840 51015840-cttctctgtggcaacatcataaccagcc GenomeWalkingSP2copA2 51015840 51015840-acgcatgtggcgcaatgcattcc GenomeWalkingSP1copT1 51015840 51015840-cattcctcgcaccagcttgcacactctc GenomeWalkingSP1copT2 51015840 51015840-cctatgaatactagatcttttccctgaac GenomeWalkingSP2copT2 51015840 51015840-aacagcttataacactcgtcactttggc GenomeWalkingcopM2 RT F 51015840-gatgaaaaaagccaatataagac RT-PCRcopA2 Rv 51015840-gaacactaactaacatcgcc RT-PCRcopM1 RT Fw 51015840-ctatcgtttttgttccgaagcttg RT-PCRcopA1 Rv 51015840-cagcagcaagaacagagacgcc RT-PCRlowastSM16Sf 51015840-acgctctaaaaaggcgtgggaata RT-qPCRlowastSM16Sr 51015840-ttgagctcggggtctttaagcagtg RT-qPCRcopA1Sm qRT F1 51015840-gctaaggtaatagagagcgg RT-qPCRcopA1Sm qRT R1 51015840-tgaacaggaatggacagg RT-qPCRcopA2Sm qRT F 51015840-tgtgcttgtctccttagcgt RT-qPCRcopA2Sm qRT R 51015840-actcttccgtctttcggagt RT-qPCRcopT1Sm qRT F 51015840-tgtaggagagtgtgcaagct RT-qPCRlcopT1Sm qRT R 51015840-tcgcaagtgagggttatggt RT-qPCRcopT2Sm qRT F 51015840-gtgttacggagcttgca RT-qPCRcopT2Sm qRT R 51015840-acactcgtcactttggc RT-qPCRAll oligonucleotides were synthesized by Invitrogen lowastOligonucleotides designed by Bathe and Norris [39]

of 10 120583L The ligation reaction was incubated at 16∘C during16 h

Two consecutive amplification reactions were then per-formedThefirst PCR reactionwas donewith 1x Elongasemixbuffer 19mM MgCl

2 02mM dNTPs 05120583M first specific

primer (SP1) (designed from the known sequence of thetarget gene a forward primer was used to amplify the 31015840end a reverse primer for 51015840 end amplification) 5 120583L of theligatedDNAdiluted 10-fold and 1 120583L of Elongase (Invitrogen)and water to a final volume of 50120583L The thermal cyclingconditions were as follows 1 cycle at 94∘C for 1min 20cycles of 94∘C for 32 s and 68∘C for 5min and one final

additional cycle at 70∘C for 7min The PCR product wasdiluted 10 fold and 3 120583L were used as a DNA template fora second PCR which was performed using the same con-ditions as the first PCR with 05 120583M second specific primer(SP2) and 02 120583Moligo-cassette-specific primer AdaptF2 (51015840-CACGCGTCGACTAGTACTAGCTT-31015840) and 1 120583L of Elon-gase The thermal cycling conditions were 1 cycle at 94∘C for1min 35 cycles of 94∘C for 32 s and 68∘C for 5min and 1final additional cycle at 70∘C for 7min PCR products wereexcised purified TA-cloned into pCR21-TOPO (Invitrogen)vector and finally sequenced

Archaea 5

28 Isolation of Total RNA Cell pellets (10mg wet weight)were collected and diluted in 60 120583L TEN buffer (20mMTris-HCl pH 80 1mM EDTA 100mM NaCl) followedby addition of 60120583L TENST buffer (20mM Tris-HCl pH80 1mM EDTA 100mM NaCl 16 Na-lauroyl sarcosineand 012 Triton X-100) This suspension was incubated atroom temperature for 15min to allow cell lysis Total RNAwas then extracted using the TRIzol reagent (Invitrogen)as recommended by the manufacturer DNA contaminationin RNA preparations was removed by DNase I treatment(Roche) following the manufacturerrsquos instructions RNAwas then purified precipitated and finally resuspended indiethylpyrocarbonate (DEPC)-treated water Total RNA con-centrations were estimated by spectrophotometric measure-ments (OD

260) and its quality was evaluated by determining

the ratio of absorption at 260 nm and 280 nm which waswithin the preferred range of 18ndash21

29 Northern Blotting For differential gene expression byNorthern blotting experiments the media were supple-mented with either different concentrations of Cu (5ndash50mM) Cd (5mM) NiSO

4(15mM) ZnSO

4(50mM) or

Ag2SO4(008mM) respectively as indicated For some

experiments elemental sulfur was replaced by a chalcopyrite(CuFeS

2) concentrate which was used as the only energy

source at 1 (wv)Total RNA (5 120583g) was separated by electrophoresis

on a 1 formaldehyde agarose gel followed by blottingonto Hybond-N nylon membranes (Amersham PharmaciaBiotech Bucking-hamshire UK) Hybridization was con-ducted as described by Sambrook et al [40] DNA probeswere labeled by randomprimerDNA labeling kit (Fermentas)using [120572-32 p] dCTP The hybridization signal was detectedand analyzed by using the molecular Imager FX system andQuantity One software The primer sequences for amplifica-tion of these gene probes are listed in Table 1

210 Cotranscriptional Analysis by RT-PCR To study theexpression of adjacent genes copM and copA cDNA wassynthesized by using 08120583g of total RNA from a S metallicusculture grown in the presence of 20mM Cu and a reverseprimer hybridizing to copA sequence A forward primerannealing on copM sequence was used for the PCR reactions(Table 1) PCR amplifications were performed with 1 120583L ofa 110 dilution of the cDNA and 25 pmol of each primerAmplification conditions included an initial 3min of denat-uration at 95∘C followed by 35 cycles of 30 s at 95∘C 30 sat 55∘C and 15min at 72∘C and finished by 10min at 72∘CA reverse transcriptase reaction without enzyme was carriedout in order to exclude amplification due to genomic DNAcontamination

211 Quantitative RT-PCR Single stranded cDNA was syn-thesized from 08 ng of DNA-free RNA samples usingrandom hexamers (Fermentas) and ImProm-II reversetranscription system (Promega) following manufacturerrsquosinstructions The software tool IDT Scitools (IntegratedDNA Technologies) was used to design primers producing

amplicons of 150ndash200 bp (Table 1) qPCRwas performedwith2 120583L of 1 10 diluted cDNA samples 125 120583L 2x QuantiFastSYBRGreenPCRMasterMix (Quiagen) 1 120583Mof each primerand water to a final volume of 25120583L The efficiency of eachprimer pair was calculated from the average slope of a linearregression curve which resulted from qPCRs using a 10-folddilution series (10 pgndash10 ng) of S metallicus chromosomalDNA as template Cq values (quantification cycle) wereautomatically determined by Real-Time Rotor-gene 6000PCR software (Corbett Life Sciences) after 40 cycles Cqvalues of each transcript of interest was standardized to theCq value of the 16S rRNA gene [39] At least 3 biologicalreplicates of each assessed condition and 2 technical replicatesper qPCR reaction were performed

3 Results and Discussion

31 Tolerance of S metallicus and S solfataricus to Cu and CdIn a previous work it was determined that S metallicus wasable to tolerate high Cu concentrations While the presenceof 100mM Cu did not affect S metallicus growth kinetics adecreased in cell biomass of only 30 was observed whenexposed to 200mM Cu [30] The high tolerance to Cu hasbeen described in other acidophilic Bacteria and Archaeacompared mostly with neutrophilic microorganisms [11 14]Here this analysis was further extended to characterize theresponse of S metallicus to Cd Thus it was determined thatS metallicus growth was not affected in the presence of either05 or 1mM Cd when compared with the control conditionin the absence of the metal (Figure 1(a)) In addition whenS metallicus was challenged with 2 and 3mM Cd it wasobserved that at the late exponential growth phase the cellnumbers decreased by 30 and 50 respectively (Figure 1(a))Moreover growth of S metallicuswas completely inhibited inthe presence of 5mM Cd (Figure 1(a)) On the other hand Ssolfataricuswas not able to grow at Cd concentrations greaterthan 005mM (Figure 1(b)) At 001mM Cd S solfataricuscell numbers decreased around 35 (Figure 1(b)) Theseresults are in agreement with previous reports in which Ssolfataricus was shown to be able to grow in up to 001mMCd [41] Other acidophilic archaeons involved in bioleachingprocesses such as Metallosphaera sedula and Ferroplasmaacidarmanus were found to be able to tolerate up to 09and 9mM Cd respectively [18 42] The minimal inhibitoryconcentration (MIC) to Cd has been described to be nothigher than 1mM for several thermophilic and neutrophilicThermococcus species [43] Recently it was reported that themost radioresistant archaeon Thermococcus gammatoleransstands Cd concentrations with an MIC of 2mM [27] Todate despite the heavy metals tolerance showed by someof these microorganisms strategies to withstand stress fromtransition metals have been most widely studied only inhaloarchaea [44]

32 Effect of Cu andCd on theGlobal Proteome of SmetallicusThe proteomic response to Cu and Cd was analyzed toidentify proteins which could be involved in heavy metalsresistance in S metallicus Early stationary phase growing

6 Archaea

14

12

10

8

6

4

2

00 2 4 6 8 10 12

Time (days)

107timescellsm

L

(a)

120

100

80

60

40

20

00 2 4 6 8 10

Time (days)

107timescellsm

L

(b)

Figure 1 Growth of S metallicus (a) and S solfataricus (b) in the presence of Cd Cells were inoculated in their respective growth media inabsence of added Cd (∙) or were supplemented with 0005mM () 001mM () 005mM (diams) 01mM () 05mM () 1mM () 2mM() 3mM (times) or 5mM (+) Cd and cells were counted daily

100

7060

50

30

20

3 10MW

33 31

32

A

B

C

20

10

(a)

100

7060

50

30

20

12

3 10MW

204

21

33 31

32

A

B

C

15

93

10

27 28

23 24

29

25 2630

1614 16

13

52

7

2219

8

20

10

11

(b)

Figure 2 Changes in the proteome of S metallicus grown in the presence of Cu Cells were incubated in the absence of any added metal(a) or in the presence of 100mM Cu (b) Arrows indicate the spots that were downregulated (a) or upregulated (b) in the presence of CuNumbers indicate the spots with increased intensity in cells treated with copper The dashed boxes are shown as enlarged areas that includesome proteins upregulated in the presence of Cu and Cd (Figure 3) The bottom panels show low molecular weight proteins upregulated inthe presence of Cu separated by using a 15 polyacrylamide gel in the second dimension

cells were untreated or treated with either 100 or 200mM Cuor 1mM Cd during 24 h and analyzed by comparative two-dimensional gel electrophoresis as indicated in experimentalprocedures Twenty-three proteins were found to be down-regulated after 100mMCu treatment (Figure 2(a)) A similar

pattern was obtained at 200mM Cu (not shown) Further-more by using the Delta 2D software (Decodon) eleven ofthese proteins were found to be completely absent in the gelsand more than 8 proteins decreased their intensity in therange from 15-to 5-fold compared with control cells grown

Archaea 7

13 13

11

13

1

Control Copper Cadmium

(a)

99 9

333

(b)

3030

30

2020

20 212121

(c)

Figure 3 Comparison of selected proteins of S metallicus cells treated with Cu or Cd Protein extracts were obtained from cells treatedwithout metals and with 100mM Cu or 1mM Cd for 24 h and the proteins were separated by 2D-PAGE The segments (a) (b) and (c) arethe enlarged dashed boxes in Figure 2 under the three conditions indicated Numbers show some of the spots with increased intensity in cellstreated with Cu or Cd

in the absence of Cu (Figure 2(a)) Therefore these resultsshow that a number of proteins became non-detectable ordecreased their levels when S metallicus faced Cu This kindof response was also seen when the microorganism wasexposed to Cd (data not shown) This behavior has alsobeen observed in similar studies where F acidarmanus waschallenged by either As(III) or Cu [16 18] In addition theexpression of 30 other proteins was found to be upregulatedafter Cu treatment (100 or 200mM) (Figure 2(b)) Most ofthese proteins could be identified only in the condition withCu and spots 5 13 and 15 were induced 26 34 and 55-fold respectively compared to cells without Cu treatmentMoreover 3 proteins of lowmolecular weight (spots 31 3 and33) that increased their expression when cells were exposedto 100mM Cu were also identified (Figure 2 bottom panels)These proteins were only detected when 15 polyacrylamidewas used during the second-dimensional separation (SDS-PAGE) On the other hand a large decrease in overallexpression of proteins after 1mMCd treatment was observed(not shown) Interestingly 10 out of the 13 proteins identifiedas up-regulated when cells were exposed to Cd and alsoshowed increased levels after Cu treatment Some of them areshown in Figure 3

33 Identification of Proteins Upregulated in Cells Treated withCu and Cd in S metallicus A total of 18 S metallicus proteinswhose levels were found to be up-regulated in responseto Cu were analyzed by mass spectrometry The identifiedproteins included functions related to the production andtransport of energy biosynthesis of amino acids stressresponses and transcription regulation (Table 2) Amongstthe proteins related to production and transport of energya putative ATP synthase subunit B (spot 23) that has beendescribed as playing a fundamental role in ATP synthesiswas identified (Table 2) When cells are subjected to somestressing conditions such as the presence of heavy metalsa greater cellular demand for energy has been reported tooccur [18] Most likely this phenomenon might be due toATP-driven Cu transport via ATPases that have a substantialinterplay during metal detoxification [45] Furthermore theATP synthase subunit B was also identified as up-regulatedwhen S metallicus was treated with Cd (Table 2)

Two other proteins corresponded to putative oxidoreduc-tases such as ferredoxin oxidoreductase (spot 3) and alcoholdehydrogenase (spot 4) that have been generally involvedin electron transporter chains and use NAD+ as an electronacceptor The levels of ferredoxin oxidoreductase were also

8 Archaea

Table 2 Proteins upregulated in S metallicus cells exposed to 100mM Cu or 1mM Cd

Spot Molecular weight (kDa) Cu induction levels Putative function Microorganism related Accesion number1dagger 266 infin Proteasome subunit S solfataricus AAK410343dagger 701 infin Ferredoxin oxidoreductase S solfataricus AAK429264dagger 429 infin Alcohol dehydrogenase S solfataricus AAK431545 35 26 Hypothetical protein S acidocaldarius YP 2549766 27 infin Hypothetical protein P aerophilum NP 5598899dagger 57 infin Phosphoglycerate dehydrogenase M thermautotrophicus AAB8546613dagger 27 34 Unknown mdash mdash14 28 infin Unknown mdash mdash15 35 55 Unknown mdash mdash20dagger 461 infin Glutamate dehydrogenase S solfataricus AAK4223021dagger 45 infin Hypothetical protein M acetivorans NP 618380122 49 infin Hypothetical protein P torridus YP 02280723dagger 51 infin ATP synthase subunit B S solfataricus AAK4088024 585 infin HSP60 subunit S shibatae AAG37273130dagger 48 infin Unknown mdash mdash31 16 ND Unknown mdash mdash32 144 ND Putative transcription regulator S solfataricus AAK4041333 138 ND Transcription factor nusA S solfataricus AAK40563Spots refer to those numbered in Figure 2 Proteins in Figure 2 which are not included in this table were not subjected to sequencing due to their low intensitiesin the gelsdaggerSpot up-regulated in cells exposed to Cu and CdND not determinedInfinity symbol indicates that proteins were expressed only in presence of copper or cadmiumM thermoautotrophicus Methanobacterium thermoautotrophicus M acetivorans Methanosarcina acetivorans S shibatae Sulfolobus shibatae P torridusPicrophilus torridus P aerophilum Pyrobaculum aerophilum S acidocaldarius Sulfolobus acidocaldarius

up-regulated in response to Cd (Table 2) Several previ-ous reports have suggested that oxidoreductases contributeto an oxidative protection in both Bacteria and Archaeain response to heavy metals [46ndash48] In the neutrophilicmicroorganisms Escherichia coli and Staphylococcus aureusit has been described that oxidases and dehydrogenasescontribute to oxidative protection Cu homeostasis and stressresponses [46 47] Furthermore some proteins involved inoxidative damage repair such as NADH-dependent oxidasesand thioredoxin reductases were expressed in cells of Facidarmanus exposed to As(III) [16] The expression of thisgroup of proteins has also been observed when the samemicroorganism was exposed to Cu [18] Therefore oxidore-ductases play an important role against oxidative stress andmay eliminate reactive oxygen species which constitute themajor component of the stress caused by transition metals[44 49 50]

The enzymes related to biosynthesis of amino acids werefound as commonly up-regulated either in response to Cu orCdThey corresponded to a phosphoglycerate dehydrogenase(spot 9) and a glutamate dehydrogenase (spot 20) Theformer one catalyzes the NAD+-dependent oxidation of 3-phosphoglycerate into 3-phosphohydroxypyruvate a branchpoint from the glycolytic pathway and the initial reactionin L-serine biosynthesis [51] Glutamate dehydrogenase cat-alyzes the oxidative deamination of glutamate to produce 2-oxoglutarate and ammonia with reduction of NAD+ [52]Proteins involved in amino acids synthesis were expressed

in cells of F acidarmanus exposed to As(III) [16] Moreoverincreased levels of proteins involved in the biosynthesisof sulfur-containing amino acids have been observed inSaccharomyces cerevisiae cells exposed to Cd [24] suggestingthat these proteins might also have a role in the cellularresponse to adverse conditions

Two proteins showed identity to components of the stressresponse mechanism such as one subunit of the HSP60chaperonin (spot 24) and one subunit of the proteasome (spot1) In Archaea the stress protein HSP60 is directly involvedin protein folding processes [53] Proteomic analysis of Facidarmanus cells exposed to As(III) and Cd revealed theexpression of proteins involved in protein folding and DNArepair including HSP60 chaperonin and DnaK heat-shockprotein (HSP70) thereby the authors suggested that thismicroorganismusesmultiplemechanisms to resist high levelsof Cu [16 18] On the other hand proteasomes are describedas nonspecific proteolytic nanomachines associated withprotein catabolism Proteasomes are known to be closelyinvolved in maintaining protein quality control by degradingmiss folded and denatured proteins in response to cell stressin all three domains of life [54] A proteomic analysis inthe thermoacidophilic archaeon Thermoplasma acidophilumshowed that proteasomes and chaperone-related proteinswere highly induced against stress conditions indicatinga high turnover rate of proteins [55] In P furiosus theexpression of the 1205731 subunit of the proteasome was inducedin cells subjected to heat shock stress [56] Additionally the

Archaea 9

150pb

(a)

1016 pb

(b)

2000 pb

(c)

Figure 4 CODEHOP-based PCR for the amplification of the putative genes copA and copM from S metallicus Amplification products(a) using degenerate PCR primers (DOP) copM degF and copM degR designed from amino acid sequence conserved blocks of SulfolobalesCopM proteins (b) using primers copA cdegF and copA cdegR designed by CODEHOP strategy for amplification of a copA-like gene (c)Primers copM degF and copA cdegRwere assesed for the amplification of a copMA-likeDNA regionThose primerswere testedwith genomicS metallicus DNA samples Amplicons expected sizes were as follows ca 150 bp for copM ca 950 bp for copA and ca 2000 bp for copMAPCR products were excised and cloned for later sequencing

proteasome subunit was also found to be up-regulated whenS metallicus was subjected to Cd stress (Table 2)

The transcriptional regulation-related proteins corre-sponded to a putative transcription regulator (spot 32) andthe transcription factor NusA (spot 33) involved in theintrinsic termination of transcription [57] NusA has beenalso reported to display important functions in the cellularresponse to adverse factors [58] The expression of twotranscriptional regulators related to amino acids biosynthesisand metal-dependent genetic repression were found to beinduced in P furiosus cells subjected to oxidative stress[48] Moreover the expression of several genes encodingpredicted transcriptional regulators were induced followingCd exposure in T gammatolerans cells [27]

Spots 5 6 13 14 15 21 22 30 and 31 (Table 2) did notshow significant scores with any protein sequences currentlyavailable in the data bases Interestingly spots 2 4 9 13 21and 30 were found to be commonly upregulated when Smetallicus cells were treated with either Cu or Cd (Figure 3Table 2)

The results presented here are consistent with previ-ous work describing global protein expression profiles inresponse to heavy metals as reported for F acidarmanus [1618] microbial-biofilm communities in an acid mine drainagesite [59] and other prokaryotic and eukaryotic microorgan-isms [24 60]Therefore a coordinated expression of differentgroups of genes suggests the existence of regulatory networkssuch as stress response mechanisms and respiratory chainadjustments to cope with the presence of heavy metal ions[61]

34 Cu-Resistance (Cop) Genes Cluster Is Duplicated in Smetallicus Genome A Cu-resistance (cop) locus has beendescribed to be highly conserved in archaeal genomeswhich consists of genes encoding a new type of archaealtranscriptional regulator (CopT) a metal-binding chaperone(CopM) and a Cu-transporting P-type ATPase (CopA) [19]Since the genome sequence of Smetallicus is not yet availablewe searched for the presence of cop genes by using consen-sus degenerate hybrid oligonucleotide primers-based PCR(CODEHOP-PCR) CopA and CopM amino acid sequencealignments from S acidocaldarius S tokodaii and S solfa-taricus were used to identify conserved sequence blocks andtherefore to design either CODEHOP or DOP (degeneratehybrid oligonucleotide) primers (Figure S1 Table 1) TherebyPCR assays using S metallicus genomic DNA as templateyielded amplicons of ca 150 bp and ca 950 bp for copMand copA CODEHOP pair primers respectively (Figures4(a) 4(b)) As estimated from primer pairrsquos position onamino acid sequence alignments the obtained PCR productscorresponded to the expected sizes suggesting the presenceof copM- and copA-like genes in S metallicus genome Inorder to determine the identity of the amplified DNA frag-ments they were TA-cloned and sequenced Blastx sequenceanalysis showed that the 150 bp DNA fragment coded for anincomplete ORF sharing 63 homology with S solfataricusCopM (SSO10823) whereas the analysis of the 950 pb DNAfragment yielded a partial ORF sharing 54 homology withS solfataricus CopA protein (SSO2651)

The genomic organization of the putative cop genes in Smetallicus was analyzed to find out its similarity with those

10 Archaea

Table 3 Sequence identity comparison of S metallicus Cop proteins to those in other Sulfolobales

S metallicus protein Identity to homologue in other SulfolobalesS solfataricus P2 S acidocaldarius S tokodaii M sedula

CopA1 51 43 44 45CopA2 88 46 49 50CopM1 63 69 71 71CopM2 89 72 70 77CopT1 36 27 30 32CopT2lowast 90 52 52 53Boldface type indicates to which organism the S metallicus homologue shows the highest sequence identitylowastRefers to the analysis obtained using the uncompleted amino acid sequence of CopT2 which represents sim90 in length when compared with S solfataricusCopT sequence

previously described in S solfataricus P2 and F acidarmanuswhere the cop cluster consists of tandem-orientated genesas copTMA [18 19] Thus using copM degF (forward) andcopA cdegR (reverse) primers we attempted to amplify aputative copMA DNA region This PCR yielded a product ofca 2000 pb corresponding to the expected size and resultedin 2 ORFs showing high sequence homology with CopMand CopA from S solfataricus (Figure 4(c)) Interestinglywhen aligning this copMA sequence with the copA and copMsequences obtained previously they were not identical whichstrongly indicated the presence of duplicated putative copgenes encoding for both Cu-ATPases (copA1 and copA2) andmetallochaperones (copM1 and copM2) in the S metallicusgenome

To isolate the entire cop gene sequences the genomewalking method described by Acevedo et al [38] was usedSix different DNA libraries were constructed from S metalli-cus genomic DNA digested with several restriction enzymesincluding 51015840 overhang and blunt end restriction enzymesThe DNA libraries were used as templates in 2 successivePCR reactions From the previously known sequences twospecific sense primers for the 31015840 end amplification and twospecific antisense primers for the 51015840 end amplification weredesigned (abbreviated SP1 and SP2) Therefore the 51015840 and 31015840ends of the putative genes copA1 and copA2 that had beenpartially identified through the genome walking techniquewere amplified

By overlapping the sequence isolated by degenerate PCRand the lateral sequences obtained by genome walking(Figure S2) it was possible to complete the whole nucleotidesequence of the putative gene copA1 Additionally it waspossible to confirm the presence of the upstream copM1 geneanddetermine the occurrence of a partial copT1 gene tandem-orientated upstream of copM1 (Figure 5)Through additionalgenome walking experiments the partial copT1 sequencecould be further completedThe samewas truewhen isolatingthe whole nucleotide sequence of the putative gene copA2as the presence of copM2 and copT2 genes were confirmedupstream of copA2 However it was not possible to completethe whole 51015840 end of the putative gene copT2 as its determinedlength corresponded to only 90 of the full length whencompared with S solfataricus copT gene (Figure 5)

In conclusion degenerate PCR together with genomewalking experiments allowed to describe the occurrence inS metallicus genome of 2 cop genes loci (named as locus cop1and locus cop2) coding for paralogous genes whose productsmay be involved in Cu-resistance (Figure 5) Although somearchaeal genomes exhibit two copies of putative Cu-P-typeATPases as described for S solfataricus [62] the genomicarrangement displayed as a cop gene cluster (copTMA) hasbeen reported to be represented in only one copy for manyarchaeal genomes [10 19 21] Moreover we further updatedthis analysis by searching for duplications of the cop genescluster in all available archaeal genome sequences up to date(October 2012) retrieving only one cop locus in each caseThereby we propose that the discovery of this cop gene clusterduplication in the genome of S metallicus constitutes so faran unprecedented feature for a representative of the Archaeadomain and might contribute to its high metal resistance

In this context it has been widely reported that increasedgene copy number can increase gene expression allowingprokaryotic to thrive under growth limiting conditions [1363ndash68] However alongside the augmented gene copy onecannot exclude that the cop operon duplication in S metal-licus may offer among others a wider repertoire of Cu-ATPases which might differ in terms of metal affinitiesandor specificity efflux rates andor differences in theirabundance regulation

Throughout amino acid sequence analysis it was furtherdetermined that each polypeptide encoded by S metallicuswith the exception of CopT1 showed to be highly homol-ogous to their orthologous counterparts encoded by othersSulfolobales (Table 3) Moreover the 3 ORFs encoded by thelocus cop2 shared about 90 identity with the correspondingS solfataricus orthologous peptides (Table 3) S metallicuscop genes products showed characteristic domains referredto as critical for their respective proposed biological activitiesS metallicus paralogous gene products CopA1 (749 aa1015840)and CopA2 (747 aa1015840) shared 513 of identity These twoputative Cu-ATPases (CopA1 and CopA2) contain the aminoacid sequence motif CPCALGLA which has been proposedto confer Cu-transporting specificity in CPx-ATPases [48]and has been also found in other CopA-like proteins frombiomining Bacteria and Archaea [14]

Archaea 11

HindIII HindIII HindIII

HindIII

CopT1Locus cop1 M1

EcoRIEcoRV

513 bp 219 bp 2250 bp

CopA1

(a)

Locus cop2 M2

EcoRI

CopA2

453 bp 171 bp 2244 bp

CopT2

PstIHindIII HindIII HindIII

(b)

Figure 5 Schematic representation of the two cop gene clusters isolated from S metallicus genome Each cluster codes for anArchaea-specifictranscriptional regulator (copT) a metallochaperone (copM) and a P-type Cu-exporting ATPase (copA) Lengths of each gene are indicatedLocus cop1 corresponds to 4346 sequenced base pairs and shows an intergenic region copT1-M1 of 232 base pairs Locus cop2 correspondsto 3435 base pairs and shows 89 base pairs copT2-M2 intergenic region Genes copM2 and copA2 overlapped in 11 base pairs copT2 was notfully isolated

Control CdSO4 Ag2SO4 NiSO4 ZnSO4 CuSO4

kb3kb2kb1

02 kb

(a)

kb3kb2kb1

02 kb

(b)

(c)

Figure 6 Northern blot analysis to determine the expression of S metallicus copA1 (a) and copA2 (b) putative genes in response to variousmetals S metallicus total RNAwas extracted from cultures grown either in the absence (control) or the presence of CuSO

4(50mM) Ag

2SO4

(008mM) NiSO4(15mM) ZnSO

4(50mM) and CdSO

4(2mM) P32-radioactivelly labelled DNA fragments annealing to copA1 and copA2

sequences respectively were used as probes (c) shows rRNAs as total RNA loading control

On the other hand CopM1 (71 aa) and CopM2 (56 aa)sharing 66 of identity present the proposed metal bindingdomain TRASH (trafficking resistance and sensing of heavymetals) [10] Additionally when analyzing the putative tran-scriptional regulators the presence of a C-terminal TRASHdomain in both CopT1 and CopT2 was also found CopT1sequence also contains an entire N-terminal helix-turn-helix (HTH) motif (not shown) that resembles the DNA-bindingmotifs of prokaryotic transcriptional regulators suchas Lrp-like proteins [67] The HTH motif was only partiallyidentified in CopT2 since the 51015840 region of copT1 gene has notbeen yet fully isolated

35 Transcriptional Analysis of S metallicus Cop Genes Toget insight into the role of the two cop loci the expressionof the corresponding genes was analyzed under variousconditions S metallicus was first grown in presence ofdifferent heavy metals (Cu Zn Cd Ni o Ag) at a givenconcentration that did not affect growth kinetics [30 42]Subsequently total RNA was isolated from late exponentiallygrown cultures and Northern blot experiments were carriedout in order to determine the expression of both copA1 andcopA2 genes As depicted in Figure 6 transcription of bothcopA1 and copA2messengers were specifically induced in thepresence of Cu and Cd ions This gene expression pattern

12 Archaea

954pb

M1 CopA1

(a)

1438 pb

M2 CopA2

(b)

1000 pb

800pb

+ minus

(c)

+ minus

1500 pb

1000 pb

(d)

Figure 7 Cotranscription analysis of copMA1 and copMA2 genes cDNA was synthesized with a reverse primer (dotted arrows) hybridizingtoward the 31015840 end of either copA1 (a) or copA2 (b) S metallicus total RNA was extracted at the late exponential phase from a culture growingin presence of 20mMCu PCR amplifications were carried out with these cDNAs and each corresponding primer pair (black arrows) as listedin Table 1 (c) and (d) show RT-PCR products obtained for the copMA1 and copMA2 intergenic regions respectively Reverse transcriptasereactions with (+) and without (minus) the Improm II reverse transcriptase enzyme were carried out in order to exclude amplification due togenomic DNA contamination Expected sizes (in base pairs) for the corresponding PCR products are given in (a) and (b)

is in good agreement with what has been described for Ssolfataricus where the bicistronic copMA transcript levelswere also found to be up-regulated in response to both Cuand Cd [19]

Northern blot analysis showed that the sizes seen for bothcopA transcripts (sim25 Kb) did not correspond exactly withthe individual genes length (each of about 225Kb) (Figure 6)This strongly suggested that the transcripts could also includethe respective copM gene located upstream of copA in eachcop loci To confirm this assumption a co-transcriptionexperiment was done (Figure 7) in which the cDNAs wereobtained by using RNA extracted from a culture grownin the presence of 20mM Cu and using a reverse primerhybridizing with copA1 (Figure 7(a)) or copA2 (Figure 7(b))gene sequence respectively PCR amplifications were carriedout by using the corresponding cDNAs as templates and eachpair of primers lying in adjacent genes (copM) The presenceof an amplicon of the expected size in each case indicatedthe adjacent genes were part of polycistronic messengers(Figure 7) These results clearly show that in S metallicuseach couple of copMA genes was expressed in the form oftranscriptional units as reported in F acidarmanus and Ssolfataricus [18 19] The coexpression of gene pairrsquos copMAmay suggest a coordinated and dependent function for therespective encoded proteins In the bacterium E hirae themetallochaperone CopZ (CopM in S metallicus) fulfills apivotal role in the mechanism of Cu homeostasis [45] Thusit was shown that this protein interacts directly with the Cu-ATPase CopB (CopA in S metallicus) handing the Cu forsubsequent removal In this context one might expect thatproteins CopM1 and CopM2 from S metallicus have a similarrole to that described for E hirae Furthermore quantitativeRT-PCR experiments were carried out in order to determinedifferential expression of the cop genes in response to differentCu concentrations The expression of copA1 copA2 copT1and copT2 was tested relative to the transcript levels ofrRNA 16S gene since its levels were not significantly affected

in all assessed conditions (data not shown) As shown inFigure 8(a) copA2 and copA1 transcript levels were foundto be concomitantly increased with the increasing Cu con-centrations present in the medium Higher transcripts levelsof copA1 were seen in all tested conditions when comparedwith copA2 levels copA1 transcript levels were found to be325-fold up-regulated when comparing 50mMCu conditionversuscontrol (absence of metal) whereas copA2 transcriptlevels showed an increment of only 175-fold (Figure 8(a))The finding that Cu-ATPases mRNA levels were significantlyincreased in response to Cu ions exposure suggests that thetransport system may operate for Cu efflux in S metallicus

Moreover copA2 and copA1 gene expression was quan-tified when S metallicus was grown using chalcopyrite(CuFeS

2) ore as an oxidizable substrate (Figure 8(a)) Mineral

oxidation mediated by the microorganism generates a pro-gressive increase in Cu ions concentration in the medium Tofind out whether the amount of solubilized Cu induced theexpression of the Cu-ATPases genes total RNAwas extractedfrom a S metallicus culture grown to late exponential phaseand in the presence of 1 CuFeS

2 It was clear that an

up-regulation of the Cu-ATPases encoding genes also tookplace when S metallicus was grown in the presence ofCuFeS

2(Figure 8(a)) copA1 transcript levels increased 14-fold

compared with the control condition in the absence of Cuwhile copA2 gene expression showed an increase of 76 foldAlong with this bymeans of atomic absorption spectrometry(AAS) analysis an overall amount of solubilized Cu ions(Cu2+Cu1+) of 144plusmn21mMwas determined to be present inthemedium indicating that copA2 and copA1 gene expressionwas most likely in response to the Cu present in the culturedue to CuFeS

2microbial-solubilization

The finding that copA1 was highly expressed comparedwith copA2 in all tested conditions may suggest a possiblephysiological hierarchy between the two ATPases whenovercoming either Cu or Cd stress In this regard by meansof a genetic approach it was demonstrated in S solfataricus

Archaea 13

700

600

500

400

300

200

100

00

5 20 50 CuFeS2CuSO4 concentration (mM)

Relativ

egenee

xpression

CopA2CopA1

(a)

8

6

7

5

4

3

2

1

00

5 20 50 CuFeS2CuSO4 concentration (mM)

Relativ

egenee

xpression

CopT1CopT2

(b)

Figure 8 Relative expression levels of S metallicus genes copA1 and copA2 (a) copT1 and copT2 (b) Expression of S metallicus cop geneswasassessed by qRT-PCR and normalized against 16S rRNA gene expression in individual cultures Mean values and standard deviations arefrom analyses of three independent cultures grown in the presence of 0 5 20 50mMCu or in 1 of a chalcopyrite ore (CuFeS

2) concentrate

that while CopA was an effective Cu efflux transporterat low and high Cu concentrations the other Cu-ATPase(CopB) only appeared to be a low-affinity Cu export ATPase[68] Moreover by using a M sedula copA deletion mutantit was demonstrated that this strain compromised metalresistance and consequently abolished chalcopyrite oxidation[22] highlighting the role of Cu detoxification mechanismsduring a given bioleaching process Our attempts to showthe functionality of CopA1 and CopA2 from S metallicusby using E coli as a heterologous host were not successfulApparently the overexpression of these ORFs had a toxiceffect on E coli that compromised its viability Althoughgene disruption tools have not been yet developed for Smetallicus the functional role of both copTMA locimight befurther studied by cross-species complementation of a coppersensitive S solfataricus copR mutant as it was described byMaezato et al [22] It will be of great interest to establishin future studies the possible functionality of the isolatedputative transporters from S metallicus

Likewise quantitative RT-PCR experiments showed thatcopT1 transcript levels increased concomitantly with increas-ing Cu concentrations whereas copT2 showed relativelysimilar transcripts levels most likely indicating a constitutiveexpression profile (Figure 8(b)) Furthermore whereas copT1transcript levels were found to be increased 36 fold inCuFeS

2

grown cultures copT2 levels remained unchanged in com-parison with the control in the absence of Cu (Figure 8(b))The results obtained for S metallicus copT2 gene expressionare consistent with those reported for S solfataricus CopTtranscriptional regulator has been proposed to function as arepressor in S solfataricus showing a constitutive expressionthat in the presence of Cu loses its affinity for the promoterregion of copMA allowing the expression of this polycistron[19] In contrast the increased transcripts levels of both copA1and copT1 concomitant with higher Cu concentrations in the

environment suggest that the putative transcriptional regula-tor CopT1 may act by activating both copMA1 and probablyitself (Figure 8) Regarding this it was recently reported thatCopR (CopT in S metallicus) from S solfataricus strain 982acts as an activator of copT (copM in S metallicus) and copAexpression [69] Nevertheless additional experiments wouldbe required to test this possibility in S metallicus

4 Concluding Remarks

We previously reported that S metallicus resists extremelyhigh Cu concentrations which was mediated to some extentby the use of a possible metal resistance system based oninorganic polyphosphate hydrolysis and consequently Cu-PO4

2minusefflux [15 29 30] Here we have addressed the questionwhether this microorganism coded for other determinantsthat might help to explain its high heavy metal toleranceAs the genomic sequence of this microorganism is notyet available we jointly used CODEHOP-PCR and genomewalking approaches and were able to establish the occurrenceof two nonidentical homologous cop loci into the genome ofS metallicus (cop1 and cop2) Each cop locus codes for anarchaeal transcriptional regulator (CopT) a metal-bindingchaperone (CopM) and a Cu-transporting P-type ATPase(CopA) High levels of the polycistronic mRNAscopMA ofeach cop locus were observed after treatment with either Cuor Cd suggesting that the encoded ATPases efflux heavymetals out in order to detoxify the intracellular environmentAltogether previous reports and the results obtained in thisstudy allow us to suggest that some key elements that mayexplain the high resistance to Cu in S metallicus is the dupli-cation of the Cu resistance cop genes a defensive responseto stress and a polyP-based accumulation mechanism Inthe case of Cd although some similar stress responses were

14 Archaea

observed whether comparable Cu-responsive elements alsoparticipate in Cd responses remains to be seen

Conflict of Interests

The authors declare that the research was done in the absenceof any commercial or financial relationships that could beconstrued as a potential conflict of interests

Acknowledgments

This work was supported by Grant no FONDECYT 1110214and in part by ICM P-05-001-F project

References

[1] A Schippers ldquoMicroorganisms involved in bioleaching andnucleic acid-based molecular methods for their identificationand quantificationrdquo in Microbial Processing of Metal SulfidesR E Donati and W Sand Eds pp 3ndash33 Elsevier BerlinGermany 2007

[2] H R Watling ldquoThe bioleaching of sulphide minerals withemphasis on copper sulphides a reviewrdquo Hydrometallurgy vol84 no 1-2 pp 81ndash108 2006

[3] D E Rawlings D Dew and C Du Plessis ldquoBiomineralizationof metal-containing ores and concentratesrdquo Trends in Biotech-nology vol 21 no 1 pp 38ndash44 2003

[4] L Valenzuela A Chi S Beard et al ldquoGenomics metagenomicsand proteomics in biomining microorganismsrdquo BiotechnologyAdvances vol 24 no 2 pp 197ndash211 2006

[5] C A Jerez ldquoBioleaching and biomining for the industrialrecovery of metalsrdquo in Comprehensive Biotechnology M Moo-Young Ed vol 3 pp 717ndash729 Elsevier Amstaerdam TheNetherlands 2nd edition 2011

[6] P R Norris and J P Owen ldquoMineral sulphide oxidationby enrichment cultures of novel thermoacidophilic bacteriardquoFEMS Microbiology Reviews vol 11 no 1ndash3 pp 51ndash56 1993

[7] D E Rawlings and D B Johnson ldquoThe microbiology ofbiomining development and optimization ofmineral-oxidizingmicrobial consortiardquo Microbiology vol 153 no 2 pp 315ndash3242007

[8] P R Norris N P Burton and N A M Foulis ldquoAcidophiles inbioreactor mineral processingrdquo Extremophiles vol 4 no 2 pp71ndash76 2000

[9] M R Bruins S Kapil and F W Oehme ldquoMicrobial resistancetometals in the environmentrdquo Ecotoxicology and EnvironmentalSafety vol 45 no 3 pp 198ndash207 2000

[10] T J G Ettema M A Huynen W M De Vos and J Van DerOost ldquoTRASH a novel metal-binding domain predicted to beinvolved in heavy-metal sensing trafficking and resistancerdquoTrends in Biochemical Sciences vol 28 no 4 pp 170ndash173 2003

[11] M Dopson C Baker-Austin P R Koppineedi and P L BondldquoGrowth in sulfidic mineral environments metal resistancemechanisms in acidophilic micro-organismsrdquo Microbiologyvol 149 no 8 pp 1959ndash1970 2003

[12] S Franke and C Rensing ldquoAcidophiles Mechanisms to toleratemetal and acid toxicityrdquo in Physiology and Biochemistry ofExtremophiles C Gerday and N Glansdorff Eds pp 271ndash278ASM Press Washington DC USA 2007

[13] C A Navarro L H Orellana C Mauriaca and C A JerezldquoTranscriptional and functional studies of Acidithiobacillusferrooxidans genes related to survival in the presence of copperrdquoApplied and Environmental Microbiology vol 75 no 19 pp6102ndash6109 2009

[14] A Orell C A Navarro R Arancibia J C Mobarec andC A Jerez ldquoLife in blue copper resistance mechanisms ofbacteria and Archaea used in industrial biomining of mineralsrdquoBiotechnology Advances vol 28 no 6 pp 839ndash848 2010

[15] A Orell C A Navarro M Rivero J S Aguilar and C A JerezldquoInorganic polyphosphates in extremophiles and their posiblefunctionsrdquo Extremophiles vol 16 no 4 pp 573ndash583 2012

[16] C Baker-Austin M Dopson M Wexler et al ldquoExtremearsenic resistance by the acidophilic archaeon ldquoFerroplasmaacidarmanusrdquo Fer1rdquo Extremophiles vol 11 no 3 pp 425ndash4342007

[17] E Pedone S Bartolucci and G Fiorentino ldquoSensing andadapting to environmental stress the archaeal tacticrdquo Frontiersin Bioscience vol 9 pp 2909ndash2926 2004

[18] C Baker-Austin M Dopson M Wexler R G Sawers and PL Bond ldquoMolecular insight into extreme copper resistance inthe extremophilic archaeon ldquoFerroplasma acidarmanusrdquo Fer1rdquoMicrobiology vol 151 no 8 pp 2637ndash2646 2005

[19] T J G Ettema A B Brinkman P P Lamers N G Kornet WM de Vos and J van der Oost ldquoMolecular characterization ofa conserved archaeal copper resistance (cop) gene cluster andits copper-responsive regulator in Sulfolobus solfataricus P2rdquoMicrobiology vol 152 no 7 pp 1969ndash1979 2006

[20] A A Villafane Y VoskoboynikM Cuebas I Ruhl and E BinildquoResponse to excess copper in the hyperthermophile Sulfolobussolfataricus strain 982rdquo Biochemical and Biophysical ResearchCommunications vol 385 no 1 pp 67ndash71 2009

[21] K S Auernik Y Maezato P H Blum and R M KellyldquoThe genome sequence of the metal-mobilizing extremelythermoacidophilic archaeon Metallosphaera sedula providesinsights into bioleaching-associated metabolismrdquo Applied andEnvironmental Microbiology vol 74 no 3 pp 682ndash692 2008

[22] Y Maezato T Johnson S McCarthy K Dana and P BlumldquoMetal resistance and lithoautotrophy in the extreme ther-moacidophile Metalosphaera sedulardquo Journal of Bacteriologyvol 194 no 24 pp 6856ndash6863 2012

[23] D H Nies ldquoMicrobial heavy-metal resistancerdquo Applied Micro-biology and Biotechnology vol 51 no 6 pp 730ndash750 1999

[24] K Vido D Spector G Lagniel S Lopez M B Toledano andJ Labarre ldquoA proteome analysis of the cadmium response inSaccharomyces cerevisiaerdquo The Journal of Biological Chemistryvol 276 no 11 pp 8469ndash8474 2001

[25] G Bertin and D Averbeck ldquoCadmium cellular effects mod-ifications of biomolecules modulation of DNA repair andgenotoxic consequences (a review)rdquo Biochimie vol 88 no 11pp 1549ndash1559 2006

[26] M H Joe S W Jung S H Im et al ldquoGenome-wide responseof Deinococcus radiodurans on cadmium toxicityrdquo Journal ofMicrobiology and Biotechnology vol 21 no 4 pp 438ndash447 2011

[27] A Lagorce A Fourcans M Dutertre B Bouyssiere YZivanovic and F Confalonieri ldquoGenome-wide transcriptionalresponse of the archeon Thermococcus gammatolerans to cad-miumrdquo PLOS ONE vol 7 no 7 Article ID e41935 2012

[28] A Kornberg N N Rao and D Ault-Riche ldquoInorganicpolyphosphate a molecule of many functionsrdquo Annual Reviewof Biochemistry vol 68 pp 89ndash125 1999

Archaea 15

[29] J D Keasling ldquoRegulation of intracellular toxic metals andother cations by hydrolysis of polyphosphaterdquoAnnals of theNewYork Academy of Sciences vol 829 pp 242ndash249 1997

[30] F Remonsellez A Orell and C A Jerez ldquoCopper tolerance ofthe thermoacidophilic archaeon Sulfolobus metallicus possiblerole of polyphosphate metabolismrdquoMicrobiology vol 152 no 1pp 59ndash66 2006

[31] V Hatzimanikatis L H Choe and K H Lee ldquoProteomicstheoretical and experimental considerationsrdquo BiotechnologyProgress vol 15 no 3 pp 312ndash318 1999

[32] L H Choe and K H Lee ldquoA comparison of three commerciallyavailable isoelectric focusing units for proteome analysis theMultiphor the IPGphor and the Protean IEF cellrdquo Electrophore-sis vol 21 no 5 pp 993ndash1000 2000

[33] U K Laemmli ldquoCleavage of structural proteins during theassembly of the head of bacteriophage T4rdquo Nature vol 227 no5259 pp 680ndash685 1970

[34] A Shevchenko M Wilm O Vorm and M Mann ldquoMassspectrometric sequencing of proteins from silver-stained poly-acrylamide gelsrdquo Analytical Chemistry vol 68 no 5 pp 850ndash858 1996

[35] P Giavalisco E Nordhoff T Kreitler et al ldquoProteome analysisof Arabidopsis thaliana by two-dimensional gel electrophoresisand matrix-assisted laser desorptionionisation-time of flightmass spectrometryrdquo Proteomics vol 5 no 7 pp 1902ndash19132005

[36] T M Rose E R Schultz J G Henikoff S Pietrokovski CM McCallum and S Henikoff ldquoConsensus-degenerate hybridoligonucleotide primers for amplification of distantly relatedsequencesrdquo Nucleic Acids Research vol 26 no 7 pp 1628ndash16351998

[37] T M Rose J G Henikoff and S Henikoff ldquoCODEHOP(Consensus-Degenerate Hybrid Oligonucleotide Primer) PCRprimer designrdquo Nucleic Acids Research vol 31 no 13 pp 3763ndash3766 2003

[38] J P Acevedo F Reyes L P Parra O Salazar B A Andrews andJ A Asenjo ldquoCloning of complete genes for novel hydrolyticenzymes from Antarctic sea water bacteria by use of animproved genomewalking techniquerdquo Journal of Biotechnologyvol 133 no 3 pp 277ndash286 2008

[39] S Bathe and P R Norris ldquoFerrous iron- and sulfur-inducedgenes in Sulfolobus metallicusrdquo Applied and EnvironmentalMicrobiology vol 73 no 8 pp 2491ndash2497 2007

[40] J Sambrook E F Fritsch and TManiatisMolecular Cloning ALaboratory Manual Cold Spring Harbor Press New York NYUSA 1989

[41] K W Miller S Sass Risanico and J B Risatti ldquoDifferentialtolerance of Sulfolobus strains to transition metalsrdquo FEMSMicrobiology Letters vol 93 no 1 pp 69ndash73 1992

[42] G Huber C Spinnler A Gambacorta and K O Stetter ldquoMet-allosphaera sedula gen and sp nov represents a new genus ofaerobic metal-mobilizing thermoacidophilic archaebacteriardquoSystematic and Applied Microbiology vol 12 pp 38ndash47 1989

[43] J Llanos C Capasso E Parisi D Prieur and C JeanthonldquoSusceptibility to heavy metals and cadmium accumulation inaerobic and anaerobic thermophilic microorganisms isolatedfrom deep-sea hydrothermal ventsrdquo Current Microbiology vol41 no 3 pp 201ndash205 2000

[44] A Kaur M Pan M Meislin M T Facciotti R El-Gewelyand N S Baliga ldquoA systems view of haloarchaeal strategies towithstand stress from transition metalsrdquo Genome Research vol16 no 7 pp 841ndash854 2006

[45] M Solioz and J V Stoyanov ldquoCopper homeostasis in Entero-coccus hiraerdquo FEMS Microbiology Reviews vol 27 no 2-3 pp183ndash195 2003

[46] S Sitthisak K Howieson C Amezola and R K JayaswalldquoCharacterization of a multicopper oxidase gene from Staphy-lococcus aureusrdquo Applied and Environmental Microbiology vol71 no 9 pp 5650ndash5653 2005

[47] L Rodrıguez-Montelongo S I Volentini R N Farıas E MMassa and V A Rapisarda ldquoThe Cu(II)-reductase NADHdehydrogenase-2 of Escherichia coli improves the bacterialgrowth in extreme copper concentrations and increases theresistance to the damage caused by copper and hydroperoxiderdquoArchives of Biochemistry and Biophysics vol 451 no 1 pp 1ndash72006

[48] E Williams T M Lowe J Savas and J DiRuggiero ldquoMicroar-ray analysis of the hyperthermophilic archaeon Pyrococcusfuriosus exposed to gamma irradiationrdquo Extremophiles vol 11no 1 pp 19ndash29 2007

[49] L M Gaetke and C K Chow ldquoCopper toxicity oxidative stressand antioxidant nutrientsrdquo Toxicology vol 189 no 1-2 pp 147ndash163 2003

[50] M J Davies ldquoThe oxidative environment and protein damagerdquoBiochimica et Biophysica Acta vol 1703 no 2 pp 93ndash109 2005

[51] J R Thompson J K Bell J Bratt G A Grant and LJ Banaszak ldquoVmax regulation through domain and subunitchanges The active form of phosphoglycerate dehydrogenaserdquoBiochemistry vol 44 no 15 pp 5763ndash5773 2005

[52] V Consalvi R Chiaraluce L Politi A Gambacorta MDe Rosa and R Scandurra ldquoGlutamate dehydrogenase fromthe thermoacidophilic archaebacterium Sulfolobus solfataricusrdquoEuropean Journal of Biochemistry vol 196 no 2 pp 459ndash4671991

[53] A J L Macario M Lange B K Ahring and E Conway DeMacario ldquoStress genes and proteins in the archaeardquo Microbiol-ogy and Molecular Biology Reviews vol 63 no 4 pp 923ndash9671999

[54] J AMaupin-FurlowM A Gil M A Humbard et al ldquoArchaealproteasomes and other regulatory proteasesrdquo Current Opinionin Microbiology vol 8 no 6 pp 720ndash728 2005

[55] N Sun F Beck R Wilhelm Knispel et al ldquoProteomics analysisof Thermoplasma acidophilum with a focus on protein com-plexesrdquoMolecular and Cellular Proteomics vol 6 no 3 pp 492ndash502 2007

[56] L S Madding J K Michel K R Shockley et al ldquoRole of the 1205731subunit in the function and stability of the 20S proteasome inthe hyperthermophilic Archaeon Pyrococcus furiosusrdquo Journalof Bacteriology vol 189 no 2 pp 583ndash590 2007

[57] R Shibata Y Bessho A Shinkai et al ldquoCrystal structureand RNA-binding analysis of the archaeal transcription factorNusArdquo Biochemical and Biophysical Research Communicationsvol 355 no 1 pp 122ndash128 2007

[58] W Bae B Xia M Inouye and K Severinov ldquoEscherichia coliCspA-family RNA chaperones are transcription antitermina-torsrdquo Proceedings of the National Academy of Sciences of theUnited States of America vol 97 no 14 pp 7784ndash7789 2000

[59] R J Ram N C VerBerkmoes M P Thelen et al ldquoMicrobi-ology community proteomics of a natural microbial biofilmrdquoScience vol 308 no 5730 pp 1915ndash1920 2005

[60] I Noel-Georis T Vallaeys R Chauvaux et al ldquoGlobal analysisof the Ralstonia metallidurans proteome prelude for the large-scale study of heavy metal responserdquo Proteomics vol 4 no 1pp 151ndash179 2004

16 Archaea

[61] M TMNovo A CDa Silva RMoreto et al ldquoThiobacillus fer-rooxidans response to copper and other heavy metals growthprotein synthesis and protein phosphorylationrdquo Antonie vanLeeuwenhoek vol 77 no 2 pp 187ndash195 2000

[62] Q She R K Singh F Confalonieri et al ldquoThe complete genomeof the crenarchaeon Sulfolobus solfataricus P2rdquo Proceedings ofthe National Academy of Sciences of the United States of Americavol 98 no 14 pp 7835ndash7840 2001

[63] R P Anderson and J R Roth ldquoTandem genetic duplications inphage and bacteriardquo Annual Review of Microbiology vol 31 pp473ndash505 1977

[64] D Gevers K Vandepoele C Simillion and Y Van De PeerldquoGene duplication and biased functional retention of paralogsin bacterial genomesrdquo Trends in Microbiology vol 12 no 4 pp148ndash154 2004

[65] A B Reams and E L Neidle ldquoSelection for gene clustering bytandemduplicationrdquoAnnual Review ofMicrobiology vol 58 pp119ndash142 2004

[66] L H Orellana and C A Jerez ldquoA genomic island providesAcidithiobacillus ferrooxidans ATCC 53993 additional copperresistance a possible competitive advantagerdquo Applied Microbi-ology and Biotechnology vol 92 no 4 pp 761ndash767 2011

[67] A B Brinkman T J G Ettema W M de Vos and J van derOost ldquoThe Lrp family of transcriptional regulatorsrdquo MolecularMicrobiology vol 48 no 2 pp 287ndash294 2003

[68] C Vollmecke S L Drees J Reimann S V Albers and MLubben ldquoThe ATPases CopA and CopB both contribute tocopper resistance of the thermoacidophilic archaeon SulfolobussolfataricusrdquoMicrobiology vol 158 no 6 pp 1622ndash1633 2012

[69] A Villafane Y Voskoboynik I Ruhl et al ldquoCopR of Sulfolobussolfataricus represents a novel class of archaeal-specific copper-responsive activators of transcriptionrdquo Microbiology vol 157no 10 pp 2808ndash2817 2011

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Anatomy Research International

PeptidesInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporation httpwwwhindawicom

International Journal of

Volume 2014

Zoology

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Molecular Biology International

GenomicsInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioinformaticsAdvances in

Marine BiologyJournal of

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Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Signal TransductionJournal of

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BioMed Research International

Evolutionary BiologyInternational Journal of

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Biochemistry Research International

ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

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International Journal of

Microbiology

2 Archaea

which includes genes encoding a new type of archaealtranscriptional regulator (CopT) a putative metal-bindingchaperone (CopM) and a putative Cu-transporting P-typeATPase (CopA) [10] The same Cu-resistance mechanismwas described in Sulfolobus solfataricus P2 and Ferroplasmaacidarmanus In both microorganisms the putative metalchaperones and the ATPase are cotranscribed and theirtranscriptional levels increase significantly in response to Cuexposure suggesting that the transport system is operatingfor Cu efflux [18 19] Recently it was described that thecopRTA operon from S solfataricus strain 982 (copTMAin S solfataricus P2) is cotranscribed at low levels fromthe copR promoter under all conditions whereas increasedtranscription from the copTA promoter took place in thepresence of Cu excess These authors proposed a model forCu homeostasis in Sulfolobus which relies on Cu efflux andsequestration [20]

In silico studies have further identified a CPx-ATPasewhich most likely mediates the efflux of heavy metalcations in the biomining archaeon Metallosphaera sedula[21] This putative protein has significant identity to a P-type ATPase from S solfataricus (CopA) [19] MoreoverM sedula contains ORFs with significant similarity to bothCopM (Msed0491) andCopT (Msed0492) from S solfataricus[21] Very recently Maezato et al [22] have reported a geneticapproach to investigate the specific relationship betweenmetal resistance and lithoautotrophy during biotransforma-tion of chalcopyrite by M sedula The functional role of itscopRTA operon was demonstrated by cross-species comple-mentation of a Cu-sensitive S solfataricus copRmutant [22]

Cadmium (Cd) is very toxic and probably carcinogenicat low concentrations However the biological effects of thismetal and the mechanism of its toxicity are not yet clearlyunderstood [23ndash26] In some neutrophilic microorganismsCd is taken up via the magnesium or manganese uptakesystems [23] Although the mechanisms in acidophiles havenot been elucidated putative Cd resistance operons in somesequenced genomes from acidophilic microorganisms havebeen identified The species with the highest homologyto the cadA motif were Acidithiobacillus ferrooxidans andThermoplasma spp These high similarities suggest that Cdexport may be a common resistance mechanism amongacidophiles [11] Recently a time-dependent transcriptomicanalysis using microarrays in the radioresistant archaeonThermococcus gammatolerans cells exposed to Cd showedthe induction of genes related to metal homeostasis drugdetoxification reoxidation of cofactors ATP production andDNA repair [27]

One alternative mechanism proposed for metal tolerancein microorganisms is the sequestration of metal cations byinorganic polyphosphates (polyP) [28] and at the same timethe intracellular cations concentration would regulate thehydrolysis of this polymer [29] S metallicus can toleratevery high concentrations of copper and accumulates highamounts of polyP granules [14 30] Furthermore the levels ofintracellular polyP are greatly decreased when this archaeonis either grown in 200mM Cu or shifted to 100mM Cu[14 30] An increase in exopolyphosphatase (PPX) activ-ity and Pi efflux due to the presence of Cu suggests a

metal tolerance mechanismmediated through polyP [14 30]Actual evidence suggests that polyPmay providemechanisticalternatives in tuning microbial fitness for the adaptationunder stressful environmental situations and may be of cru-cial relevance amongst extremophiles The genes involved inpolyP metabolism in Crenarchaeota have been only partiallyelucidated as long as a polyP synthase activity is still to bereported and characterized in this kingdom [15]

Thus far there are no studies on the prospective geneticand biochemical mechanisms that enable S metallicus tothrive in such high concentrations of Cu and other metalsUnderstanding these mechanisms could be particularly use-ful in potential improvement of the bioleaching microorgan-isms which could likely increase the efficiency of bioleachingprocesses in due course Since the genome of S metallicusis not currently available possible genes involved in Curesistance were searched by using a CODEHOP and ldquogenomewalkingrdquo approaches and the transcriptional expression ofthese isolated genes was assessed by real-time RT-PCRFurthermore a proteomic approach was used to identifypossible proteins involved in resistance to Cu and Cd inS metallicus To our knowledge this is the first paper thatshows the occurrence of a duplicated version of the cop genecluster in Archaea and gives insights into the molecular Cuand Cd resistance determinants in a thermophilic archaeonemployed for industrial biomining

2 Materials and Methods

21 Strains and Growth Conditions S metallicus DSM 6482was grown at 65∘C in medium 88 (Deutsche Sammlung vonMikroorganismen und Zellkulturen) containing 005 (wv)elemental sulfur and 002 (wv) yeast extract S solfataricusDSM 1617 was grown at 70∘C in medium 182 (DeutscheSammlung von Mikroorganismen und Zellkulturen) with01 (wv) yeast extract and with 01 (wv) Casamino acidsTo study Cd tolerance of S metallicus and S solfataricusthe microoganisms were grown in their respective mediaexcept that different concentrations of Cd (0005ndash5mM)were present initially as indicated in the correspondingexperiment

For differential expression assays S metallicus cells weregrown in the absence of Cu or Cd to the early stationaryphase and after removing the medium from the cells bycentrifugation they were then shifted to a new mediumcontaining 100ndash200mM CuSO

4(Cu from now on) or 1mM

CdSO4(Cd from now on) during 24 h After this time cells

were treated to obtain protein extracts Growth was moni-tored by measuring unstained cells numbers by means of aPetroff-Hausser chamber under a phase contrast microscope

22 Preparation of Protein Extracts from S metallicus Cellsfrom 800mL of a control culture grown to 108 cellsmL(early stationary phase) or cultures shifted to 100ndash200mMCu or 1mM were harvested by centrifugation at 7700 g for15min The pellets were washed with medium 88 to removethe sulfur Cells were then resuspended in 50mM Tris-HClpH 815 10mM EDTA 100 120583gmL PMSF buffer (20120583L per

Archaea 3

mg wet weight) frozen and sonicated six times for 30 seach time The lysates were centrifuged at 4300 g for 5minto eliminate cellular debris The protein concentration ofsupernatants was determined by the method of Bradford(Coomasie Plus protein assay reagent Pierce) Between 120and 500120583g of proteins from the protein extracts were mixedwith rehydration IEF buffer as described by Hatzimanikatiset al [31] and Choe and Lee [32] with some modificationsincluding urea 8M thiourea 2M CHAPS 2 (wv) Bio-Lyte3ndash10 027 (vv) Bio-Lyte 5ndash8 013 (vv) and bromophenolblue 0001 (wv) followed by incubation at 25∘C for 30minDTT (003 g) and sterile nanopure water were then added tocomplete a final volume of 300 120583L The samples were thenincubated at room temperature for 1 h

23 Isoelectric Focussing (IEF) Each sample (300 120583L) wasloaded onto the pH 3ndash10 (nonlinear) 17 cm IPG strips(Biorad) in the first dimension chamber and was incubatedat room temperature for 1 h Mineral oil (25mL) was thenadded to prevent evaporation of the sample and precipitationof urea and strips were passively rehydrated for 18 h Theisoelectric focusing was performed with the PROTEAN IEF(Biorad) using the following conditions 250V for 15min2000V for 2 h 8000V for 4 h 10000V for 11 h and 50Vfor 4 h reaching a total of 120000Vh

24 SDS-PAGE Prior to the second-dimensional elec-trophoresis strips were equilibrated as described by Hatz-imanikatis et al [31] with some modifications Strips wereincubated for 15min with a solution containing 6M urea156mM DTT 30 (vv) glycerol 2 (wv) SDS and 24mMTris-HCl pH 68 and subsequently for 15 more min in a solu-tion containing 6M urea 135mM iodoacetamide 30 (vv)glycerol 2 (pv) SDS and 24mM Tris-HCl pH 68 Finallystrips were incubated with electrophoresis buffer containing192mM glycine 1 (wv) SDS and 250mM Tris-HCl pH 83until the second-dimensional run SDS-PAGEwas performedusing the PROTEAN II xi cell as described by Laemmli [33]and gels consisted of 115 or 15 (wv) polyacrylamide Stripswere then overlaid onto the second-dimensional gels sealedwith 1 (wv) agarose in electrophoresis buffer containinga trace amount of bromophenol blue Electrophoresis wascarried out at constant 70V for 15 h All experiments wereperformed in triplicate Gels were stained with silver orCoomasie Blue G-250 as described by Shevchenko et al [34]and Giavalisco et al [35] respectively

25 Gels Analysis and Mass Spectrometry Gel images weredigitized by scanning (Epson) and analyzed with the Delta2D software (Decodon) to identify the spots differentiallyexpressed due to the presence of toxic metals An estimateof relative quantitative changes was made on the basis ofthe change in percent volume among silver stained gelsSpots of interest were recovered from Coomasie Blue G-250stained gels manually and were sent to electron spray tandemionizationmass spectrometric analysis (tandemMS-MS ESI-QUAD-TOF) The results obtained were analyzed with Mas-cot algorithm (httpwwwmatrixsciencecomindexhtml)

by using the MSMS Ion search and all genomes availableat databases were used as queries The entire MS analysiswas performed at the Cambridge Center for ProteomicsUniversity of Cambridge UK

26 CODEHOP-PCR CopA and CopM amino acid sequen-ces from S acidocaldarius S tokodaii and S solfataricuswereobtained fromNCBI (httpwwwncbinihgov) After align-ing the sequences by using the CLUSTAL X program toidentify areas of homology consensus-degenerate PCR pri-mers were designed according to the CODEHOP strategy[36 37] using the WWW access at httpblocksfhcrcorgblockscodehophtml Two regions of high sequence sim-ilarity were identified for both CopA and CopM seq-uences respectively (Figure S1 see Supplementry Materi-al available online at httpdxdoiorg1011552013289236)and used to design the consensus-degenerate hybrid oligonu-cleotide primers (Table 1) Consensus-degenerate hybridoligonucleotide primers were designed for the N- and C-terminus of CopA while a pair of degenerate hybrid oligonu-cleotide primers was designed for CopM (Figure S1)

Amplification reactions contained 1x thermophilic DNApolymerase buffer with 2mMMgCl

2 02mMdNTPs 05 120583M

of each primer 5U Taq DNA polymerase 40 ng of S metal-licus genomic DNA as a template and water to a final volumeof 50120583L The thermal cycling conditions were 3min at 95∘Cfollowing 30 cycles of 95∘C for 30 s 50∘C for 30 s and 72∘Cfor 1min 1 final additional cycle at 72∘C for 10min Productsof amplification were applied onto 10 (wv) agarose gelsand main amplification bands were excised purified andTA-cloned into pCR21-TOPO (Invitrogen) vector and finallysequenced

27 GenomeWalking Experiments Genome walking strategywas performed as described by Acevedo et al [38] Thusa double-stranded oligo-cassette AdaptT adapter was con-structed by annealing of the two unphosphorylated primersAdaptF (51015840-CTAGGCCACGCGTCGACTAGTACTA-G-CTT-31015840) and AdaptR (51015840-AGCTAGTACTAGTCGACGCG-TGGCCTAG-31015840) Annealing was performed by heating theprimers (10 120583M) in a boiling water bath for 5min and thenslowly cooling to room temperature

Six different DNA libraries were constructed by meansof digesting 1120583g of S metallicus genomic DNA with the fol-lowing restriction enzymes HindIII BamHI EcoRI EcoRVNcoI and PstI respectively DNA digestion reactions werecarried out using 10U of restriction enzyme 2120583L of thecorresponding enzyme reaction buffer in 20120583L of totalreaction volume The reaction mix was incubated at 16∘Cduring 16 h To complete the 31015840 recessive end of the DNAfragments and to add a 31015840 overhanging adenine 500 ng ofthe digested and purified DNA were incubated with 5U ofTaq DNA polymerase 1 120583L of 10mM dNTPs mix and 5 120583Lof 10x thermophilic DNA polymerase buffer in 50120583L totalvolume at 70∘C for 45min Seven 120583L of thismixture was thenincubatedwith 15 pmol of AdaptT oligo-cassette 1 U T4DNAligase (Promega) and 2120583L of 5x ligase buffer in a total volume

4 Archaea

Table 1 Oligonucleotides

Name Sequence DescriptioncopA cdegF 51015840-gatgtagtaatagtaaaaactggagaaataataccngcngaygg CODEHOP-PCRcopA cdegR 51015840-tcatcagcaaaattagaagaatctccngtngcdat CODEHOP-PCRcopT cdegF1 51015840-ctcaaatagaatataaagtattacaaatgttaaaagargaywsnmg CODEHOP-PCRcopT cdegR1 51015840-ggattaccatttatttcatttccacartartcrca CODEHOP-PCRcopT cdegR2 51015840-cttatcatattcataaatcttccatctattaatttrtarcaytc CODEHOP-PCRcopT degF1 51015840-gartgytayaarctnat DOP-PCRcopT degR1 51015840-atnagyttrtarcayct DOP-PCRcopM degF 51015840-gayccngtntgyggnatgga DOP-PCRcopM degR 51015840-ccnggnttyccntacgg DOP-PCRAdaptF2 51015840-cacgcgtcgactagtactagctt Genome WalkingSP1copA1 31015840 51015840-aaggatgagggggaccttatgg GenomeWalkingSP2copA1 31015840 51015840-ggagataagaaatggggtaaaagag GenomeWalkingSP1copA1 51015840 51015840-tgataccatcatggaacctgtcag GenomeWalkingSP2copA1 51015840 51015840-tcctccacaatcccatccgctg GenomeWalkingSP1copA1 51015840 2 51015840-gattgtagctaagttaacctcggcctcg GenomeWalkingSP2copA1 51015840 2 51015840-cttctcaccctcagtctggttgg GenomeWalkingSP1copA2 31015840 51015840-gaaagaggaatatatgcaagggtaaacgg GenomeWalkingSP2copA2 31015840 51015840-gtgttaatgggagagctggaggg GenomeWalkingSP1copA2 51015840 51015840-cttctctgtggcaacatcataaccagcc GenomeWalkingSP2copA2 51015840 51015840-acgcatgtggcgcaatgcattcc GenomeWalkingSP1copT1 51015840 51015840-cattcctcgcaccagcttgcacactctc GenomeWalkingSP1copT2 51015840 51015840-cctatgaatactagatcttttccctgaac GenomeWalkingSP2copT2 51015840 51015840-aacagcttataacactcgtcactttggc GenomeWalkingcopM2 RT F 51015840-gatgaaaaaagccaatataagac RT-PCRcopA2 Rv 51015840-gaacactaactaacatcgcc RT-PCRcopM1 RT Fw 51015840-ctatcgtttttgttccgaagcttg RT-PCRcopA1 Rv 51015840-cagcagcaagaacagagacgcc RT-PCRlowastSM16Sf 51015840-acgctctaaaaaggcgtgggaata RT-qPCRlowastSM16Sr 51015840-ttgagctcggggtctttaagcagtg RT-qPCRcopA1Sm qRT F1 51015840-gctaaggtaatagagagcgg RT-qPCRcopA1Sm qRT R1 51015840-tgaacaggaatggacagg RT-qPCRcopA2Sm qRT F 51015840-tgtgcttgtctccttagcgt RT-qPCRcopA2Sm qRT R 51015840-actcttccgtctttcggagt RT-qPCRcopT1Sm qRT F 51015840-tgtaggagagtgtgcaagct RT-qPCRlcopT1Sm qRT R 51015840-tcgcaagtgagggttatggt RT-qPCRcopT2Sm qRT F 51015840-gtgttacggagcttgca RT-qPCRcopT2Sm qRT R 51015840-acactcgtcactttggc RT-qPCRAll oligonucleotides were synthesized by Invitrogen lowastOligonucleotides designed by Bathe and Norris [39]

of 10 120583L The ligation reaction was incubated at 16∘C during16 h

Two consecutive amplification reactions were then per-formedThefirst PCR reactionwas donewith 1x Elongasemixbuffer 19mM MgCl

2 02mM dNTPs 05120583M first specific

primer (SP1) (designed from the known sequence of thetarget gene a forward primer was used to amplify the 31015840end a reverse primer for 51015840 end amplification) 5 120583L of theligatedDNAdiluted 10-fold and 1 120583L of Elongase (Invitrogen)and water to a final volume of 50120583L The thermal cyclingconditions were as follows 1 cycle at 94∘C for 1min 20cycles of 94∘C for 32 s and 68∘C for 5min and one final

additional cycle at 70∘C for 7min The PCR product wasdiluted 10 fold and 3 120583L were used as a DNA template fora second PCR which was performed using the same con-ditions as the first PCR with 05 120583M second specific primer(SP2) and 02 120583Moligo-cassette-specific primer AdaptF2 (51015840-CACGCGTCGACTAGTACTAGCTT-31015840) and 1 120583L of Elon-gase The thermal cycling conditions were 1 cycle at 94∘C for1min 35 cycles of 94∘C for 32 s and 68∘C for 5min and 1final additional cycle at 70∘C for 7min PCR products wereexcised purified TA-cloned into pCR21-TOPO (Invitrogen)vector and finally sequenced

Archaea 5

28 Isolation of Total RNA Cell pellets (10mg wet weight)were collected and diluted in 60 120583L TEN buffer (20mMTris-HCl pH 80 1mM EDTA 100mM NaCl) followedby addition of 60120583L TENST buffer (20mM Tris-HCl pH80 1mM EDTA 100mM NaCl 16 Na-lauroyl sarcosineand 012 Triton X-100) This suspension was incubated atroom temperature for 15min to allow cell lysis Total RNAwas then extracted using the TRIzol reagent (Invitrogen)as recommended by the manufacturer DNA contaminationin RNA preparations was removed by DNase I treatment(Roche) following the manufacturerrsquos instructions RNAwas then purified precipitated and finally resuspended indiethylpyrocarbonate (DEPC)-treated water Total RNA con-centrations were estimated by spectrophotometric measure-ments (OD

260) and its quality was evaluated by determining

the ratio of absorption at 260 nm and 280 nm which waswithin the preferred range of 18ndash21

29 Northern Blotting For differential gene expression byNorthern blotting experiments the media were supple-mented with either different concentrations of Cu (5ndash50mM) Cd (5mM) NiSO

4(15mM) ZnSO

4(50mM) or

Ag2SO4(008mM) respectively as indicated For some

experiments elemental sulfur was replaced by a chalcopyrite(CuFeS

2) concentrate which was used as the only energy

source at 1 (wv)Total RNA (5 120583g) was separated by electrophoresis

on a 1 formaldehyde agarose gel followed by blottingonto Hybond-N nylon membranes (Amersham PharmaciaBiotech Bucking-hamshire UK) Hybridization was con-ducted as described by Sambrook et al [40] DNA probeswere labeled by randomprimerDNA labeling kit (Fermentas)using [120572-32 p] dCTP The hybridization signal was detectedand analyzed by using the molecular Imager FX system andQuantity One software The primer sequences for amplifica-tion of these gene probes are listed in Table 1

210 Cotranscriptional Analysis by RT-PCR To study theexpression of adjacent genes copM and copA cDNA wassynthesized by using 08120583g of total RNA from a S metallicusculture grown in the presence of 20mM Cu and a reverseprimer hybridizing to copA sequence A forward primerannealing on copM sequence was used for the PCR reactions(Table 1) PCR amplifications were performed with 1 120583L ofa 110 dilution of the cDNA and 25 pmol of each primerAmplification conditions included an initial 3min of denat-uration at 95∘C followed by 35 cycles of 30 s at 95∘C 30 sat 55∘C and 15min at 72∘C and finished by 10min at 72∘CA reverse transcriptase reaction without enzyme was carriedout in order to exclude amplification due to genomic DNAcontamination

211 Quantitative RT-PCR Single stranded cDNA was syn-thesized from 08 ng of DNA-free RNA samples usingrandom hexamers (Fermentas) and ImProm-II reversetranscription system (Promega) following manufacturerrsquosinstructions The software tool IDT Scitools (IntegratedDNA Technologies) was used to design primers producing

amplicons of 150ndash200 bp (Table 1) qPCRwas performedwith2 120583L of 1 10 diluted cDNA samples 125 120583L 2x QuantiFastSYBRGreenPCRMasterMix (Quiagen) 1 120583Mof each primerand water to a final volume of 25120583L The efficiency of eachprimer pair was calculated from the average slope of a linearregression curve which resulted from qPCRs using a 10-folddilution series (10 pgndash10 ng) of S metallicus chromosomalDNA as template Cq values (quantification cycle) wereautomatically determined by Real-Time Rotor-gene 6000PCR software (Corbett Life Sciences) after 40 cycles Cqvalues of each transcript of interest was standardized to theCq value of the 16S rRNA gene [39] At least 3 biologicalreplicates of each assessed condition and 2 technical replicatesper qPCR reaction were performed

3 Results and Discussion

31 Tolerance of S metallicus and S solfataricus to Cu and CdIn a previous work it was determined that S metallicus wasable to tolerate high Cu concentrations While the presenceof 100mM Cu did not affect S metallicus growth kinetics adecreased in cell biomass of only 30 was observed whenexposed to 200mM Cu [30] The high tolerance to Cu hasbeen described in other acidophilic Bacteria and Archaeacompared mostly with neutrophilic microorganisms [11 14]Here this analysis was further extended to characterize theresponse of S metallicus to Cd Thus it was determined thatS metallicus growth was not affected in the presence of either05 or 1mM Cd when compared with the control conditionin the absence of the metal (Figure 1(a)) In addition whenS metallicus was challenged with 2 and 3mM Cd it wasobserved that at the late exponential growth phase the cellnumbers decreased by 30 and 50 respectively (Figure 1(a))Moreover growth of S metallicuswas completely inhibited inthe presence of 5mM Cd (Figure 1(a)) On the other hand Ssolfataricuswas not able to grow at Cd concentrations greaterthan 005mM (Figure 1(b)) At 001mM Cd S solfataricuscell numbers decreased around 35 (Figure 1(b)) Theseresults are in agreement with previous reports in which Ssolfataricus was shown to be able to grow in up to 001mMCd [41] Other acidophilic archaeons involved in bioleachingprocesses such as Metallosphaera sedula and Ferroplasmaacidarmanus were found to be able to tolerate up to 09and 9mM Cd respectively [18 42] The minimal inhibitoryconcentration (MIC) to Cd has been described to be nothigher than 1mM for several thermophilic and neutrophilicThermococcus species [43] Recently it was reported that themost radioresistant archaeon Thermococcus gammatoleransstands Cd concentrations with an MIC of 2mM [27] Todate despite the heavy metals tolerance showed by someof these microorganisms strategies to withstand stress fromtransition metals have been most widely studied only inhaloarchaea [44]

32 Effect of Cu andCd on theGlobal Proteome of SmetallicusThe proteomic response to Cu and Cd was analyzed toidentify proteins which could be involved in heavy metalsresistance in S metallicus Early stationary phase growing

6 Archaea

14

12

10

8

6

4

2

00 2 4 6 8 10 12

Time (days)

107timescellsm

L

(a)

120

100

80

60

40

20

00 2 4 6 8 10

Time (days)

107timescellsm

L

(b)

Figure 1 Growth of S metallicus (a) and S solfataricus (b) in the presence of Cd Cells were inoculated in their respective growth media inabsence of added Cd (∙) or were supplemented with 0005mM () 001mM () 005mM (diams) 01mM () 05mM () 1mM () 2mM() 3mM (times) or 5mM (+) Cd and cells were counted daily

100

7060

50

30

20

3 10MW

33 31

32

A

B

C

20

10

(a)

100

7060

50

30

20

12

3 10MW

204

21

33 31

32

A

B

C

15

93

10

27 28

23 24

29

25 2630

1614 16

13

52

7

2219

8

20

10

11

(b)

Figure 2 Changes in the proteome of S metallicus grown in the presence of Cu Cells were incubated in the absence of any added metal(a) or in the presence of 100mM Cu (b) Arrows indicate the spots that were downregulated (a) or upregulated (b) in the presence of CuNumbers indicate the spots with increased intensity in cells treated with copper The dashed boxes are shown as enlarged areas that includesome proteins upregulated in the presence of Cu and Cd (Figure 3) The bottom panels show low molecular weight proteins upregulated inthe presence of Cu separated by using a 15 polyacrylamide gel in the second dimension

cells were untreated or treated with either 100 or 200mM Cuor 1mM Cd during 24 h and analyzed by comparative two-dimensional gel electrophoresis as indicated in experimentalprocedures Twenty-three proteins were found to be down-regulated after 100mMCu treatment (Figure 2(a)) A similar

pattern was obtained at 200mM Cu (not shown) Further-more by using the Delta 2D software (Decodon) eleven ofthese proteins were found to be completely absent in the gelsand more than 8 proteins decreased their intensity in therange from 15-to 5-fold compared with control cells grown

Archaea 7

13 13

11

13

1

Control Copper Cadmium

(a)

99 9

333

(b)

3030

30

2020

20 212121

(c)

Figure 3 Comparison of selected proteins of S metallicus cells treated with Cu or Cd Protein extracts were obtained from cells treatedwithout metals and with 100mM Cu or 1mM Cd for 24 h and the proteins were separated by 2D-PAGE The segments (a) (b) and (c) arethe enlarged dashed boxes in Figure 2 under the three conditions indicated Numbers show some of the spots with increased intensity in cellstreated with Cu or Cd

in the absence of Cu (Figure 2(a)) Therefore these resultsshow that a number of proteins became non-detectable ordecreased their levels when S metallicus faced Cu This kindof response was also seen when the microorganism wasexposed to Cd (data not shown) This behavior has alsobeen observed in similar studies where F acidarmanus waschallenged by either As(III) or Cu [16 18] In addition theexpression of 30 other proteins was found to be upregulatedafter Cu treatment (100 or 200mM) (Figure 2(b)) Most ofthese proteins could be identified only in the condition withCu and spots 5 13 and 15 were induced 26 34 and 55-fold respectively compared to cells without Cu treatmentMoreover 3 proteins of lowmolecular weight (spots 31 3 and33) that increased their expression when cells were exposedto 100mM Cu were also identified (Figure 2 bottom panels)These proteins were only detected when 15 polyacrylamidewas used during the second-dimensional separation (SDS-PAGE) On the other hand a large decrease in overallexpression of proteins after 1mMCd treatment was observed(not shown) Interestingly 10 out of the 13 proteins identifiedas up-regulated when cells were exposed to Cd and alsoshowed increased levels after Cu treatment Some of them areshown in Figure 3

33 Identification of Proteins Upregulated in Cells Treated withCu and Cd in S metallicus A total of 18 S metallicus proteinswhose levels were found to be up-regulated in responseto Cu were analyzed by mass spectrometry The identifiedproteins included functions related to the production andtransport of energy biosynthesis of amino acids stressresponses and transcription regulation (Table 2) Amongstthe proteins related to production and transport of energya putative ATP synthase subunit B (spot 23) that has beendescribed as playing a fundamental role in ATP synthesiswas identified (Table 2) When cells are subjected to somestressing conditions such as the presence of heavy metalsa greater cellular demand for energy has been reported tooccur [18] Most likely this phenomenon might be due toATP-driven Cu transport via ATPases that have a substantialinterplay during metal detoxification [45] Furthermore theATP synthase subunit B was also identified as up-regulatedwhen S metallicus was treated with Cd (Table 2)

Two other proteins corresponded to putative oxidoreduc-tases such as ferredoxin oxidoreductase (spot 3) and alcoholdehydrogenase (spot 4) that have been generally involvedin electron transporter chains and use NAD+ as an electronacceptor The levels of ferredoxin oxidoreductase were also

8 Archaea

Table 2 Proteins upregulated in S metallicus cells exposed to 100mM Cu or 1mM Cd

Spot Molecular weight (kDa) Cu induction levels Putative function Microorganism related Accesion number1dagger 266 infin Proteasome subunit S solfataricus AAK410343dagger 701 infin Ferredoxin oxidoreductase S solfataricus AAK429264dagger 429 infin Alcohol dehydrogenase S solfataricus AAK431545 35 26 Hypothetical protein S acidocaldarius YP 2549766 27 infin Hypothetical protein P aerophilum NP 5598899dagger 57 infin Phosphoglycerate dehydrogenase M thermautotrophicus AAB8546613dagger 27 34 Unknown mdash mdash14 28 infin Unknown mdash mdash15 35 55 Unknown mdash mdash20dagger 461 infin Glutamate dehydrogenase S solfataricus AAK4223021dagger 45 infin Hypothetical protein M acetivorans NP 618380122 49 infin Hypothetical protein P torridus YP 02280723dagger 51 infin ATP synthase subunit B S solfataricus AAK4088024 585 infin HSP60 subunit S shibatae AAG37273130dagger 48 infin Unknown mdash mdash31 16 ND Unknown mdash mdash32 144 ND Putative transcription regulator S solfataricus AAK4041333 138 ND Transcription factor nusA S solfataricus AAK40563Spots refer to those numbered in Figure 2 Proteins in Figure 2 which are not included in this table were not subjected to sequencing due to their low intensitiesin the gelsdaggerSpot up-regulated in cells exposed to Cu and CdND not determinedInfinity symbol indicates that proteins were expressed only in presence of copper or cadmiumM thermoautotrophicus Methanobacterium thermoautotrophicus M acetivorans Methanosarcina acetivorans S shibatae Sulfolobus shibatae P torridusPicrophilus torridus P aerophilum Pyrobaculum aerophilum S acidocaldarius Sulfolobus acidocaldarius

up-regulated in response to Cd (Table 2) Several previ-ous reports have suggested that oxidoreductases contributeto an oxidative protection in both Bacteria and Archaeain response to heavy metals [46ndash48] In the neutrophilicmicroorganisms Escherichia coli and Staphylococcus aureusit has been described that oxidases and dehydrogenasescontribute to oxidative protection Cu homeostasis and stressresponses [46 47] Furthermore some proteins involved inoxidative damage repair such as NADH-dependent oxidasesand thioredoxin reductases were expressed in cells of Facidarmanus exposed to As(III) [16] The expression of thisgroup of proteins has also been observed when the samemicroorganism was exposed to Cu [18] Therefore oxidore-ductases play an important role against oxidative stress andmay eliminate reactive oxygen species which constitute themajor component of the stress caused by transition metals[44 49 50]

The enzymes related to biosynthesis of amino acids werefound as commonly up-regulated either in response to Cu orCdThey corresponded to a phosphoglycerate dehydrogenase(spot 9) and a glutamate dehydrogenase (spot 20) Theformer one catalyzes the NAD+-dependent oxidation of 3-phosphoglycerate into 3-phosphohydroxypyruvate a branchpoint from the glycolytic pathway and the initial reactionin L-serine biosynthesis [51] Glutamate dehydrogenase cat-alyzes the oxidative deamination of glutamate to produce 2-oxoglutarate and ammonia with reduction of NAD+ [52]Proteins involved in amino acids synthesis were expressed

in cells of F acidarmanus exposed to As(III) [16] Moreoverincreased levels of proteins involved in the biosynthesisof sulfur-containing amino acids have been observed inSaccharomyces cerevisiae cells exposed to Cd [24] suggestingthat these proteins might also have a role in the cellularresponse to adverse conditions

Two proteins showed identity to components of the stressresponse mechanism such as one subunit of the HSP60chaperonin (spot 24) and one subunit of the proteasome (spot1) In Archaea the stress protein HSP60 is directly involvedin protein folding processes [53] Proteomic analysis of Facidarmanus cells exposed to As(III) and Cd revealed theexpression of proteins involved in protein folding and DNArepair including HSP60 chaperonin and DnaK heat-shockprotein (HSP70) thereby the authors suggested that thismicroorganismusesmultiplemechanisms to resist high levelsof Cu [16 18] On the other hand proteasomes are describedas nonspecific proteolytic nanomachines associated withprotein catabolism Proteasomes are known to be closelyinvolved in maintaining protein quality control by degradingmiss folded and denatured proteins in response to cell stressin all three domains of life [54] A proteomic analysis inthe thermoacidophilic archaeon Thermoplasma acidophilumshowed that proteasomes and chaperone-related proteinswere highly induced against stress conditions indicatinga high turnover rate of proteins [55] In P furiosus theexpression of the 1205731 subunit of the proteasome was inducedin cells subjected to heat shock stress [56] Additionally the

Archaea 9

150pb

(a)

1016 pb

(b)

2000 pb

(c)

Figure 4 CODEHOP-based PCR for the amplification of the putative genes copA and copM from S metallicus Amplification products(a) using degenerate PCR primers (DOP) copM degF and copM degR designed from amino acid sequence conserved blocks of SulfolobalesCopM proteins (b) using primers copA cdegF and copA cdegR designed by CODEHOP strategy for amplification of a copA-like gene (c)Primers copM degF and copA cdegRwere assesed for the amplification of a copMA-likeDNA regionThose primerswere testedwith genomicS metallicus DNA samples Amplicons expected sizes were as follows ca 150 bp for copM ca 950 bp for copA and ca 2000 bp for copMAPCR products were excised and cloned for later sequencing

proteasome subunit was also found to be up-regulated whenS metallicus was subjected to Cd stress (Table 2)

The transcriptional regulation-related proteins corre-sponded to a putative transcription regulator (spot 32) andthe transcription factor NusA (spot 33) involved in theintrinsic termination of transcription [57] NusA has beenalso reported to display important functions in the cellularresponse to adverse factors [58] The expression of twotranscriptional regulators related to amino acids biosynthesisand metal-dependent genetic repression were found to beinduced in P furiosus cells subjected to oxidative stress[48] Moreover the expression of several genes encodingpredicted transcriptional regulators were induced followingCd exposure in T gammatolerans cells [27]

Spots 5 6 13 14 15 21 22 30 and 31 (Table 2) did notshow significant scores with any protein sequences currentlyavailable in the data bases Interestingly spots 2 4 9 13 21and 30 were found to be commonly upregulated when Smetallicus cells were treated with either Cu or Cd (Figure 3Table 2)

The results presented here are consistent with previ-ous work describing global protein expression profiles inresponse to heavy metals as reported for F acidarmanus [1618] microbial-biofilm communities in an acid mine drainagesite [59] and other prokaryotic and eukaryotic microorgan-isms [24 60]Therefore a coordinated expression of differentgroups of genes suggests the existence of regulatory networkssuch as stress response mechanisms and respiratory chainadjustments to cope with the presence of heavy metal ions[61]

34 Cu-Resistance (Cop) Genes Cluster Is Duplicated in Smetallicus Genome A Cu-resistance (cop) locus has beendescribed to be highly conserved in archaeal genomeswhich consists of genes encoding a new type of archaealtranscriptional regulator (CopT) a metal-binding chaperone(CopM) and a Cu-transporting P-type ATPase (CopA) [19]Since the genome sequence of Smetallicus is not yet availablewe searched for the presence of cop genes by using consen-sus degenerate hybrid oligonucleotide primers-based PCR(CODEHOP-PCR) CopA and CopM amino acid sequencealignments from S acidocaldarius S tokodaii and S solfa-taricus were used to identify conserved sequence blocks andtherefore to design either CODEHOP or DOP (degeneratehybrid oligonucleotide) primers (Figure S1 Table 1) TherebyPCR assays using S metallicus genomic DNA as templateyielded amplicons of ca 150 bp and ca 950 bp for copMand copA CODEHOP pair primers respectively (Figures4(a) 4(b)) As estimated from primer pairrsquos position onamino acid sequence alignments the obtained PCR productscorresponded to the expected sizes suggesting the presenceof copM- and copA-like genes in S metallicus genome Inorder to determine the identity of the amplified DNA frag-ments they were TA-cloned and sequenced Blastx sequenceanalysis showed that the 150 bp DNA fragment coded for anincomplete ORF sharing 63 homology with S solfataricusCopM (SSO10823) whereas the analysis of the 950 pb DNAfragment yielded a partial ORF sharing 54 homology withS solfataricus CopA protein (SSO2651)

The genomic organization of the putative cop genes in Smetallicus was analyzed to find out its similarity with those

10 Archaea

Table 3 Sequence identity comparison of S metallicus Cop proteins to those in other Sulfolobales

S metallicus protein Identity to homologue in other SulfolobalesS solfataricus P2 S acidocaldarius S tokodaii M sedula

CopA1 51 43 44 45CopA2 88 46 49 50CopM1 63 69 71 71CopM2 89 72 70 77CopT1 36 27 30 32CopT2lowast 90 52 52 53Boldface type indicates to which organism the S metallicus homologue shows the highest sequence identitylowastRefers to the analysis obtained using the uncompleted amino acid sequence of CopT2 which represents sim90 in length when compared with S solfataricusCopT sequence

previously described in S solfataricus P2 and F acidarmanuswhere the cop cluster consists of tandem-orientated genesas copTMA [18 19] Thus using copM degF (forward) andcopA cdegR (reverse) primers we attempted to amplify aputative copMA DNA region This PCR yielded a product ofca 2000 pb corresponding to the expected size and resultedin 2 ORFs showing high sequence homology with CopMand CopA from S solfataricus (Figure 4(c)) Interestinglywhen aligning this copMA sequence with the copA and copMsequences obtained previously they were not identical whichstrongly indicated the presence of duplicated putative copgenes encoding for both Cu-ATPases (copA1 and copA2) andmetallochaperones (copM1 and copM2) in the S metallicusgenome

To isolate the entire cop gene sequences the genomewalking method described by Acevedo et al [38] was usedSix different DNA libraries were constructed from S metalli-cus genomic DNA digested with several restriction enzymesincluding 51015840 overhang and blunt end restriction enzymesThe DNA libraries were used as templates in 2 successivePCR reactions From the previously known sequences twospecific sense primers for the 31015840 end amplification and twospecific antisense primers for the 51015840 end amplification weredesigned (abbreviated SP1 and SP2) Therefore the 51015840 and 31015840ends of the putative genes copA1 and copA2 that had beenpartially identified through the genome walking techniquewere amplified

By overlapping the sequence isolated by degenerate PCRand the lateral sequences obtained by genome walking(Figure S2) it was possible to complete the whole nucleotidesequence of the putative gene copA1 Additionally it waspossible to confirm the presence of the upstream copM1 geneanddetermine the occurrence of a partial copT1 gene tandem-orientated upstream of copM1 (Figure 5)Through additionalgenome walking experiments the partial copT1 sequencecould be further completedThe samewas truewhen isolatingthe whole nucleotide sequence of the putative gene copA2as the presence of copM2 and copT2 genes were confirmedupstream of copA2 However it was not possible to completethe whole 51015840 end of the putative gene copT2 as its determinedlength corresponded to only 90 of the full length whencompared with S solfataricus copT gene (Figure 5)

In conclusion degenerate PCR together with genomewalking experiments allowed to describe the occurrence inS metallicus genome of 2 cop genes loci (named as locus cop1and locus cop2) coding for paralogous genes whose productsmay be involved in Cu-resistance (Figure 5) Although somearchaeal genomes exhibit two copies of putative Cu-P-typeATPases as described for S solfataricus [62] the genomicarrangement displayed as a cop gene cluster (copTMA) hasbeen reported to be represented in only one copy for manyarchaeal genomes [10 19 21] Moreover we further updatedthis analysis by searching for duplications of the cop genescluster in all available archaeal genome sequences up to date(October 2012) retrieving only one cop locus in each caseThereby we propose that the discovery of this cop gene clusterduplication in the genome of S metallicus constitutes so faran unprecedented feature for a representative of the Archaeadomain and might contribute to its high metal resistance

In this context it has been widely reported that increasedgene copy number can increase gene expression allowingprokaryotic to thrive under growth limiting conditions [1363ndash68] However alongside the augmented gene copy onecannot exclude that the cop operon duplication in S metal-licus may offer among others a wider repertoire of Cu-ATPases which might differ in terms of metal affinitiesandor specificity efflux rates andor differences in theirabundance regulation

Throughout amino acid sequence analysis it was furtherdetermined that each polypeptide encoded by S metallicuswith the exception of CopT1 showed to be highly homol-ogous to their orthologous counterparts encoded by othersSulfolobales (Table 3) Moreover the 3 ORFs encoded by thelocus cop2 shared about 90 identity with the correspondingS solfataricus orthologous peptides (Table 3) S metallicuscop genes products showed characteristic domains referredto as critical for their respective proposed biological activitiesS metallicus paralogous gene products CopA1 (749 aa1015840)and CopA2 (747 aa1015840) shared 513 of identity These twoputative Cu-ATPases (CopA1 and CopA2) contain the aminoacid sequence motif CPCALGLA which has been proposedto confer Cu-transporting specificity in CPx-ATPases [48]and has been also found in other CopA-like proteins frombiomining Bacteria and Archaea [14]

Archaea 11

HindIII HindIII HindIII

HindIII

CopT1Locus cop1 M1

EcoRIEcoRV

513 bp 219 bp 2250 bp

CopA1

(a)

Locus cop2 M2

EcoRI

CopA2

453 bp 171 bp 2244 bp

CopT2

PstIHindIII HindIII HindIII

(b)

Figure 5 Schematic representation of the two cop gene clusters isolated from S metallicus genome Each cluster codes for anArchaea-specifictranscriptional regulator (copT) a metallochaperone (copM) and a P-type Cu-exporting ATPase (copA) Lengths of each gene are indicatedLocus cop1 corresponds to 4346 sequenced base pairs and shows an intergenic region copT1-M1 of 232 base pairs Locus cop2 correspondsto 3435 base pairs and shows 89 base pairs copT2-M2 intergenic region Genes copM2 and copA2 overlapped in 11 base pairs copT2 was notfully isolated

Control CdSO4 Ag2SO4 NiSO4 ZnSO4 CuSO4

kb3kb2kb1

02 kb

(a)

kb3kb2kb1

02 kb

(b)

(c)

Figure 6 Northern blot analysis to determine the expression of S metallicus copA1 (a) and copA2 (b) putative genes in response to variousmetals S metallicus total RNAwas extracted from cultures grown either in the absence (control) or the presence of CuSO

4(50mM) Ag

2SO4

(008mM) NiSO4(15mM) ZnSO

4(50mM) and CdSO

4(2mM) P32-radioactivelly labelled DNA fragments annealing to copA1 and copA2

sequences respectively were used as probes (c) shows rRNAs as total RNA loading control

On the other hand CopM1 (71 aa) and CopM2 (56 aa)sharing 66 of identity present the proposed metal bindingdomain TRASH (trafficking resistance and sensing of heavymetals) [10] Additionally when analyzing the putative tran-scriptional regulators the presence of a C-terminal TRASHdomain in both CopT1 and CopT2 was also found CopT1sequence also contains an entire N-terminal helix-turn-helix (HTH) motif (not shown) that resembles the DNA-bindingmotifs of prokaryotic transcriptional regulators suchas Lrp-like proteins [67] The HTH motif was only partiallyidentified in CopT2 since the 51015840 region of copT1 gene has notbeen yet fully isolated

35 Transcriptional Analysis of S metallicus Cop Genes Toget insight into the role of the two cop loci the expressionof the corresponding genes was analyzed under variousconditions S metallicus was first grown in presence ofdifferent heavy metals (Cu Zn Cd Ni o Ag) at a givenconcentration that did not affect growth kinetics [30 42]Subsequently total RNA was isolated from late exponentiallygrown cultures and Northern blot experiments were carriedout in order to determine the expression of both copA1 andcopA2 genes As depicted in Figure 6 transcription of bothcopA1 and copA2messengers were specifically induced in thepresence of Cu and Cd ions This gene expression pattern

12 Archaea

954pb

M1 CopA1

(a)

1438 pb

M2 CopA2

(b)

1000 pb

800pb

+ minus

(c)

+ minus

1500 pb

1000 pb

(d)

Figure 7 Cotranscription analysis of copMA1 and copMA2 genes cDNA was synthesized with a reverse primer (dotted arrows) hybridizingtoward the 31015840 end of either copA1 (a) or copA2 (b) S metallicus total RNA was extracted at the late exponential phase from a culture growingin presence of 20mMCu PCR amplifications were carried out with these cDNAs and each corresponding primer pair (black arrows) as listedin Table 1 (c) and (d) show RT-PCR products obtained for the copMA1 and copMA2 intergenic regions respectively Reverse transcriptasereactions with (+) and without (minus) the Improm II reverse transcriptase enzyme were carried out in order to exclude amplification due togenomic DNA contamination Expected sizes (in base pairs) for the corresponding PCR products are given in (a) and (b)

is in good agreement with what has been described for Ssolfataricus where the bicistronic copMA transcript levelswere also found to be up-regulated in response to both Cuand Cd [19]

Northern blot analysis showed that the sizes seen for bothcopA transcripts (sim25 Kb) did not correspond exactly withthe individual genes length (each of about 225Kb) (Figure 6)This strongly suggested that the transcripts could also includethe respective copM gene located upstream of copA in eachcop loci To confirm this assumption a co-transcriptionexperiment was done (Figure 7) in which the cDNAs wereobtained by using RNA extracted from a culture grownin the presence of 20mM Cu and using a reverse primerhybridizing with copA1 (Figure 7(a)) or copA2 (Figure 7(b))gene sequence respectively PCR amplifications were carriedout by using the corresponding cDNAs as templates and eachpair of primers lying in adjacent genes (copM) The presenceof an amplicon of the expected size in each case indicatedthe adjacent genes were part of polycistronic messengers(Figure 7) These results clearly show that in S metallicuseach couple of copMA genes was expressed in the form oftranscriptional units as reported in F acidarmanus and Ssolfataricus [18 19] The coexpression of gene pairrsquos copMAmay suggest a coordinated and dependent function for therespective encoded proteins In the bacterium E hirae themetallochaperone CopZ (CopM in S metallicus) fulfills apivotal role in the mechanism of Cu homeostasis [45] Thusit was shown that this protein interacts directly with the Cu-ATPase CopB (CopA in S metallicus) handing the Cu forsubsequent removal In this context one might expect thatproteins CopM1 and CopM2 from S metallicus have a similarrole to that described for E hirae Furthermore quantitativeRT-PCR experiments were carried out in order to determinedifferential expression of the cop genes in response to differentCu concentrations The expression of copA1 copA2 copT1and copT2 was tested relative to the transcript levels ofrRNA 16S gene since its levels were not significantly affected

in all assessed conditions (data not shown) As shown inFigure 8(a) copA2 and copA1 transcript levels were foundto be concomitantly increased with the increasing Cu con-centrations present in the medium Higher transcripts levelsof copA1 were seen in all tested conditions when comparedwith copA2 levels copA1 transcript levels were found to be325-fold up-regulated when comparing 50mMCu conditionversuscontrol (absence of metal) whereas copA2 transcriptlevels showed an increment of only 175-fold (Figure 8(a))The finding that Cu-ATPases mRNA levels were significantlyincreased in response to Cu ions exposure suggests that thetransport system may operate for Cu efflux in S metallicus

Moreover copA2 and copA1 gene expression was quan-tified when S metallicus was grown using chalcopyrite(CuFeS

2) ore as an oxidizable substrate (Figure 8(a)) Mineral

oxidation mediated by the microorganism generates a pro-gressive increase in Cu ions concentration in the medium Tofind out whether the amount of solubilized Cu induced theexpression of the Cu-ATPases genes total RNAwas extractedfrom a S metallicus culture grown to late exponential phaseand in the presence of 1 CuFeS

2 It was clear that an

up-regulation of the Cu-ATPases encoding genes also tookplace when S metallicus was grown in the presence ofCuFeS

2(Figure 8(a)) copA1 transcript levels increased 14-fold

compared with the control condition in the absence of Cuwhile copA2 gene expression showed an increase of 76 foldAlong with this bymeans of atomic absorption spectrometry(AAS) analysis an overall amount of solubilized Cu ions(Cu2+Cu1+) of 144plusmn21mMwas determined to be present inthemedium indicating that copA2 and copA1 gene expressionwas most likely in response to the Cu present in the culturedue to CuFeS

2microbial-solubilization

The finding that copA1 was highly expressed comparedwith copA2 in all tested conditions may suggest a possiblephysiological hierarchy between the two ATPases whenovercoming either Cu or Cd stress In this regard by meansof a genetic approach it was demonstrated in S solfataricus

Archaea 13

700

600

500

400

300

200

100

00

5 20 50 CuFeS2CuSO4 concentration (mM)

Relativ

egenee

xpression

CopA2CopA1

(a)

8

6

7

5

4

3

2

1

00

5 20 50 CuFeS2CuSO4 concentration (mM)

Relativ

egenee

xpression

CopT1CopT2

(b)

Figure 8 Relative expression levels of S metallicus genes copA1 and copA2 (a) copT1 and copT2 (b) Expression of S metallicus cop geneswasassessed by qRT-PCR and normalized against 16S rRNA gene expression in individual cultures Mean values and standard deviations arefrom analyses of three independent cultures grown in the presence of 0 5 20 50mMCu or in 1 of a chalcopyrite ore (CuFeS

2) concentrate

that while CopA was an effective Cu efflux transporterat low and high Cu concentrations the other Cu-ATPase(CopB) only appeared to be a low-affinity Cu export ATPase[68] Moreover by using a M sedula copA deletion mutantit was demonstrated that this strain compromised metalresistance and consequently abolished chalcopyrite oxidation[22] highlighting the role of Cu detoxification mechanismsduring a given bioleaching process Our attempts to showthe functionality of CopA1 and CopA2 from S metallicusby using E coli as a heterologous host were not successfulApparently the overexpression of these ORFs had a toxiceffect on E coli that compromised its viability Althoughgene disruption tools have not been yet developed for Smetallicus the functional role of both copTMA locimight befurther studied by cross-species complementation of a coppersensitive S solfataricus copR mutant as it was described byMaezato et al [22] It will be of great interest to establishin future studies the possible functionality of the isolatedputative transporters from S metallicus

Likewise quantitative RT-PCR experiments showed thatcopT1 transcript levels increased concomitantly with increas-ing Cu concentrations whereas copT2 showed relativelysimilar transcripts levels most likely indicating a constitutiveexpression profile (Figure 8(b)) Furthermore whereas copT1transcript levels were found to be increased 36 fold inCuFeS

2

grown cultures copT2 levels remained unchanged in com-parison with the control in the absence of Cu (Figure 8(b))The results obtained for S metallicus copT2 gene expressionare consistent with those reported for S solfataricus CopTtranscriptional regulator has been proposed to function as arepressor in S solfataricus showing a constitutive expressionthat in the presence of Cu loses its affinity for the promoterregion of copMA allowing the expression of this polycistron[19] In contrast the increased transcripts levels of both copA1and copT1 concomitant with higher Cu concentrations in the

environment suggest that the putative transcriptional regula-tor CopT1 may act by activating both copMA1 and probablyitself (Figure 8) Regarding this it was recently reported thatCopR (CopT in S metallicus) from S solfataricus strain 982acts as an activator of copT (copM in S metallicus) and copAexpression [69] Nevertheless additional experiments wouldbe required to test this possibility in S metallicus

4 Concluding Remarks

We previously reported that S metallicus resists extremelyhigh Cu concentrations which was mediated to some extentby the use of a possible metal resistance system based oninorganic polyphosphate hydrolysis and consequently Cu-PO4

2minusefflux [15 29 30] Here we have addressed the questionwhether this microorganism coded for other determinantsthat might help to explain its high heavy metal toleranceAs the genomic sequence of this microorganism is notyet available we jointly used CODEHOP-PCR and genomewalking approaches and were able to establish the occurrenceof two nonidentical homologous cop loci into the genome ofS metallicus (cop1 and cop2) Each cop locus codes for anarchaeal transcriptional regulator (CopT) a metal-bindingchaperone (CopM) and a Cu-transporting P-type ATPase(CopA) High levels of the polycistronic mRNAscopMA ofeach cop locus were observed after treatment with either Cuor Cd suggesting that the encoded ATPases efflux heavymetals out in order to detoxify the intracellular environmentAltogether previous reports and the results obtained in thisstudy allow us to suggest that some key elements that mayexplain the high resistance to Cu in S metallicus is the dupli-cation of the Cu resistance cop genes a defensive responseto stress and a polyP-based accumulation mechanism Inthe case of Cd although some similar stress responses were

14 Archaea

observed whether comparable Cu-responsive elements alsoparticipate in Cd responses remains to be seen

Conflict of Interests

The authors declare that the research was done in the absenceof any commercial or financial relationships that could beconstrued as a potential conflict of interests

Acknowledgments

This work was supported by Grant no FONDECYT 1110214and in part by ICM P-05-001-F project

References

[1] A Schippers ldquoMicroorganisms involved in bioleaching andnucleic acid-based molecular methods for their identificationand quantificationrdquo in Microbial Processing of Metal SulfidesR E Donati and W Sand Eds pp 3ndash33 Elsevier BerlinGermany 2007

[2] H R Watling ldquoThe bioleaching of sulphide minerals withemphasis on copper sulphides a reviewrdquo Hydrometallurgy vol84 no 1-2 pp 81ndash108 2006

[3] D E Rawlings D Dew and C Du Plessis ldquoBiomineralizationof metal-containing ores and concentratesrdquo Trends in Biotech-nology vol 21 no 1 pp 38ndash44 2003

[4] L Valenzuela A Chi S Beard et al ldquoGenomics metagenomicsand proteomics in biomining microorganismsrdquo BiotechnologyAdvances vol 24 no 2 pp 197ndash211 2006

[5] C A Jerez ldquoBioleaching and biomining for the industrialrecovery of metalsrdquo in Comprehensive Biotechnology M Moo-Young Ed vol 3 pp 717ndash729 Elsevier Amstaerdam TheNetherlands 2nd edition 2011

[6] P R Norris and J P Owen ldquoMineral sulphide oxidationby enrichment cultures of novel thermoacidophilic bacteriardquoFEMS Microbiology Reviews vol 11 no 1ndash3 pp 51ndash56 1993

[7] D E Rawlings and D B Johnson ldquoThe microbiology ofbiomining development and optimization ofmineral-oxidizingmicrobial consortiardquo Microbiology vol 153 no 2 pp 315ndash3242007

[8] P R Norris N P Burton and N A M Foulis ldquoAcidophiles inbioreactor mineral processingrdquo Extremophiles vol 4 no 2 pp71ndash76 2000

[9] M R Bruins S Kapil and F W Oehme ldquoMicrobial resistancetometals in the environmentrdquo Ecotoxicology and EnvironmentalSafety vol 45 no 3 pp 198ndash207 2000

[10] T J G Ettema M A Huynen W M De Vos and J Van DerOost ldquoTRASH a novel metal-binding domain predicted to beinvolved in heavy-metal sensing trafficking and resistancerdquoTrends in Biochemical Sciences vol 28 no 4 pp 170ndash173 2003

[11] M Dopson C Baker-Austin P R Koppineedi and P L BondldquoGrowth in sulfidic mineral environments metal resistancemechanisms in acidophilic micro-organismsrdquo Microbiologyvol 149 no 8 pp 1959ndash1970 2003

[12] S Franke and C Rensing ldquoAcidophiles Mechanisms to toleratemetal and acid toxicityrdquo in Physiology and Biochemistry ofExtremophiles C Gerday and N Glansdorff Eds pp 271ndash278ASM Press Washington DC USA 2007

[13] C A Navarro L H Orellana C Mauriaca and C A JerezldquoTranscriptional and functional studies of Acidithiobacillusferrooxidans genes related to survival in the presence of copperrdquoApplied and Environmental Microbiology vol 75 no 19 pp6102ndash6109 2009

[14] A Orell C A Navarro R Arancibia J C Mobarec andC A Jerez ldquoLife in blue copper resistance mechanisms ofbacteria and Archaea used in industrial biomining of mineralsrdquoBiotechnology Advances vol 28 no 6 pp 839ndash848 2010

[15] A Orell C A Navarro M Rivero J S Aguilar and C A JerezldquoInorganic polyphosphates in extremophiles and their posiblefunctionsrdquo Extremophiles vol 16 no 4 pp 573ndash583 2012

[16] C Baker-Austin M Dopson M Wexler et al ldquoExtremearsenic resistance by the acidophilic archaeon ldquoFerroplasmaacidarmanusrdquo Fer1rdquo Extremophiles vol 11 no 3 pp 425ndash4342007

[17] E Pedone S Bartolucci and G Fiorentino ldquoSensing andadapting to environmental stress the archaeal tacticrdquo Frontiersin Bioscience vol 9 pp 2909ndash2926 2004

[18] C Baker-Austin M Dopson M Wexler R G Sawers and PL Bond ldquoMolecular insight into extreme copper resistance inthe extremophilic archaeon ldquoFerroplasma acidarmanusrdquo Fer1rdquoMicrobiology vol 151 no 8 pp 2637ndash2646 2005

[19] T J G Ettema A B Brinkman P P Lamers N G Kornet WM de Vos and J van der Oost ldquoMolecular characterization ofa conserved archaeal copper resistance (cop) gene cluster andits copper-responsive regulator in Sulfolobus solfataricus P2rdquoMicrobiology vol 152 no 7 pp 1969ndash1979 2006

[20] A A Villafane Y VoskoboynikM Cuebas I Ruhl and E BinildquoResponse to excess copper in the hyperthermophile Sulfolobussolfataricus strain 982rdquo Biochemical and Biophysical ResearchCommunications vol 385 no 1 pp 67ndash71 2009

[21] K S Auernik Y Maezato P H Blum and R M KellyldquoThe genome sequence of the metal-mobilizing extremelythermoacidophilic archaeon Metallosphaera sedula providesinsights into bioleaching-associated metabolismrdquo Applied andEnvironmental Microbiology vol 74 no 3 pp 682ndash692 2008

[22] Y Maezato T Johnson S McCarthy K Dana and P BlumldquoMetal resistance and lithoautotrophy in the extreme ther-moacidophile Metalosphaera sedulardquo Journal of Bacteriologyvol 194 no 24 pp 6856ndash6863 2012

[23] D H Nies ldquoMicrobial heavy-metal resistancerdquo Applied Micro-biology and Biotechnology vol 51 no 6 pp 730ndash750 1999

[24] K Vido D Spector G Lagniel S Lopez M B Toledano andJ Labarre ldquoA proteome analysis of the cadmium response inSaccharomyces cerevisiaerdquo The Journal of Biological Chemistryvol 276 no 11 pp 8469ndash8474 2001

[25] G Bertin and D Averbeck ldquoCadmium cellular effects mod-ifications of biomolecules modulation of DNA repair andgenotoxic consequences (a review)rdquo Biochimie vol 88 no 11pp 1549ndash1559 2006

[26] M H Joe S W Jung S H Im et al ldquoGenome-wide responseof Deinococcus radiodurans on cadmium toxicityrdquo Journal ofMicrobiology and Biotechnology vol 21 no 4 pp 438ndash447 2011

[27] A Lagorce A Fourcans M Dutertre B Bouyssiere YZivanovic and F Confalonieri ldquoGenome-wide transcriptionalresponse of the archeon Thermococcus gammatolerans to cad-miumrdquo PLOS ONE vol 7 no 7 Article ID e41935 2012

[28] A Kornberg N N Rao and D Ault-Riche ldquoInorganicpolyphosphate a molecule of many functionsrdquo Annual Reviewof Biochemistry vol 68 pp 89ndash125 1999

Archaea 15

[29] J D Keasling ldquoRegulation of intracellular toxic metals andother cations by hydrolysis of polyphosphaterdquoAnnals of theNewYork Academy of Sciences vol 829 pp 242ndash249 1997

[30] F Remonsellez A Orell and C A Jerez ldquoCopper tolerance ofthe thermoacidophilic archaeon Sulfolobus metallicus possiblerole of polyphosphate metabolismrdquoMicrobiology vol 152 no 1pp 59ndash66 2006

[31] V Hatzimanikatis L H Choe and K H Lee ldquoProteomicstheoretical and experimental considerationsrdquo BiotechnologyProgress vol 15 no 3 pp 312ndash318 1999

[32] L H Choe and K H Lee ldquoA comparison of three commerciallyavailable isoelectric focusing units for proteome analysis theMultiphor the IPGphor and the Protean IEF cellrdquo Electrophore-sis vol 21 no 5 pp 993ndash1000 2000

[33] U K Laemmli ldquoCleavage of structural proteins during theassembly of the head of bacteriophage T4rdquo Nature vol 227 no5259 pp 680ndash685 1970

[34] A Shevchenko M Wilm O Vorm and M Mann ldquoMassspectrometric sequencing of proteins from silver-stained poly-acrylamide gelsrdquo Analytical Chemistry vol 68 no 5 pp 850ndash858 1996

[35] P Giavalisco E Nordhoff T Kreitler et al ldquoProteome analysisof Arabidopsis thaliana by two-dimensional gel electrophoresisand matrix-assisted laser desorptionionisation-time of flightmass spectrometryrdquo Proteomics vol 5 no 7 pp 1902ndash19132005

[36] T M Rose E R Schultz J G Henikoff S Pietrokovski CM McCallum and S Henikoff ldquoConsensus-degenerate hybridoligonucleotide primers for amplification of distantly relatedsequencesrdquo Nucleic Acids Research vol 26 no 7 pp 1628ndash16351998

[37] T M Rose J G Henikoff and S Henikoff ldquoCODEHOP(Consensus-Degenerate Hybrid Oligonucleotide Primer) PCRprimer designrdquo Nucleic Acids Research vol 31 no 13 pp 3763ndash3766 2003

[38] J P Acevedo F Reyes L P Parra O Salazar B A Andrews andJ A Asenjo ldquoCloning of complete genes for novel hydrolyticenzymes from Antarctic sea water bacteria by use of animproved genomewalking techniquerdquo Journal of Biotechnologyvol 133 no 3 pp 277ndash286 2008

[39] S Bathe and P R Norris ldquoFerrous iron- and sulfur-inducedgenes in Sulfolobus metallicusrdquo Applied and EnvironmentalMicrobiology vol 73 no 8 pp 2491ndash2497 2007

[40] J Sambrook E F Fritsch and TManiatisMolecular Cloning ALaboratory Manual Cold Spring Harbor Press New York NYUSA 1989

[41] K W Miller S Sass Risanico and J B Risatti ldquoDifferentialtolerance of Sulfolobus strains to transition metalsrdquo FEMSMicrobiology Letters vol 93 no 1 pp 69ndash73 1992

[42] G Huber C Spinnler A Gambacorta and K O Stetter ldquoMet-allosphaera sedula gen and sp nov represents a new genus ofaerobic metal-mobilizing thermoacidophilic archaebacteriardquoSystematic and Applied Microbiology vol 12 pp 38ndash47 1989

[43] J Llanos C Capasso E Parisi D Prieur and C JeanthonldquoSusceptibility to heavy metals and cadmium accumulation inaerobic and anaerobic thermophilic microorganisms isolatedfrom deep-sea hydrothermal ventsrdquo Current Microbiology vol41 no 3 pp 201ndash205 2000

[44] A Kaur M Pan M Meislin M T Facciotti R El-Gewelyand N S Baliga ldquoA systems view of haloarchaeal strategies towithstand stress from transition metalsrdquo Genome Research vol16 no 7 pp 841ndash854 2006

[45] M Solioz and J V Stoyanov ldquoCopper homeostasis in Entero-coccus hiraerdquo FEMS Microbiology Reviews vol 27 no 2-3 pp183ndash195 2003

[46] S Sitthisak K Howieson C Amezola and R K JayaswalldquoCharacterization of a multicopper oxidase gene from Staphy-lococcus aureusrdquo Applied and Environmental Microbiology vol71 no 9 pp 5650ndash5653 2005

[47] L Rodrıguez-Montelongo S I Volentini R N Farıas E MMassa and V A Rapisarda ldquoThe Cu(II)-reductase NADHdehydrogenase-2 of Escherichia coli improves the bacterialgrowth in extreme copper concentrations and increases theresistance to the damage caused by copper and hydroperoxiderdquoArchives of Biochemistry and Biophysics vol 451 no 1 pp 1ndash72006

[48] E Williams T M Lowe J Savas and J DiRuggiero ldquoMicroar-ray analysis of the hyperthermophilic archaeon Pyrococcusfuriosus exposed to gamma irradiationrdquo Extremophiles vol 11no 1 pp 19ndash29 2007

[49] L M Gaetke and C K Chow ldquoCopper toxicity oxidative stressand antioxidant nutrientsrdquo Toxicology vol 189 no 1-2 pp 147ndash163 2003

[50] M J Davies ldquoThe oxidative environment and protein damagerdquoBiochimica et Biophysica Acta vol 1703 no 2 pp 93ndash109 2005

[51] J R Thompson J K Bell J Bratt G A Grant and LJ Banaszak ldquoVmax regulation through domain and subunitchanges The active form of phosphoglycerate dehydrogenaserdquoBiochemistry vol 44 no 15 pp 5763ndash5773 2005

[52] V Consalvi R Chiaraluce L Politi A Gambacorta MDe Rosa and R Scandurra ldquoGlutamate dehydrogenase fromthe thermoacidophilic archaebacterium Sulfolobus solfataricusrdquoEuropean Journal of Biochemistry vol 196 no 2 pp 459ndash4671991

[53] A J L Macario M Lange B K Ahring and E Conway DeMacario ldquoStress genes and proteins in the archaeardquo Microbiol-ogy and Molecular Biology Reviews vol 63 no 4 pp 923ndash9671999

[54] J AMaupin-FurlowM A Gil M A Humbard et al ldquoArchaealproteasomes and other regulatory proteasesrdquo Current Opinionin Microbiology vol 8 no 6 pp 720ndash728 2005

[55] N Sun F Beck R Wilhelm Knispel et al ldquoProteomics analysisof Thermoplasma acidophilum with a focus on protein com-plexesrdquoMolecular and Cellular Proteomics vol 6 no 3 pp 492ndash502 2007

[56] L S Madding J K Michel K R Shockley et al ldquoRole of the 1205731subunit in the function and stability of the 20S proteasome inthe hyperthermophilic Archaeon Pyrococcus furiosusrdquo Journalof Bacteriology vol 189 no 2 pp 583ndash590 2007

[57] R Shibata Y Bessho A Shinkai et al ldquoCrystal structureand RNA-binding analysis of the archaeal transcription factorNusArdquo Biochemical and Biophysical Research Communicationsvol 355 no 1 pp 122ndash128 2007

[58] W Bae B Xia M Inouye and K Severinov ldquoEscherichia coliCspA-family RNA chaperones are transcription antitermina-torsrdquo Proceedings of the National Academy of Sciences of theUnited States of America vol 97 no 14 pp 7784ndash7789 2000

[59] R J Ram N C VerBerkmoes M P Thelen et al ldquoMicrobi-ology community proteomics of a natural microbial biofilmrdquoScience vol 308 no 5730 pp 1915ndash1920 2005

[60] I Noel-Georis T Vallaeys R Chauvaux et al ldquoGlobal analysisof the Ralstonia metallidurans proteome prelude for the large-scale study of heavy metal responserdquo Proteomics vol 4 no 1pp 151ndash179 2004

16 Archaea

[61] M TMNovo A CDa Silva RMoreto et al ldquoThiobacillus fer-rooxidans response to copper and other heavy metals growthprotein synthesis and protein phosphorylationrdquo Antonie vanLeeuwenhoek vol 77 no 2 pp 187ndash195 2000

[62] Q She R K Singh F Confalonieri et al ldquoThe complete genomeof the crenarchaeon Sulfolobus solfataricus P2rdquo Proceedings ofthe National Academy of Sciences of the United States of Americavol 98 no 14 pp 7835ndash7840 2001

[63] R P Anderson and J R Roth ldquoTandem genetic duplications inphage and bacteriardquo Annual Review of Microbiology vol 31 pp473ndash505 1977

[64] D Gevers K Vandepoele C Simillion and Y Van De PeerldquoGene duplication and biased functional retention of paralogsin bacterial genomesrdquo Trends in Microbiology vol 12 no 4 pp148ndash154 2004

[65] A B Reams and E L Neidle ldquoSelection for gene clustering bytandemduplicationrdquoAnnual Review ofMicrobiology vol 58 pp119ndash142 2004

[66] L H Orellana and C A Jerez ldquoA genomic island providesAcidithiobacillus ferrooxidans ATCC 53993 additional copperresistance a possible competitive advantagerdquo Applied Microbi-ology and Biotechnology vol 92 no 4 pp 761ndash767 2011

[67] A B Brinkman T J G Ettema W M de Vos and J van derOost ldquoThe Lrp family of transcriptional regulatorsrdquo MolecularMicrobiology vol 48 no 2 pp 287ndash294 2003

[68] C Vollmecke S L Drees J Reimann S V Albers and MLubben ldquoThe ATPases CopA and CopB both contribute tocopper resistance of the thermoacidophilic archaeon SulfolobussolfataricusrdquoMicrobiology vol 158 no 6 pp 1622ndash1633 2012

[69] A Villafane Y Voskoboynik I Ruhl et al ldquoCopR of Sulfolobussolfataricus represents a novel class of archaeal-specific copper-responsive activators of transcriptionrdquo Microbiology vol 157no 10 pp 2808ndash2817 2011

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Anatomy Research International

PeptidesInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporation httpwwwhindawicom

International Journal of

Volume 2014

Zoology

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Molecular Biology International

GenomicsInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioinformaticsAdvances in

Marine BiologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Signal TransductionJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

Evolutionary BiologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Biochemistry Research International

ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Genetics Research International

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Advances in

Virolog y

Hindawi Publishing Corporationhttpwwwhindawicom

Nucleic AcidsJournal of

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Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

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Enzyme Research

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

International Journal of

Microbiology

Archaea 3

mg wet weight) frozen and sonicated six times for 30 seach time The lysates were centrifuged at 4300 g for 5minto eliminate cellular debris The protein concentration ofsupernatants was determined by the method of Bradford(Coomasie Plus protein assay reagent Pierce) Between 120and 500120583g of proteins from the protein extracts were mixedwith rehydration IEF buffer as described by Hatzimanikatiset al [31] and Choe and Lee [32] with some modificationsincluding urea 8M thiourea 2M CHAPS 2 (wv) Bio-Lyte3ndash10 027 (vv) Bio-Lyte 5ndash8 013 (vv) and bromophenolblue 0001 (wv) followed by incubation at 25∘C for 30minDTT (003 g) and sterile nanopure water were then added tocomplete a final volume of 300 120583L The samples were thenincubated at room temperature for 1 h

23 Isoelectric Focussing (IEF) Each sample (300 120583L) wasloaded onto the pH 3ndash10 (nonlinear) 17 cm IPG strips(Biorad) in the first dimension chamber and was incubatedat room temperature for 1 h Mineral oil (25mL) was thenadded to prevent evaporation of the sample and precipitationof urea and strips were passively rehydrated for 18 h Theisoelectric focusing was performed with the PROTEAN IEF(Biorad) using the following conditions 250V for 15min2000V for 2 h 8000V for 4 h 10000V for 11 h and 50Vfor 4 h reaching a total of 120000Vh

24 SDS-PAGE Prior to the second-dimensional elec-trophoresis strips were equilibrated as described by Hatz-imanikatis et al [31] with some modifications Strips wereincubated for 15min with a solution containing 6M urea156mM DTT 30 (vv) glycerol 2 (wv) SDS and 24mMTris-HCl pH 68 and subsequently for 15 more min in a solu-tion containing 6M urea 135mM iodoacetamide 30 (vv)glycerol 2 (pv) SDS and 24mM Tris-HCl pH 68 Finallystrips were incubated with electrophoresis buffer containing192mM glycine 1 (wv) SDS and 250mM Tris-HCl pH 83until the second-dimensional run SDS-PAGEwas performedusing the PROTEAN II xi cell as described by Laemmli [33]and gels consisted of 115 or 15 (wv) polyacrylamide Stripswere then overlaid onto the second-dimensional gels sealedwith 1 (wv) agarose in electrophoresis buffer containinga trace amount of bromophenol blue Electrophoresis wascarried out at constant 70V for 15 h All experiments wereperformed in triplicate Gels were stained with silver orCoomasie Blue G-250 as described by Shevchenko et al [34]and Giavalisco et al [35] respectively

25 Gels Analysis and Mass Spectrometry Gel images weredigitized by scanning (Epson) and analyzed with the Delta2D software (Decodon) to identify the spots differentiallyexpressed due to the presence of toxic metals An estimateof relative quantitative changes was made on the basis ofthe change in percent volume among silver stained gelsSpots of interest were recovered from Coomasie Blue G-250stained gels manually and were sent to electron spray tandemionizationmass spectrometric analysis (tandemMS-MS ESI-QUAD-TOF) The results obtained were analyzed with Mas-cot algorithm (httpwwwmatrixsciencecomindexhtml)

by using the MSMS Ion search and all genomes availableat databases were used as queries The entire MS analysiswas performed at the Cambridge Center for ProteomicsUniversity of Cambridge UK

26 CODEHOP-PCR CopA and CopM amino acid sequen-ces from S acidocaldarius S tokodaii and S solfataricuswereobtained fromNCBI (httpwwwncbinihgov) After align-ing the sequences by using the CLUSTAL X program toidentify areas of homology consensus-degenerate PCR pri-mers were designed according to the CODEHOP strategy[36 37] using the WWW access at httpblocksfhcrcorgblockscodehophtml Two regions of high sequence sim-ilarity were identified for both CopA and CopM seq-uences respectively (Figure S1 see Supplementry Materi-al available online at httpdxdoiorg1011552013289236)and used to design the consensus-degenerate hybrid oligonu-cleotide primers (Table 1) Consensus-degenerate hybridoligonucleotide primers were designed for the N- and C-terminus of CopA while a pair of degenerate hybrid oligonu-cleotide primers was designed for CopM (Figure S1)

Amplification reactions contained 1x thermophilic DNApolymerase buffer with 2mMMgCl

2 02mMdNTPs 05 120583M

of each primer 5U Taq DNA polymerase 40 ng of S metal-licus genomic DNA as a template and water to a final volumeof 50120583L The thermal cycling conditions were 3min at 95∘Cfollowing 30 cycles of 95∘C for 30 s 50∘C for 30 s and 72∘Cfor 1min 1 final additional cycle at 72∘C for 10min Productsof amplification were applied onto 10 (wv) agarose gelsand main amplification bands were excised purified andTA-cloned into pCR21-TOPO (Invitrogen) vector and finallysequenced

27 GenomeWalking Experiments Genome walking strategywas performed as described by Acevedo et al [38] Thusa double-stranded oligo-cassette AdaptT adapter was con-structed by annealing of the two unphosphorylated primersAdaptF (51015840-CTAGGCCACGCGTCGACTAGTACTA-G-CTT-31015840) and AdaptR (51015840-AGCTAGTACTAGTCGACGCG-TGGCCTAG-31015840) Annealing was performed by heating theprimers (10 120583M) in a boiling water bath for 5min and thenslowly cooling to room temperature

Six different DNA libraries were constructed by meansof digesting 1120583g of S metallicus genomic DNA with the fol-lowing restriction enzymes HindIII BamHI EcoRI EcoRVNcoI and PstI respectively DNA digestion reactions werecarried out using 10U of restriction enzyme 2120583L of thecorresponding enzyme reaction buffer in 20120583L of totalreaction volume The reaction mix was incubated at 16∘Cduring 16 h To complete the 31015840 recessive end of the DNAfragments and to add a 31015840 overhanging adenine 500 ng ofthe digested and purified DNA were incubated with 5U ofTaq DNA polymerase 1 120583L of 10mM dNTPs mix and 5 120583Lof 10x thermophilic DNA polymerase buffer in 50120583L totalvolume at 70∘C for 45min Seven 120583L of thismixture was thenincubatedwith 15 pmol of AdaptT oligo-cassette 1 U T4DNAligase (Promega) and 2120583L of 5x ligase buffer in a total volume

4 Archaea

Table 1 Oligonucleotides

Name Sequence DescriptioncopA cdegF 51015840-gatgtagtaatagtaaaaactggagaaataataccngcngaygg CODEHOP-PCRcopA cdegR 51015840-tcatcagcaaaattagaagaatctccngtngcdat CODEHOP-PCRcopT cdegF1 51015840-ctcaaatagaatataaagtattacaaatgttaaaagargaywsnmg CODEHOP-PCRcopT cdegR1 51015840-ggattaccatttatttcatttccacartartcrca CODEHOP-PCRcopT cdegR2 51015840-cttatcatattcataaatcttccatctattaatttrtarcaytc CODEHOP-PCRcopT degF1 51015840-gartgytayaarctnat DOP-PCRcopT degR1 51015840-atnagyttrtarcayct DOP-PCRcopM degF 51015840-gayccngtntgyggnatgga DOP-PCRcopM degR 51015840-ccnggnttyccntacgg DOP-PCRAdaptF2 51015840-cacgcgtcgactagtactagctt Genome WalkingSP1copA1 31015840 51015840-aaggatgagggggaccttatgg GenomeWalkingSP2copA1 31015840 51015840-ggagataagaaatggggtaaaagag GenomeWalkingSP1copA1 51015840 51015840-tgataccatcatggaacctgtcag GenomeWalkingSP2copA1 51015840 51015840-tcctccacaatcccatccgctg GenomeWalkingSP1copA1 51015840 2 51015840-gattgtagctaagttaacctcggcctcg GenomeWalkingSP2copA1 51015840 2 51015840-cttctcaccctcagtctggttgg GenomeWalkingSP1copA2 31015840 51015840-gaaagaggaatatatgcaagggtaaacgg GenomeWalkingSP2copA2 31015840 51015840-gtgttaatgggagagctggaggg GenomeWalkingSP1copA2 51015840 51015840-cttctctgtggcaacatcataaccagcc GenomeWalkingSP2copA2 51015840 51015840-acgcatgtggcgcaatgcattcc GenomeWalkingSP1copT1 51015840 51015840-cattcctcgcaccagcttgcacactctc GenomeWalkingSP1copT2 51015840 51015840-cctatgaatactagatcttttccctgaac GenomeWalkingSP2copT2 51015840 51015840-aacagcttataacactcgtcactttggc GenomeWalkingcopM2 RT F 51015840-gatgaaaaaagccaatataagac RT-PCRcopA2 Rv 51015840-gaacactaactaacatcgcc RT-PCRcopM1 RT Fw 51015840-ctatcgtttttgttccgaagcttg RT-PCRcopA1 Rv 51015840-cagcagcaagaacagagacgcc RT-PCRlowastSM16Sf 51015840-acgctctaaaaaggcgtgggaata RT-qPCRlowastSM16Sr 51015840-ttgagctcggggtctttaagcagtg RT-qPCRcopA1Sm qRT F1 51015840-gctaaggtaatagagagcgg RT-qPCRcopA1Sm qRT R1 51015840-tgaacaggaatggacagg RT-qPCRcopA2Sm qRT F 51015840-tgtgcttgtctccttagcgt RT-qPCRcopA2Sm qRT R 51015840-actcttccgtctttcggagt RT-qPCRcopT1Sm qRT F 51015840-tgtaggagagtgtgcaagct RT-qPCRlcopT1Sm qRT R 51015840-tcgcaagtgagggttatggt RT-qPCRcopT2Sm qRT F 51015840-gtgttacggagcttgca RT-qPCRcopT2Sm qRT R 51015840-acactcgtcactttggc RT-qPCRAll oligonucleotides were synthesized by Invitrogen lowastOligonucleotides designed by Bathe and Norris [39]

of 10 120583L The ligation reaction was incubated at 16∘C during16 h

Two consecutive amplification reactions were then per-formedThefirst PCR reactionwas donewith 1x Elongasemixbuffer 19mM MgCl

2 02mM dNTPs 05120583M first specific

primer (SP1) (designed from the known sequence of thetarget gene a forward primer was used to amplify the 31015840end a reverse primer for 51015840 end amplification) 5 120583L of theligatedDNAdiluted 10-fold and 1 120583L of Elongase (Invitrogen)and water to a final volume of 50120583L The thermal cyclingconditions were as follows 1 cycle at 94∘C for 1min 20cycles of 94∘C for 32 s and 68∘C for 5min and one final

additional cycle at 70∘C for 7min The PCR product wasdiluted 10 fold and 3 120583L were used as a DNA template fora second PCR which was performed using the same con-ditions as the first PCR with 05 120583M second specific primer(SP2) and 02 120583Moligo-cassette-specific primer AdaptF2 (51015840-CACGCGTCGACTAGTACTAGCTT-31015840) and 1 120583L of Elon-gase The thermal cycling conditions were 1 cycle at 94∘C for1min 35 cycles of 94∘C for 32 s and 68∘C for 5min and 1final additional cycle at 70∘C for 7min PCR products wereexcised purified TA-cloned into pCR21-TOPO (Invitrogen)vector and finally sequenced

Archaea 5

28 Isolation of Total RNA Cell pellets (10mg wet weight)were collected and diluted in 60 120583L TEN buffer (20mMTris-HCl pH 80 1mM EDTA 100mM NaCl) followedby addition of 60120583L TENST buffer (20mM Tris-HCl pH80 1mM EDTA 100mM NaCl 16 Na-lauroyl sarcosineand 012 Triton X-100) This suspension was incubated atroom temperature for 15min to allow cell lysis Total RNAwas then extracted using the TRIzol reagent (Invitrogen)as recommended by the manufacturer DNA contaminationin RNA preparations was removed by DNase I treatment(Roche) following the manufacturerrsquos instructions RNAwas then purified precipitated and finally resuspended indiethylpyrocarbonate (DEPC)-treated water Total RNA con-centrations were estimated by spectrophotometric measure-ments (OD

260) and its quality was evaluated by determining

the ratio of absorption at 260 nm and 280 nm which waswithin the preferred range of 18ndash21

29 Northern Blotting For differential gene expression byNorthern blotting experiments the media were supple-mented with either different concentrations of Cu (5ndash50mM) Cd (5mM) NiSO

4(15mM) ZnSO

4(50mM) or

Ag2SO4(008mM) respectively as indicated For some

experiments elemental sulfur was replaced by a chalcopyrite(CuFeS

2) concentrate which was used as the only energy

source at 1 (wv)Total RNA (5 120583g) was separated by electrophoresis

on a 1 formaldehyde agarose gel followed by blottingonto Hybond-N nylon membranes (Amersham PharmaciaBiotech Bucking-hamshire UK) Hybridization was con-ducted as described by Sambrook et al [40] DNA probeswere labeled by randomprimerDNA labeling kit (Fermentas)using [120572-32 p] dCTP The hybridization signal was detectedand analyzed by using the molecular Imager FX system andQuantity One software The primer sequences for amplifica-tion of these gene probes are listed in Table 1

210 Cotranscriptional Analysis by RT-PCR To study theexpression of adjacent genes copM and copA cDNA wassynthesized by using 08120583g of total RNA from a S metallicusculture grown in the presence of 20mM Cu and a reverseprimer hybridizing to copA sequence A forward primerannealing on copM sequence was used for the PCR reactions(Table 1) PCR amplifications were performed with 1 120583L ofa 110 dilution of the cDNA and 25 pmol of each primerAmplification conditions included an initial 3min of denat-uration at 95∘C followed by 35 cycles of 30 s at 95∘C 30 sat 55∘C and 15min at 72∘C and finished by 10min at 72∘CA reverse transcriptase reaction without enzyme was carriedout in order to exclude amplification due to genomic DNAcontamination

211 Quantitative RT-PCR Single stranded cDNA was syn-thesized from 08 ng of DNA-free RNA samples usingrandom hexamers (Fermentas) and ImProm-II reversetranscription system (Promega) following manufacturerrsquosinstructions The software tool IDT Scitools (IntegratedDNA Technologies) was used to design primers producing

amplicons of 150ndash200 bp (Table 1) qPCRwas performedwith2 120583L of 1 10 diluted cDNA samples 125 120583L 2x QuantiFastSYBRGreenPCRMasterMix (Quiagen) 1 120583Mof each primerand water to a final volume of 25120583L The efficiency of eachprimer pair was calculated from the average slope of a linearregression curve which resulted from qPCRs using a 10-folddilution series (10 pgndash10 ng) of S metallicus chromosomalDNA as template Cq values (quantification cycle) wereautomatically determined by Real-Time Rotor-gene 6000PCR software (Corbett Life Sciences) after 40 cycles Cqvalues of each transcript of interest was standardized to theCq value of the 16S rRNA gene [39] At least 3 biologicalreplicates of each assessed condition and 2 technical replicatesper qPCR reaction were performed

3 Results and Discussion

31 Tolerance of S metallicus and S solfataricus to Cu and CdIn a previous work it was determined that S metallicus wasable to tolerate high Cu concentrations While the presenceof 100mM Cu did not affect S metallicus growth kinetics adecreased in cell biomass of only 30 was observed whenexposed to 200mM Cu [30] The high tolerance to Cu hasbeen described in other acidophilic Bacteria and Archaeacompared mostly with neutrophilic microorganisms [11 14]Here this analysis was further extended to characterize theresponse of S metallicus to Cd Thus it was determined thatS metallicus growth was not affected in the presence of either05 or 1mM Cd when compared with the control conditionin the absence of the metal (Figure 1(a)) In addition whenS metallicus was challenged with 2 and 3mM Cd it wasobserved that at the late exponential growth phase the cellnumbers decreased by 30 and 50 respectively (Figure 1(a))Moreover growth of S metallicuswas completely inhibited inthe presence of 5mM Cd (Figure 1(a)) On the other hand Ssolfataricuswas not able to grow at Cd concentrations greaterthan 005mM (Figure 1(b)) At 001mM Cd S solfataricuscell numbers decreased around 35 (Figure 1(b)) Theseresults are in agreement with previous reports in which Ssolfataricus was shown to be able to grow in up to 001mMCd [41] Other acidophilic archaeons involved in bioleachingprocesses such as Metallosphaera sedula and Ferroplasmaacidarmanus were found to be able to tolerate up to 09and 9mM Cd respectively [18 42] The minimal inhibitoryconcentration (MIC) to Cd has been described to be nothigher than 1mM for several thermophilic and neutrophilicThermococcus species [43] Recently it was reported that themost radioresistant archaeon Thermococcus gammatoleransstands Cd concentrations with an MIC of 2mM [27] Todate despite the heavy metals tolerance showed by someof these microorganisms strategies to withstand stress fromtransition metals have been most widely studied only inhaloarchaea [44]

32 Effect of Cu andCd on theGlobal Proteome of SmetallicusThe proteomic response to Cu and Cd was analyzed toidentify proteins which could be involved in heavy metalsresistance in S metallicus Early stationary phase growing

6 Archaea

14

12

10

8

6

4

2

00 2 4 6 8 10 12

Time (days)

107timescellsm

L

(a)

120

100

80

60

40

20

00 2 4 6 8 10

Time (days)

107timescellsm

L

(b)

Figure 1 Growth of S metallicus (a) and S solfataricus (b) in the presence of Cd Cells were inoculated in their respective growth media inabsence of added Cd (∙) or were supplemented with 0005mM () 001mM () 005mM (diams) 01mM () 05mM () 1mM () 2mM() 3mM (times) or 5mM (+) Cd and cells were counted daily

100

7060

50

30

20

3 10MW

33 31

32

A

B

C

20

10

(a)

100

7060

50

30

20

12

3 10MW

204

21

33 31

32

A

B

C

15

93

10

27 28

23 24

29

25 2630

1614 16

13

52

7

2219

8

20

10

11

(b)

Figure 2 Changes in the proteome of S metallicus grown in the presence of Cu Cells were incubated in the absence of any added metal(a) or in the presence of 100mM Cu (b) Arrows indicate the spots that were downregulated (a) or upregulated (b) in the presence of CuNumbers indicate the spots with increased intensity in cells treated with copper The dashed boxes are shown as enlarged areas that includesome proteins upregulated in the presence of Cu and Cd (Figure 3) The bottom panels show low molecular weight proteins upregulated inthe presence of Cu separated by using a 15 polyacrylamide gel in the second dimension

cells were untreated or treated with either 100 or 200mM Cuor 1mM Cd during 24 h and analyzed by comparative two-dimensional gel electrophoresis as indicated in experimentalprocedures Twenty-three proteins were found to be down-regulated after 100mMCu treatment (Figure 2(a)) A similar

pattern was obtained at 200mM Cu (not shown) Further-more by using the Delta 2D software (Decodon) eleven ofthese proteins were found to be completely absent in the gelsand more than 8 proteins decreased their intensity in therange from 15-to 5-fold compared with control cells grown

Archaea 7

13 13

11

13

1

Control Copper Cadmium

(a)

99 9

333

(b)

3030

30

2020

20 212121

(c)

Figure 3 Comparison of selected proteins of S metallicus cells treated with Cu or Cd Protein extracts were obtained from cells treatedwithout metals and with 100mM Cu or 1mM Cd for 24 h and the proteins were separated by 2D-PAGE The segments (a) (b) and (c) arethe enlarged dashed boxes in Figure 2 under the three conditions indicated Numbers show some of the spots with increased intensity in cellstreated with Cu or Cd

in the absence of Cu (Figure 2(a)) Therefore these resultsshow that a number of proteins became non-detectable ordecreased their levels when S metallicus faced Cu This kindof response was also seen when the microorganism wasexposed to Cd (data not shown) This behavior has alsobeen observed in similar studies where F acidarmanus waschallenged by either As(III) or Cu [16 18] In addition theexpression of 30 other proteins was found to be upregulatedafter Cu treatment (100 or 200mM) (Figure 2(b)) Most ofthese proteins could be identified only in the condition withCu and spots 5 13 and 15 were induced 26 34 and 55-fold respectively compared to cells without Cu treatmentMoreover 3 proteins of lowmolecular weight (spots 31 3 and33) that increased their expression when cells were exposedto 100mM Cu were also identified (Figure 2 bottom panels)These proteins were only detected when 15 polyacrylamidewas used during the second-dimensional separation (SDS-PAGE) On the other hand a large decrease in overallexpression of proteins after 1mMCd treatment was observed(not shown) Interestingly 10 out of the 13 proteins identifiedas up-regulated when cells were exposed to Cd and alsoshowed increased levels after Cu treatment Some of them areshown in Figure 3

33 Identification of Proteins Upregulated in Cells Treated withCu and Cd in S metallicus A total of 18 S metallicus proteinswhose levels were found to be up-regulated in responseto Cu were analyzed by mass spectrometry The identifiedproteins included functions related to the production andtransport of energy biosynthesis of amino acids stressresponses and transcription regulation (Table 2) Amongstthe proteins related to production and transport of energya putative ATP synthase subunit B (spot 23) that has beendescribed as playing a fundamental role in ATP synthesiswas identified (Table 2) When cells are subjected to somestressing conditions such as the presence of heavy metalsa greater cellular demand for energy has been reported tooccur [18] Most likely this phenomenon might be due toATP-driven Cu transport via ATPases that have a substantialinterplay during metal detoxification [45] Furthermore theATP synthase subunit B was also identified as up-regulatedwhen S metallicus was treated with Cd (Table 2)

Two other proteins corresponded to putative oxidoreduc-tases such as ferredoxin oxidoreductase (spot 3) and alcoholdehydrogenase (spot 4) that have been generally involvedin electron transporter chains and use NAD+ as an electronacceptor The levels of ferredoxin oxidoreductase were also

8 Archaea

Table 2 Proteins upregulated in S metallicus cells exposed to 100mM Cu or 1mM Cd

Spot Molecular weight (kDa) Cu induction levels Putative function Microorganism related Accesion number1dagger 266 infin Proteasome subunit S solfataricus AAK410343dagger 701 infin Ferredoxin oxidoreductase S solfataricus AAK429264dagger 429 infin Alcohol dehydrogenase S solfataricus AAK431545 35 26 Hypothetical protein S acidocaldarius YP 2549766 27 infin Hypothetical protein P aerophilum NP 5598899dagger 57 infin Phosphoglycerate dehydrogenase M thermautotrophicus AAB8546613dagger 27 34 Unknown mdash mdash14 28 infin Unknown mdash mdash15 35 55 Unknown mdash mdash20dagger 461 infin Glutamate dehydrogenase S solfataricus AAK4223021dagger 45 infin Hypothetical protein M acetivorans NP 618380122 49 infin Hypothetical protein P torridus YP 02280723dagger 51 infin ATP synthase subunit B S solfataricus AAK4088024 585 infin HSP60 subunit S shibatae AAG37273130dagger 48 infin Unknown mdash mdash31 16 ND Unknown mdash mdash32 144 ND Putative transcription regulator S solfataricus AAK4041333 138 ND Transcription factor nusA S solfataricus AAK40563Spots refer to those numbered in Figure 2 Proteins in Figure 2 which are not included in this table were not subjected to sequencing due to their low intensitiesin the gelsdaggerSpot up-regulated in cells exposed to Cu and CdND not determinedInfinity symbol indicates that proteins were expressed only in presence of copper or cadmiumM thermoautotrophicus Methanobacterium thermoautotrophicus M acetivorans Methanosarcina acetivorans S shibatae Sulfolobus shibatae P torridusPicrophilus torridus P aerophilum Pyrobaculum aerophilum S acidocaldarius Sulfolobus acidocaldarius

up-regulated in response to Cd (Table 2) Several previ-ous reports have suggested that oxidoreductases contributeto an oxidative protection in both Bacteria and Archaeain response to heavy metals [46ndash48] In the neutrophilicmicroorganisms Escherichia coli and Staphylococcus aureusit has been described that oxidases and dehydrogenasescontribute to oxidative protection Cu homeostasis and stressresponses [46 47] Furthermore some proteins involved inoxidative damage repair such as NADH-dependent oxidasesand thioredoxin reductases were expressed in cells of Facidarmanus exposed to As(III) [16] The expression of thisgroup of proteins has also been observed when the samemicroorganism was exposed to Cu [18] Therefore oxidore-ductases play an important role against oxidative stress andmay eliminate reactive oxygen species which constitute themajor component of the stress caused by transition metals[44 49 50]

The enzymes related to biosynthesis of amino acids werefound as commonly up-regulated either in response to Cu orCdThey corresponded to a phosphoglycerate dehydrogenase(spot 9) and a glutamate dehydrogenase (spot 20) Theformer one catalyzes the NAD+-dependent oxidation of 3-phosphoglycerate into 3-phosphohydroxypyruvate a branchpoint from the glycolytic pathway and the initial reactionin L-serine biosynthesis [51] Glutamate dehydrogenase cat-alyzes the oxidative deamination of glutamate to produce 2-oxoglutarate and ammonia with reduction of NAD+ [52]Proteins involved in amino acids synthesis were expressed

in cells of F acidarmanus exposed to As(III) [16] Moreoverincreased levels of proteins involved in the biosynthesisof sulfur-containing amino acids have been observed inSaccharomyces cerevisiae cells exposed to Cd [24] suggestingthat these proteins might also have a role in the cellularresponse to adverse conditions

Two proteins showed identity to components of the stressresponse mechanism such as one subunit of the HSP60chaperonin (spot 24) and one subunit of the proteasome (spot1) In Archaea the stress protein HSP60 is directly involvedin protein folding processes [53] Proteomic analysis of Facidarmanus cells exposed to As(III) and Cd revealed theexpression of proteins involved in protein folding and DNArepair including HSP60 chaperonin and DnaK heat-shockprotein (HSP70) thereby the authors suggested that thismicroorganismusesmultiplemechanisms to resist high levelsof Cu [16 18] On the other hand proteasomes are describedas nonspecific proteolytic nanomachines associated withprotein catabolism Proteasomes are known to be closelyinvolved in maintaining protein quality control by degradingmiss folded and denatured proteins in response to cell stressin all three domains of life [54] A proteomic analysis inthe thermoacidophilic archaeon Thermoplasma acidophilumshowed that proteasomes and chaperone-related proteinswere highly induced against stress conditions indicatinga high turnover rate of proteins [55] In P furiosus theexpression of the 1205731 subunit of the proteasome was inducedin cells subjected to heat shock stress [56] Additionally the

Archaea 9

150pb

(a)

1016 pb

(b)

2000 pb

(c)

Figure 4 CODEHOP-based PCR for the amplification of the putative genes copA and copM from S metallicus Amplification products(a) using degenerate PCR primers (DOP) copM degF and copM degR designed from amino acid sequence conserved blocks of SulfolobalesCopM proteins (b) using primers copA cdegF and copA cdegR designed by CODEHOP strategy for amplification of a copA-like gene (c)Primers copM degF and copA cdegRwere assesed for the amplification of a copMA-likeDNA regionThose primerswere testedwith genomicS metallicus DNA samples Amplicons expected sizes were as follows ca 150 bp for copM ca 950 bp for copA and ca 2000 bp for copMAPCR products were excised and cloned for later sequencing

proteasome subunit was also found to be up-regulated whenS metallicus was subjected to Cd stress (Table 2)

The transcriptional regulation-related proteins corre-sponded to a putative transcription regulator (spot 32) andthe transcription factor NusA (spot 33) involved in theintrinsic termination of transcription [57] NusA has beenalso reported to display important functions in the cellularresponse to adverse factors [58] The expression of twotranscriptional regulators related to amino acids biosynthesisand metal-dependent genetic repression were found to beinduced in P furiosus cells subjected to oxidative stress[48] Moreover the expression of several genes encodingpredicted transcriptional regulators were induced followingCd exposure in T gammatolerans cells [27]

Spots 5 6 13 14 15 21 22 30 and 31 (Table 2) did notshow significant scores with any protein sequences currentlyavailable in the data bases Interestingly spots 2 4 9 13 21and 30 were found to be commonly upregulated when Smetallicus cells were treated with either Cu or Cd (Figure 3Table 2)

The results presented here are consistent with previ-ous work describing global protein expression profiles inresponse to heavy metals as reported for F acidarmanus [1618] microbial-biofilm communities in an acid mine drainagesite [59] and other prokaryotic and eukaryotic microorgan-isms [24 60]Therefore a coordinated expression of differentgroups of genes suggests the existence of regulatory networkssuch as stress response mechanisms and respiratory chainadjustments to cope with the presence of heavy metal ions[61]

34 Cu-Resistance (Cop) Genes Cluster Is Duplicated in Smetallicus Genome A Cu-resistance (cop) locus has beendescribed to be highly conserved in archaeal genomeswhich consists of genes encoding a new type of archaealtranscriptional regulator (CopT) a metal-binding chaperone(CopM) and a Cu-transporting P-type ATPase (CopA) [19]Since the genome sequence of Smetallicus is not yet availablewe searched for the presence of cop genes by using consen-sus degenerate hybrid oligonucleotide primers-based PCR(CODEHOP-PCR) CopA and CopM amino acid sequencealignments from S acidocaldarius S tokodaii and S solfa-taricus were used to identify conserved sequence blocks andtherefore to design either CODEHOP or DOP (degeneratehybrid oligonucleotide) primers (Figure S1 Table 1) TherebyPCR assays using S metallicus genomic DNA as templateyielded amplicons of ca 150 bp and ca 950 bp for copMand copA CODEHOP pair primers respectively (Figures4(a) 4(b)) As estimated from primer pairrsquos position onamino acid sequence alignments the obtained PCR productscorresponded to the expected sizes suggesting the presenceof copM- and copA-like genes in S metallicus genome Inorder to determine the identity of the amplified DNA frag-ments they were TA-cloned and sequenced Blastx sequenceanalysis showed that the 150 bp DNA fragment coded for anincomplete ORF sharing 63 homology with S solfataricusCopM (SSO10823) whereas the analysis of the 950 pb DNAfragment yielded a partial ORF sharing 54 homology withS solfataricus CopA protein (SSO2651)

The genomic organization of the putative cop genes in Smetallicus was analyzed to find out its similarity with those

10 Archaea

Table 3 Sequence identity comparison of S metallicus Cop proteins to those in other Sulfolobales

S metallicus protein Identity to homologue in other SulfolobalesS solfataricus P2 S acidocaldarius S tokodaii M sedula

CopA1 51 43 44 45CopA2 88 46 49 50CopM1 63 69 71 71CopM2 89 72 70 77CopT1 36 27 30 32CopT2lowast 90 52 52 53Boldface type indicates to which organism the S metallicus homologue shows the highest sequence identitylowastRefers to the analysis obtained using the uncompleted amino acid sequence of CopT2 which represents sim90 in length when compared with S solfataricusCopT sequence

previously described in S solfataricus P2 and F acidarmanuswhere the cop cluster consists of tandem-orientated genesas copTMA [18 19] Thus using copM degF (forward) andcopA cdegR (reverse) primers we attempted to amplify aputative copMA DNA region This PCR yielded a product ofca 2000 pb corresponding to the expected size and resultedin 2 ORFs showing high sequence homology with CopMand CopA from S solfataricus (Figure 4(c)) Interestinglywhen aligning this copMA sequence with the copA and copMsequences obtained previously they were not identical whichstrongly indicated the presence of duplicated putative copgenes encoding for both Cu-ATPases (copA1 and copA2) andmetallochaperones (copM1 and copM2) in the S metallicusgenome

To isolate the entire cop gene sequences the genomewalking method described by Acevedo et al [38] was usedSix different DNA libraries were constructed from S metalli-cus genomic DNA digested with several restriction enzymesincluding 51015840 overhang and blunt end restriction enzymesThe DNA libraries were used as templates in 2 successivePCR reactions From the previously known sequences twospecific sense primers for the 31015840 end amplification and twospecific antisense primers for the 51015840 end amplification weredesigned (abbreviated SP1 and SP2) Therefore the 51015840 and 31015840ends of the putative genes copA1 and copA2 that had beenpartially identified through the genome walking techniquewere amplified

By overlapping the sequence isolated by degenerate PCRand the lateral sequences obtained by genome walking(Figure S2) it was possible to complete the whole nucleotidesequence of the putative gene copA1 Additionally it waspossible to confirm the presence of the upstream copM1 geneanddetermine the occurrence of a partial copT1 gene tandem-orientated upstream of copM1 (Figure 5)Through additionalgenome walking experiments the partial copT1 sequencecould be further completedThe samewas truewhen isolatingthe whole nucleotide sequence of the putative gene copA2as the presence of copM2 and copT2 genes were confirmedupstream of copA2 However it was not possible to completethe whole 51015840 end of the putative gene copT2 as its determinedlength corresponded to only 90 of the full length whencompared with S solfataricus copT gene (Figure 5)

In conclusion degenerate PCR together with genomewalking experiments allowed to describe the occurrence inS metallicus genome of 2 cop genes loci (named as locus cop1and locus cop2) coding for paralogous genes whose productsmay be involved in Cu-resistance (Figure 5) Although somearchaeal genomes exhibit two copies of putative Cu-P-typeATPases as described for S solfataricus [62] the genomicarrangement displayed as a cop gene cluster (copTMA) hasbeen reported to be represented in only one copy for manyarchaeal genomes [10 19 21] Moreover we further updatedthis analysis by searching for duplications of the cop genescluster in all available archaeal genome sequences up to date(October 2012) retrieving only one cop locus in each caseThereby we propose that the discovery of this cop gene clusterduplication in the genome of S metallicus constitutes so faran unprecedented feature for a representative of the Archaeadomain and might contribute to its high metal resistance

In this context it has been widely reported that increasedgene copy number can increase gene expression allowingprokaryotic to thrive under growth limiting conditions [1363ndash68] However alongside the augmented gene copy onecannot exclude that the cop operon duplication in S metal-licus may offer among others a wider repertoire of Cu-ATPases which might differ in terms of metal affinitiesandor specificity efflux rates andor differences in theirabundance regulation

Throughout amino acid sequence analysis it was furtherdetermined that each polypeptide encoded by S metallicuswith the exception of CopT1 showed to be highly homol-ogous to their orthologous counterparts encoded by othersSulfolobales (Table 3) Moreover the 3 ORFs encoded by thelocus cop2 shared about 90 identity with the correspondingS solfataricus orthologous peptides (Table 3) S metallicuscop genes products showed characteristic domains referredto as critical for their respective proposed biological activitiesS metallicus paralogous gene products CopA1 (749 aa1015840)and CopA2 (747 aa1015840) shared 513 of identity These twoputative Cu-ATPases (CopA1 and CopA2) contain the aminoacid sequence motif CPCALGLA which has been proposedto confer Cu-transporting specificity in CPx-ATPases [48]and has been also found in other CopA-like proteins frombiomining Bacteria and Archaea [14]

Archaea 11

HindIII HindIII HindIII

HindIII

CopT1Locus cop1 M1

EcoRIEcoRV

513 bp 219 bp 2250 bp

CopA1

(a)

Locus cop2 M2

EcoRI

CopA2

453 bp 171 bp 2244 bp

CopT2

PstIHindIII HindIII HindIII

(b)

Figure 5 Schematic representation of the two cop gene clusters isolated from S metallicus genome Each cluster codes for anArchaea-specifictranscriptional regulator (copT) a metallochaperone (copM) and a P-type Cu-exporting ATPase (copA) Lengths of each gene are indicatedLocus cop1 corresponds to 4346 sequenced base pairs and shows an intergenic region copT1-M1 of 232 base pairs Locus cop2 correspondsto 3435 base pairs and shows 89 base pairs copT2-M2 intergenic region Genes copM2 and copA2 overlapped in 11 base pairs copT2 was notfully isolated

Control CdSO4 Ag2SO4 NiSO4 ZnSO4 CuSO4

kb3kb2kb1

02 kb

(a)

kb3kb2kb1

02 kb

(b)

(c)

Figure 6 Northern blot analysis to determine the expression of S metallicus copA1 (a) and copA2 (b) putative genes in response to variousmetals S metallicus total RNAwas extracted from cultures grown either in the absence (control) or the presence of CuSO

4(50mM) Ag

2SO4

(008mM) NiSO4(15mM) ZnSO

4(50mM) and CdSO

4(2mM) P32-radioactivelly labelled DNA fragments annealing to copA1 and copA2

sequences respectively were used as probes (c) shows rRNAs as total RNA loading control

On the other hand CopM1 (71 aa) and CopM2 (56 aa)sharing 66 of identity present the proposed metal bindingdomain TRASH (trafficking resistance and sensing of heavymetals) [10] Additionally when analyzing the putative tran-scriptional regulators the presence of a C-terminal TRASHdomain in both CopT1 and CopT2 was also found CopT1sequence also contains an entire N-terminal helix-turn-helix (HTH) motif (not shown) that resembles the DNA-bindingmotifs of prokaryotic transcriptional regulators suchas Lrp-like proteins [67] The HTH motif was only partiallyidentified in CopT2 since the 51015840 region of copT1 gene has notbeen yet fully isolated

35 Transcriptional Analysis of S metallicus Cop Genes Toget insight into the role of the two cop loci the expressionof the corresponding genes was analyzed under variousconditions S metallicus was first grown in presence ofdifferent heavy metals (Cu Zn Cd Ni o Ag) at a givenconcentration that did not affect growth kinetics [30 42]Subsequently total RNA was isolated from late exponentiallygrown cultures and Northern blot experiments were carriedout in order to determine the expression of both copA1 andcopA2 genes As depicted in Figure 6 transcription of bothcopA1 and copA2messengers were specifically induced in thepresence of Cu and Cd ions This gene expression pattern

12 Archaea

954pb

M1 CopA1

(a)

1438 pb

M2 CopA2

(b)

1000 pb

800pb

+ minus

(c)

+ minus

1500 pb

1000 pb

(d)

Figure 7 Cotranscription analysis of copMA1 and copMA2 genes cDNA was synthesized with a reverse primer (dotted arrows) hybridizingtoward the 31015840 end of either copA1 (a) or copA2 (b) S metallicus total RNA was extracted at the late exponential phase from a culture growingin presence of 20mMCu PCR amplifications were carried out with these cDNAs and each corresponding primer pair (black arrows) as listedin Table 1 (c) and (d) show RT-PCR products obtained for the copMA1 and copMA2 intergenic regions respectively Reverse transcriptasereactions with (+) and without (minus) the Improm II reverse transcriptase enzyme were carried out in order to exclude amplification due togenomic DNA contamination Expected sizes (in base pairs) for the corresponding PCR products are given in (a) and (b)

is in good agreement with what has been described for Ssolfataricus where the bicistronic copMA transcript levelswere also found to be up-regulated in response to both Cuand Cd [19]

Northern blot analysis showed that the sizes seen for bothcopA transcripts (sim25 Kb) did not correspond exactly withthe individual genes length (each of about 225Kb) (Figure 6)This strongly suggested that the transcripts could also includethe respective copM gene located upstream of copA in eachcop loci To confirm this assumption a co-transcriptionexperiment was done (Figure 7) in which the cDNAs wereobtained by using RNA extracted from a culture grownin the presence of 20mM Cu and using a reverse primerhybridizing with copA1 (Figure 7(a)) or copA2 (Figure 7(b))gene sequence respectively PCR amplifications were carriedout by using the corresponding cDNAs as templates and eachpair of primers lying in adjacent genes (copM) The presenceof an amplicon of the expected size in each case indicatedthe adjacent genes were part of polycistronic messengers(Figure 7) These results clearly show that in S metallicuseach couple of copMA genes was expressed in the form oftranscriptional units as reported in F acidarmanus and Ssolfataricus [18 19] The coexpression of gene pairrsquos copMAmay suggest a coordinated and dependent function for therespective encoded proteins In the bacterium E hirae themetallochaperone CopZ (CopM in S metallicus) fulfills apivotal role in the mechanism of Cu homeostasis [45] Thusit was shown that this protein interacts directly with the Cu-ATPase CopB (CopA in S metallicus) handing the Cu forsubsequent removal In this context one might expect thatproteins CopM1 and CopM2 from S metallicus have a similarrole to that described for E hirae Furthermore quantitativeRT-PCR experiments were carried out in order to determinedifferential expression of the cop genes in response to differentCu concentrations The expression of copA1 copA2 copT1and copT2 was tested relative to the transcript levels ofrRNA 16S gene since its levels were not significantly affected

in all assessed conditions (data not shown) As shown inFigure 8(a) copA2 and copA1 transcript levels were foundto be concomitantly increased with the increasing Cu con-centrations present in the medium Higher transcripts levelsof copA1 were seen in all tested conditions when comparedwith copA2 levels copA1 transcript levels were found to be325-fold up-regulated when comparing 50mMCu conditionversuscontrol (absence of metal) whereas copA2 transcriptlevels showed an increment of only 175-fold (Figure 8(a))The finding that Cu-ATPases mRNA levels were significantlyincreased in response to Cu ions exposure suggests that thetransport system may operate for Cu efflux in S metallicus

Moreover copA2 and copA1 gene expression was quan-tified when S metallicus was grown using chalcopyrite(CuFeS

2) ore as an oxidizable substrate (Figure 8(a)) Mineral

oxidation mediated by the microorganism generates a pro-gressive increase in Cu ions concentration in the medium Tofind out whether the amount of solubilized Cu induced theexpression of the Cu-ATPases genes total RNAwas extractedfrom a S metallicus culture grown to late exponential phaseand in the presence of 1 CuFeS

2 It was clear that an

up-regulation of the Cu-ATPases encoding genes also tookplace when S metallicus was grown in the presence ofCuFeS

2(Figure 8(a)) copA1 transcript levels increased 14-fold

compared with the control condition in the absence of Cuwhile copA2 gene expression showed an increase of 76 foldAlong with this bymeans of atomic absorption spectrometry(AAS) analysis an overall amount of solubilized Cu ions(Cu2+Cu1+) of 144plusmn21mMwas determined to be present inthemedium indicating that copA2 and copA1 gene expressionwas most likely in response to the Cu present in the culturedue to CuFeS

2microbial-solubilization

The finding that copA1 was highly expressed comparedwith copA2 in all tested conditions may suggest a possiblephysiological hierarchy between the two ATPases whenovercoming either Cu or Cd stress In this regard by meansof a genetic approach it was demonstrated in S solfataricus

Archaea 13

700

600

500

400

300

200

100

00

5 20 50 CuFeS2CuSO4 concentration (mM)

Relativ

egenee

xpression

CopA2CopA1

(a)

8

6

7

5

4

3

2

1

00

5 20 50 CuFeS2CuSO4 concentration (mM)

Relativ

egenee

xpression

CopT1CopT2

(b)

Figure 8 Relative expression levels of S metallicus genes copA1 and copA2 (a) copT1 and copT2 (b) Expression of S metallicus cop geneswasassessed by qRT-PCR and normalized against 16S rRNA gene expression in individual cultures Mean values and standard deviations arefrom analyses of three independent cultures grown in the presence of 0 5 20 50mMCu or in 1 of a chalcopyrite ore (CuFeS

2) concentrate

that while CopA was an effective Cu efflux transporterat low and high Cu concentrations the other Cu-ATPase(CopB) only appeared to be a low-affinity Cu export ATPase[68] Moreover by using a M sedula copA deletion mutantit was demonstrated that this strain compromised metalresistance and consequently abolished chalcopyrite oxidation[22] highlighting the role of Cu detoxification mechanismsduring a given bioleaching process Our attempts to showthe functionality of CopA1 and CopA2 from S metallicusby using E coli as a heterologous host were not successfulApparently the overexpression of these ORFs had a toxiceffect on E coli that compromised its viability Althoughgene disruption tools have not been yet developed for Smetallicus the functional role of both copTMA locimight befurther studied by cross-species complementation of a coppersensitive S solfataricus copR mutant as it was described byMaezato et al [22] It will be of great interest to establishin future studies the possible functionality of the isolatedputative transporters from S metallicus

Likewise quantitative RT-PCR experiments showed thatcopT1 transcript levels increased concomitantly with increas-ing Cu concentrations whereas copT2 showed relativelysimilar transcripts levels most likely indicating a constitutiveexpression profile (Figure 8(b)) Furthermore whereas copT1transcript levels were found to be increased 36 fold inCuFeS

2

grown cultures copT2 levels remained unchanged in com-parison with the control in the absence of Cu (Figure 8(b))The results obtained for S metallicus copT2 gene expressionare consistent with those reported for S solfataricus CopTtranscriptional regulator has been proposed to function as arepressor in S solfataricus showing a constitutive expressionthat in the presence of Cu loses its affinity for the promoterregion of copMA allowing the expression of this polycistron[19] In contrast the increased transcripts levels of both copA1and copT1 concomitant with higher Cu concentrations in the

environment suggest that the putative transcriptional regula-tor CopT1 may act by activating both copMA1 and probablyitself (Figure 8) Regarding this it was recently reported thatCopR (CopT in S metallicus) from S solfataricus strain 982acts as an activator of copT (copM in S metallicus) and copAexpression [69] Nevertheless additional experiments wouldbe required to test this possibility in S metallicus

4 Concluding Remarks

We previously reported that S metallicus resists extremelyhigh Cu concentrations which was mediated to some extentby the use of a possible metal resistance system based oninorganic polyphosphate hydrolysis and consequently Cu-PO4

2minusefflux [15 29 30] Here we have addressed the questionwhether this microorganism coded for other determinantsthat might help to explain its high heavy metal toleranceAs the genomic sequence of this microorganism is notyet available we jointly used CODEHOP-PCR and genomewalking approaches and were able to establish the occurrenceof two nonidentical homologous cop loci into the genome ofS metallicus (cop1 and cop2) Each cop locus codes for anarchaeal transcriptional regulator (CopT) a metal-bindingchaperone (CopM) and a Cu-transporting P-type ATPase(CopA) High levels of the polycistronic mRNAscopMA ofeach cop locus were observed after treatment with either Cuor Cd suggesting that the encoded ATPases efflux heavymetals out in order to detoxify the intracellular environmentAltogether previous reports and the results obtained in thisstudy allow us to suggest that some key elements that mayexplain the high resistance to Cu in S metallicus is the dupli-cation of the Cu resistance cop genes a defensive responseto stress and a polyP-based accumulation mechanism Inthe case of Cd although some similar stress responses were

14 Archaea

observed whether comparable Cu-responsive elements alsoparticipate in Cd responses remains to be seen

Conflict of Interests

The authors declare that the research was done in the absenceof any commercial or financial relationships that could beconstrued as a potential conflict of interests

Acknowledgments

This work was supported by Grant no FONDECYT 1110214and in part by ICM P-05-001-F project

References

[1] A Schippers ldquoMicroorganisms involved in bioleaching andnucleic acid-based molecular methods for their identificationand quantificationrdquo in Microbial Processing of Metal SulfidesR E Donati and W Sand Eds pp 3ndash33 Elsevier BerlinGermany 2007

[2] H R Watling ldquoThe bioleaching of sulphide minerals withemphasis on copper sulphides a reviewrdquo Hydrometallurgy vol84 no 1-2 pp 81ndash108 2006

[3] D E Rawlings D Dew and C Du Plessis ldquoBiomineralizationof metal-containing ores and concentratesrdquo Trends in Biotech-nology vol 21 no 1 pp 38ndash44 2003

[4] L Valenzuela A Chi S Beard et al ldquoGenomics metagenomicsand proteomics in biomining microorganismsrdquo BiotechnologyAdvances vol 24 no 2 pp 197ndash211 2006

[5] C A Jerez ldquoBioleaching and biomining for the industrialrecovery of metalsrdquo in Comprehensive Biotechnology M Moo-Young Ed vol 3 pp 717ndash729 Elsevier Amstaerdam TheNetherlands 2nd edition 2011

[6] P R Norris and J P Owen ldquoMineral sulphide oxidationby enrichment cultures of novel thermoacidophilic bacteriardquoFEMS Microbiology Reviews vol 11 no 1ndash3 pp 51ndash56 1993

[7] D E Rawlings and D B Johnson ldquoThe microbiology ofbiomining development and optimization ofmineral-oxidizingmicrobial consortiardquo Microbiology vol 153 no 2 pp 315ndash3242007

[8] P R Norris N P Burton and N A M Foulis ldquoAcidophiles inbioreactor mineral processingrdquo Extremophiles vol 4 no 2 pp71ndash76 2000

[9] M R Bruins S Kapil and F W Oehme ldquoMicrobial resistancetometals in the environmentrdquo Ecotoxicology and EnvironmentalSafety vol 45 no 3 pp 198ndash207 2000

[10] T J G Ettema M A Huynen W M De Vos and J Van DerOost ldquoTRASH a novel metal-binding domain predicted to beinvolved in heavy-metal sensing trafficking and resistancerdquoTrends in Biochemical Sciences vol 28 no 4 pp 170ndash173 2003

[11] M Dopson C Baker-Austin P R Koppineedi and P L BondldquoGrowth in sulfidic mineral environments metal resistancemechanisms in acidophilic micro-organismsrdquo Microbiologyvol 149 no 8 pp 1959ndash1970 2003

[12] S Franke and C Rensing ldquoAcidophiles Mechanisms to toleratemetal and acid toxicityrdquo in Physiology and Biochemistry ofExtremophiles C Gerday and N Glansdorff Eds pp 271ndash278ASM Press Washington DC USA 2007

[13] C A Navarro L H Orellana C Mauriaca and C A JerezldquoTranscriptional and functional studies of Acidithiobacillusferrooxidans genes related to survival in the presence of copperrdquoApplied and Environmental Microbiology vol 75 no 19 pp6102ndash6109 2009

[14] A Orell C A Navarro R Arancibia J C Mobarec andC A Jerez ldquoLife in blue copper resistance mechanisms ofbacteria and Archaea used in industrial biomining of mineralsrdquoBiotechnology Advances vol 28 no 6 pp 839ndash848 2010

[15] A Orell C A Navarro M Rivero J S Aguilar and C A JerezldquoInorganic polyphosphates in extremophiles and their posiblefunctionsrdquo Extremophiles vol 16 no 4 pp 573ndash583 2012

[16] C Baker-Austin M Dopson M Wexler et al ldquoExtremearsenic resistance by the acidophilic archaeon ldquoFerroplasmaacidarmanusrdquo Fer1rdquo Extremophiles vol 11 no 3 pp 425ndash4342007

[17] E Pedone S Bartolucci and G Fiorentino ldquoSensing andadapting to environmental stress the archaeal tacticrdquo Frontiersin Bioscience vol 9 pp 2909ndash2926 2004

[18] C Baker-Austin M Dopson M Wexler R G Sawers and PL Bond ldquoMolecular insight into extreme copper resistance inthe extremophilic archaeon ldquoFerroplasma acidarmanusrdquo Fer1rdquoMicrobiology vol 151 no 8 pp 2637ndash2646 2005

[19] T J G Ettema A B Brinkman P P Lamers N G Kornet WM de Vos and J van der Oost ldquoMolecular characterization ofa conserved archaeal copper resistance (cop) gene cluster andits copper-responsive regulator in Sulfolobus solfataricus P2rdquoMicrobiology vol 152 no 7 pp 1969ndash1979 2006

[20] A A Villafane Y VoskoboynikM Cuebas I Ruhl and E BinildquoResponse to excess copper in the hyperthermophile Sulfolobussolfataricus strain 982rdquo Biochemical and Biophysical ResearchCommunications vol 385 no 1 pp 67ndash71 2009

[21] K S Auernik Y Maezato P H Blum and R M KellyldquoThe genome sequence of the metal-mobilizing extremelythermoacidophilic archaeon Metallosphaera sedula providesinsights into bioleaching-associated metabolismrdquo Applied andEnvironmental Microbiology vol 74 no 3 pp 682ndash692 2008

[22] Y Maezato T Johnson S McCarthy K Dana and P BlumldquoMetal resistance and lithoautotrophy in the extreme ther-moacidophile Metalosphaera sedulardquo Journal of Bacteriologyvol 194 no 24 pp 6856ndash6863 2012

[23] D H Nies ldquoMicrobial heavy-metal resistancerdquo Applied Micro-biology and Biotechnology vol 51 no 6 pp 730ndash750 1999

[24] K Vido D Spector G Lagniel S Lopez M B Toledano andJ Labarre ldquoA proteome analysis of the cadmium response inSaccharomyces cerevisiaerdquo The Journal of Biological Chemistryvol 276 no 11 pp 8469ndash8474 2001

[25] G Bertin and D Averbeck ldquoCadmium cellular effects mod-ifications of biomolecules modulation of DNA repair andgenotoxic consequences (a review)rdquo Biochimie vol 88 no 11pp 1549ndash1559 2006

[26] M H Joe S W Jung S H Im et al ldquoGenome-wide responseof Deinococcus radiodurans on cadmium toxicityrdquo Journal ofMicrobiology and Biotechnology vol 21 no 4 pp 438ndash447 2011

[27] A Lagorce A Fourcans M Dutertre B Bouyssiere YZivanovic and F Confalonieri ldquoGenome-wide transcriptionalresponse of the archeon Thermococcus gammatolerans to cad-miumrdquo PLOS ONE vol 7 no 7 Article ID e41935 2012

[28] A Kornberg N N Rao and D Ault-Riche ldquoInorganicpolyphosphate a molecule of many functionsrdquo Annual Reviewof Biochemistry vol 68 pp 89ndash125 1999

Archaea 15

[29] J D Keasling ldquoRegulation of intracellular toxic metals andother cations by hydrolysis of polyphosphaterdquoAnnals of theNewYork Academy of Sciences vol 829 pp 242ndash249 1997

[30] F Remonsellez A Orell and C A Jerez ldquoCopper tolerance ofthe thermoacidophilic archaeon Sulfolobus metallicus possiblerole of polyphosphate metabolismrdquoMicrobiology vol 152 no 1pp 59ndash66 2006

[31] V Hatzimanikatis L H Choe and K H Lee ldquoProteomicstheoretical and experimental considerationsrdquo BiotechnologyProgress vol 15 no 3 pp 312ndash318 1999

[32] L H Choe and K H Lee ldquoA comparison of three commerciallyavailable isoelectric focusing units for proteome analysis theMultiphor the IPGphor and the Protean IEF cellrdquo Electrophore-sis vol 21 no 5 pp 993ndash1000 2000

[33] U K Laemmli ldquoCleavage of structural proteins during theassembly of the head of bacteriophage T4rdquo Nature vol 227 no5259 pp 680ndash685 1970

[34] A Shevchenko M Wilm O Vorm and M Mann ldquoMassspectrometric sequencing of proteins from silver-stained poly-acrylamide gelsrdquo Analytical Chemistry vol 68 no 5 pp 850ndash858 1996

[35] P Giavalisco E Nordhoff T Kreitler et al ldquoProteome analysisof Arabidopsis thaliana by two-dimensional gel electrophoresisand matrix-assisted laser desorptionionisation-time of flightmass spectrometryrdquo Proteomics vol 5 no 7 pp 1902ndash19132005

[36] T M Rose E R Schultz J G Henikoff S Pietrokovski CM McCallum and S Henikoff ldquoConsensus-degenerate hybridoligonucleotide primers for amplification of distantly relatedsequencesrdquo Nucleic Acids Research vol 26 no 7 pp 1628ndash16351998

[37] T M Rose J G Henikoff and S Henikoff ldquoCODEHOP(Consensus-Degenerate Hybrid Oligonucleotide Primer) PCRprimer designrdquo Nucleic Acids Research vol 31 no 13 pp 3763ndash3766 2003

[38] J P Acevedo F Reyes L P Parra O Salazar B A Andrews andJ A Asenjo ldquoCloning of complete genes for novel hydrolyticenzymes from Antarctic sea water bacteria by use of animproved genomewalking techniquerdquo Journal of Biotechnologyvol 133 no 3 pp 277ndash286 2008

[39] S Bathe and P R Norris ldquoFerrous iron- and sulfur-inducedgenes in Sulfolobus metallicusrdquo Applied and EnvironmentalMicrobiology vol 73 no 8 pp 2491ndash2497 2007

[40] J Sambrook E F Fritsch and TManiatisMolecular Cloning ALaboratory Manual Cold Spring Harbor Press New York NYUSA 1989

[41] K W Miller S Sass Risanico and J B Risatti ldquoDifferentialtolerance of Sulfolobus strains to transition metalsrdquo FEMSMicrobiology Letters vol 93 no 1 pp 69ndash73 1992

[42] G Huber C Spinnler A Gambacorta and K O Stetter ldquoMet-allosphaera sedula gen and sp nov represents a new genus ofaerobic metal-mobilizing thermoacidophilic archaebacteriardquoSystematic and Applied Microbiology vol 12 pp 38ndash47 1989

[43] J Llanos C Capasso E Parisi D Prieur and C JeanthonldquoSusceptibility to heavy metals and cadmium accumulation inaerobic and anaerobic thermophilic microorganisms isolatedfrom deep-sea hydrothermal ventsrdquo Current Microbiology vol41 no 3 pp 201ndash205 2000

[44] A Kaur M Pan M Meislin M T Facciotti R El-Gewelyand N S Baliga ldquoA systems view of haloarchaeal strategies towithstand stress from transition metalsrdquo Genome Research vol16 no 7 pp 841ndash854 2006

[45] M Solioz and J V Stoyanov ldquoCopper homeostasis in Entero-coccus hiraerdquo FEMS Microbiology Reviews vol 27 no 2-3 pp183ndash195 2003

[46] S Sitthisak K Howieson C Amezola and R K JayaswalldquoCharacterization of a multicopper oxidase gene from Staphy-lococcus aureusrdquo Applied and Environmental Microbiology vol71 no 9 pp 5650ndash5653 2005

[47] L Rodrıguez-Montelongo S I Volentini R N Farıas E MMassa and V A Rapisarda ldquoThe Cu(II)-reductase NADHdehydrogenase-2 of Escherichia coli improves the bacterialgrowth in extreme copper concentrations and increases theresistance to the damage caused by copper and hydroperoxiderdquoArchives of Biochemistry and Biophysics vol 451 no 1 pp 1ndash72006

[48] E Williams T M Lowe J Savas and J DiRuggiero ldquoMicroar-ray analysis of the hyperthermophilic archaeon Pyrococcusfuriosus exposed to gamma irradiationrdquo Extremophiles vol 11no 1 pp 19ndash29 2007

[49] L M Gaetke and C K Chow ldquoCopper toxicity oxidative stressand antioxidant nutrientsrdquo Toxicology vol 189 no 1-2 pp 147ndash163 2003

[50] M J Davies ldquoThe oxidative environment and protein damagerdquoBiochimica et Biophysica Acta vol 1703 no 2 pp 93ndash109 2005

[51] J R Thompson J K Bell J Bratt G A Grant and LJ Banaszak ldquoVmax regulation through domain and subunitchanges The active form of phosphoglycerate dehydrogenaserdquoBiochemistry vol 44 no 15 pp 5763ndash5773 2005

[52] V Consalvi R Chiaraluce L Politi A Gambacorta MDe Rosa and R Scandurra ldquoGlutamate dehydrogenase fromthe thermoacidophilic archaebacterium Sulfolobus solfataricusrdquoEuropean Journal of Biochemistry vol 196 no 2 pp 459ndash4671991

[53] A J L Macario M Lange B K Ahring and E Conway DeMacario ldquoStress genes and proteins in the archaeardquo Microbiol-ogy and Molecular Biology Reviews vol 63 no 4 pp 923ndash9671999

[54] J AMaupin-FurlowM A Gil M A Humbard et al ldquoArchaealproteasomes and other regulatory proteasesrdquo Current Opinionin Microbiology vol 8 no 6 pp 720ndash728 2005

[55] N Sun F Beck R Wilhelm Knispel et al ldquoProteomics analysisof Thermoplasma acidophilum with a focus on protein com-plexesrdquoMolecular and Cellular Proteomics vol 6 no 3 pp 492ndash502 2007

[56] L S Madding J K Michel K R Shockley et al ldquoRole of the 1205731subunit in the function and stability of the 20S proteasome inthe hyperthermophilic Archaeon Pyrococcus furiosusrdquo Journalof Bacteriology vol 189 no 2 pp 583ndash590 2007

[57] R Shibata Y Bessho A Shinkai et al ldquoCrystal structureand RNA-binding analysis of the archaeal transcription factorNusArdquo Biochemical and Biophysical Research Communicationsvol 355 no 1 pp 122ndash128 2007

[58] W Bae B Xia M Inouye and K Severinov ldquoEscherichia coliCspA-family RNA chaperones are transcription antitermina-torsrdquo Proceedings of the National Academy of Sciences of theUnited States of America vol 97 no 14 pp 7784ndash7789 2000

[59] R J Ram N C VerBerkmoes M P Thelen et al ldquoMicrobi-ology community proteomics of a natural microbial biofilmrdquoScience vol 308 no 5730 pp 1915ndash1920 2005

[60] I Noel-Georis T Vallaeys R Chauvaux et al ldquoGlobal analysisof the Ralstonia metallidurans proteome prelude for the large-scale study of heavy metal responserdquo Proteomics vol 4 no 1pp 151ndash179 2004

16 Archaea

[61] M TMNovo A CDa Silva RMoreto et al ldquoThiobacillus fer-rooxidans response to copper and other heavy metals growthprotein synthesis and protein phosphorylationrdquo Antonie vanLeeuwenhoek vol 77 no 2 pp 187ndash195 2000

[62] Q She R K Singh F Confalonieri et al ldquoThe complete genomeof the crenarchaeon Sulfolobus solfataricus P2rdquo Proceedings ofthe National Academy of Sciences of the United States of Americavol 98 no 14 pp 7835ndash7840 2001

[63] R P Anderson and J R Roth ldquoTandem genetic duplications inphage and bacteriardquo Annual Review of Microbiology vol 31 pp473ndash505 1977

[64] D Gevers K Vandepoele C Simillion and Y Van De PeerldquoGene duplication and biased functional retention of paralogsin bacterial genomesrdquo Trends in Microbiology vol 12 no 4 pp148ndash154 2004

[65] A B Reams and E L Neidle ldquoSelection for gene clustering bytandemduplicationrdquoAnnual Review ofMicrobiology vol 58 pp119ndash142 2004

[66] L H Orellana and C A Jerez ldquoA genomic island providesAcidithiobacillus ferrooxidans ATCC 53993 additional copperresistance a possible competitive advantagerdquo Applied Microbi-ology and Biotechnology vol 92 no 4 pp 761ndash767 2011

[67] A B Brinkman T J G Ettema W M de Vos and J van derOost ldquoThe Lrp family of transcriptional regulatorsrdquo MolecularMicrobiology vol 48 no 2 pp 287ndash294 2003

[68] C Vollmecke S L Drees J Reimann S V Albers and MLubben ldquoThe ATPases CopA and CopB both contribute tocopper resistance of the thermoacidophilic archaeon SulfolobussolfataricusrdquoMicrobiology vol 158 no 6 pp 1622ndash1633 2012

[69] A Villafane Y Voskoboynik I Ruhl et al ldquoCopR of Sulfolobussolfataricus represents a novel class of archaeal-specific copper-responsive activators of transcriptionrdquo Microbiology vol 157no 10 pp 2808ndash2817 2011

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Anatomy Research International

PeptidesInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporation httpwwwhindawicom

International Journal of

Volume 2014

Zoology

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Molecular Biology International

GenomicsInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioinformaticsAdvances in

Marine BiologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Signal TransductionJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

Evolutionary BiologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Biochemistry Research International

ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Genetics Research International

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Advances in

Virolog y

Hindawi Publishing Corporationhttpwwwhindawicom

Nucleic AcidsJournal of

Volume 2014

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

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Enzyme Research

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

International Journal of

Microbiology

4 Archaea

Table 1 Oligonucleotides

Name Sequence DescriptioncopA cdegF 51015840-gatgtagtaatagtaaaaactggagaaataataccngcngaygg CODEHOP-PCRcopA cdegR 51015840-tcatcagcaaaattagaagaatctccngtngcdat CODEHOP-PCRcopT cdegF1 51015840-ctcaaatagaatataaagtattacaaatgttaaaagargaywsnmg CODEHOP-PCRcopT cdegR1 51015840-ggattaccatttatttcatttccacartartcrca CODEHOP-PCRcopT cdegR2 51015840-cttatcatattcataaatcttccatctattaatttrtarcaytc CODEHOP-PCRcopT degF1 51015840-gartgytayaarctnat DOP-PCRcopT degR1 51015840-atnagyttrtarcayct DOP-PCRcopM degF 51015840-gayccngtntgyggnatgga DOP-PCRcopM degR 51015840-ccnggnttyccntacgg DOP-PCRAdaptF2 51015840-cacgcgtcgactagtactagctt Genome WalkingSP1copA1 31015840 51015840-aaggatgagggggaccttatgg GenomeWalkingSP2copA1 31015840 51015840-ggagataagaaatggggtaaaagag GenomeWalkingSP1copA1 51015840 51015840-tgataccatcatggaacctgtcag GenomeWalkingSP2copA1 51015840 51015840-tcctccacaatcccatccgctg GenomeWalkingSP1copA1 51015840 2 51015840-gattgtagctaagttaacctcggcctcg GenomeWalkingSP2copA1 51015840 2 51015840-cttctcaccctcagtctggttgg GenomeWalkingSP1copA2 31015840 51015840-gaaagaggaatatatgcaagggtaaacgg GenomeWalkingSP2copA2 31015840 51015840-gtgttaatgggagagctggaggg GenomeWalkingSP1copA2 51015840 51015840-cttctctgtggcaacatcataaccagcc GenomeWalkingSP2copA2 51015840 51015840-acgcatgtggcgcaatgcattcc GenomeWalkingSP1copT1 51015840 51015840-cattcctcgcaccagcttgcacactctc GenomeWalkingSP1copT2 51015840 51015840-cctatgaatactagatcttttccctgaac GenomeWalkingSP2copT2 51015840 51015840-aacagcttataacactcgtcactttggc GenomeWalkingcopM2 RT F 51015840-gatgaaaaaagccaatataagac RT-PCRcopA2 Rv 51015840-gaacactaactaacatcgcc RT-PCRcopM1 RT Fw 51015840-ctatcgtttttgttccgaagcttg RT-PCRcopA1 Rv 51015840-cagcagcaagaacagagacgcc RT-PCRlowastSM16Sf 51015840-acgctctaaaaaggcgtgggaata RT-qPCRlowastSM16Sr 51015840-ttgagctcggggtctttaagcagtg RT-qPCRcopA1Sm qRT F1 51015840-gctaaggtaatagagagcgg RT-qPCRcopA1Sm qRT R1 51015840-tgaacaggaatggacagg RT-qPCRcopA2Sm qRT F 51015840-tgtgcttgtctccttagcgt RT-qPCRcopA2Sm qRT R 51015840-actcttccgtctttcggagt RT-qPCRcopT1Sm qRT F 51015840-tgtaggagagtgtgcaagct RT-qPCRlcopT1Sm qRT R 51015840-tcgcaagtgagggttatggt RT-qPCRcopT2Sm qRT F 51015840-gtgttacggagcttgca RT-qPCRcopT2Sm qRT R 51015840-acactcgtcactttggc RT-qPCRAll oligonucleotides were synthesized by Invitrogen lowastOligonucleotides designed by Bathe and Norris [39]

of 10 120583L The ligation reaction was incubated at 16∘C during16 h

Two consecutive amplification reactions were then per-formedThefirst PCR reactionwas donewith 1x Elongasemixbuffer 19mM MgCl

2 02mM dNTPs 05120583M first specific

primer (SP1) (designed from the known sequence of thetarget gene a forward primer was used to amplify the 31015840end a reverse primer for 51015840 end amplification) 5 120583L of theligatedDNAdiluted 10-fold and 1 120583L of Elongase (Invitrogen)and water to a final volume of 50120583L The thermal cyclingconditions were as follows 1 cycle at 94∘C for 1min 20cycles of 94∘C for 32 s and 68∘C for 5min and one final

additional cycle at 70∘C for 7min The PCR product wasdiluted 10 fold and 3 120583L were used as a DNA template fora second PCR which was performed using the same con-ditions as the first PCR with 05 120583M second specific primer(SP2) and 02 120583Moligo-cassette-specific primer AdaptF2 (51015840-CACGCGTCGACTAGTACTAGCTT-31015840) and 1 120583L of Elon-gase The thermal cycling conditions were 1 cycle at 94∘C for1min 35 cycles of 94∘C for 32 s and 68∘C for 5min and 1final additional cycle at 70∘C for 7min PCR products wereexcised purified TA-cloned into pCR21-TOPO (Invitrogen)vector and finally sequenced

Archaea 5

28 Isolation of Total RNA Cell pellets (10mg wet weight)were collected and diluted in 60 120583L TEN buffer (20mMTris-HCl pH 80 1mM EDTA 100mM NaCl) followedby addition of 60120583L TENST buffer (20mM Tris-HCl pH80 1mM EDTA 100mM NaCl 16 Na-lauroyl sarcosineand 012 Triton X-100) This suspension was incubated atroom temperature for 15min to allow cell lysis Total RNAwas then extracted using the TRIzol reagent (Invitrogen)as recommended by the manufacturer DNA contaminationin RNA preparations was removed by DNase I treatment(Roche) following the manufacturerrsquos instructions RNAwas then purified precipitated and finally resuspended indiethylpyrocarbonate (DEPC)-treated water Total RNA con-centrations were estimated by spectrophotometric measure-ments (OD

260) and its quality was evaluated by determining

the ratio of absorption at 260 nm and 280 nm which waswithin the preferred range of 18ndash21

29 Northern Blotting For differential gene expression byNorthern blotting experiments the media were supple-mented with either different concentrations of Cu (5ndash50mM) Cd (5mM) NiSO

4(15mM) ZnSO

4(50mM) or

Ag2SO4(008mM) respectively as indicated For some

experiments elemental sulfur was replaced by a chalcopyrite(CuFeS

2) concentrate which was used as the only energy

source at 1 (wv)Total RNA (5 120583g) was separated by electrophoresis

on a 1 formaldehyde agarose gel followed by blottingonto Hybond-N nylon membranes (Amersham PharmaciaBiotech Bucking-hamshire UK) Hybridization was con-ducted as described by Sambrook et al [40] DNA probeswere labeled by randomprimerDNA labeling kit (Fermentas)using [120572-32 p] dCTP The hybridization signal was detectedand analyzed by using the molecular Imager FX system andQuantity One software The primer sequences for amplifica-tion of these gene probes are listed in Table 1

210 Cotranscriptional Analysis by RT-PCR To study theexpression of adjacent genes copM and copA cDNA wassynthesized by using 08120583g of total RNA from a S metallicusculture grown in the presence of 20mM Cu and a reverseprimer hybridizing to copA sequence A forward primerannealing on copM sequence was used for the PCR reactions(Table 1) PCR amplifications were performed with 1 120583L ofa 110 dilution of the cDNA and 25 pmol of each primerAmplification conditions included an initial 3min of denat-uration at 95∘C followed by 35 cycles of 30 s at 95∘C 30 sat 55∘C and 15min at 72∘C and finished by 10min at 72∘CA reverse transcriptase reaction without enzyme was carriedout in order to exclude amplification due to genomic DNAcontamination

211 Quantitative RT-PCR Single stranded cDNA was syn-thesized from 08 ng of DNA-free RNA samples usingrandom hexamers (Fermentas) and ImProm-II reversetranscription system (Promega) following manufacturerrsquosinstructions The software tool IDT Scitools (IntegratedDNA Technologies) was used to design primers producing

amplicons of 150ndash200 bp (Table 1) qPCRwas performedwith2 120583L of 1 10 diluted cDNA samples 125 120583L 2x QuantiFastSYBRGreenPCRMasterMix (Quiagen) 1 120583Mof each primerand water to a final volume of 25120583L The efficiency of eachprimer pair was calculated from the average slope of a linearregression curve which resulted from qPCRs using a 10-folddilution series (10 pgndash10 ng) of S metallicus chromosomalDNA as template Cq values (quantification cycle) wereautomatically determined by Real-Time Rotor-gene 6000PCR software (Corbett Life Sciences) after 40 cycles Cqvalues of each transcript of interest was standardized to theCq value of the 16S rRNA gene [39] At least 3 biologicalreplicates of each assessed condition and 2 technical replicatesper qPCR reaction were performed

3 Results and Discussion

31 Tolerance of S metallicus and S solfataricus to Cu and CdIn a previous work it was determined that S metallicus wasable to tolerate high Cu concentrations While the presenceof 100mM Cu did not affect S metallicus growth kinetics adecreased in cell biomass of only 30 was observed whenexposed to 200mM Cu [30] The high tolerance to Cu hasbeen described in other acidophilic Bacteria and Archaeacompared mostly with neutrophilic microorganisms [11 14]Here this analysis was further extended to characterize theresponse of S metallicus to Cd Thus it was determined thatS metallicus growth was not affected in the presence of either05 or 1mM Cd when compared with the control conditionin the absence of the metal (Figure 1(a)) In addition whenS metallicus was challenged with 2 and 3mM Cd it wasobserved that at the late exponential growth phase the cellnumbers decreased by 30 and 50 respectively (Figure 1(a))Moreover growth of S metallicuswas completely inhibited inthe presence of 5mM Cd (Figure 1(a)) On the other hand Ssolfataricuswas not able to grow at Cd concentrations greaterthan 005mM (Figure 1(b)) At 001mM Cd S solfataricuscell numbers decreased around 35 (Figure 1(b)) Theseresults are in agreement with previous reports in which Ssolfataricus was shown to be able to grow in up to 001mMCd [41] Other acidophilic archaeons involved in bioleachingprocesses such as Metallosphaera sedula and Ferroplasmaacidarmanus were found to be able to tolerate up to 09and 9mM Cd respectively [18 42] The minimal inhibitoryconcentration (MIC) to Cd has been described to be nothigher than 1mM for several thermophilic and neutrophilicThermococcus species [43] Recently it was reported that themost radioresistant archaeon Thermococcus gammatoleransstands Cd concentrations with an MIC of 2mM [27] Todate despite the heavy metals tolerance showed by someof these microorganisms strategies to withstand stress fromtransition metals have been most widely studied only inhaloarchaea [44]

32 Effect of Cu andCd on theGlobal Proteome of SmetallicusThe proteomic response to Cu and Cd was analyzed toidentify proteins which could be involved in heavy metalsresistance in S metallicus Early stationary phase growing

6 Archaea

14

12

10

8

6

4

2

00 2 4 6 8 10 12

Time (days)

107timescellsm

L

(a)

120

100

80

60

40

20

00 2 4 6 8 10

Time (days)

107timescellsm

L

(b)

Figure 1 Growth of S metallicus (a) and S solfataricus (b) in the presence of Cd Cells were inoculated in their respective growth media inabsence of added Cd (∙) or were supplemented with 0005mM () 001mM () 005mM (diams) 01mM () 05mM () 1mM () 2mM() 3mM (times) or 5mM (+) Cd and cells were counted daily

100

7060

50

30

20

3 10MW

33 31

32

A

B

C

20

10

(a)

100

7060

50

30

20

12

3 10MW

204

21

33 31

32

A

B

C

15

93

10

27 28

23 24

29

25 2630

1614 16

13

52

7

2219

8

20

10

11

(b)

Figure 2 Changes in the proteome of S metallicus grown in the presence of Cu Cells were incubated in the absence of any added metal(a) or in the presence of 100mM Cu (b) Arrows indicate the spots that were downregulated (a) or upregulated (b) in the presence of CuNumbers indicate the spots with increased intensity in cells treated with copper The dashed boxes are shown as enlarged areas that includesome proteins upregulated in the presence of Cu and Cd (Figure 3) The bottom panels show low molecular weight proteins upregulated inthe presence of Cu separated by using a 15 polyacrylamide gel in the second dimension

cells were untreated or treated with either 100 or 200mM Cuor 1mM Cd during 24 h and analyzed by comparative two-dimensional gel electrophoresis as indicated in experimentalprocedures Twenty-three proteins were found to be down-regulated after 100mMCu treatment (Figure 2(a)) A similar

pattern was obtained at 200mM Cu (not shown) Further-more by using the Delta 2D software (Decodon) eleven ofthese proteins were found to be completely absent in the gelsand more than 8 proteins decreased their intensity in therange from 15-to 5-fold compared with control cells grown

Archaea 7

13 13

11

13

1

Control Copper Cadmium

(a)

99 9

333

(b)

3030

30

2020

20 212121

(c)

Figure 3 Comparison of selected proteins of S metallicus cells treated with Cu or Cd Protein extracts were obtained from cells treatedwithout metals and with 100mM Cu or 1mM Cd for 24 h and the proteins were separated by 2D-PAGE The segments (a) (b) and (c) arethe enlarged dashed boxes in Figure 2 under the three conditions indicated Numbers show some of the spots with increased intensity in cellstreated with Cu or Cd

in the absence of Cu (Figure 2(a)) Therefore these resultsshow that a number of proteins became non-detectable ordecreased their levels when S metallicus faced Cu This kindof response was also seen when the microorganism wasexposed to Cd (data not shown) This behavior has alsobeen observed in similar studies where F acidarmanus waschallenged by either As(III) or Cu [16 18] In addition theexpression of 30 other proteins was found to be upregulatedafter Cu treatment (100 or 200mM) (Figure 2(b)) Most ofthese proteins could be identified only in the condition withCu and spots 5 13 and 15 were induced 26 34 and 55-fold respectively compared to cells without Cu treatmentMoreover 3 proteins of lowmolecular weight (spots 31 3 and33) that increased their expression when cells were exposedto 100mM Cu were also identified (Figure 2 bottom panels)These proteins were only detected when 15 polyacrylamidewas used during the second-dimensional separation (SDS-PAGE) On the other hand a large decrease in overallexpression of proteins after 1mMCd treatment was observed(not shown) Interestingly 10 out of the 13 proteins identifiedas up-regulated when cells were exposed to Cd and alsoshowed increased levels after Cu treatment Some of them areshown in Figure 3

33 Identification of Proteins Upregulated in Cells Treated withCu and Cd in S metallicus A total of 18 S metallicus proteinswhose levels were found to be up-regulated in responseto Cu were analyzed by mass spectrometry The identifiedproteins included functions related to the production andtransport of energy biosynthesis of amino acids stressresponses and transcription regulation (Table 2) Amongstthe proteins related to production and transport of energya putative ATP synthase subunit B (spot 23) that has beendescribed as playing a fundamental role in ATP synthesiswas identified (Table 2) When cells are subjected to somestressing conditions such as the presence of heavy metalsa greater cellular demand for energy has been reported tooccur [18] Most likely this phenomenon might be due toATP-driven Cu transport via ATPases that have a substantialinterplay during metal detoxification [45] Furthermore theATP synthase subunit B was also identified as up-regulatedwhen S metallicus was treated with Cd (Table 2)

Two other proteins corresponded to putative oxidoreduc-tases such as ferredoxin oxidoreductase (spot 3) and alcoholdehydrogenase (spot 4) that have been generally involvedin electron transporter chains and use NAD+ as an electronacceptor The levels of ferredoxin oxidoreductase were also

8 Archaea

Table 2 Proteins upregulated in S metallicus cells exposed to 100mM Cu or 1mM Cd

Spot Molecular weight (kDa) Cu induction levels Putative function Microorganism related Accesion number1dagger 266 infin Proteasome subunit S solfataricus AAK410343dagger 701 infin Ferredoxin oxidoreductase S solfataricus AAK429264dagger 429 infin Alcohol dehydrogenase S solfataricus AAK431545 35 26 Hypothetical protein S acidocaldarius YP 2549766 27 infin Hypothetical protein P aerophilum NP 5598899dagger 57 infin Phosphoglycerate dehydrogenase M thermautotrophicus AAB8546613dagger 27 34 Unknown mdash mdash14 28 infin Unknown mdash mdash15 35 55 Unknown mdash mdash20dagger 461 infin Glutamate dehydrogenase S solfataricus AAK4223021dagger 45 infin Hypothetical protein M acetivorans NP 618380122 49 infin Hypothetical protein P torridus YP 02280723dagger 51 infin ATP synthase subunit B S solfataricus AAK4088024 585 infin HSP60 subunit S shibatae AAG37273130dagger 48 infin Unknown mdash mdash31 16 ND Unknown mdash mdash32 144 ND Putative transcription regulator S solfataricus AAK4041333 138 ND Transcription factor nusA S solfataricus AAK40563Spots refer to those numbered in Figure 2 Proteins in Figure 2 which are not included in this table were not subjected to sequencing due to their low intensitiesin the gelsdaggerSpot up-regulated in cells exposed to Cu and CdND not determinedInfinity symbol indicates that proteins were expressed only in presence of copper or cadmiumM thermoautotrophicus Methanobacterium thermoautotrophicus M acetivorans Methanosarcina acetivorans S shibatae Sulfolobus shibatae P torridusPicrophilus torridus P aerophilum Pyrobaculum aerophilum S acidocaldarius Sulfolobus acidocaldarius

up-regulated in response to Cd (Table 2) Several previ-ous reports have suggested that oxidoreductases contributeto an oxidative protection in both Bacteria and Archaeain response to heavy metals [46ndash48] In the neutrophilicmicroorganisms Escherichia coli and Staphylococcus aureusit has been described that oxidases and dehydrogenasescontribute to oxidative protection Cu homeostasis and stressresponses [46 47] Furthermore some proteins involved inoxidative damage repair such as NADH-dependent oxidasesand thioredoxin reductases were expressed in cells of Facidarmanus exposed to As(III) [16] The expression of thisgroup of proteins has also been observed when the samemicroorganism was exposed to Cu [18] Therefore oxidore-ductases play an important role against oxidative stress andmay eliminate reactive oxygen species which constitute themajor component of the stress caused by transition metals[44 49 50]

The enzymes related to biosynthesis of amino acids werefound as commonly up-regulated either in response to Cu orCdThey corresponded to a phosphoglycerate dehydrogenase(spot 9) and a glutamate dehydrogenase (spot 20) Theformer one catalyzes the NAD+-dependent oxidation of 3-phosphoglycerate into 3-phosphohydroxypyruvate a branchpoint from the glycolytic pathway and the initial reactionin L-serine biosynthesis [51] Glutamate dehydrogenase cat-alyzes the oxidative deamination of glutamate to produce 2-oxoglutarate and ammonia with reduction of NAD+ [52]Proteins involved in amino acids synthesis were expressed

in cells of F acidarmanus exposed to As(III) [16] Moreoverincreased levels of proteins involved in the biosynthesisof sulfur-containing amino acids have been observed inSaccharomyces cerevisiae cells exposed to Cd [24] suggestingthat these proteins might also have a role in the cellularresponse to adverse conditions

Two proteins showed identity to components of the stressresponse mechanism such as one subunit of the HSP60chaperonin (spot 24) and one subunit of the proteasome (spot1) In Archaea the stress protein HSP60 is directly involvedin protein folding processes [53] Proteomic analysis of Facidarmanus cells exposed to As(III) and Cd revealed theexpression of proteins involved in protein folding and DNArepair including HSP60 chaperonin and DnaK heat-shockprotein (HSP70) thereby the authors suggested that thismicroorganismusesmultiplemechanisms to resist high levelsof Cu [16 18] On the other hand proteasomes are describedas nonspecific proteolytic nanomachines associated withprotein catabolism Proteasomes are known to be closelyinvolved in maintaining protein quality control by degradingmiss folded and denatured proteins in response to cell stressin all three domains of life [54] A proteomic analysis inthe thermoacidophilic archaeon Thermoplasma acidophilumshowed that proteasomes and chaperone-related proteinswere highly induced against stress conditions indicatinga high turnover rate of proteins [55] In P furiosus theexpression of the 1205731 subunit of the proteasome was inducedin cells subjected to heat shock stress [56] Additionally the

Archaea 9

150pb

(a)

1016 pb

(b)

2000 pb

(c)

Figure 4 CODEHOP-based PCR for the amplification of the putative genes copA and copM from S metallicus Amplification products(a) using degenerate PCR primers (DOP) copM degF and copM degR designed from amino acid sequence conserved blocks of SulfolobalesCopM proteins (b) using primers copA cdegF and copA cdegR designed by CODEHOP strategy for amplification of a copA-like gene (c)Primers copM degF and copA cdegRwere assesed for the amplification of a copMA-likeDNA regionThose primerswere testedwith genomicS metallicus DNA samples Amplicons expected sizes were as follows ca 150 bp for copM ca 950 bp for copA and ca 2000 bp for copMAPCR products were excised and cloned for later sequencing

proteasome subunit was also found to be up-regulated whenS metallicus was subjected to Cd stress (Table 2)

The transcriptional regulation-related proteins corre-sponded to a putative transcription regulator (spot 32) andthe transcription factor NusA (spot 33) involved in theintrinsic termination of transcription [57] NusA has beenalso reported to display important functions in the cellularresponse to adverse factors [58] The expression of twotranscriptional regulators related to amino acids biosynthesisand metal-dependent genetic repression were found to beinduced in P furiosus cells subjected to oxidative stress[48] Moreover the expression of several genes encodingpredicted transcriptional regulators were induced followingCd exposure in T gammatolerans cells [27]

Spots 5 6 13 14 15 21 22 30 and 31 (Table 2) did notshow significant scores with any protein sequences currentlyavailable in the data bases Interestingly spots 2 4 9 13 21and 30 were found to be commonly upregulated when Smetallicus cells were treated with either Cu or Cd (Figure 3Table 2)

The results presented here are consistent with previ-ous work describing global protein expression profiles inresponse to heavy metals as reported for F acidarmanus [1618] microbial-biofilm communities in an acid mine drainagesite [59] and other prokaryotic and eukaryotic microorgan-isms [24 60]Therefore a coordinated expression of differentgroups of genes suggests the existence of regulatory networkssuch as stress response mechanisms and respiratory chainadjustments to cope with the presence of heavy metal ions[61]

34 Cu-Resistance (Cop) Genes Cluster Is Duplicated in Smetallicus Genome A Cu-resistance (cop) locus has beendescribed to be highly conserved in archaeal genomeswhich consists of genes encoding a new type of archaealtranscriptional regulator (CopT) a metal-binding chaperone(CopM) and a Cu-transporting P-type ATPase (CopA) [19]Since the genome sequence of Smetallicus is not yet availablewe searched for the presence of cop genes by using consen-sus degenerate hybrid oligonucleotide primers-based PCR(CODEHOP-PCR) CopA and CopM amino acid sequencealignments from S acidocaldarius S tokodaii and S solfa-taricus were used to identify conserved sequence blocks andtherefore to design either CODEHOP or DOP (degeneratehybrid oligonucleotide) primers (Figure S1 Table 1) TherebyPCR assays using S metallicus genomic DNA as templateyielded amplicons of ca 150 bp and ca 950 bp for copMand copA CODEHOP pair primers respectively (Figures4(a) 4(b)) As estimated from primer pairrsquos position onamino acid sequence alignments the obtained PCR productscorresponded to the expected sizes suggesting the presenceof copM- and copA-like genes in S metallicus genome Inorder to determine the identity of the amplified DNA frag-ments they were TA-cloned and sequenced Blastx sequenceanalysis showed that the 150 bp DNA fragment coded for anincomplete ORF sharing 63 homology with S solfataricusCopM (SSO10823) whereas the analysis of the 950 pb DNAfragment yielded a partial ORF sharing 54 homology withS solfataricus CopA protein (SSO2651)

The genomic organization of the putative cop genes in Smetallicus was analyzed to find out its similarity with those

10 Archaea

Table 3 Sequence identity comparison of S metallicus Cop proteins to those in other Sulfolobales

S metallicus protein Identity to homologue in other SulfolobalesS solfataricus P2 S acidocaldarius S tokodaii M sedula

CopA1 51 43 44 45CopA2 88 46 49 50CopM1 63 69 71 71CopM2 89 72 70 77CopT1 36 27 30 32CopT2lowast 90 52 52 53Boldface type indicates to which organism the S metallicus homologue shows the highest sequence identitylowastRefers to the analysis obtained using the uncompleted amino acid sequence of CopT2 which represents sim90 in length when compared with S solfataricusCopT sequence

previously described in S solfataricus P2 and F acidarmanuswhere the cop cluster consists of tandem-orientated genesas copTMA [18 19] Thus using copM degF (forward) andcopA cdegR (reverse) primers we attempted to amplify aputative copMA DNA region This PCR yielded a product ofca 2000 pb corresponding to the expected size and resultedin 2 ORFs showing high sequence homology with CopMand CopA from S solfataricus (Figure 4(c)) Interestinglywhen aligning this copMA sequence with the copA and copMsequences obtained previously they were not identical whichstrongly indicated the presence of duplicated putative copgenes encoding for both Cu-ATPases (copA1 and copA2) andmetallochaperones (copM1 and copM2) in the S metallicusgenome

To isolate the entire cop gene sequences the genomewalking method described by Acevedo et al [38] was usedSix different DNA libraries were constructed from S metalli-cus genomic DNA digested with several restriction enzymesincluding 51015840 overhang and blunt end restriction enzymesThe DNA libraries were used as templates in 2 successivePCR reactions From the previously known sequences twospecific sense primers for the 31015840 end amplification and twospecific antisense primers for the 51015840 end amplification weredesigned (abbreviated SP1 and SP2) Therefore the 51015840 and 31015840ends of the putative genes copA1 and copA2 that had beenpartially identified through the genome walking techniquewere amplified

By overlapping the sequence isolated by degenerate PCRand the lateral sequences obtained by genome walking(Figure S2) it was possible to complete the whole nucleotidesequence of the putative gene copA1 Additionally it waspossible to confirm the presence of the upstream copM1 geneanddetermine the occurrence of a partial copT1 gene tandem-orientated upstream of copM1 (Figure 5)Through additionalgenome walking experiments the partial copT1 sequencecould be further completedThe samewas truewhen isolatingthe whole nucleotide sequence of the putative gene copA2as the presence of copM2 and copT2 genes were confirmedupstream of copA2 However it was not possible to completethe whole 51015840 end of the putative gene copT2 as its determinedlength corresponded to only 90 of the full length whencompared with S solfataricus copT gene (Figure 5)

In conclusion degenerate PCR together with genomewalking experiments allowed to describe the occurrence inS metallicus genome of 2 cop genes loci (named as locus cop1and locus cop2) coding for paralogous genes whose productsmay be involved in Cu-resistance (Figure 5) Although somearchaeal genomes exhibit two copies of putative Cu-P-typeATPases as described for S solfataricus [62] the genomicarrangement displayed as a cop gene cluster (copTMA) hasbeen reported to be represented in only one copy for manyarchaeal genomes [10 19 21] Moreover we further updatedthis analysis by searching for duplications of the cop genescluster in all available archaeal genome sequences up to date(October 2012) retrieving only one cop locus in each caseThereby we propose that the discovery of this cop gene clusterduplication in the genome of S metallicus constitutes so faran unprecedented feature for a representative of the Archaeadomain and might contribute to its high metal resistance

In this context it has been widely reported that increasedgene copy number can increase gene expression allowingprokaryotic to thrive under growth limiting conditions [1363ndash68] However alongside the augmented gene copy onecannot exclude that the cop operon duplication in S metal-licus may offer among others a wider repertoire of Cu-ATPases which might differ in terms of metal affinitiesandor specificity efflux rates andor differences in theirabundance regulation

Throughout amino acid sequence analysis it was furtherdetermined that each polypeptide encoded by S metallicuswith the exception of CopT1 showed to be highly homol-ogous to their orthologous counterparts encoded by othersSulfolobales (Table 3) Moreover the 3 ORFs encoded by thelocus cop2 shared about 90 identity with the correspondingS solfataricus orthologous peptides (Table 3) S metallicuscop genes products showed characteristic domains referredto as critical for their respective proposed biological activitiesS metallicus paralogous gene products CopA1 (749 aa1015840)and CopA2 (747 aa1015840) shared 513 of identity These twoputative Cu-ATPases (CopA1 and CopA2) contain the aminoacid sequence motif CPCALGLA which has been proposedto confer Cu-transporting specificity in CPx-ATPases [48]and has been also found in other CopA-like proteins frombiomining Bacteria and Archaea [14]

Archaea 11

HindIII HindIII HindIII

HindIII

CopT1Locus cop1 M1

EcoRIEcoRV

513 bp 219 bp 2250 bp

CopA1

(a)

Locus cop2 M2

EcoRI

CopA2

453 bp 171 bp 2244 bp

CopT2

PstIHindIII HindIII HindIII

(b)

Figure 5 Schematic representation of the two cop gene clusters isolated from S metallicus genome Each cluster codes for anArchaea-specifictranscriptional regulator (copT) a metallochaperone (copM) and a P-type Cu-exporting ATPase (copA) Lengths of each gene are indicatedLocus cop1 corresponds to 4346 sequenced base pairs and shows an intergenic region copT1-M1 of 232 base pairs Locus cop2 correspondsto 3435 base pairs and shows 89 base pairs copT2-M2 intergenic region Genes copM2 and copA2 overlapped in 11 base pairs copT2 was notfully isolated

Control CdSO4 Ag2SO4 NiSO4 ZnSO4 CuSO4

kb3kb2kb1

02 kb

(a)

kb3kb2kb1

02 kb

(b)

(c)

Figure 6 Northern blot analysis to determine the expression of S metallicus copA1 (a) and copA2 (b) putative genes in response to variousmetals S metallicus total RNAwas extracted from cultures grown either in the absence (control) or the presence of CuSO

4(50mM) Ag

2SO4

(008mM) NiSO4(15mM) ZnSO

4(50mM) and CdSO

4(2mM) P32-radioactivelly labelled DNA fragments annealing to copA1 and copA2

sequences respectively were used as probes (c) shows rRNAs as total RNA loading control

On the other hand CopM1 (71 aa) and CopM2 (56 aa)sharing 66 of identity present the proposed metal bindingdomain TRASH (trafficking resistance and sensing of heavymetals) [10] Additionally when analyzing the putative tran-scriptional regulators the presence of a C-terminal TRASHdomain in both CopT1 and CopT2 was also found CopT1sequence also contains an entire N-terminal helix-turn-helix (HTH) motif (not shown) that resembles the DNA-bindingmotifs of prokaryotic transcriptional regulators suchas Lrp-like proteins [67] The HTH motif was only partiallyidentified in CopT2 since the 51015840 region of copT1 gene has notbeen yet fully isolated

35 Transcriptional Analysis of S metallicus Cop Genes Toget insight into the role of the two cop loci the expressionof the corresponding genes was analyzed under variousconditions S metallicus was first grown in presence ofdifferent heavy metals (Cu Zn Cd Ni o Ag) at a givenconcentration that did not affect growth kinetics [30 42]Subsequently total RNA was isolated from late exponentiallygrown cultures and Northern blot experiments were carriedout in order to determine the expression of both copA1 andcopA2 genes As depicted in Figure 6 transcription of bothcopA1 and copA2messengers were specifically induced in thepresence of Cu and Cd ions This gene expression pattern

12 Archaea

954pb

M1 CopA1

(a)

1438 pb

M2 CopA2

(b)

1000 pb

800pb

+ minus

(c)

+ minus

1500 pb

1000 pb

(d)

Figure 7 Cotranscription analysis of copMA1 and copMA2 genes cDNA was synthesized with a reverse primer (dotted arrows) hybridizingtoward the 31015840 end of either copA1 (a) or copA2 (b) S metallicus total RNA was extracted at the late exponential phase from a culture growingin presence of 20mMCu PCR amplifications were carried out with these cDNAs and each corresponding primer pair (black arrows) as listedin Table 1 (c) and (d) show RT-PCR products obtained for the copMA1 and copMA2 intergenic regions respectively Reverse transcriptasereactions with (+) and without (minus) the Improm II reverse transcriptase enzyme were carried out in order to exclude amplification due togenomic DNA contamination Expected sizes (in base pairs) for the corresponding PCR products are given in (a) and (b)

is in good agreement with what has been described for Ssolfataricus where the bicistronic copMA transcript levelswere also found to be up-regulated in response to both Cuand Cd [19]

Northern blot analysis showed that the sizes seen for bothcopA transcripts (sim25 Kb) did not correspond exactly withthe individual genes length (each of about 225Kb) (Figure 6)This strongly suggested that the transcripts could also includethe respective copM gene located upstream of copA in eachcop loci To confirm this assumption a co-transcriptionexperiment was done (Figure 7) in which the cDNAs wereobtained by using RNA extracted from a culture grownin the presence of 20mM Cu and using a reverse primerhybridizing with copA1 (Figure 7(a)) or copA2 (Figure 7(b))gene sequence respectively PCR amplifications were carriedout by using the corresponding cDNAs as templates and eachpair of primers lying in adjacent genes (copM) The presenceof an amplicon of the expected size in each case indicatedthe adjacent genes were part of polycistronic messengers(Figure 7) These results clearly show that in S metallicuseach couple of copMA genes was expressed in the form oftranscriptional units as reported in F acidarmanus and Ssolfataricus [18 19] The coexpression of gene pairrsquos copMAmay suggest a coordinated and dependent function for therespective encoded proteins In the bacterium E hirae themetallochaperone CopZ (CopM in S metallicus) fulfills apivotal role in the mechanism of Cu homeostasis [45] Thusit was shown that this protein interacts directly with the Cu-ATPase CopB (CopA in S metallicus) handing the Cu forsubsequent removal In this context one might expect thatproteins CopM1 and CopM2 from S metallicus have a similarrole to that described for E hirae Furthermore quantitativeRT-PCR experiments were carried out in order to determinedifferential expression of the cop genes in response to differentCu concentrations The expression of copA1 copA2 copT1and copT2 was tested relative to the transcript levels ofrRNA 16S gene since its levels were not significantly affected

in all assessed conditions (data not shown) As shown inFigure 8(a) copA2 and copA1 transcript levels were foundto be concomitantly increased with the increasing Cu con-centrations present in the medium Higher transcripts levelsof copA1 were seen in all tested conditions when comparedwith copA2 levels copA1 transcript levels were found to be325-fold up-regulated when comparing 50mMCu conditionversuscontrol (absence of metal) whereas copA2 transcriptlevels showed an increment of only 175-fold (Figure 8(a))The finding that Cu-ATPases mRNA levels were significantlyincreased in response to Cu ions exposure suggests that thetransport system may operate for Cu efflux in S metallicus

Moreover copA2 and copA1 gene expression was quan-tified when S metallicus was grown using chalcopyrite(CuFeS

2) ore as an oxidizable substrate (Figure 8(a)) Mineral

oxidation mediated by the microorganism generates a pro-gressive increase in Cu ions concentration in the medium Tofind out whether the amount of solubilized Cu induced theexpression of the Cu-ATPases genes total RNAwas extractedfrom a S metallicus culture grown to late exponential phaseand in the presence of 1 CuFeS

2 It was clear that an

up-regulation of the Cu-ATPases encoding genes also tookplace when S metallicus was grown in the presence ofCuFeS

2(Figure 8(a)) copA1 transcript levels increased 14-fold

compared with the control condition in the absence of Cuwhile copA2 gene expression showed an increase of 76 foldAlong with this bymeans of atomic absorption spectrometry(AAS) analysis an overall amount of solubilized Cu ions(Cu2+Cu1+) of 144plusmn21mMwas determined to be present inthemedium indicating that copA2 and copA1 gene expressionwas most likely in response to the Cu present in the culturedue to CuFeS

2microbial-solubilization

The finding that copA1 was highly expressed comparedwith copA2 in all tested conditions may suggest a possiblephysiological hierarchy between the two ATPases whenovercoming either Cu or Cd stress In this regard by meansof a genetic approach it was demonstrated in S solfataricus

Archaea 13

700

600

500

400

300

200

100

00

5 20 50 CuFeS2CuSO4 concentration (mM)

Relativ

egenee

xpression

CopA2CopA1

(a)

8

6

7

5

4

3

2

1

00

5 20 50 CuFeS2CuSO4 concentration (mM)

Relativ

egenee

xpression

CopT1CopT2

(b)

Figure 8 Relative expression levels of S metallicus genes copA1 and copA2 (a) copT1 and copT2 (b) Expression of S metallicus cop geneswasassessed by qRT-PCR and normalized against 16S rRNA gene expression in individual cultures Mean values and standard deviations arefrom analyses of three independent cultures grown in the presence of 0 5 20 50mMCu or in 1 of a chalcopyrite ore (CuFeS

2) concentrate

that while CopA was an effective Cu efflux transporterat low and high Cu concentrations the other Cu-ATPase(CopB) only appeared to be a low-affinity Cu export ATPase[68] Moreover by using a M sedula copA deletion mutantit was demonstrated that this strain compromised metalresistance and consequently abolished chalcopyrite oxidation[22] highlighting the role of Cu detoxification mechanismsduring a given bioleaching process Our attempts to showthe functionality of CopA1 and CopA2 from S metallicusby using E coli as a heterologous host were not successfulApparently the overexpression of these ORFs had a toxiceffect on E coli that compromised its viability Althoughgene disruption tools have not been yet developed for Smetallicus the functional role of both copTMA locimight befurther studied by cross-species complementation of a coppersensitive S solfataricus copR mutant as it was described byMaezato et al [22] It will be of great interest to establishin future studies the possible functionality of the isolatedputative transporters from S metallicus

Likewise quantitative RT-PCR experiments showed thatcopT1 transcript levels increased concomitantly with increas-ing Cu concentrations whereas copT2 showed relativelysimilar transcripts levels most likely indicating a constitutiveexpression profile (Figure 8(b)) Furthermore whereas copT1transcript levels were found to be increased 36 fold inCuFeS

2

grown cultures copT2 levels remained unchanged in com-parison with the control in the absence of Cu (Figure 8(b))The results obtained for S metallicus copT2 gene expressionare consistent with those reported for S solfataricus CopTtranscriptional regulator has been proposed to function as arepressor in S solfataricus showing a constitutive expressionthat in the presence of Cu loses its affinity for the promoterregion of copMA allowing the expression of this polycistron[19] In contrast the increased transcripts levels of both copA1and copT1 concomitant with higher Cu concentrations in the

environment suggest that the putative transcriptional regula-tor CopT1 may act by activating both copMA1 and probablyitself (Figure 8) Regarding this it was recently reported thatCopR (CopT in S metallicus) from S solfataricus strain 982acts as an activator of copT (copM in S metallicus) and copAexpression [69] Nevertheless additional experiments wouldbe required to test this possibility in S metallicus

4 Concluding Remarks

We previously reported that S metallicus resists extremelyhigh Cu concentrations which was mediated to some extentby the use of a possible metal resistance system based oninorganic polyphosphate hydrolysis and consequently Cu-PO4

2minusefflux [15 29 30] Here we have addressed the questionwhether this microorganism coded for other determinantsthat might help to explain its high heavy metal toleranceAs the genomic sequence of this microorganism is notyet available we jointly used CODEHOP-PCR and genomewalking approaches and were able to establish the occurrenceof two nonidentical homologous cop loci into the genome ofS metallicus (cop1 and cop2) Each cop locus codes for anarchaeal transcriptional regulator (CopT) a metal-bindingchaperone (CopM) and a Cu-transporting P-type ATPase(CopA) High levels of the polycistronic mRNAscopMA ofeach cop locus were observed after treatment with either Cuor Cd suggesting that the encoded ATPases efflux heavymetals out in order to detoxify the intracellular environmentAltogether previous reports and the results obtained in thisstudy allow us to suggest that some key elements that mayexplain the high resistance to Cu in S metallicus is the dupli-cation of the Cu resistance cop genes a defensive responseto stress and a polyP-based accumulation mechanism Inthe case of Cd although some similar stress responses were

14 Archaea

observed whether comparable Cu-responsive elements alsoparticipate in Cd responses remains to be seen

Conflict of Interests

The authors declare that the research was done in the absenceof any commercial or financial relationships that could beconstrued as a potential conflict of interests

Acknowledgments

This work was supported by Grant no FONDECYT 1110214and in part by ICM P-05-001-F project

References

[1] A Schippers ldquoMicroorganisms involved in bioleaching andnucleic acid-based molecular methods for their identificationand quantificationrdquo in Microbial Processing of Metal SulfidesR E Donati and W Sand Eds pp 3ndash33 Elsevier BerlinGermany 2007

[2] H R Watling ldquoThe bioleaching of sulphide minerals withemphasis on copper sulphides a reviewrdquo Hydrometallurgy vol84 no 1-2 pp 81ndash108 2006

[3] D E Rawlings D Dew and C Du Plessis ldquoBiomineralizationof metal-containing ores and concentratesrdquo Trends in Biotech-nology vol 21 no 1 pp 38ndash44 2003

[4] L Valenzuela A Chi S Beard et al ldquoGenomics metagenomicsand proteomics in biomining microorganismsrdquo BiotechnologyAdvances vol 24 no 2 pp 197ndash211 2006

[5] C A Jerez ldquoBioleaching and biomining for the industrialrecovery of metalsrdquo in Comprehensive Biotechnology M Moo-Young Ed vol 3 pp 717ndash729 Elsevier Amstaerdam TheNetherlands 2nd edition 2011

[6] P R Norris and J P Owen ldquoMineral sulphide oxidationby enrichment cultures of novel thermoacidophilic bacteriardquoFEMS Microbiology Reviews vol 11 no 1ndash3 pp 51ndash56 1993

[7] D E Rawlings and D B Johnson ldquoThe microbiology ofbiomining development and optimization ofmineral-oxidizingmicrobial consortiardquo Microbiology vol 153 no 2 pp 315ndash3242007

[8] P R Norris N P Burton and N A M Foulis ldquoAcidophiles inbioreactor mineral processingrdquo Extremophiles vol 4 no 2 pp71ndash76 2000

[9] M R Bruins S Kapil and F W Oehme ldquoMicrobial resistancetometals in the environmentrdquo Ecotoxicology and EnvironmentalSafety vol 45 no 3 pp 198ndash207 2000

[10] T J G Ettema M A Huynen W M De Vos and J Van DerOost ldquoTRASH a novel metal-binding domain predicted to beinvolved in heavy-metal sensing trafficking and resistancerdquoTrends in Biochemical Sciences vol 28 no 4 pp 170ndash173 2003

[11] M Dopson C Baker-Austin P R Koppineedi and P L BondldquoGrowth in sulfidic mineral environments metal resistancemechanisms in acidophilic micro-organismsrdquo Microbiologyvol 149 no 8 pp 1959ndash1970 2003

[12] S Franke and C Rensing ldquoAcidophiles Mechanisms to toleratemetal and acid toxicityrdquo in Physiology and Biochemistry ofExtremophiles C Gerday and N Glansdorff Eds pp 271ndash278ASM Press Washington DC USA 2007

[13] C A Navarro L H Orellana C Mauriaca and C A JerezldquoTranscriptional and functional studies of Acidithiobacillusferrooxidans genes related to survival in the presence of copperrdquoApplied and Environmental Microbiology vol 75 no 19 pp6102ndash6109 2009

[14] A Orell C A Navarro R Arancibia J C Mobarec andC A Jerez ldquoLife in blue copper resistance mechanisms ofbacteria and Archaea used in industrial biomining of mineralsrdquoBiotechnology Advances vol 28 no 6 pp 839ndash848 2010

[15] A Orell C A Navarro M Rivero J S Aguilar and C A JerezldquoInorganic polyphosphates in extremophiles and their posiblefunctionsrdquo Extremophiles vol 16 no 4 pp 573ndash583 2012

[16] C Baker-Austin M Dopson M Wexler et al ldquoExtremearsenic resistance by the acidophilic archaeon ldquoFerroplasmaacidarmanusrdquo Fer1rdquo Extremophiles vol 11 no 3 pp 425ndash4342007

[17] E Pedone S Bartolucci and G Fiorentino ldquoSensing andadapting to environmental stress the archaeal tacticrdquo Frontiersin Bioscience vol 9 pp 2909ndash2926 2004

[18] C Baker-Austin M Dopson M Wexler R G Sawers and PL Bond ldquoMolecular insight into extreme copper resistance inthe extremophilic archaeon ldquoFerroplasma acidarmanusrdquo Fer1rdquoMicrobiology vol 151 no 8 pp 2637ndash2646 2005

[19] T J G Ettema A B Brinkman P P Lamers N G Kornet WM de Vos and J van der Oost ldquoMolecular characterization ofa conserved archaeal copper resistance (cop) gene cluster andits copper-responsive regulator in Sulfolobus solfataricus P2rdquoMicrobiology vol 152 no 7 pp 1969ndash1979 2006

[20] A A Villafane Y VoskoboynikM Cuebas I Ruhl and E BinildquoResponse to excess copper in the hyperthermophile Sulfolobussolfataricus strain 982rdquo Biochemical and Biophysical ResearchCommunications vol 385 no 1 pp 67ndash71 2009

[21] K S Auernik Y Maezato P H Blum and R M KellyldquoThe genome sequence of the metal-mobilizing extremelythermoacidophilic archaeon Metallosphaera sedula providesinsights into bioleaching-associated metabolismrdquo Applied andEnvironmental Microbiology vol 74 no 3 pp 682ndash692 2008

[22] Y Maezato T Johnson S McCarthy K Dana and P BlumldquoMetal resistance and lithoautotrophy in the extreme ther-moacidophile Metalosphaera sedulardquo Journal of Bacteriologyvol 194 no 24 pp 6856ndash6863 2012

[23] D H Nies ldquoMicrobial heavy-metal resistancerdquo Applied Micro-biology and Biotechnology vol 51 no 6 pp 730ndash750 1999

[24] K Vido D Spector G Lagniel S Lopez M B Toledano andJ Labarre ldquoA proteome analysis of the cadmium response inSaccharomyces cerevisiaerdquo The Journal of Biological Chemistryvol 276 no 11 pp 8469ndash8474 2001

[25] G Bertin and D Averbeck ldquoCadmium cellular effects mod-ifications of biomolecules modulation of DNA repair andgenotoxic consequences (a review)rdquo Biochimie vol 88 no 11pp 1549ndash1559 2006

[26] M H Joe S W Jung S H Im et al ldquoGenome-wide responseof Deinococcus radiodurans on cadmium toxicityrdquo Journal ofMicrobiology and Biotechnology vol 21 no 4 pp 438ndash447 2011

[27] A Lagorce A Fourcans M Dutertre B Bouyssiere YZivanovic and F Confalonieri ldquoGenome-wide transcriptionalresponse of the archeon Thermococcus gammatolerans to cad-miumrdquo PLOS ONE vol 7 no 7 Article ID e41935 2012

[28] A Kornberg N N Rao and D Ault-Riche ldquoInorganicpolyphosphate a molecule of many functionsrdquo Annual Reviewof Biochemistry vol 68 pp 89ndash125 1999

Archaea 15

[29] J D Keasling ldquoRegulation of intracellular toxic metals andother cations by hydrolysis of polyphosphaterdquoAnnals of theNewYork Academy of Sciences vol 829 pp 242ndash249 1997

[30] F Remonsellez A Orell and C A Jerez ldquoCopper tolerance ofthe thermoacidophilic archaeon Sulfolobus metallicus possiblerole of polyphosphate metabolismrdquoMicrobiology vol 152 no 1pp 59ndash66 2006

[31] V Hatzimanikatis L H Choe and K H Lee ldquoProteomicstheoretical and experimental considerationsrdquo BiotechnologyProgress vol 15 no 3 pp 312ndash318 1999

[32] L H Choe and K H Lee ldquoA comparison of three commerciallyavailable isoelectric focusing units for proteome analysis theMultiphor the IPGphor and the Protean IEF cellrdquo Electrophore-sis vol 21 no 5 pp 993ndash1000 2000

[33] U K Laemmli ldquoCleavage of structural proteins during theassembly of the head of bacteriophage T4rdquo Nature vol 227 no5259 pp 680ndash685 1970

[34] A Shevchenko M Wilm O Vorm and M Mann ldquoMassspectrometric sequencing of proteins from silver-stained poly-acrylamide gelsrdquo Analytical Chemistry vol 68 no 5 pp 850ndash858 1996

[35] P Giavalisco E Nordhoff T Kreitler et al ldquoProteome analysisof Arabidopsis thaliana by two-dimensional gel electrophoresisand matrix-assisted laser desorptionionisation-time of flightmass spectrometryrdquo Proteomics vol 5 no 7 pp 1902ndash19132005

[36] T M Rose E R Schultz J G Henikoff S Pietrokovski CM McCallum and S Henikoff ldquoConsensus-degenerate hybridoligonucleotide primers for amplification of distantly relatedsequencesrdquo Nucleic Acids Research vol 26 no 7 pp 1628ndash16351998

[37] T M Rose J G Henikoff and S Henikoff ldquoCODEHOP(Consensus-Degenerate Hybrid Oligonucleotide Primer) PCRprimer designrdquo Nucleic Acids Research vol 31 no 13 pp 3763ndash3766 2003

[38] J P Acevedo F Reyes L P Parra O Salazar B A Andrews andJ A Asenjo ldquoCloning of complete genes for novel hydrolyticenzymes from Antarctic sea water bacteria by use of animproved genomewalking techniquerdquo Journal of Biotechnologyvol 133 no 3 pp 277ndash286 2008

[39] S Bathe and P R Norris ldquoFerrous iron- and sulfur-inducedgenes in Sulfolobus metallicusrdquo Applied and EnvironmentalMicrobiology vol 73 no 8 pp 2491ndash2497 2007

[40] J Sambrook E F Fritsch and TManiatisMolecular Cloning ALaboratory Manual Cold Spring Harbor Press New York NYUSA 1989

[41] K W Miller S Sass Risanico and J B Risatti ldquoDifferentialtolerance of Sulfolobus strains to transition metalsrdquo FEMSMicrobiology Letters vol 93 no 1 pp 69ndash73 1992

[42] G Huber C Spinnler A Gambacorta and K O Stetter ldquoMet-allosphaera sedula gen and sp nov represents a new genus ofaerobic metal-mobilizing thermoacidophilic archaebacteriardquoSystematic and Applied Microbiology vol 12 pp 38ndash47 1989

[43] J Llanos C Capasso E Parisi D Prieur and C JeanthonldquoSusceptibility to heavy metals and cadmium accumulation inaerobic and anaerobic thermophilic microorganisms isolatedfrom deep-sea hydrothermal ventsrdquo Current Microbiology vol41 no 3 pp 201ndash205 2000

[44] A Kaur M Pan M Meislin M T Facciotti R El-Gewelyand N S Baliga ldquoA systems view of haloarchaeal strategies towithstand stress from transition metalsrdquo Genome Research vol16 no 7 pp 841ndash854 2006

[45] M Solioz and J V Stoyanov ldquoCopper homeostasis in Entero-coccus hiraerdquo FEMS Microbiology Reviews vol 27 no 2-3 pp183ndash195 2003

[46] S Sitthisak K Howieson C Amezola and R K JayaswalldquoCharacterization of a multicopper oxidase gene from Staphy-lococcus aureusrdquo Applied and Environmental Microbiology vol71 no 9 pp 5650ndash5653 2005

[47] L Rodrıguez-Montelongo S I Volentini R N Farıas E MMassa and V A Rapisarda ldquoThe Cu(II)-reductase NADHdehydrogenase-2 of Escherichia coli improves the bacterialgrowth in extreme copper concentrations and increases theresistance to the damage caused by copper and hydroperoxiderdquoArchives of Biochemistry and Biophysics vol 451 no 1 pp 1ndash72006

[48] E Williams T M Lowe J Savas and J DiRuggiero ldquoMicroar-ray analysis of the hyperthermophilic archaeon Pyrococcusfuriosus exposed to gamma irradiationrdquo Extremophiles vol 11no 1 pp 19ndash29 2007

[49] L M Gaetke and C K Chow ldquoCopper toxicity oxidative stressand antioxidant nutrientsrdquo Toxicology vol 189 no 1-2 pp 147ndash163 2003

[50] M J Davies ldquoThe oxidative environment and protein damagerdquoBiochimica et Biophysica Acta vol 1703 no 2 pp 93ndash109 2005

[51] J R Thompson J K Bell J Bratt G A Grant and LJ Banaszak ldquoVmax regulation through domain and subunitchanges The active form of phosphoglycerate dehydrogenaserdquoBiochemistry vol 44 no 15 pp 5763ndash5773 2005

[52] V Consalvi R Chiaraluce L Politi A Gambacorta MDe Rosa and R Scandurra ldquoGlutamate dehydrogenase fromthe thermoacidophilic archaebacterium Sulfolobus solfataricusrdquoEuropean Journal of Biochemistry vol 196 no 2 pp 459ndash4671991

[53] A J L Macario M Lange B K Ahring and E Conway DeMacario ldquoStress genes and proteins in the archaeardquo Microbiol-ogy and Molecular Biology Reviews vol 63 no 4 pp 923ndash9671999

[54] J AMaupin-FurlowM A Gil M A Humbard et al ldquoArchaealproteasomes and other regulatory proteasesrdquo Current Opinionin Microbiology vol 8 no 6 pp 720ndash728 2005

[55] N Sun F Beck R Wilhelm Knispel et al ldquoProteomics analysisof Thermoplasma acidophilum with a focus on protein com-plexesrdquoMolecular and Cellular Proteomics vol 6 no 3 pp 492ndash502 2007

[56] L S Madding J K Michel K R Shockley et al ldquoRole of the 1205731subunit in the function and stability of the 20S proteasome inthe hyperthermophilic Archaeon Pyrococcus furiosusrdquo Journalof Bacteriology vol 189 no 2 pp 583ndash590 2007

[57] R Shibata Y Bessho A Shinkai et al ldquoCrystal structureand RNA-binding analysis of the archaeal transcription factorNusArdquo Biochemical and Biophysical Research Communicationsvol 355 no 1 pp 122ndash128 2007

[58] W Bae B Xia M Inouye and K Severinov ldquoEscherichia coliCspA-family RNA chaperones are transcription antitermina-torsrdquo Proceedings of the National Academy of Sciences of theUnited States of America vol 97 no 14 pp 7784ndash7789 2000

[59] R J Ram N C VerBerkmoes M P Thelen et al ldquoMicrobi-ology community proteomics of a natural microbial biofilmrdquoScience vol 308 no 5730 pp 1915ndash1920 2005

[60] I Noel-Georis T Vallaeys R Chauvaux et al ldquoGlobal analysisof the Ralstonia metallidurans proteome prelude for the large-scale study of heavy metal responserdquo Proteomics vol 4 no 1pp 151ndash179 2004

16 Archaea

[61] M TMNovo A CDa Silva RMoreto et al ldquoThiobacillus fer-rooxidans response to copper and other heavy metals growthprotein synthesis and protein phosphorylationrdquo Antonie vanLeeuwenhoek vol 77 no 2 pp 187ndash195 2000

[62] Q She R K Singh F Confalonieri et al ldquoThe complete genomeof the crenarchaeon Sulfolobus solfataricus P2rdquo Proceedings ofthe National Academy of Sciences of the United States of Americavol 98 no 14 pp 7835ndash7840 2001

[63] R P Anderson and J R Roth ldquoTandem genetic duplications inphage and bacteriardquo Annual Review of Microbiology vol 31 pp473ndash505 1977

[64] D Gevers K Vandepoele C Simillion and Y Van De PeerldquoGene duplication and biased functional retention of paralogsin bacterial genomesrdquo Trends in Microbiology vol 12 no 4 pp148ndash154 2004

[65] A B Reams and E L Neidle ldquoSelection for gene clustering bytandemduplicationrdquoAnnual Review ofMicrobiology vol 58 pp119ndash142 2004

[66] L H Orellana and C A Jerez ldquoA genomic island providesAcidithiobacillus ferrooxidans ATCC 53993 additional copperresistance a possible competitive advantagerdquo Applied Microbi-ology and Biotechnology vol 92 no 4 pp 761ndash767 2011

[67] A B Brinkman T J G Ettema W M de Vos and J van derOost ldquoThe Lrp family of transcriptional regulatorsrdquo MolecularMicrobiology vol 48 no 2 pp 287ndash294 2003

[68] C Vollmecke S L Drees J Reimann S V Albers and MLubben ldquoThe ATPases CopA and CopB both contribute tocopper resistance of the thermoacidophilic archaeon SulfolobussolfataricusrdquoMicrobiology vol 158 no 6 pp 1622ndash1633 2012

[69] A Villafane Y Voskoboynik I Ruhl et al ldquoCopR of Sulfolobussolfataricus represents a novel class of archaeal-specific copper-responsive activators of transcriptionrdquo Microbiology vol 157no 10 pp 2808ndash2817 2011

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Anatomy Research International

PeptidesInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporation httpwwwhindawicom

International Journal of

Volume 2014

Zoology

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Molecular Biology International

GenomicsInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioinformaticsAdvances in

Marine BiologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Signal TransductionJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

Evolutionary BiologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Biochemistry Research International

ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Genetics Research International

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Advances in

Virolog y

Hindawi Publishing Corporationhttpwwwhindawicom

Nucleic AcidsJournal of

Volume 2014

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

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Enzyme Research

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

International Journal of

Microbiology

Archaea 5

28 Isolation of Total RNA Cell pellets (10mg wet weight)were collected and diluted in 60 120583L TEN buffer (20mMTris-HCl pH 80 1mM EDTA 100mM NaCl) followedby addition of 60120583L TENST buffer (20mM Tris-HCl pH80 1mM EDTA 100mM NaCl 16 Na-lauroyl sarcosineand 012 Triton X-100) This suspension was incubated atroom temperature for 15min to allow cell lysis Total RNAwas then extracted using the TRIzol reagent (Invitrogen)as recommended by the manufacturer DNA contaminationin RNA preparations was removed by DNase I treatment(Roche) following the manufacturerrsquos instructions RNAwas then purified precipitated and finally resuspended indiethylpyrocarbonate (DEPC)-treated water Total RNA con-centrations were estimated by spectrophotometric measure-ments (OD

260) and its quality was evaluated by determining

the ratio of absorption at 260 nm and 280 nm which waswithin the preferred range of 18ndash21

29 Northern Blotting For differential gene expression byNorthern blotting experiments the media were supple-mented with either different concentrations of Cu (5ndash50mM) Cd (5mM) NiSO

4(15mM) ZnSO

4(50mM) or

Ag2SO4(008mM) respectively as indicated For some

experiments elemental sulfur was replaced by a chalcopyrite(CuFeS

2) concentrate which was used as the only energy

source at 1 (wv)Total RNA (5 120583g) was separated by electrophoresis

on a 1 formaldehyde agarose gel followed by blottingonto Hybond-N nylon membranes (Amersham PharmaciaBiotech Bucking-hamshire UK) Hybridization was con-ducted as described by Sambrook et al [40] DNA probeswere labeled by randomprimerDNA labeling kit (Fermentas)using [120572-32 p] dCTP The hybridization signal was detectedand analyzed by using the molecular Imager FX system andQuantity One software The primer sequences for amplifica-tion of these gene probes are listed in Table 1

210 Cotranscriptional Analysis by RT-PCR To study theexpression of adjacent genes copM and copA cDNA wassynthesized by using 08120583g of total RNA from a S metallicusculture grown in the presence of 20mM Cu and a reverseprimer hybridizing to copA sequence A forward primerannealing on copM sequence was used for the PCR reactions(Table 1) PCR amplifications were performed with 1 120583L ofa 110 dilution of the cDNA and 25 pmol of each primerAmplification conditions included an initial 3min of denat-uration at 95∘C followed by 35 cycles of 30 s at 95∘C 30 sat 55∘C and 15min at 72∘C and finished by 10min at 72∘CA reverse transcriptase reaction without enzyme was carriedout in order to exclude amplification due to genomic DNAcontamination

211 Quantitative RT-PCR Single stranded cDNA was syn-thesized from 08 ng of DNA-free RNA samples usingrandom hexamers (Fermentas) and ImProm-II reversetranscription system (Promega) following manufacturerrsquosinstructions The software tool IDT Scitools (IntegratedDNA Technologies) was used to design primers producing

amplicons of 150ndash200 bp (Table 1) qPCRwas performedwith2 120583L of 1 10 diluted cDNA samples 125 120583L 2x QuantiFastSYBRGreenPCRMasterMix (Quiagen) 1 120583Mof each primerand water to a final volume of 25120583L The efficiency of eachprimer pair was calculated from the average slope of a linearregression curve which resulted from qPCRs using a 10-folddilution series (10 pgndash10 ng) of S metallicus chromosomalDNA as template Cq values (quantification cycle) wereautomatically determined by Real-Time Rotor-gene 6000PCR software (Corbett Life Sciences) after 40 cycles Cqvalues of each transcript of interest was standardized to theCq value of the 16S rRNA gene [39] At least 3 biologicalreplicates of each assessed condition and 2 technical replicatesper qPCR reaction were performed

3 Results and Discussion

31 Tolerance of S metallicus and S solfataricus to Cu and CdIn a previous work it was determined that S metallicus wasable to tolerate high Cu concentrations While the presenceof 100mM Cu did not affect S metallicus growth kinetics adecreased in cell biomass of only 30 was observed whenexposed to 200mM Cu [30] The high tolerance to Cu hasbeen described in other acidophilic Bacteria and Archaeacompared mostly with neutrophilic microorganisms [11 14]Here this analysis was further extended to characterize theresponse of S metallicus to Cd Thus it was determined thatS metallicus growth was not affected in the presence of either05 or 1mM Cd when compared with the control conditionin the absence of the metal (Figure 1(a)) In addition whenS metallicus was challenged with 2 and 3mM Cd it wasobserved that at the late exponential growth phase the cellnumbers decreased by 30 and 50 respectively (Figure 1(a))Moreover growth of S metallicuswas completely inhibited inthe presence of 5mM Cd (Figure 1(a)) On the other hand Ssolfataricuswas not able to grow at Cd concentrations greaterthan 005mM (Figure 1(b)) At 001mM Cd S solfataricuscell numbers decreased around 35 (Figure 1(b)) Theseresults are in agreement with previous reports in which Ssolfataricus was shown to be able to grow in up to 001mMCd [41] Other acidophilic archaeons involved in bioleachingprocesses such as Metallosphaera sedula and Ferroplasmaacidarmanus were found to be able to tolerate up to 09and 9mM Cd respectively [18 42] The minimal inhibitoryconcentration (MIC) to Cd has been described to be nothigher than 1mM for several thermophilic and neutrophilicThermococcus species [43] Recently it was reported that themost radioresistant archaeon Thermococcus gammatoleransstands Cd concentrations with an MIC of 2mM [27] Todate despite the heavy metals tolerance showed by someof these microorganisms strategies to withstand stress fromtransition metals have been most widely studied only inhaloarchaea [44]

32 Effect of Cu andCd on theGlobal Proteome of SmetallicusThe proteomic response to Cu and Cd was analyzed toidentify proteins which could be involved in heavy metalsresistance in S metallicus Early stationary phase growing

6 Archaea

14

12

10

8

6

4

2

00 2 4 6 8 10 12

Time (days)

107timescellsm

L

(a)

120

100

80

60

40

20

00 2 4 6 8 10

Time (days)

107timescellsm

L

(b)

Figure 1 Growth of S metallicus (a) and S solfataricus (b) in the presence of Cd Cells were inoculated in their respective growth media inabsence of added Cd (∙) or were supplemented with 0005mM () 001mM () 005mM (diams) 01mM () 05mM () 1mM () 2mM() 3mM (times) or 5mM (+) Cd and cells were counted daily

100

7060

50

30

20

3 10MW

33 31

32

A

B

C

20

10

(a)

100

7060

50

30

20

12

3 10MW

204

21

33 31

32

A

B

C

15

93

10

27 28

23 24

29

25 2630

1614 16

13

52

7

2219

8

20

10

11

(b)

Figure 2 Changes in the proteome of S metallicus grown in the presence of Cu Cells were incubated in the absence of any added metal(a) or in the presence of 100mM Cu (b) Arrows indicate the spots that were downregulated (a) or upregulated (b) in the presence of CuNumbers indicate the spots with increased intensity in cells treated with copper The dashed boxes are shown as enlarged areas that includesome proteins upregulated in the presence of Cu and Cd (Figure 3) The bottom panels show low molecular weight proteins upregulated inthe presence of Cu separated by using a 15 polyacrylamide gel in the second dimension

cells were untreated or treated with either 100 or 200mM Cuor 1mM Cd during 24 h and analyzed by comparative two-dimensional gel electrophoresis as indicated in experimentalprocedures Twenty-three proteins were found to be down-regulated after 100mMCu treatment (Figure 2(a)) A similar

pattern was obtained at 200mM Cu (not shown) Further-more by using the Delta 2D software (Decodon) eleven ofthese proteins were found to be completely absent in the gelsand more than 8 proteins decreased their intensity in therange from 15-to 5-fold compared with control cells grown

Archaea 7

13 13

11

13

1

Control Copper Cadmium

(a)

99 9

333

(b)

3030

30

2020

20 212121

(c)

Figure 3 Comparison of selected proteins of S metallicus cells treated with Cu or Cd Protein extracts were obtained from cells treatedwithout metals and with 100mM Cu or 1mM Cd for 24 h and the proteins were separated by 2D-PAGE The segments (a) (b) and (c) arethe enlarged dashed boxes in Figure 2 under the three conditions indicated Numbers show some of the spots with increased intensity in cellstreated with Cu or Cd

in the absence of Cu (Figure 2(a)) Therefore these resultsshow that a number of proteins became non-detectable ordecreased their levels when S metallicus faced Cu This kindof response was also seen when the microorganism wasexposed to Cd (data not shown) This behavior has alsobeen observed in similar studies where F acidarmanus waschallenged by either As(III) or Cu [16 18] In addition theexpression of 30 other proteins was found to be upregulatedafter Cu treatment (100 or 200mM) (Figure 2(b)) Most ofthese proteins could be identified only in the condition withCu and spots 5 13 and 15 were induced 26 34 and 55-fold respectively compared to cells without Cu treatmentMoreover 3 proteins of lowmolecular weight (spots 31 3 and33) that increased their expression when cells were exposedto 100mM Cu were also identified (Figure 2 bottom panels)These proteins were only detected when 15 polyacrylamidewas used during the second-dimensional separation (SDS-PAGE) On the other hand a large decrease in overallexpression of proteins after 1mMCd treatment was observed(not shown) Interestingly 10 out of the 13 proteins identifiedas up-regulated when cells were exposed to Cd and alsoshowed increased levels after Cu treatment Some of them areshown in Figure 3

33 Identification of Proteins Upregulated in Cells Treated withCu and Cd in S metallicus A total of 18 S metallicus proteinswhose levels were found to be up-regulated in responseto Cu were analyzed by mass spectrometry The identifiedproteins included functions related to the production andtransport of energy biosynthesis of amino acids stressresponses and transcription regulation (Table 2) Amongstthe proteins related to production and transport of energya putative ATP synthase subunit B (spot 23) that has beendescribed as playing a fundamental role in ATP synthesiswas identified (Table 2) When cells are subjected to somestressing conditions such as the presence of heavy metalsa greater cellular demand for energy has been reported tooccur [18] Most likely this phenomenon might be due toATP-driven Cu transport via ATPases that have a substantialinterplay during metal detoxification [45] Furthermore theATP synthase subunit B was also identified as up-regulatedwhen S metallicus was treated with Cd (Table 2)

Two other proteins corresponded to putative oxidoreduc-tases such as ferredoxin oxidoreductase (spot 3) and alcoholdehydrogenase (spot 4) that have been generally involvedin electron transporter chains and use NAD+ as an electronacceptor The levels of ferredoxin oxidoreductase were also

8 Archaea

Table 2 Proteins upregulated in S metallicus cells exposed to 100mM Cu or 1mM Cd

Spot Molecular weight (kDa) Cu induction levels Putative function Microorganism related Accesion number1dagger 266 infin Proteasome subunit S solfataricus AAK410343dagger 701 infin Ferredoxin oxidoreductase S solfataricus AAK429264dagger 429 infin Alcohol dehydrogenase S solfataricus AAK431545 35 26 Hypothetical protein S acidocaldarius YP 2549766 27 infin Hypothetical protein P aerophilum NP 5598899dagger 57 infin Phosphoglycerate dehydrogenase M thermautotrophicus AAB8546613dagger 27 34 Unknown mdash mdash14 28 infin Unknown mdash mdash15 35 55 Unknown mdash mdash20dagger 461 infin Glutamate dehydrogenase S solfataricus AAK4223021dagger 45 infin Hypothetical protein M acetivorans NP 618380122 49 infin Hypothetical protein P torridus YP 02280723dagger 51 infin ATP synthase subunit B S solfataricus AAK4088024 585 infin HSP60 subunit S shibatae AAG37273130dagger 48 infin Unknown mdash mdash31 16 ND Unknown mdash mdash32 144 ND Putative transcription regulator S solfataricus AAK4041333 138 ND Transcription factor nusA S solfataricus AAK40563Spots refer to those numbered in Figure 2 Proteins in Figure 2 which are not included in this table were not subjected to sequencing due to their low intensitiesin the gelsdaggerSpot up-regulated in cells exposed to Cu and CdND not determinedInfinity symbol indicates that proteins were expressed only in presence of copper or cadmiumM thermoautotrophicus Methanobacterium thermoautotrophicus M acetivorans Methanosarcina acetivorans S shibatae Sulfolobus shibatae P torridusPicrophilus torridus P aerophilum Pyrobaculum aerophilum S acidocaldarius Sulfolobus acidocaldarius

up-regulated in response to Cd (Table 2) Several previ-ous reports have suggested that oxidoreductases contributeto an oxidative protection in both Bacteria and Archaeain response to heavy metals [46ndash48] In the neutrophilicmicroorganisms Escherichia coli and Staphylococcus aureusit has been described that oxidases and dehydrogenasescontribute to oxidative protection Cu homeostasis and stressresponses [46 47] Furthermore some proteins involved inoxidative damage repair such as NADH-dependent oxidasesand thioredoxin reductases were expressed in cells of Facidarmanus exposed to As(III) [16] The expression of thisgroup of proteins has also been observed when the samemicroorganism was exposed to Cu [18] Therefore oxidore-ductases play an important role against oxidative stress andmay eliminate reactive oxygen species which constitute themajor component of the stress caused by transition metals[44 49 50]

The enzymes related to biosynthesis of amino acids werefound as commonly up-regulated either in response to Cu orCdThey corresponded to a phosphoglycerate dehydrogenase(spot 9) and a glutamate dehydrogenase (spot 20) Theformer one catalyzes the NAD+-dependent oxidation of 3-phosphoglycerate into 3-phosphohydroxypyruvate a branchpoint from the glycolytic pathway and the initial reactionin L-serine biosynthesis [51] Glutamate dehydrogenase cat-alyzes the oxidative deamination of glutamate to produce 2-oxoglutarate and ammonia with reduction of NAD+ [52]Proteins involved in amino acids synthesis were expressed

in cells of F acidarmanus exposed to As(III) [16] Moreoverincreased levels of proteins involved in the biosynthesisof sulfur-containing amino acids have been observed inSaccharomyces cerevisiae cells exposed to Cd [24] suggestingthat these proteins might also have a role in the cellularresponse to adverse conditions

Two proteins showed identity to components of the stressresponse mechanism such as one subunit of the HSP60chaperonin (spot 24) and one subunit of the proteasome (spot1) In Archaea the stress protein HSP60 is directly involvedin protein folding processes [53] Proteomic analysis of Facidarmanus cells exposed to As(III) and Cd revealed theexpression of proteins involved in protein folding and DNArepair including HSP60 chaperonin and DnaK heat-shockprotein (HSP70) thereby the authors suggested that thismicroorganismusesmultiplemechanisms to resist high levelsof Cu [16 18] On the other hand proteasomes are describedas nonspecific proteolytic nanomachines associated withprotein catabolism Proteasomes are known to be closelyinvolved in maintaining protein quality control by degradingmiss folded and denatured proteins in response to cell stressin all three domains of life [54] A proteomic analysis inthe thermoacidophilic archaeon Thermoplasma acidophilumshowed that proteasomes and chaperone-related proteinswere highly induced against stress conditions indicatinga high turnover rate of proteins [55] In P furiosus theexpression of the 1205731 subunit of the proteasome was inducedin cells subjected to heat shock stress [56] Additionally the

Archaea 9

150pb

(a)

1016 pb

(b)

2000 pb

(c)

Figure 4 CODEHOP-based PCR for the amplification of the putative genes copA and copM from S metallicus Amplification products(a) using degenerate PCR primers (DOP) copM degF and copM degR designed from amino acid sequence conserved blocks of SulfolobalesCopM proteins (b) using primers copA cdegF and copA cdegR designed by CODEHOP strategy for amplification of a copA-like gene (c)Primers copM degF and copA cdegRwere assesed for the amplification of a copMA-likeDNA regionThose primerswere testedwith genomicS metallicus DNA samples Amplicons expected sizes were as follows ca 150 bp for copM ca 950 bp for copA and ca 2000 bp for copMAPCR products were excised and cloned for later sequencing

proteasome subunit was also found to be up-regulated whenS metallicus was subjected to Cd stress (Table 2)

The transcriptional regulation-related proteins corre-sponded to a putative transcription regulator (spot 32) andthe transcription factor NusA (spot 33) involved in theintrinsic termination of transcription [57] NusA has beenalso reported to display important functions in the cellularresponse to adverse factors [58] The expression of twotranscriptional regulators related to amino acids biosynthesisand metal-dependent genetic repression were found to beinduced in P furiosus cells subjected to oxidative stress[48] Moreover the expression of several genes encodingpredicted transcriptional regulators were induced followingCd exposure in T gammatolerans cells [27]

Spots 5 6 13 14 15 21 22 30 and 31 (Table 2) did notshow significant scores with any protein sequences currentlyavailable in the data bases Interestingly spots 2 4 9 13 21and 30 were found to be commonly upregulated when Smetallicus cells were treated with either Cu or Cd (Figure 3Table 2)

The results presented here are consistent with previ-ous work describing global protein expression profiles inresponse to heavy metals as reported for F acidarmanus [1618] microbial-biofilm communities in an acid mine drainagesite [59] and other prokaryotic and eukaryotic microorgan-isms [24 60]Therefore a coordinated expression of differentgroups of genes suggests the existence of regulatory networkssuch as stress response mechanisms and respiratory chainadjustments to cope with the presence of heavy metal ions[61]

34 Cu-Resistance (Cop) Genes Cluster Is Duplicated in Smetallicus Genome A Cu-resistance (cop) locus has beendescribed to be highly conserved in archaeal genomeswhich consists of genes encoding a new type of archaealtranscriptional regulator (CopT) a metal-binding chaperone(CopM) and a Cu-transporting P-type ATPase (CopA) [19]Since the genome sequence of Smetallicus is not yet availablewe searched for the presence of cop genes by using consen-sus degenerate hybrid oligonucleotide primers-based PCR(CODEHOP-PCR) CopA and CopM amino acid sequencealignments from S acidocaldarius S tokodaii and S solfa-taricus were used to identify conserved sequence blocks andtherefore to design either CODEHOP or DOP (degeneratehybrid oligonucleotide) primers (Figure S1 Table 1) TherebyPCR assays using S metallicus genomic DNA as templateyielded amplicons of ca 150 bp and ca 950 bp for copMand copA CODEHOP pair primers respectively (Figures4(a) 4(b)) As estimated from primer pairrsquos position onamino acid sequence alignments the obtained PCR productscorresponded to the expected sizes suggesting the presenceof copM- and copA-like genes in S metallicus genome Inorder to determine the identity of the amplified DNA frag-ments they were TA-cloned and sequenced Blastx sequenceanalysis showed that the 150 bp DNA fragment coded for anincomplete ORF sharing 63 homology with S solfataricusCopM (SSO10823) whereas the analysis of the 950 pb DNAfragment yielded a partial ORF sharing 54 homology withS solfataricus CopA protein (SSO2651)

The genomic organization of the putative cop genes in Smetallicus was analyzed to find out its similarity with those

10 Archaea

Table 3 Sequence identity comparison of S metallicus Cop proteins to those in other Sulfolobales

S metallicus protein Identity to homologue in other SulfolobalesS solfataricus P2 S acidocaldarius S tokodaii M sedula

CopA1 51 43 44 45CopA2 88 46 49 50CopM1 63 69 71 71CopM2 89 72 70 77CopT1 36 27 30 32CopT2lowast 90 52 52 53Boldface type indicates to which organism the S metallicus homologue shows the highest sequence identitylowastRefers to the analysis obtained using the uncompleted amino acid sequence of CopT2 which represents sim90 in length when compared with S solfataricusCopT sequence

previously described in S solfataricus P2 and F acidarmanuswhere the cop cluster consists of tandem-orientated genesas copTMA [18 19] Thus using copM degF (forward) andcopA cdegR (reverse) primers we attempted to amplify aputative copMA DNA region This PCR yielded a product ofca 2000 pb corresponding to the expected size and resultedin 2 ORFs showing high sequence homology with CopMand CopA from S solfataricus (Figure 4(c)) Interestinglywhen aligning this copMA sequence with the copA and copMsequences obtained previously they were not identical whichstrongly indicated the presence of duplicated putative copgenes encoding for both Cu-ATPases (copA1 and copA2) andmetallochaperones (copM1 and copM2) in the S metallicusgenome

To isolate the entire cop gene sequences the genomewalking method described by Acevedo et al [38] was usedSix different DNA libraries were constructed from S metalli-cus genomic DNA digested with several restriction enzymesincluding 51015840 overhang and blunt end restriction enzymesThe DNA libraries were used as templates in 2 successivePCR reactions From the previously known sequences twospecific sense primers for the 31015840 end amplification and twospecific antisense primers for the 51015840 end amplification weredesigned (abbreviated SP1 and SP2) Therefore the 51015840 and 31015840ends of the putative genes copA1 and copA2 that had beenpartially identified through the genome walking techniquewere amplified

By overlapping the sequence isolated by degenerate PCRand the lateral sequences obtained by genome walking(Figure S2) it was possible to complete the whole nucleotidesequence of the putative gene copA1 Additionally it waspossible to confirm the presence of the upstream copM1 geneanddetermine the occurrence of a partial copT1 gene tandem-orientated upstream of copM1 (Figure 5)Through additionalgenome walking experiments the partial copT1 sequencecould be further completedThe samewas truewhen isolatingthe whole nucleotide sequence of the putative gene copA2as the presence of copM2 and copT2 genes were confirmedupstream of copA2 However it was not possible to completethe whole 51015840 end of the putative gene copT2 as its determinedlength corresponded to only 90 of the full length whencompared with S solfataricus copT gene (Figure 5)

In conclusion degenerate PCR together with genomewalking experiments allowed to describe the occurrence inS metallicus genome of 2 cop genes loci (named as locus cop1and locus cop2) coding for paralogous genes whose productsmay be involved in Cu-resistance (Figure 5) Although somearchaeal genomes exhibit two copies of putative Cu-P-typeATPases as described for S solfataricus [62] the genomicarrangement displayed as a cop gene cluster (copTMA) hasbeen reported to be represented in only one copy for manyarchaeal genomes [10 19 21] Moreover we further updatedthis analysis by searching for duplications of the cop genescluster in all available archaeal genome sequences up to date(October 2012) retrieving only one cop locus in each caseThereby we propose that the discovery of this cop gene clusterduplication in the genome of S metallicus constitutes so faran unprecedented feature for a representative of the Archaeadomain and might contribute to its high metal resistance

In this context it has been widely reported that increasedgene copy number can increase gene expression allowingprokaryotic to thrive under growth limiting conditions [1363ndash68] However alongside the augmented gene copy onecannot exclude that the cop operon duplication in S metal-licus may offer among others a wider repertoire of Cu-ATPases which might differ in terms of metal affinitiesandor specificity efflux rates andor differences in theirabundance regulation

Throughout amino acid sequence analysis it was furtherdetermined that each polypeptide encoded by S metallicuswith the exception of CopT1 showed to be highly homol-ogous to their orthologous counterparts encoded by othersSulfolobales (Table 3) Moreover the 3 ORFs encoded by thelocus cop2 shared about 90 identity with the correspondingS solfataricus orthologous peptides (Table 3) S metallicuscop genes products showed characteristic domains referredto as critical for their respective proposed biological activitiesS metallicus paralogous gene products CopA1 (749 aa1015840)and CopA2 (747 aa1015840) shared 513 of identity These twoputative Cu-ATPases (CopA1 and CopA2) contain the aminoacid sequence motif CPCALGLA which has been proposedto confer Cu-transporting specificity in CPx-ATPases [48]and has been also found in other CopA-like proteins frombiomining Bacteria and Archaea [14]

Archaea 11

HindIII HindIII HindIII

HindIII

CopT1Locus cop1 M1

EcoRIEcoRV

513 bp 219 bp 2250 bp

CopA1

(a)

Locus cop2 M2

EcoRI

CopA2

453 bp 171 bp 2244 bp

CopT2

PstIHindIII HindIII HindIII

(b)

Figure 5 Schematic representation of the two cop gene clusters isolated from S metallicus genome Each cluster codes for anArchaea-specifictranscriptional regulator (copT) a metallochaperone (copM) and a P-type Cu-exporting ATPase (copA) Lengths of each gene are indicatedLocus cop1 corresponds to 4346 sequenced base pairs and shows an intergenic region copT1-M1 of 232 base pairs Locus cop2 correspondsto 3435 base pairs and shows 89 base pairs copT2-M2 intergenic region Genes copM2 and copA2 overlapped in 11 base pairs copT2 was notfully isolated

Control CdSO4 Ag2SO4 NiSO4 ZnSO4 CuSO4

kb3kb2kb1

02 kb

(a)

kb3kb2kb1

02 kb

(b)

(c)

Figure 6 Northern blot analysis to determine the expression of S metallicus copA1 (a) and copA2 (b) putative genes in response to variousmetals S metallicus total RNAwas extracted from cultures grown either in the absence (control) or the presence of CuSO

4(50mM) Ag

2SO4

(008mM) NiSO4(15mM) ZnSO

4(50mM) and CdSO

4(2mM) P32-radioactivelly labelled DNA fragments annealing to copA1 and copA2

sequences respectively were used as probes (c) shows rRNAs as total RNA loading control

On the other hand CopM1 (71 aa) and CopM2 (56 aa)sharing 66 of identity present the proposed metal bindingdomain TRASH (trafficking resistance and sensing of heavymetals) [10] Additionally when analyzing the putative tran-scriptional regulators the presence of a C-terminal TRASHdomain in both CopT1 and CopT2 was also found CopT1sequence also contains an entire N-terminal helix-turn-helix (HTH) motif (not shown) that resembles the DNA-bindingmotifs of prokaryotic transcriptional regulators suchas Lrp-like proteins [67] The HTH motif was only partiallyidentified in CopT2 since the 51015840 region of copT1 gene has notbeen yet fully isolated

35 Transcriptional Analysis of S metallicus Cop Genes Toget insight into the role of the two cop loci the expressionof the corresponding genes was analyzed under variousconditions S metallicus was first grown in presence ofdifferent heavy metals (Cu Zn Cd Ni o Ag) at a givenconcentration that did not affect growth kinetics [30 42]Subsequently total RNA was isolated from late exponentiallygrown cultures and Northern blot experiments were carriedout in order to determine the expression of both copA1 andcopA2 genes As depicted in Figure 6 transcription of bothcopA1 and copA2messengers were specifically induced in thepresence of Cu and Cd ions This gene expression pattern

12 Archaea

954pb

M1 CopA1

(a)

1438 pb

M2 CopA2

(b)

1000 pb

800pb

+ minus

(c)

+ minus

1500 pb

1000 pb

(d)

Figure 7 Cotranscription analysis of copMA1 and copMA2 genes cDNA was synthesized with a reverse primer (dotted arrows) hybridizingtoward the 31015840 end of either copA1 (a) or copA2 (b) S metallicus total RNA was extracted at the late exponential phase from a culture growingin presence of 20mMCu PCR amplifications were carried out with these cDNAs and each corresponding primer pair (black arrows) as listedin Table 1 (c) and (d) show RT-PCR products obtained for the copMA1 and copMA2 intergenic regions respectively Reverse transcriptasereactions with (+) and without (minus) the Improm II reverse transcriptase enzyme were carried out in order to exclude amplification due togenomic DNA contamination Expected sizes (in base pairs) for the corresponding PCR products are given in (a) and (b)

is in good agreement with what has been described for Ssolfataricus where the bicistronic copMA transcript levelswere also found to be up-regulated in response to both Cuand Cd [19]

Northern blot analysis showed that the sizes seen for bothcopA transcripts (sim25 Kb) did not correspond exactly withthe individual genes length (each of about 225Kb) (Figure 6)This strongly suggested that the transcripts could also includethe respective copM gene located upstream of copA in eachcop loci To confirm this assumption a co-transcriptionexperiment was done (Figure 7) in which the cDNAs wereobtained by using RNA extracted from a culture grownin the presence of 20mM Cu and using a reverse primerhybridizing with copA1 (Figure 7(a)) or copA2 (Figure 7(b))gene sequence respectively PCR amplifications were carriedout by using the corresponding cDNAs as templates and eachpair of primers lying in adjacent genes (copM) The presenceof an amplicon of the expected size in each case indicatedthe adjacent genes were part of polycistronic messengers(Figure 7) These results clearly show that in S metallicuseach couple of copMA genes was expressed in the form oftranscriptional units as reported in F acidarmanus and Ssolfataricus [18 19] The coexpression of gene pairrsquos copMAmay suggest a coordinated and dependent function for therespective encoded proteins In the bacterium E hirae themetallochaperone CopZ (CopM in S metallicus) fulfills apivotal role in the mechanism of Cu homeostasis [45] Thusit was shown that this protein interacts directly with the Cu-ATPase CopB (CopA in S metallicus) handing the Cu forsubsequent removal In this context one might expect thatproteins CopM1 and CopM2 from S metallicus have a similarrole to that described for E hirae Furthermore quantitativeRT-PCR experiments were carried out in order to determinedifferential expression of the cop genes in response to differentCu concentrations The expression of copA1 copA2 copT1and copT2 was tested relative to the transcript levels ofrRNA 16S gene since its levels were not significantly affected

in all assessed conditions (data not shown) As shown inFigure 8(a) copA2 and copA1 transcript levels were foundto be concomitantly increased with the increasing Cu con-centrations present in the medium Higher transcripts levelsof copA1 were seen in all tested conditions when comparedwith copA2 levels copA1 transcript levels were found to be325-fold up-regulated when comparing 50mMCu conditionversuscontrol (absence of metal) whereas copA2 transcriptlevels showed an increment of only 175-fold (Figure 8(a))The finding that Cu-ATPases mRNA levels were significantlyincreased in response to Cu ions exposure suggests that thetransport system may operate for Cu efflux in S metallicus

Moreover copA2 and copA1 gene expression was quan-tified when S metallicus was grown using chalcopyrite(CuFeS

2) ore as an oxidizable substrate (Figure 8(a)) Mineral

oxidation mediated by the microorganism generates a pro-gressive increase in Cu ions concentration in the medium Tofind out whether the amount of solubilized Cu induced theexpression of the Cu-ATPases genes total RNAwas extractedfrom a S metallicus culture grown to late exponential phaseand in the presence of 1 CuFeS

2 It was clear that an

up-regulation of the Cu-ATPases encoding genes also tookplace when S metallicus was grown in the presence ofCuFeS

2(Figure 8(a)) copA1 transcript levels increased 14-fold

compared with the control condition in the absence of Cuwhile copA2 gene expression showed an increase of 76 foldAlong with this bymeans of atomic absorption spectrometry(AAS) analysis an overall amount of solubilized Cu ions(Cu2+Cu1+) of 144plusmn21mMwas determined to be present inthemedium indicating that copA2 and copA1 gene expressionwas most likely in response to the Cu present in the culturedue to CuFeS

2microbial-solubilization

The finding that copA1 was highly expressed comparedwith copA2 in all tested conditions may suggest a possiblephysiological hierarchy between the two ATPases whenovercoming either Cu or Cd stress In this regard by meansof a genetic approach it was demonstrated in S solfataricus

Archaea 13

700

600

500

400

300

200

100

00

5 20 50 CuFeS2CuSO4 concentration (mM)

Relativ

egenee

xpression

CopA2CopA1

(a)

8

6

7

5

4

3

2

1

00

5 20 50 CuFeS2CuSO4 concentration (mM)

Relativ

egenee

xpression

CopT1CopT2

(b)

Figure 8 Relative expression levels of S metallicus genes copA1 and copA2 (a) copT1 and copT2 (b) Expression of S metallicus cop geneswasassessed by qRT-PCR and normalized against 16S rRNA gene expression in individual cultures Mean values and standard deviations arefrom analyses of three independent cultures grown in the presence of 0 5 20 50mMCu or in 1 of a chalcopyrite ore (CuFeS

2) concentrate

that while CopA was an effective Cu efflux transporterat low and high Cu concentrations the other Cu-ATPase(CopB) only appeared to be a low-affinity Cu export ATPase[68] Moreover by using a M sedula copA deletion mutantit was demonstrated that this strain compromised metalresistance and consequently abolished chalcopyrite oxidation[22] highlighting the role of Cu detoxification mechanismsduring a given bioleaching process Our attempts to showthe functionality of CopA1 and CopA2 from S metallicusby using E coli as a heterologous host were not successfulApparently the overexpression of these ORFs had a toxiceffect on E coli that compromised its viability Althoughgene disruption tools have not been yet developed for Smetallicus the functional role of both copTMA locimight befurther studied by cross-species complementation of a coppersensitive S solfataricus copR mutant as it was described byMaezato et al [22] It will be of great interest to establishin future studies the possible functionality of the isolatedputative transporters from S metallicus

Likewise quantitative RT-PCR experiments showed thatcopT1 transcript levels increased concomitantly with increas-ing Cu concentrations whereas copT2 showed relativelysimilar transcripts levels most likely indicating a constitutiveexpression profile (Figure 8(b)) Furthermore whereas copT1transcript levels were found to be increased 36 fold inCuFeS

2

grown cultures copT2 levels remained unchanged in com-parison with the control in the absence of Cu (Figure 8(b))The results obtained for S metallicus copT2 gene expressionare consistent with those reported for S solfataricus CopTtranscriptional regulator has been proposed to function as arepressor in S solfataricus showing a constitutive expressionthat in the presence of Cu loses its affinity for the promoterregion of copMA allowing the expression of this polycistron[19] In contrast the increased transcripts levels of both copA1and copT1 concomitant with higher Cu concentrations in the

environment suggest that the putative transcriptional regula-tor CopT1 may act by activating both copMA1 and probablyitself (Figure 8) Regarding this it was recently reported thatCopR (CopT in S metallicus) from S solfataricus strain 982acts as an activator of copT (copM in S metallicus) and copAexpression [69] Nevertheless additional experiments wouldbe required to test this possibility in S metallicus

4 Concluding Remarks

We previously reported that S metallicus resists extremelyhigh Cu concentrations which was mediated to some extentby the use of a possible metal resistance system based oninorganic polyphosphate hydrolysis and consequently Cu-PO4

2minusefflux [15 29 30] Here we have addressed the questionwhether this microorganism coded for other determinantsthat might help to explain its high heavy metal toleranceAs the genomic sequence of this microorganism is notyet available we jointly used CODEHOP-PCR and genomewalking approaches and were able to establish the occurrenceof two nonidentical homologous cop loci into the genome ofS metallicus (cop1 and cop2) Each cop locus codes for anarchaeal transcriptional regulator (CopT) a metal-bindingchaperone (CopM) and a Cu-transporting P-type ATPase(CopA) High levels of the polycistronic mRNAscopMA ofeach cop locus were observed after treatment with either Cuor Cd suggesting that the encoded ATPases efflux heavymetals out in order to detoxify the intracellular environmentAltogether previous reports and the results obtained in thisstudy allow us to suggest that some key elements that mayexplain the high resistance to Cu in S metallicus is the dupli-cation of the Cu resistance cop genes a defensive responseto stress and a polyP-based accumulation mechanism Inthe case of Cd although some similar stress responses were

14 Archaea

observed whether comparable Cu-responsive elements alsoparticipate in Cd responses remains to be seen

Conflict of Interests

The authors declare that the research was done in the absenceof any commercial or financial relationships that could beconstrued as a potential conflict of interests

Acknowledgments

This work was supported by Grant no FONDECYT 1110214and in part by ICM P-05-001-F project

References

[1] A Schippers ldquoMicroorganisms involved in bioleaching andnucleic acid-based molecular methods for their identificationand quantificationrdquo in Microbial Processing of Metal SulfidesR E Donati and W Sand Eds pp 3ndash33 Elsevier BerlinGermany 2007

[2] H R Watling ldquoThe bioleaching of sulphide minerals withemphasis on copper sulphides a reviewrdquo Hydrometallurgy vol84 no 1-2 pp 81ndash108 2006

[3] D E Rawlings D Dew and C Du Plessis ldquoBiomineralizationof metal-containing ores and concentratesrdquo Trends in Biotech-nology vol 21 no 1 pp 38ndash44 2003

[4] L Valenzuela A Chi S Beard et al ldquoGenomics metagenomicsand proteomics in biomining microorganismsrdquo BiotechnologyAdvances vol 24 no 2 pp 197ndash211 2006

[5] C A Jerez ldquoBioleaching and biomining for the industrialrecovery of metalsrdquo in Comprehensive Biotechnology M Moo-Young Ed vol 3 pp 717ndash729 Elsevier Amstaerdam TheNetherlands 2nd edition 2011

[6] P R Norris and J P Owen ldquoMineral sulphide oxidationby enrichment cultures of novel thermoacidophilic bacteriardquoFEMS Microbiology Reviews vol 11 no 1ndash3 pp 51ndash56 1993

[7] D E Rawlings and D B Johnson ldquoThe microbiology ofbiomining development and optimization ofmineral-oxidizingmicrobial consortiardquo Microbiology vol 153 no 2 pp 315ndash3242007

[8] P R Norris N P Burton and N A M Foulis ldquoAcidophiles inbioreactor mineral processingrdquo Extremophiles vol 4 no 2 pp71ndash76 2000

[9] M R Bruins S Kapil and F W Oehme ldquoMicrobial resistancetometals in the environmentrdquo Ecotoxicology and EnvironmentalSafety vol 45 no 3 pp 198ndash207 2000

[10] T J G Ettema M A Huynen W M De Vos and J Van DerOost ldquoTRASH a novel metal-binding domain predicted to beinvolved in heavy-metal sensing trafficking and resistancerdquoTrends in Biochemical Sciences vol 28 no 4 pp 170ndash173 2003

[11] M Dopson C Baker-Austin P R Koppineedi and P L BondldquoGrowth in sulfidic mineral environments metal resistancemechanisms in acidophilic micro-organismsrdquo Microbiologyvol 149 no 8 pp 1959ndash1970 2003

[12] S Franke and C Rensing ldquoAcidophiles Mechanisms to toleratemetal and acid toxicityrdquo in Physiology and Biochemistry ofExtremophiles C Gerday and N Glansdorff Eds pp 271ndash278ASM Press Washington DC USA 2007

[13] C A Navarro L H Orellana C Mauriaca and C A JerezldquoTranscriptional and functional studies of Acidithiobacillusferrooxidans genes related to survival in the presence of copperrdquoApplied and Environmental Microbiology vol 75 no 19 pp6102ndash6109 2009

[14] A Orell C A Navarro R Arancibia J C Mobarec andC A Jerez ldquoLife in blue copper resistance mechanisms ofbacteria and Archaea used in industrial biomining of mineralsrdquoBiotechnology Advances vol 28 no 6 pp 839ndash848 2010

[15] A Orell C A Navarro M Rivero J S Aguilar and C A JerezldquoInorganic polyphosphates in extremophiles and their posiblefunctionsrdquo Extremophiles vol 16 no 4 pp 573ndash583 2012

[16] C Baker-Austin M Dopson M Wexler et al ldquoExtremearsenic resistance by the acidophilic archaeon ldquoFerroplasmaacidarmanusrdquo Fer1rdquo Extremophiles vol 11 no 3 pp 425ndash4342007

[17] E Pedone S Bartolucci and G Fiorentino ldquoSensing andadapting to environmental stress the archaeal tacticrdquo Frontiersin Bioscience vol 9 pp 2909ndash2926 2004

[18] C Baker-Austin M Dopson M Wexler R G Sawers and PL Bond ldquoMolecular insight into extreme copper resistance inthe extremophilic archaeon ldquoFerroplasma acidarmanusrdquo Fer1rdquoMicrobiology vol 151 no 8 pp 2637ndash2646 2005

[19] T J G Ettema A B Brinkman P P Lamers N G Kornet WM de Vos and J van der Oost ldquoMolecular characterization ofa conserved archaeal copper resistance (cop) gene cluster andits copper-responsive regulator in Sulfolobus solfataricus P2rdquoMicrobiology vol 152 no 7 pp 1969ndash1979 2006

[20] A A Villafane Y VoskoboynikM Cuebas I Ruhl and E BinildquoResponse to excess copper in the hyperthermophile Sulfolobussolfataricus strain 982rdquo Biochemical and Biophysical ResearchCommunications vol 385 no 1 pp 67ndash71 2009

[21] K S Auernik Y Maezato P H Blum and R M KellyldquoThe genome sequence of the metal-mobilizing extremelythermoacidophilic archaeon Metallosphaera sedula providesinsights into bioleaching-associated metabolismrdquo Applied andEnvironmental Microbiology vol 74 no 3 pp 682ndash692 2008

[22] Y Maezato T Johnson S McCarthy K Dana and P BlumldquoMetal resistance and lithoautotrophy in the extreme ther-moacidophile Metalosphaera sedulardquo Journal of Bacteriologyvol 194 no 24 pp 6856ndash6863 2012

[23] D H Nies ldquoMicrobial heavy-metal resistancerdquo Applied Micro-biology and Biotechnology vol 51 no 6 pp 730ndash750 1999

[24] K Vido D Spector G Lagniel S Lopez M B Toledano andJ Labarre ldquoA proteome analysis of the cadmium response inSaccharomyces cerevisiaerdquo The Journal of Biological Chemistryvol 276 no 11 pp 8469ndash8474 2001

[25] G Bertin and D Averbeck ldquoCadmium cellular effects mod-ifications of biomolecules modulation of DNA repair andgenotoxic consequences (a review)rdquo Biochimie vol 88 no 11pp 1549ndash1559 2006

[26] M H Joe S W Jung S H Im et al ldquoGenome-wide responseof Deinococcus radiodurans on cadmium toxicityrdquo Journal ofMicrobiology and Biotechnology vol 21 no 4 pp 438ndash447 2011

[27] A Lagorce A Fourcans M Dutertre B Bouyssiere YZivanovic and F Confalonieri ldquoGenome-wide transcriptionalresponse of the archeon Thermococcus gammatolerans to cad-miumrdquo PLOS ONE vol 7 no 7 Article ID e41935 2012

[28] A Kornberg N N Rao and D Ault-Riche ldquoInorganicpolyphosphate a molecule of many functionsrdquo Annual Reviewof Biochemistry vol 68 pp 89ndash125 1999

Archaea 15

[29] J D Keasling ldquoRegulation of intracellular toxic metals andother cations by hydrolysis of polyphosphaterdquoAnnals of theNewYork Academy of Sciences vol 829 pp 242ndash249 1997

[30] F Remonsellez A Orell and C A Jerez ldquoCopper tolerance ofthe thermoacidophilic archaeon Sulfolobus metallicus possiblerole of polyphosphate metabolismrdquoMicrobiology vol 152 no 1pp 59ndash66 2006

[31] V Hatzimanikatis L H Choe and K H Lee ldquoProteomicstheoretical and experimental considerationsrdquo BiotechnologyProgress vol 15 no 3 pp 312ndash318 1999

[32] L H Choe and K H Lee ldquoA comparison of three commerciallyavailable isoelectric focusing units for proteome analysis theMultiphor the IPGphor and the Protean IEF cellrdquo Electrophore-sis vol 21 no 5 pp 993ndash1000 2000

[33] U K Laemmli ldquoCleavage of structural proteins during theassembly of the head of bacteriophage T4rdquo Nature vol 227 no5259 pp 680ndash685 1970

[34] A Shevchenko M Wilm O Vorm and M Mann ldquoMassspectrometric sequencing of proteins from silver-stained poly-acrylamide gelsrdquo Analytical Chemistry vol 68 no 5 pp 850ndash858 1996

[35] P Giavalisco E Nordhoff T Kreitler et al ldquoProteome analysisof Arabidopsis thaliana by two-dimensional gel electrophoresisand matrix-assisted laser desorptionionisation-time of flightmass spectrometryrdquo Proteomics vol 5 no 7 pp 1902ndash19132005

[36] T M Rose E R Schultz J G Henikoff S Pietrokovski CM McCallum and S Henikoff ldquoConsensus-degenerate hybridoligonucleotide primers for amplification of distantly relatedsequencesrdquo Nucleic Acids Research vol 26 no 7 pp 1628ndash16351998

[37] T M Rose J G Henikoff and S Henikoff ldquoCODEHOP(Consensus-Degenerate Hybrid Oligonucleotide Primer) PCRprimer designrdquo Nucleic Acids Research vol 31 no 13 pp 3763ndash3766 2003

[38] J P Acevedo F Reyes L P Parra O Salazar B A Andrews andJ A Asenjo ldquoCloning of complete genes for novel hydrolyticenzymes from Antarctic sea water bacteria by use of animproved genomewalking techniquerdquo Journal of Biotechnologyvol 133 no 3 pp 277ndash286 2008

[39] S Bathe and P R Norris ldquoFerrous iron- and sulfur-inducedgenes in Sulfolobus metallicusrdquo Applied and EnvironmentalMicrobiology vol 73 no 8 pp 2491ndash2497 2007

[40] J Sambrook E F Fritsch and TManiatisMolecular Cloning ALaboratory Manual Cold Spring Harbor Press New York NYUSA 1989

[41] K W Miller S Sass Risanico and J B Risatti ldquoDifferentialtolerance of Sulfolobus strains to transition metalsrdquo FEMSMicrobiology Letters vol 93 no 1 pp 69ndash73 1992

[42] G Huber C Spinnler A Gambacorta and K O Stetter ldquoMet-allosphaera sedula gen and sp nov represents a new genus ofaerobic metal-mobilizing thermoacidophilic archaebacteriardquoSystematic and Applied Microbiology vol 12 pp 38ndash47 1989

[43] J Llanos C Capasso E Parisi D Prieur and C JeanthonldquoSusceptibility to heavy metals and cadmium accumulation inaerobic and anaerobic thermophilic microorganisms isolatedfrom deep-sea hydrothermal ventsrdquo Current Microbiology vol41 no 3 pp 201ndash205 2000

[44] A Kaur M Pan M Meislin M T Facciotti R El-Gewelyand N S Baliga ldquoA systems view of haloarchaeal strategies towithstand stress from transition metalsrdquo Genome Research vol16 no 7 pp 841ndash854 2006

[45] M Solioz and J V Stoyanov ldquoCopper homeostasis in Entero-coccus hiraerdquo FEMS Microbiology Reviews vol 27 no 2-3 pp183ndash195 2003

[46] S Sitthisak K Howieson C Amezola and R K JayaswalldquoCharacterization of a multicopper oxidase gene from Staphy-lococcus aureusrdquo Applied and Environmental Microbiology vol71 no 9 pp 5650ndash5653 2005

[47] L Rodrıguez-Montelongo S I Volentini R N Farıas E MMassa and V A Rapisarda ldquoThe Cu(II)-reductase NADHdehydrogenase-2 of Escherichia coli improves the bacterialgrowth in extreme copper concentrations and increases theresistance to the damage caused by copper and hydroperoxiderdquoArchives of Biochemistry and Biophysics vol 451 no 1 pp 1ndash72006

[48] E Williams T M Lowe J Savas and J DiRuggiero ldquoMicroar-ray analysis of the hyperthermophilic archaeon Pyrococcusfuriosus exposed to gamma irradiationrdquo Extremophiles vol 11no 1 pp 19ndash29 2007

[49] L M Gaetke and C K Chow ldquoCopper toxicity oxidative stressand antioxidant nutrientsrdquo Toxicology vol 189 no 1-2 pp 147ndash163 2003

[50] M J Davies ldquoThe oxidative environment and protein damagerdquoBiochimica et Biophysica Acta vol 1703 no 2 pp 93ndash109 2005

[51] J R Thompson J K Bell J Bratt G A Grant and LJ Banaszak ldquoVmax regulation through domain and subunitchanges The active form of phosphoglycerate dehydrogenaserdquoBiochemistry vol 44 no 15 pp 5763ndash5773 2005

[52] V Consalvi R Chiaraluce L Politi A Gambacorta MDe Rosa and R Scandurra ldquoGlutamate dehydrogenase fromthe thermoacidophilic archaebacterium Sulfolobus solfataricusrdquoEuropean Journal of Biochemistry vol 196 no 2 pp 459ndash4671991

[53] A J L Macario M Lange B K Ahring and E Conway DeMacario ldquoStress genes and proteins in the archaeardquo Microbiol-ogy and Molecular Biology Reviews vol 63 no 4 pp 923ndash9671999

[54] J AMaupin-FurlowM A Gil M A Humbard et al ldquoArchaealproteasomes and other regulatory proteasesrdquo Current Opinionin Microbiology vol 8 no 6 pp 720ndash728 2005

[55] N Sun F Beck R Wilhelm Knispel et al ldquoProteomics analysisof Thermoplasma acidophilum with a focus on protein com-plexesrdquoMolecular and Cellular Proteomics vol 6 no 3 pp 492ndash502 2007

[56] L S Madding J K Michel K R Shockley et al ldquoRole of the 1205731subunit in the function and stability of the 20S proteasome inthe hyperthermophilic Archaeon Pyrococcus furiosusrdquo Journalof Bacteriology vol 189 no 2 pp 583ndash590 2007

[57] R Shibata Y Bessho A Shinkai et al ldquoCrystal structureand RNA-binding analysis of the archaeal transcription factorNusArdquo Biochemical and Biophysical Research Communicationsvol 355 no 1 pp 122ndash128 2007

[58] W Bae B Xia M Inouye and K Severinov ldquoEscherichia coliCspA-family RNA chaperones are transcription antitermina-torsrdquo Proceedings of the National Academy of Sciences of theUnited States of America vol 97 no 14 pp 7784ndash7789 2000

[59] R J Ram N C VerBerkmoes M P Thelen et al ldquoMicrobi-ology community proteomics of a natural microbial biofilmrdquoScience vol 308 no 5730 pp 1915ndash1920 2005

[60] I Noel-Georis T Vallaeys R Chauvaux et al ldquoGlobal analysisof the Ralstonia metallidurans proteome prelude for the large-scale study of heavy metal responserdquo Proteomics vol 4 no 1pp 151ndash179 2004

16 Archaea

[61] M TMNovo A CDa Silva RMoreto et al ldquoThiobacillus fer-rooxidans response to copper and other heavy metals growthprotein synthesis and protein phosphorylationrdquo Antonie vanLeeuwenhoek vol 77 no 2 pp 187ndash195 2000

[62] Q She R K Singh F Confalonieri et al ldquoThe complete genomeof the crenarchaeon Sulfolobus solfataricus P2rdquo Proceedings ofthe National Academy of Sciences of the United States of Americavol 98 no 14 pp 7835ndash7840 2001

[63] R P Anderson and J R Roth ldquoTandem genetic duplications inphage and bacteriardquo Annual Review of Microbiology vol 31 pp473ndash505 1977

[64] D Gevers K Vandepoele C Simillion and Y Van De PeerldquoGene duplication and biased functional retention of paralogsin bacterial genomesrdquo Trends in Microbiology vol 12 no 4 pp148ndash154 2004

[65] A B Reams and E L Neidle ldquoSelection for gene clustering bytandemduplicationrdquoAnnual Review ofMicrobiology vol 58 pp119ndash142 2004

[66] L H Orellana and C A Jerez ldquoA genomic island providesAcidithiobacillus ferrooxidans ATCC 53993 additional copperresistance a possible competitive advantagerdquo Applied Microbi-ology and Biotechnology vol 92 no 4 pp 761ndash767 2011

[67] A B Brinkman T J G Ettema W M de Vos and J van derOost ldquoThe Lrp family of transcriptional regulatorsrdquo MolecularMicrobiology vol 48 no 2 pp 287ndash294 2003

[68] C Vollmecke S L Drees J Reimann S V Albers and MLubben ldquoThe ATPases CopA and CopB both contribute tocopper resistance of the thermoacidophilic archaeon SulfolobussolfataricusrdquoMicrobiology vol 158 no 6 pp 1622ndash1633 2012

[69] A Villafane Y Voskoboynik I Ruhl et al ldquoCopR of Sulfolobussolfataricus represents a novel class of archaeal-specific copper-responsive activators of transcriptionrdquo Microbiology vol 157no 10 pp 2808ndash2817 2011

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

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International Journal of

Volume 2014

Zoology

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Enzyme Research

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International Journal of

Microbiology

6 Archaea

14

12

10

8

6

4

2

00 2 4 6 8 10 12

Time (days)

107timescellsm

L

(a)

120

100

80

60

40

20

00 2 4 6 8 10

Time (days)

107timescellsm

L

(b)

Figure 1 Growth of S metallicus (a) and S solfataricus (b) in the presence of Cd Cells were inoculated in their respective growth media inabsence of added Cd (∙) or were supplemented with 0005mM () 001mM () 005mM (diams) 01mM () 05mM () 1mM () 2mM() 3mM (times) or 5mM (+) Cd and cells were counted daily

100

7060

50

30

20

3 10MW

33 31

32

A

B

C

20

10

(a)

100

7060

50

30

20

12

3 10MW

204

21

33 31

32

A

B

C

15

93

10

27 28

23 24

29

25 2630

1614 16

13

52

7

2219

8

20

10

11

(b)

Figure 2 Changes in the proteome of S metallicus grown in the presence of Cu Cells were incubated in the absence of any added metal(a) or in the presence of 100mM Cu (b) Arrows indicate the spots that were downregulated (a) or upregulated (b) in the presence of CuNumbers indicate the spots with increased intensity in cells treated with copper The dashed boxes are shown as enlarged areas that includesome proteins upregulated in the presence of Cu and Cd (Figure 3) The bottom panels show low molecular weight proteins upregulated inthe presence of Cu separated by using a 15 polyacrylamide gel in the second dimension

cells were untreated or treated with either 100 or 200mM Cuor 1mM Cd during 24 h and analyzed by comparative two-dimensional gel electrophoresis as indicated in experimentalprocedures Twenty-three proteins were found to be down-regulated after 100mMCu treatment (Figure 2(a)) A similar

pattern was obtained at 200mM Cu (not shown) Further-more by using the Delta 2D software (Decodon) eleven ofthese proteins were found to be completely absent in the gelsand more than 8 proteins decreased their intensity in therange from 15-to 5-fold compared with control cells grown

Archaea 7

13 13

11

13

1

Control Copper Cadmium

(a)

99 9

333

(b)

3030

30

2020

20 212121

(c)

Figure 3 Comparison of selected proteins of S metallicus cells treated with Cu or Cd Protein extracts were obtained from cells treatedwithout metals and with 100mM Cu or 1mM Cd for 24 h and the proteins were separated by 2D-PAGE The segments (a) (b) and (c) arethe enlarged dashed boxes in Figure 2 under the three conditions indicated Numbers show some of the spots with increased intensity in cellstreated with Cu or Cd

in the absence of Cu (Figure 2(a)) Therefore these resultsshow that a number of proteins became non-detectable ordecreased their levels when S metallicus faced Cu This kindof response was also seen when the microorganism wasexposed to Cd (data not shown) This behavior has alsobeen observed in similar studies where F acidarmanus waschallenged by either As(III) or Cu [16 18] In addition theexpression of 30 other proteins was found to be upregulatedafter Cu treatment (100 or 200mM) (Figure 2(b)) Most ofthese proteins could be identified only in the condition withCu and spots 5 13 and 15 were induced 26 34 and 55-fold respectively compared to cells without Cu treatmentMoreover 3 proteins of lowmolecular weight (spots 31 3 and33) that increased their expression when cells were exposedto 100mM Cu were also identified (Figure 2 bottom panels)These proteins were only detected when 15 polyacrylamidewas used during the second-dimensional separation (SDS-PAGE) On the other hand a large decrease in overallexpression of proteins after 1mMCd treatment was observed(not shown) Interestingly 10 out of the 13 proteins identifiedas up-regulated when cells were exposed to Cd and alsoshowed increased levels after Cu treatment Some of them areshown in Figure 3

33 Identification of Proteins Upregulated in Cells Treated withCu and Cd in S metallicus A total of 18 S metallicus proteinswhose levels were found to be up-regulated in responseto Cu were analyzed by mass spectrometry The identifiedproteins included functions related to the production andtransport of energy biosynthesis of amino acids stressresponses and transcription regulation (Table 2) Amongstthe proteins related to production and transport of energya putative ATP synthase subunit B (spot 23) that has beendescribed as playing a fundamental role in ATP synthesiswas identified (Table 2) When cells are subjected to somestressing conditions such as the presence of heavy metalsa greater cellular demand for energy has been reported tooccur [18] Most likely this phenomenon might be due toATP-driven Cu transport via ATPases that have a substantialinterplay during metal detoxification [45] Furthermore theATP synthase subunit B was also identified as up-regulatedwhen S metallicus was treated with Cd (Table 2)

Two other proteins corresponded to putative oxidoreduc-tases such as ferredoxin oxidoreductase (spot 3) and alcoholdehydrogenase (spot 4) that have been generally involvedin electron transporter chains and use NAD+ as an electronacceptor The levels of ferredoxin oxidoreductase were also

8 Archaea

Table 2 Proteins upregulated in S metallicus cells exposed to 100mM Cu or 1mM Cd

Spot Molecular weight (kDa) Cu induction levels Putative function Microorganism related Accesion number1dagger 266 infin Proteasome subunit S solfataricus AAK410343dagger 701 infin Ferredoxin oxidoreductase S solfataricus AAK429264dagger 429 infin Alcohol dehydrogenase S solfataricus AAK431545 35 26 Hypothetical protein S acidocaldarius YP 2549766 27 infin Hypothetical protein P aerophilum NP 5598899dagger 57 infin Phosphoglycerate dehydrogenase M thermautotrophicus AAB8546613dagger 27 34 Unknown mdash mdash14 28 infin Unknown mdash mdash15 35 55 Unknown mdash mdash20dagger 461 infin Glutamate dehydrogenase S solfataricus AAK4223021dagger 45 infin Hypothetical protein M acetivorans NP 618380122 49 infin Hypothetical protein P torridus YP 02280723dagger 51 infin ATP synthase subunit B S solfataricus AAK4088024 585 infin HSP60 subunit S shibatae AAG37273130dagger 48 infin Unknown mdash mdash31 16 ND Unknown mdash mdash32 144 ND Putative transcription regulator S solfataricus AAK4041333 138 ND Transcription factor nusA S solfataricus AAK40563Spots refer to those numbered in Figure 2 Proteins in Figure 2 which are not included in this table were not subjected to sequencing due to their low intensitiesin the gelsdaggerSpot up-regulated in cells exposed to Cu and CdND not determinedInfinity symbol indicates that proteins were expressed only in presence of copper or cadmiumM thermoautotrophicus Methanobacterium thermoautotrophicus M acetivorans Methanosarcina acetivorans S shibatae Sulfolobus shibatae P torridusPicrophilus torridus P aerophilum Pyrobaculum aerophilum S acidocaldarius Sulfolobus acidocaldarius

up-regulated in response to Cd (Table 2) Several previ-ous reports have suggested that oxidoreductases contributeto an oxidative protection in both Bacteria and Archaeain response to heavy metals [46ndash48] In the neutrophilicmicroorganisms Escherichia coli and Staphylococcus aureusit has been described that oxidases and dehydrogenasescontribute to oxidative protection Cu homeostasis and stressresponses [46 47] Furthermore some proteins involved inoxidative damage repair such as NADH-dependent oxidasesand thioredoxin reductases were expressed in cells of Facidarmanus exposed to As(III) [16] The expression of thisgroup of proteins has also been observed when the samemicroorganism was exposed to Cu [18] Therefore oxidore-ductases play an important role against oxidative stress andmay eliminate reactive oxygen species which constitute themajor component of the stress caused by transition metals[44 49 50]

The enzymes related to biosynthesis of amino acids werefound as commonly up-regulated either in response to Cu orCdThey corresponded to a phosphoglycerate dehydrogenase(spot 9) and a glutamate dehydrogenase (spot 20) Theformer one catalyzes the NAD+-dependent oxidation of 3-phosphoglycerate into 3-phosphohydroxypyruvate a branchpoint from the glycolytic pathway and the initial reactionin L-serine biosynthesis [51] Glutamate dehydrogenase cat-alyzes the oxidative deamination of glutamate to produce 2-oxoglutarate and ammonia with reduction of NAD+ [52]Proteins involved in amino acids synthesis were expressed

in cells of F acidarmanus exposed to As(III) [16] Moreoverincreased levels of proteins involved in the biosynthesisof sulfur-containing amino acids have been observed inSaccharomyces cerevisiae cells exposed to Cd [24] suggestingthat these proteins might also have a role in the cellularresponse to adverse conditions

Two proteins showed identity to components of the stressresponse mechanism such as one subunit of the HSP60chaperonin (spot 24) and one subunit of the proteasome (spot1) In Archaea the stress protein HSP60 is directly involvedin protein folding processes [53] Proteomic analysis of Facidarmanus cells exposed to As(III) and Cd revealed theexpression of proteins involved in protein folding and DNArepair including HSP60 chaperonin and DnaK heat-shockprotein (HSP70) thereby the authors suggested that thismicroorganismusesmultiplemechanisms to resist high levelsof Cu [16 18] On the other hand proteasomes are describedas nonspecific proteolytic nanomachines associated withprotein catabolism Proteasomes are known to be closelyinvolved in maintaining protein quality control by degradingmiss folded and denatured proteins in response to cell stressin all three domains of life [54] A proteomic analysis inthe thermoacidophilic archaeon Thermoplasma acidophilumshowed that proteasomes and chaperone-related proteinswere highly induced against stress conditions indicatinga high turnover rate of proteins [55] In P furiosus theexpression of the 1205731 subunit of the proteasome was inducedin cells subjected to heat shock stress [56] Additionally the

Archaea 9

150pb

(a)

1016 pb

(b)

2000 pb

(c)

Figure 4 CODEHOP-based PCR for the amplification of the putative genes copA and copM from S metallicus Amplification products(a) using degenerate PCR primers (DOP) copM degF and copM degR designed from amino acid sequence conserved blocks of SulfolobalesCopM proteins (b) using primers copA cdegF and copA cdegR designed by CODEHOP strategy for amplification of a copA-like gene (c)Primers copM degF and copA cdegRwere assesed for the amplification of a copMA-likeDNA regionThose primerswere testedwith genomicS metallicus DNA samples Amplicons expected sizes were as follows ca 150 bp for copM ca 950 bp for copA and ca 2000 bp for copMAPCR products were excised and cloned for later sequencing

proteasome subunit was also found to be up-regulated whenS metallicus was subjected to Cd stress (Table 2)

The transcriptional regulation-related proteins corre-sponded to a putative transcription regulator (spot 32) andthe transcription factor NusA (spot 33) involved in theintrinsic termination of transcription [57] NusA has beenalso reported to display important functions in the cellularresponse to adverse factors [58] The expression of twotranscriptional regulators related to amino acids biosynthesisand metal-dependent genetic repression were found to beinduced in P furiosus cells subjected to oxidative stress[48] Moreover the expression of several genes encodingpredicted transcriptional regulators were induced followingCd exposure in T gammatolerans cells [27]

Spots 5 6 13 14 15 21 22 30 and 31 (Table 2) did notshow significant scores with any protein sequences currentlyavailable in the data bases Interestingly spots 2 4 9 13 21and 30 were found to be commonly upregulated when Smetallicus cells were treated with either Cu or Cd (Figure 3Table 2)

The results presented here are consistent with previ-ous work describing global protein expression profiles inresponse to heavy metals as reported for F acidarmanus [1618] microbial-biofilm communities in an acid mine drainagesite [59] and other prokaryotic and eukaryotic microorgan-isms [24 60]Therefore a coordinated expression of differentgroups of genes suggests the existence of regulatory networkssuch as stress response mechanisms and respiratory chainadjustments to cope with the presence of heavy metal ions[61]

34 Cu-Resistance (Cop) Genes Cluster Is Duplicated in Smetallicus Genome A Cu-resistance (cop) locus has beendescribed to be highly conserved in archaeal genomeswhich consists of genes encoding a new type of archaealtranscriptional regulator (CopT) a metal-binding chaperone(CopM) and a Cu-transporting P-type ATPase (CopA) [19]Since the genome sequence of Smetallicus is not yet availablewe searched for the presence of cop genes by using consen-sus degenerate hybrid oligonucleotide primers-based PCR(CODEHOP-PCR) CopA and CopM amino acid sequencealignments from S acidocaldarius S tokodaii and S solfa-taricus were used to identify conserved sequence blocks andtherefore to design either CODEHOP or DOP (degeneratehybrid oligonucleotide) primers (Figure S1 Table 1) TherebyPCR assays using S metallicus genomic DNA as templateyielded amplicons of ca 150 bp and ca 950 bp for copMand copA CODEHOP pair primers respectively (Figures4(a) 4(b)) As estimated from primer pairrsquos position onamino acid sequence alignments the obtained PCR productscorresponded to the expected sizes suggesting the presenceof copM- and copA-like genes in S metallicus genome Inorder to determine the identity of the amplified DNA frag-ments they were TA-cloned and sequenced Blastx sequenceanalysis showed that the 150 bp DNA fragment coded for anincomplete ORF sharing 63 homology with S solfataricusCopM (SSO10823) whereas the analysis of the 950 pb DNAfragment yielded a partial ORF sharing 54 homology withS solfataricus CopA protein (SSO2651)

The genomic organization of the putative cop genes in Smetallicus was analyzed to find out its similarity with those

10 Archaea

Table 3 Sequence identity comparison of S metallicus Cop proteins to those in other Sulfolobales

S metallicus protein Identity to homologue in other SulfolobalesS solfataricus P2 S acidocaldarius S tokodaii M sedula

CopA1 51 43 44 45CopA2 88 46 49 50CopM1 63 69 71 71CopM2 89 72 70 77CopT1 36 27 30 32CopT2lowast 90 52 52 53Boldface type indicates to which organism the S metallicus homologue shows the highest sequence identitylowastRefers to the analysis obtained using the uncompleted amino acid sequence of CopT2 which represents sim90 in length when compared with S solfataricusCopT sequence

previously described in S solfataricus P2 and F acidarmanuswhere the cop cluster consists of tandem-orientated genesas copTMA [18 19] Thus using copM degF (forward) andcopA cdegR (reverse) primers we attempted to amplify aputative copMA DNA region This PCR yielded a product ofca 2000 pb corresponding to the expected size and resultedin 2 ORFs showing high sequence homology with CopMand CopA from S solfataricus (Figure 4(c)) Interestinglywhen aligning this copMA sequence with the copA and copMsequences obtained previously they were not identical whichstrongly indicated the presence of duplicated putative copgenes encoding for both Cu-ATPases (copA1 and copA2) andmetallochaperones (copM1 and copM2) in the S metallicusgenome

To isolate the entire cop gene sequences the genomewalking method described by Acevedo et al [38] was usedSix different DNA libraries were constructed from S metalli-cus genomic DNA digested with several restriction enzymesincluding 51015840 overhang and blunt end restriction enzymesThe DNA libraries were used as templates in 2 successivePCR reactions From the previously known sequences twospecific sense primers for the 31015840 end amplification and twospecific antisense primers for the 51015840 end amplification weredesigned (abbreviated SP1 and SP2) Therefore the 51015840 and 31015840ends of the putative genes copA1 and copA2 that had beenpartially identified through the genome walking techniquewere amplified

By overlapping the sequence isolated by degenerate PCRand the lateral sequences obtained by genome walking(Figure S2) it was possible to complete the whole nucleotidesequence of the putative gene copA1 Additionally it waspossible to confirm the presence of the upstream copM1 geneanddetermine the occurrence of a partial copT1 gene tandem-orientated upstream of copM1 (Figure 5)Through additionalgenome walking experiments the partial copT1 sequencecould be further completedThe samewas truewhen isolatingthe whole nucleotide sequence of the putative gene copA2as the presence of copM2 and copT2 genes were confirmedupstream of copA2 However it was not possible to completethe whole 51015840 end of the putative gene copT2 as its determinedlength corresponded to only 90 of the full length whencompared with S solfataricus copT gene (Figure 5)

In conclusion degenerate PCR together with genomewalking experiments allowed to describe the occurrence inS metallicus genome of 2 cop genes loci (named as locus cop1and locus cop2) coding for paralogous genes whose productsmay be involved in Cu-resistance (Figure 5) Although somearchaeal genomes exhibit two copies of putative Cu-P-typeATPases as described for S solfataricus [62] the genomicarrangement displayed as a cop gene cluster (copTMA) hasbeen reported to be represented in only one copy for manyarchaeal genomes [10 19 21] Moreover we further updatedthis analysis by searching for duplications of the cop genescluster in all available archaeal genome sequences up to date(October 2012) retrieving only one cop locus in each caseThereby we propose that the discovery of this cop gene clusterduplication in the genome of S metallicus constitutes so faran unprecedented feature for a representative of the Archaeadomain and might contribute to its high metal resistance

In this context it has been widely reported that increasedgene copy number can increase gene expression allowingprokaryotic to thrive under growth limiting conditions [1363ndash68] However alongside the augmented gene copy onecannot exclude that the cop operon duplication in S metal-licus may offer among others a wider repertoire of Cu-ATPases which might differ in terms of metal affinitiesandor specificity efflux rates andor differences in theirabundance regulation

Throughout amino acid sequence analysis it was furtherdetermined that each polypeptide encoded by S metallicuswith the exception of CopT1 showed to be highly homol-ogous to their orthologous counterparts encoded by othersSulfolobales (Table 3) Moreover the 3 ORFs encoded by thelocus cop2 shared about 90 identity with the correspondingS solfataricus orthologous peptides (Table 3) S metallicuscop genes products showed characteristic domains referredto as critical for their respective proposed biological activitiesS metallicus paralogous gene products CopA1 (749 aa1015840)and CopA2 (747 aa1015840) shared 513 of identity These twoputative Cu-ATPases (CopA1 and CopA2) contain the aminoacid sequence motif CPCALGLA which has been proposedto confer Cu-transporting specificity in CPx-ATPases [48]and has been also found in other CopA-like proteins frombiomining Bacteria and Archaea [14]

Archaea 11

HindIII HindIII HindIII

HindIII

CopT1Locus cop1 M1

EcoRIEcoRV

513 bp 219 bp 2250 bp

CopA1

(a)

Locus cop2 M2

EcoRI

CopA2

453 bp 171 bp 2244 bp

CopT2

PstIHindIII HindIII HindIII

(b)

Figure 5 Schematic representation of the two cop gene clusters isolated from S metallicus genome Each cluster codes for anArchaea-specifictranscriptional regulator (copT) a metallochaperone (copM) and a P-type Cu-exporting ATPase (copA) Lengths of each gene are indicatedLocus cop1 corresponds to 4346 sequenced base pairs and shows an intergenic region copT1-M1 of 232 base pairs Locus cop2 correspondsto 3435 base pairs and shows 89 base pairs copT2-M2 intergenic region Genes copM2 and copA2 overlapped in 11 base pairs copT2 was notfully isolated

Control CdSO4 Ag2SO4 NiSO4 ZnSO4 CuSO4

kb3kb2kb1

02 kb

(a)

kb3kb2kb1

02 kb

(b)

(c)

Figure 6 Northern blot analysis to determine the expression of S metallicus copA1 (a) and copA2 (b) putative genes in response to variousmetals S metallicus total RNAwas extracted from cultures grown either in the absence (control) or the presence of CuSO

4(50mM) Ag

2SO4

(008mM) NiSO4(15mM) ZnSO

4(50mM) and CdSO

4(2mM) P32-radioactivelly labelled DNA fragments annealing to copA1 and copA2

sequences respectively were used as probes (c) shows rRNAs as total RNA loading control

On the other hand CopM1 (71 aa) and CopM2 (56 aa)sharing 66 of identity present the proposed metal bindingdomain TRASH (trafficking resistance and sensing of heavymetals) [10] Additionally when analyzing the putative tran-scriptional regulators the presence of a C-terminal TRASHdomain in both CopT1 and CopT2 was also found CopT1sequence also contains an entire N-terminal helix-turn-helix (HTH) motif (not shown) that resembles the DNA-bindingmotifs of prokaryotic transcriptional regulators suchas Lrp-like proteins [67] The HTH motif was only partiallyidentified in CopT2 since the 51015840 region of copT1 gene has notbeen yet fully isolated

35 Transcriptional Analysis of S metallicus Cop Genes Toget insight into the role of the two cop loci the expressionof the corresponding genes was analyzed under variousconditions S metallicus was first grown in presence ofdifferent heavy metals (Cu Zn Cd Ni o Ag) at a givenconcentration that did not affect growth kinetics [30 42]Subsequently total RNA was isolated from late exponentiallygrown cultures and Northern blot experiments were carriedout in order to determine the expression of both copA1 andcopA2 genes As depicted in Figure 6 transcription of bothcopA1 and copA2messengers were specifically induced in thepresence of Cu and Cd ions This gene expression pattern

12 Archaea

954pb

M1 CopA1

(a)

1438 pb

M2 CopA2

(b)

1000 pb

800pb

+ minus

(c)

+ minus

1500 pb

1000 pb

(d)

Figure 7 Cotranscription analysis of copMA1 and copMA2 genes cDNA was synthesized with a reverse primer (dotted arrows) hybridizingtoward the 31015840 end of either copA1 (a) or copA2 (b) S metallicus total RNA was extracted at the late exponential phase from a culture growingin presence of 20mMCu PCR amplifications were carried out with these cDNAs and each corresponding primer pair (black arrows) as listedin Table 1 (c) and (d) show RT-PCR products obtained for the copMA1 and copMA2 intergenic regions respectively Reverse transcriptasereactions with (+) and without (minus) the Improm II reverse transcriptase enzyme were carried out in order to exclude amplification due togenomic DNA contamination Expected sizes (in base pairs) for the corresponding PCR products are given in (a) and (b)

is in good agreement with what has been described for Ssolfataricus where the bicistronic copMA transcript levelswere also found to be up-regulated in response to both Cuand Cd [19]

Northern blot analysis showed that the sizes seen for bothcopA transcripts (sim25 Kb) did not correspond exactly withthe individual genes length (each of about 225Kb) (Figure 6)This strongly suggested that the transcripts could also includethe respective copM gene located upstream of copA in eachcop loci To confirm this assumption a co-transcriptionexperiment was done (Figure 7) in which the cDNAs wereobtained by using RNA extracted from a culture grownin the presence of 20mM Cu and using a reverse primerhybridizing with copA1 (Figure 7(a)) or copA2 (Figure 7(b))gene sequence respectively PCR amplifications were carriedout by using the corresponding cDNAs as templates and eachpair of primers lying in adjacent genes (copM) The presenceof an amplicon of the expected size in each case indicatedthe adjacent genes were part of polycistronic messengers(Figure 7) These results clearly show that in S metallicuseach couple of copMA genes was expressed in the form oftranscriptional units as reported in F acidarmanus and Ssolfataricus [18 19] The coexpression of gene pairrsquos copMAmay suggest a coordinated and dependent function for therespective encoded proteins In the bacterium E hirae themetallochaperone CopZ (CopM in S metallicus) fulfills apivotal role in the mechanism of Cu homeostasis [45] Thusit was shown that this protein interacts directly with the Cu-ATPase CopB (CopA in S metallicus) handing the Cu forsubsequent removal In this context one might expect thatproteins CopM1 and CopM2 from S metallicus have a similarrole to that described for E hirae Furthermore quantitativeRT-PCR experiments were carried out in order to determinedifferential expression of the cop genes in response to differentCu concentrations The expression of copA1 copA2 copT1and copT2 was tested relative to the transcript levels ofrRNA 16S gene since its levels were not significantly affected

in all assessed conditions (data not shown) As shown inFigure 8(a) copA2 and copA1 transcript levels were foundto be concomitantly increased with the increasing Cu con-centrations present in the medium Higher transcripts levelsof copA1 were seen in all tested conditions when comparedwith copA2 levels copA1 transcript levels were found to be325-fold up-regulated when comparing 50mMCu conditionversuscontrol (absence of metal) whereas copA2 transcriptlevels showed an increment of only 175-fold (Figure 8(a))The finding that Cu-ATPases mRNA levels were significantlyincreased in response to Cu ions exposure suggests that thetransport system may operate for Cu efflux in S metallicus

Moreover copA2 and copA1 gene expression was quan-tified when S metallicus was grown using chalcopyrite(CuFeS

2) ore as an oxidizable substrate (Figure 8(a)) Mineral

oxidation mediated by the microorganism generates a pro-gressive increase in Cu ions concentration in the medium Tofind out whether the amount of solubilized Cu induced theexpression of the Cu-ATPases genes total RNAwas extractedfrom a S metallicus culture grown to late exponential phaseand in the presence of 1 CuFeS

2 It was clear that an

up-regulation of the Cu-ATPases encoding genes also tookplace when S metallicus was grown in the presence ofCuFeS

2(Figure 8(a)) copA1 transcript levels increased 14-fold

compared with the control condition in the absence of Cuwhile copA2 gene expression showed an increase of 76 foldAlong with this bymeans of atomic absorption spectrometry(AAS) analysis an overall amount of solubilized Cu ions(Cu2+Cu1+) of 144plusmn21mMwas determined to be present inthemedium indicating that copA2 and copA1 gene expressionwas most likely in response to the Cu present in the culturedue to CuFeS

2microbial-solubilization

The finding that copA1 was highly expressed comparedwith copA2 in all tested conditions may suggest a possiblephysiological hierarchy between the two ATPases whenovercoming either Cu or Cd stress In this regard by meansof a genetic approach it was demonstrated in S solfataricus

Archaea 13

700

600

500

400

300

200

100

00

5 20 50 CuFeS2CuSO4 concentration (mM)

Relativ

egenee

xpression

CopA2CopA1

(a)

8

6

7

5

4

3

2

1

00

5 20 50 CuFeS2CuSO4 concentration (mM)

Relativ

egenee

xpression

CopT1CopT2

(b)

Figure 8 Relative expression levels of S metallicus genes copA1 and copA2 (a) copT1 and copT2 (b) Expression of S metallicus cop geneswasassessed by qRT-PCR and normalized against 16S rRNA gene expression in individual cultures Mean values and standard deviations arefrom analyses of three independent cultures grown in the presence of 0 5 20 50mMCu or in 1 of a chalcopyrite ore (CuFeS

2) concentrate

that while CopA was an effective Cu efflux transporterat low and high Cu concentrations the other Cu-ATPase(CopB) only appeared to be a low-affinity Cu export ATPase[68] Moreover by using a M sedula copA deletion mutantit was demonstrated that this strain compromised metalresistance and consequently abolished chalcopyrite oxidation[22] highlighting the role of Cu detoxification mechanismsduring a given bioleaching process Our attempts to showthe functionality of CopA1 and CopA2 from S metallicusby using E coli as a heterologous host were not successfulApparently the overexpression of these ORFs had a toxiceffect on E coli that compromised its viability Althoughgene disruption tools have not been yet developed for Smetallicus the functional role of both copTMA locimight befurther studied by cross-species complementation of a coppersensitive S solfataricus copR mutant as it was described byMaezato et al [22] It will be of great interest to establishin future studies the possible functionality of the isolatedputative transporters from S metallicus

Likewise quantitative RT-PCR experiments showed thatcopT1 transcript levels increased concomitantly with increas-ing Cu concentrations whereas copT2 showed relativelysimilar transcripts levels most likely indicating a constitutiveexpression profile (Figure 8(b)) Furthermore whereas copT1transcript levels were found to be increased 36 fold inCuFeS

2

grown cultures copT2 levels remained unchanged in com-parison with the control in the absence of Cu (Figure 8(b))The results obtained for S metallicus copT2 gene expressionare consistent with those reported for S solfataricus CopTtranscriptional regulator has been proposed to function as arepressor in S solfataricus showing a constitutive expressionthat in the presence of Cu loses its affinity for the promoterregion of copMA allowing the expression of this polycistron[19] In contrast the increased transcripts levels of both copA1and copT1 concomitant with higher Cu concentrations in the

environment suggest that the putative transcriptional regula-tor CopT1 may act by activating both copMA1 and probablyitself (Figure 8) Regarding this it was recently reported thatCopR (CopT in S metallicus) from S solfataricus strain 982acts as an activator of copT (copM in S metallicus) and copAexpression [69] Nevertheless additional experiments wouldbe required to test this possibility in S metallicus

4 Concluding Remarks

We previously reported that S metallicus resists extremelyhigh Cu concentrations which was mediated to some extentby the use of a possible metal resistance system based oninorganic polyphosphate hydrolysis and consequently Cu-PO4

2minusefflux [15 29 30] Here we have addressed the questionwhether this microorganism coded for other determinantsthat might help to explain its high heavy metal toleranceAs the genomic sequence of this microorganism is notyet available we jointly used CODEHOP-PCR and genomewalking approaches and were able to establish the occurrenceof two nonidentical homologous cop loci into the genome ofS metallicus (cop1 and cop2) Each cop locus codes for anarchaeal transcriptional regulator (CopT) a metal-bindingchaperone (CopM) and a Cu-transporting P-type ATPase(CopA) High levels of the polycistronic mRNAscopMA ofeach cop locus were observed after treatment with either Cuor Cd suggesting that the encoded ATPases efflux heavymetals out in order to detoxify the intracellular environmentAltogether previous reports and the results obtained in thisstudy allow us to suggest that some key elements that mayexplain the high resistance to Cu in S metallicus is the dupli-cation of the Cu resistance cop genes a defensive responseto stress and a polyP-based accumulation mechanism Inthe case of Cd although some similar stress responses were

14 Archaea

observed whether comparable Cu-responsive elements alsoparticipate in Cd responses remains to be seen

Conflict of Interests

The authors declare that the research was done in the absenceof any commercial or financial relationships that could beconstrued as a potential conflict of interests

Acknowledgments

This work was supported by Grant no FONDECYT 1110214and in part by ICM P-05-001-F project

References

[1] A Schippers ldquoMicroorganisms involved in bioleaching andnucleic acid-based molecular methods for their identificationand quantificationrdquo in Microbial Processing of Metal SulfidesR E Donati and W Sand Eds pp 3ndash33 Elsevier BerlinGermany 2007

[2] H R Watling ldquoThe bioleaching of sulphide minerals withemphasis on copper sulphides a reviewrdquo Hydrometallurgy vol84 no 1-2 pp 81ndash108 2006

[3] D E Rawlings D Dew and C Du Plessis ldquoBiomineralizationof metal-containing ores and concentratesrdquo Trends in Biotech-nology vol 21 no 1 pp 38ndash44 2003

[4] L Valenzuela A Chi S Beard et al ldquoGenomics metagenomicsand proteomics in biomining microorganismsrdquo BiotechnologyAdvances vol 24 no 2 pp 197ndash211 2006

[5] C A Jerez ldquoBioleaching and biomining for the industrialrecovery of metalsrdquo in Comprehensive Biotechnology M Moo-Young Ed vol 3 pp 717ndash729 Elsevier Amstaerdam TheNetherlands 2nd edition 2011

[6] P R Norris and J P Owen ldquoMineral sulphide oxidationby enrichment cultures of novel thermoacidophilic bacteriardquoFEMS Microbiology Reviews vol 11 no 1ndash3 pp 51ndash56 1993

[7] D E Rawlings and D B Johnson ldquoThe microbiology ofbiomining development and optimization ofmineral-oxidizingmicrobial consortiardquo Microbiology vol 153 no 2 pp 315ndash3242007

[8] P R Norris N P Burton and N A M Foulis ldquoAcidophiles inbioreactor mineral processingrdquo Extremophiles vol 4 no 2 pp71ndash76 2000

[9] M R Bruins S Kapil and F W Oehme ldquoMicrobial resistancetometals in the environmentrdquo Ecotoxicology and EnvironmentalSafety vol 45 no 3 pp 198ndash207 2000

[10] T J G Ettema M A Huynen W M De Vos and J Van DerOost ldquoTRASH a novel metal-binding domain predicted to beinvolved in heavy-metal sensing trafficking and resistancerdquoTrends in Biochemical Sciences vol 28 no 4 pp 170ndash173 2003

[11] M Dopson C Baker-Austin P R Koppineedi and P L BondldquoGrowth in sulfidic mineral environments metal resistancemechanisms in acidophilic micro-organismsrdquo Microbiologyvol 149 no 8 pp 1959ndash1970 2003

[12] S Franke and C Rensing ldquoAcidophiles Mechanisms to toleratemetal and acid toxicityrdquo in Physiology and Biochemistry ofExtremophiles C Gerday and N Glansdorff Eds pp 271ndash278ASM Press Washington DC USA 2007

[13] C A Navarro L H Orellana C Mauriaca and C A JerezldquoTranscriptional and functional studies of Acidithiobacillusferrooxidans genes related to survival in the presence of copperrdquoApplied and Environmental Microbiology vol 75 no 19 pp6102ndash6109 2009

[14] A Orell C A Navarro R Arancibia J C Mobarec andC A Jerez ldquoLife in blue copper resistance mechanisms ofbacteria and Archaea used in industrial biomining of mineralsrdquoBiotechnology Advances vol 28 no 6 pp 839ndash848 2010

[15] A Orell C A Navarro M Rivero J S Aguilar and C A JerezldquoInorganic polyphosphates in extremophiles and their posiblefunctionsrdquo Extremophiles vol 16 no 4 pp 573ndash583 2012

[16] C Baker-Austin M Dopson M Wexler et al ldquoExtremearsenic resistance by the acidophilic archaeon ldquoFerroplasmaacidarmanusrdquo Fer1rdquo Extremophiles vol 11 no 3 pp 425ndash4342007

[17] E Pedone S Bartolucci and G Fiorentino ldquoSensing andadapting to environmental stress the archaeal tacticrdquo Frontiersin Bioscience vol 9 pp 2909ndash2926 2004

[18] C Baker-Austin M Dopson M Wexler R G Sawers and PL Bond ldquoMolecular insight into extreme copper resistance inthe extremophilic archaeon ldquoFerroplasma acidarmanusrdquo Fer1rdquoMicrobiology vol 151 no 8 pp 2637ndash2646 2005

[19] T J G Ettema A B Brinkman P P Lamers N G Kornet WM de Vos and J van der Oost ldquoMolecular characterization ofa conserved archaeal copper resistance (cop) gene cluster andits copper-responsive regulator in Sulfolobus solfataricus P2rdquoMicrobiology vol 152 no 7 pp 1969ndash1979 2006

[20] A A Villafane Y VoskoboynikM Cuebas I Ruhl and E BinildquoResponse to excess copper in the hyperthermophile Sulfolobussolfataricus strain 982rdquo Biochemical and Biophysical ResearchCommunications vol 385 no 1 pp 67ndash71 2009

[21] K S Auernik Y Maezato P H Blum and R M KellyldquoThe genome sequence of the metal-mobilizing extremelythermoacidophilic archaeon Metallosphaera sedula providesinsights into bioleaching-associated metabolismrdquo Applied andEnvironmental Microbiology vol 74 no 3 pp 682ndash692 2008

[22] Y Maezato T Johnson S McCarthy K Dana and P BlumldquoMetal resistance and lithoautotrophy in the extreme ther-moacidophile Metalosphaera sedulardquo Journal of Bacteriologyvol 194 no 24 pp 6856ndash6863 2012

[23] D H Nies ldquoMicrobial heavy-metal resistancerdquo Applied Micro-biology and Biotechnology vol 51 no 6 pp 730ndash750 1999

[24] K Vido D Spector G Lagniel S Lopez M B Toledano andJ Labarre ldquoA proteome analysis of the cadmium response inSaccharomyces cerevisiaerdquo The Journal of Biological Chemistryvol 276 no 11 pp 8469ndash8474 2001

[25] G Bertin and D Averbeck ldquoCadmium cellular effects mod-ifications of biomolecules modulation of DNA repair andgenotoxic consequences (a review)rdquo Biochimie vol 88 no 11pp 1549ndash1559 2006

[26] M H Joe S W Jung S H Im et al ldquoGenome-wide responseof Deinococcus radiodurans on cadmium toxicityrdquo Journal ofMicrobiology and Biotechnology vol 21 no 4 pp 438ndash447 2011

[27] A Lagorce A Fourcans M Dutertre B Bouyssiere YZivanovic and F Confalonieri ldquoGenome-wide transcriptionalresponse of the archeon Thermococcus gammatolerans to cad-miumrdquo PLOS ONE vol 7 no 7 Article ID e41935 2012

[28] A Kornberg N N Rao and D Ault-Riche ldquoInorganicpolyphosphate a molecule of many functionsrdquo Annual Reviewof Biochemistry vol 68 pp 89ndash125 1999

Archaea 15

[29] J D Keasling ldquoRegulation of intracellular toxic metals andother cations by hydrolysis of polyphosphaterdquoAnnals of theNewYork Academy of Sciences vol 829 pp 242ndash249 1997

[30] F Remonsellez A Orell and C A Jerez ldquoCopper tolerance ofthe thermoacidophilic archaeon Sulfolobus metallicus possiblerole of polyphosphate metabolismrdquoMicrobiology vol 152 no 1pp 59ndash66 2006

[31] V Hatzimanikatis L H Choe and K H Lee ldquoProteomicstheoretical and experimental considerationsrdquo BiotechnologyProgress vol 15 no 3 pp 312ndash318 1999

[32] L H Choe and K H Lee ldquoA comparison of three commerciallyavailable isoelectric focusing units for proteome analysis theMultiphor the IPGphor and the Protean IEF cellrdquo Electrophore-sis vol 21 no 5 pp 993ndash1000 2000

[33] U K Laemmli ldquoCleavage of structural proteins during theassembly of the head of bacteriophage T4rdquo Nature vol 227 no5259 pp 680ndash685 1970

[34] A Shevchenko M Wilm O Vorm and M Mann ldquoMassspectrometric sequencing of proteins from silver-stained poly-acrylamide gelsrdquo Analytical Chemistry vol 68 no 5 pp 850ndash858 1996

[35] P Giavalisco E Nordhoff T Kreitler et al ldquoProteome analysisof Arabidopsis thaliana by two-dimensional gel electrophoresisand matrix-assisted laser desorptionionisation-time of flightmass spectrometryrdquo Proteomics vol 5 no 7 pp 1902ndash19132005

[36] T M Rose E R Schultz J G Henikoff S Pietrokovski CM McCallum and S Henikoff ldquoConsensus-degenerate hybridoligonucleotide primers for amplification of distantly relatedsequencesrdquo Nucleic Acids Research vol 26 no 7 pp 1628ndash16351998

[37] T M Rose J G Henikoff and S Henikoff ldquoCODEHOP(Consensus-Degenerate Hybrid Oligonucleotide Primer) PCRprimer designrdquo Nucleic Acids Research vol 31 no 13 pp 3763ndash3766 2003

[38] J P Acevedo F Reyes L P Parra O Salazar B A Andrews andJ A Asenjo ldquoCloning of complete genes for novel hydrolyticenzymes from Antarctic sea water bacteria by use of animproved genomewalking techniquerdquo Journal of Biotechnologyvol 133 no 3 pp 277ndash286 2008

[39] S Bathe and P R Norris ldquoFerrous iron- and sulfur-inducedgenes in Sulfolobus metallicusrdquo Applied and EnvironmentalMicrobiology vol 73 no 8 pp 2491ndash2497 2007

[40] J Sambrook E F Fritsch and TManiatisMolecular Cloning ALaboratory Manual Cold Spring Harbor Press New York NYUSA 1989

[41] K W Miller S Sass Risanico and J B Risatti ldquoDifferentialtolerance of Sulfolobus strains to transition metalsrdquo FEMSMicrobiology Letters vol 93 no 1 pp 69ndash73 1992

[42] G Huber C Spinnler A Gambacorta and K O Stetter ldquoMet-allosphaera sedula gen and sp nov represents a new genus ofaerobic metal-mobilizing thermoacidophilic archaebacteriardquoSystematic and Applied Microbiology vol 12 pp 38ndash47 1989

[43] J Llanos C Capasso E Parisi D Prieur and C JeanthonldquoSusceptibility to heavy metals and cadmium accumulation inaerobic and anaerobic thermophilic microorganisms isolatedfrom deep-sea hydrothermal ventsrdquo Current Microbiology vol41 no 3 pp 201ndash205 2000

[44] A Kaur M Pan M Meislin M T Facciotti R El-Gewelyand N S Baliga ldquoA systems view of haloarchaeal strategies towithstand stress from transition metalsrdquo Genome Research vol16 no 7 pp 841ndash854 2006

[45] M Solioz and J V Stoyanov ldquoCopper homeostasis in Entero-coccus hiraerdquo FEMS Microbiology Reviews vol 27 no 2-3 pp183ndash195 2003

[46] S Sitthisak K Howieson C Amezola and R K JayaswalldquoCharacterization of a multicopper oxidase gene from Staphy-lococcus aureusrdquo Applied and Environmental Microbiology vol71 no 9 pp 5650ndash5653 2005

[47] L Rodrıguez-Montelongo S I Volentini R N Farıas E MMassa and V A Rapisarda ldquoThe Cu(II)-reductase NADHdehydrogenase-2 of Escherichia coli improves the bacterialgrowth in extreme copper concentrations and increases theresistance to the damage caused by copper and hydroperoxiderdquoArchives of Biochemistry and Biophysics vol 451 no 1 pp 1ndash72006

[48] E Williams T M Lowe J Savas and J DiRuggiero ldquoMicroar-ray analysis of the hyperthermophilic archaeon Pyrococcusfuriosus exposed to gamma irradiationrdquo Extremophiles vol 11no 1 pp 19ndash29 2007

[49] L M Gaetke and C K Chow ldquoCopper toxicity oxidative stressand antioxidant nutrientsrdquo Toxicology vol 189 no 1-2 pp 147ndash163 2003

[50] M J Davies ldquoThe oxidative environment and protein damagerdquoBiochimica et Biophysica Acta vol 1703 no 2 pp 93ndash109 2005

[51] J R Thompson J K Bell J Bratt G A Grant and LJ Banaszak ldquoVmax regulation through domain and subunitchanges The active form of phosphoglycerate dehydrogenaserdquoBiochemistry vol 44 no 15 pp 5763ndash5773 2005

[52] V Consalvi R Chiaraluce L Politi A Gambacorta MDe Rosa and R Scandurra ldquoGlutamate dehydrogenase fromthe thermoacidophilic archaebacterium Sulfolobus solfataricusrdquoEuropean Journal of Biochemistry vol 196 no 2 pp 459ndash4671991

[53] A J L Macario M Lange B K Ahring and E Conway DeMacario ldquoStress genes and proteins in the archaeardquo Microbiol-ogy and Molecular Biology Reviews vol 63 no 4 pp 923ndash9671999

[54] J AMaupin-FurlowM A Gil M A Humbard et al ldquoArchaealproteasomes and other regulatory proteasesrdquo Current Opinionin Microbiology vol 8 no 6 pp 720ndash728 2005

[55] N Sun F Beck R Wilhelm Knispel et al ldquoProteomics analysisof Thermoplasma acidophilum with a focus on protein com-plexesrdquoMolecular and Cellular Proteomics vol 6 no 3 pp 492ndash502 2007

[56] L S Madding J K Michel K R Shockley et al ldquoRole of the 1205731subunit in the function and stability of the 20S proteasome inthe hyperthermophilic Archaeon Pyrococcus furiosusrdquo Journalof Bacteriology vol 189 no 2 pp 583ndash590 2007

[57] R Shibata Y Bessho A Shinkai et al ldquoCrystal structureand RNA-binding analysis of the archaeal transcription factorNusArdquo Biochemical and Biophysical Research Communicationsvol 355 no 1 pp 122ndash128 2007

[58] W Bae B Xia M Inouye and K Severinov ldquoEscherichia coliCspA-family RNA chaperones are transcription antitermina-torsrdquo Proceedings of the National Academy of Sciences of theUnited States of America vol 97 no 14 pp 7784ndash7789 2000

[59] R J Ram N C VerBerkmoes M P Thelen et al ldquoMicrobi-ology community proteomics of a natural microbial biofilmrdquoScience vol 308 no 5730 pp 1915ndash1920 2005

[60] I Noel-Georis T Vallaeys R Chauvaux et al ldquoGlobal analysisof the Ralstonia metallidurans proteome prelude for the large-scale study of heavy metal responserdquo Proteomics vol 4 no 1pp 151ndash179 2004

16 Archaea

[61] M TMNovo A CDa Silva RMoreto et al ldquoThiobacillus fer-rooxidans response to copper and other heavy metals growthprotein synthesis and protein phosphorylationrdquo Antonie vanLeeuwenhoek vol 77 no 2 pp 187ndash195 2000

[62] Q She R K Singh F Confalonieri et al ldquoThe complete genomeof the crenarchaeon Sulfolobus solfataricus P2rdquo Proceedings ofthe National Academy of Sciences of the United States of Americavol 98 no 14 pp 7835ndash7840 2001

[63] R P Anderson and J R Roth ldquoTandem genetic duplications inphage and bacteriardquo Annual Review of Microbiology vol 31 pp473ndash505 1977

[64] D Gevers K Vandepoele C Simillion and Y Van De PeerldquoGene duplication and biased functional retention of paralogsin bacterial genomesrdquo Trends in Microbiology vol 12 no 4 pp148ndash154 2004

[65] A B Reams and E L Neidle ldquoSelection for gene clustering bytandemduplicationrdquoAnnual Review ofMicrobiology vol 58 pp119ndash142 2004

[66] L H Orellana and C A Jerez ldquoA genomic island providesAcidithiobacillus ferrooxidans ATCC 53993 additional copperresistance a possible competitive advantagerdquo Applied Microbi-ology and Biotechnology vol 92 no 4 pp 761ndash767 2011

[67] A B Brinkman T J G Ettema W M de Vos and J van derOost ldquoThe Lrp family of transcriptional regulatorsrdquo MolecularMicrobiology vol 48 no 2 pp 287ndash294 2003

[68] C Vollmecke S L Drees J Reimann S V Albers and MLubben ldquoThe ATPases CopA and CopB both contribute tocopper resistance of the thermoacidophilic archaeon SulfolobussolfataricusrdquoMicrobiology vol 158 no 6 pp 1622ndash1633 2012

[69] A Villafane Y Voskoboynik I Ruhl et al ldquoCopR of Sulfolobussolfataricus represents a novel class of archaeal-specific copper-responsive activators of transcriptionrdquo Microbiology vol 157no 10 pp 2808ndash2817 2011

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Anatomy Research International

PeptidesInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporation httpwwwhindawicom

International Journal of

Volume 2014

Zoology

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Molecular Biology International

GenomicsInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioinformaticsAdvances in

Marine BiologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Signal TransductionJournal of

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BioMed Research International

Evolutionary BiologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Biochemistry Research International

ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Genetics Research International

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Advances in

Virolog y

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Nucleic AcidsJournal of

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Enzyme Research

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

International Journal of

Microbiology

Archaea 7

13 13

11

13

1

Control Copper Cadmium

(a)

99 9

333

(b)

3030

30

2020

20 212121

(c)

Figure 3 Comparison of selected proteins of S metallicus cells treated with Cu or Cd Protein extracts were obtained from cells treatedwithout metals and with 100mM Cu or 1mM Cd for 24 h and the proteins were separated by 2D-PAGE The segments (a) (b) and (c) arethe enlarged dashed boxes in Figure 2 under the three conditions indicated Numbers show some of the spots with increased intensity in cellstreated with Cu or Cd

in the absence of Cu (Figure 2(a)) Therefore these resultsshow that a number of proteins became non-detectable ordecreased their levels when S metallicus faced Cu This kindof response was also seen when the microorganism wasexposed to Cd (data not shown) This behavior has alsobeen observed in similar studies where F acidarmanus waschallenged by either As(III) or Cu [16 18] In addition theexpression of 30 other proteins was found to be upregulatedafter Cu treatment (100 or 200mM) (Figure 2(b)) Most ofthese proteins could be identified only in the condition withCu and spots 5 13 and 15 were induced 26 34 and 55-fold respectively compared to cells without Cu treatmentMoreover 3 proteins of lowmolecular weight (spots 31 3 and33) that increased their expression when cells were exposedto 100mM Cu were also identified (Figure 2 bottom panels)These proteins were only detected when 15 polyacrylamidewas used during the second-dimensional separation (SDS-PAGE) On the other hand a large decrease in overallexpression of proteins after 1mMCd treatment was observed(not shown) Interestingly 10 out of the 13 proteins identifiedas up-regulated when cells were exposed to Cd and alsoshowed increased levels after Cu treatment Some of them areshown in Figure 3

33 Identification of Proteins Upregulated in Cells Treated withCu and Cd in S metallicus A total of 18 S metallicus proteinswhose levels were found to be up-regulated in responseto Cu were analyzed by mass spectrometry The identifiedproteins included functions related to the production andtransport of energy biosynthesis of amino acids stressresponses and transcription regulation (Table 2) Amongstthe proteins related to production and transport of energya putative ATP synthase subunit B (spot 23) that has beendescribed as playing a fundamental role in ATP synthesiswas identified (Table 2) When cells are subjected to somestressing conditions such as the presence of heavy metalsa greater cellular demand for energy has been reported tooccur [18] Most likely this phenomenon might be due toATP-driven Cu transport via ATPases that have a substantialinterplay during metal detoxification [45] Furthermore theATP synthase subunit B was also identified as up-regulatedwhen S metallicus was treated with Cd (Table 2)

Two other proteins corresponded to putative oxidoreduc-tases such as ferredoxin oxidoreductase (spot 3) and alcoholdehydrogenase (spot 4) that have been generally involvedin electron transporter chains and use NAD+ as an electronacceptor The levels of ferredoxin oxidoreductase were also

8 Archaea

Table 2 Proteins upregulated in S metallicus cells exposed to 100mM Cu or 1mM Cd

Spot Molecular weight (kDa) Cu induction levels Putative function Microorganism related Accesion number1dagger 266 infin Proteasome subunit S solfataricus AAK410343dagger 701 infin Ferredoxin oxidoreductase S solfataricus AAK429264dagger 429 infin Alcohol dehydrogenase S solfataricus AAK431545 35 26 Hypothetical protein S acidocaldarius YP 2549766 27 infin Hypothetical protein P aerophilum NP 5598899dagger 57 infin Phosphoglycerate dehydrogenase M thermautotrophicus AAB8546613dagger 27 34 Unknown mdash mdash14 28 infin Unknown mdash mdash15 35 55 Unknown mdash mdash20dagger 461 infin Glutamate dehydrogenase S solfataricus AAK4223021dagger 45 infin Hypothetical protein M acetivorans NP 618380122 49 infin Hypothetical protein P torridus YP 02280723dagger 51 infin ATP synthase subunit B S solfataricus AAK4088024 585 infin HSP60 subunit S shibatae AAG37273130dagger 48 infin Unknown mdash mdash31 16 ND Unknown mdash mdash32 144 ND Putative transcription regulator S solfataricus AAK4041333 138 ND Transcription factor nusA S solfataricus AAK40563Spots refer to those numbered in Figure 2 Proteins in Figure 2 which are not included in this table were not subjected to sequencing due to their low intensitiesin the gelsdaggerSpot up-regulated in cells exposed to Cu and CdND not determinedInfinity symbol indicates that proteins were expressed only in presence of copper or cadmiumM thermoautotrophicus Methanobacterium thermoautotrophicus M acetivorans Methanosarcina acetivorans S shibatae Sulfolobus shibatae P torridusPicrophilus torridus P aerophilum Pyrobaculum aerophilum S acidocaldarius Sulfolobus acidocaldarius

up-regulated in response to Cd (Table 2) Several previ-ous reports have suggested that oxidoreductases contributeto an oxidative protection in both Bacteria and Archaeain response to heavy metals [46ndash48] In the neutrophilicmicroorganisms Escherichia coli and Staphylococcus aureusit has been described that oxidases and dehydrogenasescontribute to oxidative protection Cu homeostasis and stressresponses [46 47] Furthermore some proteins involved inoxidative damage repair such as NADH-dependent oxidasesand thioredoxin reductases were expressed in cells of Facidarmanus exposed to As(III) [16] The expression of thisgroup of proteins has also been observed when the samemicroorganism was exposed to Cu [18] Therefore oxidore-ductases play an important role against oxidative stress andmay eliminate reactive oxygen species which constitute themajor component of the stress caused by transition metals[44 49 50]

The enzymes related to biosynthesis of amino acids werefound as commonly up-regulated either in response to Cu orCdThey corresponded to a phosphoglycerate dehydrogenase(spot 9) and a glutamate dehydrogenase (spot 20) Theformer one catalyzes the NAD+-dependent oxidation of 3-phosphoglycerate into 3-phosphohydroxypyruvate a branchpoint from the glycolytic pathway and the initial reactionin L-serine biosynthesis [51] Glutamate dehydrogenase cat-alyzes the oxidative deamination of glutamate to produce 2-oxoglutarate and ammonia with reduction of NAD+ [52]Proteins involved in amino acids synthesis were expressed

in cells of F acidarmanus exposed to As(III) [16] Moreoverincreased levels of proteins involved in the biosynthesisof sulfur-containing amino acids have been observed inSaccharomyces cerevisiae cells exposed to Cd [24] suggestingthat these proteins might also have a role in the cellularresponse to adverse conditions

Two proteins showed identity to components of the stressresponse mechanism such as one subunit of the HSP60chaperonin (spot 24) and one subunit of the proteasome (spot1) In Archaea the stress protein HSP60 is directly involvedin protein folding processes [53] Proteomic analysis of Facidarmanus cells exposed to As(III) and Cd revealed theexpression of proteins involved in protein folding and DNArepair including HSP60 chaperonin and DnaK heat-shockprotein (HSP70) thereby the authors suggested that thismicroorganismusesmultiplemechanisms to resist high levelsof Cu [16 18] On the other hand proteasomes are describedas nonspecific proteolytic nanomachines associated withprotein catabolism Proteasomes are known to be closelyinvolved in maintaining protein quality control by degradingmiss folded and denatured proteins in response to cell stressin all three domains of life [54] A proteomic analysis inthe thermoacidophilic archaeon Thermoplasma acidophilumshowed that proteasomes and chaperone-related proteinswere highly induced against stress conditions indicatinga high turnover rate of proteins [55] In P furiosus theexpression of the 1205731 subunit of the proteasome was inducedin cells subjected to heat shock stress [56] Additionally the

Archaea 9

150pb

(a)

1016 pb

(b)

2000 pb

(c)

Figure 4 CODEHOP-based PCR for the amplification of the putative genes copA and copM from S metallicus Amplification products(a) using degenerate PCR primers (DOP) copM degF and copM degR designed from amino acid sequence conserved blocks of SulfolobalesCopM proteins (b) using primers copA cdegF and copA cdegR designed by CODEHOP strategy for amplification of a copA-like gene (c)Primers copM degF and copA cdegRwere assesed for the amplification of a copMA-likeDNA regionThose primerswere testedwith genomicS metallicus DNA samples Amplicons expected sizes were as follows ca 150 bp for copM ca 950 bp for copA and ca 2000 bp for copMAPCR products were excised and cloned for later sequencing

proteasome subunit was also found to be up-regulated whenS metallicus was subjected to Cd stress (Table 2)

The transcriptional regulation-related proteins corre-sponded to a putative transcription regulator (spot 32) andthe transcription factor NusA (spot 33) involved in theintrinsic termination of transcription [57] NusA has beenalso reported to display important functions in the cellularresponse to adverse factors [58] The expression of twotranscriptional regulators related to amino acids biosynthesisand metal-dependent genetic repression were found to beinduced in P furiosus cells subjected to oxidative stress[48] Moreover the expression of several genes encodingpredicted transcriptional regulators were induced followingCd exposure in T gammatolerans cells [27]

Spots 5 6 13 14 15 21 22 30 and 31 (Table 2) did notshow significant scores with any protein sequences currentlyavailable in the data bases Interestingly spots 2 4 9 13 21and 30 were found to be commonly upregulated when Smetallicus cells were treated with either Cu or Cd (Figure 3Table 2)

The results presented here are consistent with previ-ous work describing global protein expression profiles inresponse to heavy metals as reported for F acidarmanus [1618] microbial-biofilm communities in an acid mine drainagesite [59] and other prokaryotic and eukaryotic microorgan-isms [24 60]Therefore a coordinated expression of differentgroups of genes suggests the existence of regulatory networkssuch as stress response mechanisms and respiratory chainadjustments to cope with the presence of heavy metal ions[61]

34 Cu-Resistance (Cop) Genes Cluster Is Duplicated in Smetallicus Genome A Cu-resistance (cop) locus has beendescribed to be highly conserved in archaeal genomeswhich consists of genes encoding a new type of archaealtranscriptional regulator (CopT) a metal-binding chaperone(CopM) and a Cu-transporting P-type ATPase (CopA) [19]Since the genome sequence of Smetallicus is not yet availablewe searched for the presence of cop genes by using consen-sus degenerate hybrid oligonucleotide primers-based PCR(CODEHOP-PCR) CopA and CopM amino acid sequencealignments from S acidocaldarius S tokodaii and S solfa-taricus were used to identify conserved sequence blocks andtherefore to design either CODEHOP or DOP (degeneratehybrid oligonucleotide) primers (Figure S1 Table 1) TherebyPCR assays using S metallicus genomic DNA as templateyielded amplicons of ca 150 bp and ca 950 bp for copMand copA CODEHOP pair primers respectively (Figures4(a) 4(b)) As estimated from primer pairrsquos position onamino acid sequence alignments the obtained PCR productscorresponded to the expected sizes suggesting the presenceof copM- and copA-like genes in S metallicus genome Inorder to determine the identity of the amplified DNA frag-ments they were TA-cloned and sequenced Blastx sequenceanalysis showed that the 150 bp DNA fragment coded for anincomplete ORF sharing 63 homology with S solfataricusCopM (SSO10823) whereas the analysis of the 950 pb DNAfragment yielded a partial ORF sharing 54 homology withS solfataricus CopA protein (SSO2651)

The genomic organization of the putative cop genes in Smetallicus was analyzed to find out its similarity with those

10 Archaea

Table 3 Sequence identity comparison of S metallicus Cop proteins to those in other Sulfolobales

S metallicus protein Identity to homologue in other SulfolobalesS solfataricus P2 S acidocaldarius S tokodaii M sedula

CopA1 51 43 44 45CopA2 88 46 49 50CopM1 63 69 71 71CopM2 89 72 70 77CopT1 36 27 30 32CopT2lowast 90 52 52 53Boldface type indicates to which organism the S metallicus homologue shows the highest sequence identitylowastRefers to the analysis obtained using the uncompleted amino acid sequence of CopT2 which represents sim90 in length when compared with S solfataricusCopT sequence

previously described in S solfataricus P2 and F acidarmanuswhere the cop cluster consists of tandem-orientated genesas copTMA [18 19] Thus using copM degF (forward) andcopA cdegR (reverse) primers we attempted to amplify aputative copMA DNA region This PCR yielded a product ofca 2000 pb corresponding to the expected size and resultedin 2 ORFs showing high sequence homology with CopMand CopA from S solfataricus (Figure 4(c)) Interestinglywhen aligning this copMA sequence with the copA and copMsequences obtained previously they were not identical whichstrongly indicated the presence of duplicated putative copgenes encoding for both Cu-ATPases (copA1 and copA2) andmetallochaperones (copM1 and copM2) in the S metallicusgenome

To isolate the entire cop gene sequences the genomewalking method described by Acevedo et al [38] was usedSix different DNA libraries were constructed from S metalli-cus genomic DNA digested with several restriction enzymesincluding 51015840 overhang and blunt end restriction enzymesThe DNA libraries were used as templates in 2 successivePCR reactions From the previously known sequences twospecific sense primers for the 31015840 end amplification and twospecific antisense primers for the 51015840 end amplification weredesigned (abbreviated SP1 and SP2) Therefore the 51015840 and 31015840ends of the putative genes copA1 and copA2 that had beenpartially identified through the genome walking techniquewere amplified

By overlapping the sequence isolated by degenerate PCRand the lateral sequences obtained by genome walking(Figure S2) it was possible to complete the whole nucleotidesequence of the putative gene copA1 Additionally it waspossible to confirm the presence of the upstream copM1 geneanddetermine the occurrence of a partial copT1 gene tandem-orientated upstream of copM1 (Figure 5)Through additionalgenome walking experiments the partial copT1 sequencecould be further completedThe samewas truewhen isolatingthe whole nucleotide sequence of the putative gene copA2as the presence of copM2 and copT2 genes were confirmedupstream of copA2 However it was not possible to completethe whole 51015840 end of the putative gene copT2 as its determinedlength corresponded to only 90 of the full length whencompared with S solfataricus copT gene (Figure 5)

In conclusion degenerate PCR together with genomewalking experiments allowed to describe the occurrence inS metallicus genome of 2 cop genes loci (named as locus cop1and locus cop2) coding for paralogous genes whose productsmay be involved in Cu-resistance (Figure 5) Although somearchaeal genomes exhibit two copies of putative Cu-P-typeATPases as described for S solfataricus [62] the genomicarrangement displayed as a cop gene cluster (copTMA) hasbeen reported to be represented in only one copy for manyarchaeal genomes [10 19 21] Moreover we further updatedthis analysis by searching for duplications of the cop genescluster in all available archaeal genome sequences up to date(October 2012) retrieving only one cop locus in each caseThereby we propose that the discovery of this cop gene clusterduplication in the genome of S metallicus constitutes so faran unprecedented feature for a representative of the Archaeadomain and might contribute to its high metal resistance

In this context it has been widely reported that increasedgene copy number can increase gene expression allowingprokaryotic to thrive under growth limiting conditions [1363ndash68] However alongside the augmented gene copy onecannot exclude that the cop operon duplication in S metal-licus may offer among others a wider repertoire of Cu-ATPases which might differ in terms of metal affinitiesandor specificity efflux rates andor differences in theirabundance regulation

Throughout amino acid sequence analysis it was furtherdetermined that each polypeptide encoded by S metallicuswith the exception of CopT1 showed to be highly homol-ogous to their orthologous counterparts encoded by othersSulfolobales (Table 3) Moreover the 3 ORFs encoded by thelocus cop2 shared about 90 identity with the correspondingS solfataricus orthologous peptides (Table 3) S metallicuscop genes products showed characteristic domains referredto as critical for their respective proposed biological activitiesS metallicus paralogous gene products CopA1 (749 aa1015840)and CopA2 (747 aa1015840) shared 513 of identity These twoputative Cu-ATPases (CopA1 and CopA2) contain the aminoacid sequence motif CPCALGLA which has been proposedto confer Cu-transporting specificity in CPx-ATPases [48]and has been also found in other CopA-like proteins frombiomining Bacteria and Archaea [14]

Archaea 11

HindIII HindIII HindIII

HindIII

CopT1Locus cop1 M1

EcoRIEcoRV

513 bp 219 bp 2250 bp

CopA1

(a)

Locus cop2 M2

EcoRI

CopA2

453 bp 171 bp 2244 bp

CopT2

PstIHindIII HindIII HindIII

(b)

Figure 5 Schematic representation of the two cop gene clusters isolated from S metallicus genome Each cluster codes for anArchaea-specifictranscriptional regulator (copT) a metallochaperone (copM) and a P-type Cu-exporting ATPase (copA) Lengths of each gene are indicatedLocus cop1 corresponds to 4346 sequenced base pairs and shows an intergenic region copT1-M1 of 232 base pairs Locus cop2 correspondsto 3435 base pairs and shows 89 base pairs copT2-M2 intergenic region Genes copM2 and copA2 overlapped in 11 base pairs copT2 was notfully isolated

Control CdSO4 Ag2SO4 NiSO4 ZnSO4 CuSO4

kb3kb2kb1

02 kb

(a)

kb3kb2kb1

02 kb

(b)

(c)

Figure 6 Northern blot analysis to determine the expression of S metallicus copA1 (a) and copA2 (b) putative genes in response to variousmetals S metallicus total RNAwas extracted from cultures grown either in the absence (control) or the presence of CuSO

4(50mM) Ag

2SO4

(008mM) NiSO4(15mM) ZnSO

4(50mM) and CdSO

4(2mM) P32-radioactivelly labelled DNA fragments annealing to copA1 and copA2

sequences respectively were used as probes (c) shows rRNAs as total RNA loading control

On the other hand CopM1 (71 aa) and CopM2 (56 aa)sharing 66 of identity present the proposed metal bindingdomain TRASH (trafficking resistance and sensing of heavymetals) [10] Additionally when analyzing the putative tran-scriptional regulators the presence of a C-terminal TRASHdomain in both CopT1 and CopT2 was also found CopT1sequence also contains an entire N-terminal helix-turn-helix (HTH) motif (not shown) that resembles the DNA-bindingmotifs of prokaryotic transcriptional regulators suchas Lrp-like proteins [67] The HTH motif was only partiallyidentified in CopT2 since the 51015840 region of copT1 gene has notbeen yet fully isolated

35 Transcriptional Analysis of S metallicus Cop Genes Toget insight into the role of the two cop loci the expressionof the corresponding genes was analyzed under variousconditions S metallicus was first grown in presence ofdifferent heavy metals (Cu Zn Cd Ni o Ag) at a givenconcentration that did not affect growth kinetics [30 42]Subsequently total RNA was isolated from late exponentiallygrown cultures and Northern blot experiments were carriedout in order to determine the expression of both copA1 andcopA2 genes As depicted in Figure 6 transcription of bothcopA1 and copA2messengers were specifically induced in thepresence of Cu and Cd ions This gene expression pattern

12 Archaea

954pb

M1 CopA1

(a)

1438 pb

M2 CopA2

(b)

1000 pb

800pb

+ minus

(c)

+ minus

1500 pb

1000 pb

(d)

Figure 7 Cotranscription analysis of copMA1 and copMA2 genes cDNA was synthesized with a reverse primer (dotted arrows) hybridizingtoward the 31015840 end of either copA1 (a) or copA2 (b) S metallicus total RNA was extracted at the late exponential phase from a culture growingin presence of 20mMCu PCR amplifications were carried out with these cDNAs and each corresponding primer pair (black arrows) as listedin Table 1 (c) and (d) show RT-PCR products obtained for the copMA1 and copMA2 intergenic regions respectively Reverse transcriptasereactions with (+) and without (minus) the Improm II reverse transcriptase enzyme were carried out in order to exclude amplification due togenomic DNA contamination Expected sizes (in base pairs) for the corresponding PCR products are given in (a) and (b)

is in good agreement with what has been described for Ssolfataricus where the bicistronic copMA transcript levelswere also found to be up-regulated in response to both Cuand Cd [19]

Northern blot analysis showed that the sizes seen for bothcopA transcripts (sim25 Kb) did not correspond exactly withthe individual genes length (each of about 225Kb) (Figure 6)This strongly suggested that the transcripts could also includethe respective copM gene located upstream of copA in eachcop loci To confirm this assumption a co-transcriptionexperiment was done (Figure 7) in which the cDNAs wereobtained by using RNA extracted from a culture grownin the presence of 20mM Cu and using a reverse primerhybridizing with copA1 (Figure 7(a)) or copA2 (Figure 7(b))gene sequence respectively PCR amplifications were carriedout by using the corresponding cDNAs as templates and eachpair of primers lying in adjacent genes (copM) The presenceof an amplicon of the expected size in each case indicatedthe adjacent genes were part of polycistronic messengers(Figure 7) These results clearly show that in S metallicuseach couple of copMA genes was expressed in the form oftranscriptional units as reported in F acidarmanus and Ssolfataricus [18 19] The coexpression of gene pairrsquos copMAmay suggest a coordinated and dependent function for therespective encoded proteins In the bacterium E hirae themetallochaperone CopZ (CopM in S metallicus) fulfills apivotal role in the mechanism of Cu homeostasis [45] Thusit was shown that this protein interacts directly with the Cu-ATPase CopB (CopA in S metallicus) handing the Cu forsubsequent removal In this context one might expect thatproteins CopM1 and CopM2 from S metallicus have a similarrole to that described for E hirae Furthermore quantitativeRT-PCR experiments were carried out in order to determinedifferential expression of the cop genes in response to differentCu concentrations The expression of copA1 copA2 copT1and copT2 was tested relative to the transcript levels ofrRNA 16S gene since its levels were not significantly affected

in all assessed conditions (data not shown) As shown inFigure 8(a) copA2 and copA1 transcript levels were foundto be concomitantly increased with the increasing Cu con-centrations present in the medium Higher transcripts levelsof copA1 were seen in all tested conditions when comparedwith copA2 levels copA1 transcript levels were found to be325-fold up-regulated when comparing 50mMCu conditionversuscontrol (absence of metal) whereas copA2 transcriptlevels showed an increment of only 175-fold (Figure 8(a))The finding that Cu-ATPases mRNA levels were significantlyincreased in response to Cu ions exposure suggests that thetransport system may operate for Cu efflux in S metallicus

Moreover copA2 and copA1 gene expression was quan-tified when S metallicus was grown using chalcopyrite(CuFeS

2) ore as an oxidizable substrate (Figure 8(a)) Mineral

oxidation mediated by the microorganism generates a pro-gressive increase in Cu ions concentration in the medium Tofind out whether the amount of solubilized Cu induced theexpression of the Cu-ATPases genes total RNAwas extractedfrom a S metallicus culture grown to late exponential phaseand in the presence of 1 CuFeS

2 It was clear that an

up-regulation of the Cu-ATPases encoding genes also tookplace when S metallicus was grown in the presence ofCuFeS

2(Figure 8(a)) copA1 transcript levels increased 14-fold

compared with the control condition in the absence of Cuwhile copA2 gene expression showed an increase of 76 foldAlong with this bymeans of atomic absorption spectrometry(AAS) analysis an overall amount of solubilized Cu ions(Cu2+Cu1+) of 144plusmn21mMwas determined to be present inthemedium indicating that copA2 and copA1 gene expressionwas most likely in response to the Cu present in the culturedue to CuFeS

2microbial-solubilization

The finding that copA1 was highly expressed comparedwith copA2 in all tested conditions may suggest a possiblephysiological hierarchy between the two ATPases whenovercoming either Cu or Cd stress In this regard by meansof a genetic approach it was demonstrated in S solfataricus

Archaea 13

700

600

500

400

300

200

100

00

5 20 50 CuFeS2CuSO4 concentration (mM)

Relativ

egenee

xpression

CopA2CopA1

(a)

8

6

7

5

4

3

2

1

00

5 20 50 CuFeS2CuSO4 concentration (mM)

Relativ

egenee

xpression

CopT1CopT2

(b)

Figure 8 Relative expression levels of S metallicus genes copA1 and copA2 (a) copT1 and copT2 (b) Expression of S metallicus cop geneswasassessed by qRT-PCR and normalized against 16S rRNA gene expression in individual cultures Mean values and standard deviations arefrom analyses of three independent cultures grown in the presence of 0 5 20 50mMCu or in 1 of a chalcopyrite ore (CuFeS

2) concentrate

that while CopA was an effective Cu efflux transporterat low and high Cu concentrations the other Cu-ATPase(CopB) only appeared to be a low-affinity Cu export ATPase[68] Moreover by using a M sedula copA deletion mutantit was demonstrated that this strain compromised metalresistance and consequently abolished chalcopyrite oxidation[22] highlighting the role of Cu detoxification mechanismsduring a given bioleaching process Our attempts to showthe functionality of CopA1 and CopA2 from S metallicusby using E coli as a heterologous host were not successfulApparently the overexpression of these ORFs had a toxiceffect on E coli that compromised its viability Althoughgene disruption tools have not been yet developed for Smetallicus the functional role of both copTMA locimight befurther studied by cross-species complementation of a coppersensitive S solfataricus copR mutant as it was described byMaezato et al [22] It will be of great interest to establishin future studies the possible functionality of the isolatedputative transporters from S metallicus

Likewise quantitative RT-PCR experiments showed thatcopT1 transcript levels increased concomitantly with increas-ing Cu concentrations whereas copT2 showed relativelysimilar transcripts levels most likely indicating a constitutiveexpression profile (Figure 8(b)) Furthermore whereas copT1transcript levels were found to be increased 36 fold inCuFeS

2

grown cultures copT2 levels remained unchanged in com-parison with the control in the absence of Cu (Figure 8(b))The results obtained for S metallicus copT2 gene expressionare consistent with those reported for S solfataricus CopTtranscriptional regulator has been proposed to function as arepressor in S solfataricus showing a constitutive expressionthat in the presence of Cu loses its affinity for the promoterregion of copMA allowing the expression of this polycistron[19] In contrast the increased transcripts levels of both copA1and copT1 concomitant with higher Cu concentrations in the

environment suggest that the putative transcriptional regula-tor CopT1 may act by activating both copMA1 and probablyitself (Figure 8) Regarding this it was recently reported thatCopR (CopT in S metallicus) from S solfataricus strain 982acts as an activator of copT (copM in S metallicus) and copAexpression [69] Nevertheless additional experiments wouldbe required to test this possibility in S metallicus

4 Concluding Remarks

We previously reported that S metallicus resists extremelyhigh Cu concentrations which was mediated to some extentby the use of a possible metal resistance system based oninorganic polyphosphate hydrolysis and consequently Cu-PO4

2minusefflux [15 29 30] Here we have addressed the questionwhether this microorganism coded for other determinantsthat might help to explain its high heavy metal toleranceAs the genomic sequence of this microorganism is notyet available we jointly used CODEHOP-PCR and genomewalking approaches and were able to establish the occurrenceof two nonidentical homologous cop loci into the genome ofS metallicus (cop1 and cop2) Each cop locus codes for anarchaeal transcriptional regulator (CopT) a metal-bindingchaperone (CopM) and a Cu-transporting P-type ATPase(CopA) High levels of the polycistronic mRNAscopMA ofeach cop locus were observed after treatment with either Cuor Cd suggesting that the encoded ATPases efflux heavymetals out in order to detoxify the intracellular environmentAltogether previous reports and the results obtained in thisstudy allow us to suggest that some key elements that mayexplain the high resistance to Cu in S metallicus is the dupli-cation of the Cu resistance cop genes a defensive responseto stress and a polyP-based accumulation mechanism Inthe case of Cd although some similar stress responses were

14 Archaea

observed whether comparable Cu-responsive elements alsoparticipate in Cd responses remains to be seen

Conflict of Interests

The authors declare that the research was done in the absenceof any commercial or financial relationships that could beconstrued as a potential conflict of interests

Acknowledgments

This work was supported by Grant no FONDECYT 1110214and in part by ICM P-05-001-F project

References

[1] A Schippers ldquoMicroorganisms involved in bioleaching andnucleic acid-based molecular methods for their identificationand quantificationrdquo in Microbial Processing of Metal SulfidesR E Donati and W Sand Eds pp 3ndash33 Elsevier BerlinGermany 2007

[2] H R Watling ldquoThe bioleaching of sulphide minerals withemphasis on copper sulphides a reviewrdquo Hydrometallurgy vol84 no 1-2 pp 81ndash108 2006

[3] D E Rawlings D Dew and C Du Plessis ldquoBiomineralizationof metal-containing ores and concentratesrdquo Trends in Biotech-nology vol 21 no 1 pp 38ndash44 2003

[4] L Valenzuela A Chi S Beard et al ldquoGenomics metagenomicsand proteomics in biomining microorganismsrdquo BiotechnologyAdvances vol 24 no 2 pp 197ndash211 2006

[5] C A Jerez ldquoBioleaching and biomining for the industrialrecovery of metalsrdquo in Comprehensive Biotechnology M Moo-Young Ed vol 3 pp 717ndash729 Elsevier Amstaerdam TheNetherlands 2nd edition 2011

[6] P R Norris and J P Owen ldquoMineral sulphide oxidationby enrichment cultures of novel thermoacidophilic bacteriardquoFEMS Microbiology Reviews vol 11 no 1ndash3 pp 51ndash56 1993

[7] D E Rawlings and D B Johnson ldquoThe microbiology ofbiomining development and optimization ofmineral-oxidizingmicrobial consortiardquo Microbiology vol 153 no 2 pp 315ndash3242007

[8] P R Norris N P Burton and N A M Foulis ldquoAcidophiles inbioreactor mineral processingrdquo Extremophiles vol 4 no 2 pp71ndash76 2000

[9] M R Bruins S Kapil and F W Oehme ldquoMicrobial resistancetometals in the environmentrdquo Ecotoxicology and EnvironmentalSafety vol 45 no 3 pp 198ndash207 2000

[10] T J G Ettema M A Huynen W M De Vos and J Van DerOost ldquoTRASH a novel metal-binding domain predicted to beinvolved in heavy-metal sensing trafficking and resistancerdquoTrends in Biochemical Sciences vol 28 no 4 pp 170ndash173 2003

[11] M Dopson C Baker-Austin P R Koppineedi and P L BondldquoGrowth in sulfidic mineral environments metal resistancemechanisms in acidophilic micro-organismsrdquo Microbiologyvol 149 no 8 pp 1959ndash1970 2003

[12] S Franke and C Rensing ldquoAcidophiles Mechanisms to toleratemetal and acid toxicityrdquo in Physiology and Biochemistry ofExtremophiles C Gerday and N Glansdorff Eds pp 271ndash278ASM Press Washington DC USA 2007

[13] C A Navarro L H Orellana C Mauriaca and C A JerezldquoTranscriptional and functional studies of Acidithiobacillusferrooxidans genes related to survival in the presence of copperrdquoApplied and Environmental Microbiology vol 75 no 19 pp6102ndash6109 2009

[14] A Orell C A Navarro R Arancibia J C Mobarec andC A Jerez ldquoLife in blue copper resistance mechanisms ofbacteria and Archaea used in industrial biomining of mineralsrdquoBiotechnology Advances vol 28 no 6 pp 839ndash848 2010

[15] A Orell C A Navarro M Rivero J S Aguilar and C A JerezldquoInorganic polyphosphates in extremophiles and their posiblefunctionsrdquo Extremophiles vol 16 no 4 pp 573ndash583 2012

[16] C Baker-Austin M Dopson M Wexler et al ldquoExtremearsenic resistance by the acidophilic archaeon ldquoFerroplasmaacidarmanusrdquo Fer1rdquo Extremophiles vol 11 no 3 pp 425ndash4342007

[17] E Pedone S Bartolucci and G Fiorentino ldquoSensing andadapting to environmental stress the archaeal tacticrdquo Frontiersin Bioscience vol 9 pp 2909ndash2926 2004

[18] C Baker-Austin M Dopson M Wexler R G Sawers and PL Bond ldquoMolecular insight into extreme copper resistance inthe extremophilic archaeon ldquoFerroplasma acidarmanusrdquo Fer1rdquoMicrobiology vol 151 no 8 pp 2637ndash2646 2005

[19] T J G Ettema A B Brinkman P P Lamers N G Kornet WM de Vos and J van der Oost ldquoMolecular characterization ofa conserved archaeal copper resistance (cop) gene cluster andits copper-responsive regulator in Sulfolobus solfataricus P2rdquoMicrobiology vol 152 no 7 pp 1969ndash1979 2006

[20] A A Villafane Y VoskoboynikM Cuebas I Ruhl and E BinildquoResponse to excess copper in the hyperthermophile Sulfolobussolfataricus strain 982rdquo Biochemical and Biophysical ResearchCommunications vol 385 no 1 pp 67ndash71 2009

[21] K S Auernik Y Maezato P H Blum and R M KellyldquoThe genome sequence of the metal-mobilizing extremelythermoacidophilic archaeon Metallosphaera sedula providesinsights into bioleaching-associated metabolismrdquo Applied andEnvironmental Microbiology vol 74 no 3 pp 682ndash692 2008

[22] Y Maezato T Johnson S McCarthy K Dana and P BlumldquoMetal resistance and lithoautotrophy in the extreme ther-moacidophile Metalosphaera sedulardquo Journal of Bacteriologyvol 194 no 24 pp 6856ndash6863 2012

[23] D H Nies ldquoMicrobial heavy-metal resistancerdquo Applied Micro-biology and Biotechnology vol 51 no 6 pp 730ndash750 1999

[24] K Vido D Spector G Lagniel S Lopez M B Toledano andJ Labarre ldquoA proteome analysis of the cadmium response inSaccharomyces cerevisiaerdquo The Journal of Biological Chemistryvol 276 no 11 pp 8469ndash8474 2001

[25] G Bertin and D Averbeck ldquoCadmium cellular effects mod-ifications of biomolecules modulation of DNA repair andgenotoxic consequences (a review)rdquo Biochimie vol 88 no 11pp 1549ndash1559 2006

[26] M H Joe S W Jung S H Im et al ldquoGenome-wide responseof Deinococcus radiodurans on cadmium toxicityrdquo Journal ofMicrobiology and Biotechnology vol 21 no 4 pp 438ndash447 2011

[27] A Lagorce A Fourcans M Dutertre B Bouyssiere YZivanovic and F Confalonieri ldquoGenome-wide transcriptionalresponse of the archeon Thermococcus gammatolerans to cad-miumrdquo PLOS ONE vol 7 no 7 Article ID e41935 2012

[28] A Kornberg N N Rao and D Ault-Riche ldquoInorganicpolyphosphate a molecule of many functionsrdquo Annual Reviewof Biochemistry vol 68 pp 89ndash125 1999

Archaea 15

[29] J D Keasling ldquoRegulation of intracellular toxic metals andother cations by hydrolysis of polyphosphaterdquoAnnals of theNewYork Academy of Sciences vol 829 pp 242ndash249 1997

[30] F Remonsellez A Orell and C A Jerez ldquoCopper tolerance ofthe thermoacidophilic archaeon Sulfolobus metallicus possiblerole of polyphosphate metabolismrdquoMicrobiology vol 152 no 1pp 59ndash66 2006

[31] V Hatzimanikatis L H Choe and K H Lee ldquoProteomicstheoretical and experimental considerationsrdquo BiotechnologyProgress vol 15 no 3 pp 312ndash318 1999

[32] L H Choe and K H Lee ldquoA comparison of three commerciallyavailable isoelectric focusing units for proteome analysis theMultiphor the IPGphor and the Protean IEF cellrdquo Electrophore-sis vol 21 no 5 pp 993ndash1000 2000

[33] U K Laemmli ldquoCleavage of structural proteins during theassembly of the head of bacteriophage T4rdquo Nature vol 227 no5259 pp 680ndash685 1970

[34] A Shevchenko M Wilm O Vorm and M Mann ldquoMassspectrometric sequencing of proteins from silver-stained poly-acrylamide gelsrdquo Analytical Chemistry vol 68 no 5 pp 850ndash858 1996

[35] P Giavalisco E Nordhoff T Kreitler et al ldquoProteome analysisof Arabidopsis thaliana by two-dimensional gel electrophoresisand matrix-assisted laser desorptionionisation-time of flightmass spectrometryrdquo Proteomics vol 5 no 7 pp 1902ndash19132005

[36] T M Rose E R Schultz J G Henikoff S Pietrokovski CM McCallum and S Henikoff ldquoConsensus-degenerate hybridoligonucleotide primers for amplification of distantly relatedsequencesrdquo Nucleic Acids Research vol 26 no 7 pp 1628ndash16351998

[37] T M Rose J G Henikoff and S Henikoff ldquoCODEHOP(Consensus-Degenerate Hybrid Oligonucleotide Primer) PCRprimer designrdquo Nucleic Acids Research vol 31 no 13 pp 3763ndash3766 2003

[38] J P Acevedo F Reyes L P Parra O Salazar B A Andrews andJ A Asenjo ldquoCloning of complete genes for novel hydrolyticenzymes from Antarctic sea water bacteria by use of animproved genomewalking techniquerdquo Journal of Biotechnologyvol 133 no 3 pp 277ndash286 2008

[39] S Bathe and P R Norris ldquoFerrous iron- and sulfur-inducedgenes in Sulfolobus metallicusrdquo Applied and EnvironmentalMicrobiology vol 73 no 8 pp 2491ndash2497 2007

[40] J Sambrook E F Fritsch and TManiatisMolecular Cloning ALaboratory Manual Cold Spring Harbor Press New York NYUSA 1989

[41] K W Miller S Sass Risanico and J B Risatti ldquoDifferentialtolerance of Sulfolobus strains to transition metalsrdquo FEMSMicrobiology Letters vol 93 no 1 pp 69ndash73 1992

[42] G Huber C Spinnler A Gambacorta and K O Stetter ldquoMet-allosphaera sedula gen and sp nov represents a new genus ofaerobic metal-mobilizing thermoacidophilic archaebacteriardquoSystematic and Applied Microbiology vol 12 pp 38ndash47 1989

[43] J Llanos C Capasso E Parisi D Prieur and C JeanthonldquoSusceptibility to heavy metals and cadmium accumulation inaerobic and anaerobic thermophilic microorganisms isolatedfrom deep-sea hydrothermal ventsrdquo Current Microbiology vol41 no 3 pp 201ndash205 2000

[44] A Kaur M Pan M Meislin M T Facciotti R El-Gewelyand N S Baliga ldquoA systems view of haloarchaeal strategies towithstand stress from transition metalsrdquo Genome Research vol16 no 7 pp 841ndash854 2006

[45] M Solioz and J V Stoyanov ldquoCopper homeostasis in Entero-coccus hiraerdquo FEMS Microbiology Reviews vol 27 no 2-3 pp183ndash195 2003

[46] S Sitthisak K Howieson C Amezola and R K JayaswalldquoCharacterization of a multicopper oxidase gene from Staphy-lococcus aureusrdquo Applied and Environmental Microbiology vol71 no 9 pp 5650ndash5653 2005

[47] L Rodrıguez-Montelongo S I Volentini R N Farıas E MMassa and V A Rapisarda ldquoThe Cu(II)-reductase NADHdehydrogenase-2 of Escherichia coli improves the bacterialgrowth in extreme copper concentrations and increases theresistance to the damage caused by copper and hydroperoxiderdquoArchives of Biochemistry and Biophysics vol 451 no 1 pp 1ndash72006

[48] E Williams T M Lowe J Savas and J DiRuggiero ldquoMicroar-ray analysis of the hyperthermophilic archaeon Pyrococcusfuriosus exposed to gamma irradiationrdquo Extremophiles vol 11no 1 pp 19ndash29 2007

[49] L M Gaetke and C K Chow ldquoCopper toxicity oxidative stressand antioxidant nutrientsrdquo Toxicology vol 189 no 1-2 pp 147ndash163 2003

[50] M J Davies ldquoThe oxidative environment and protein damagerdquoBiochimica et Biophysica Acta vol 1703 no 2 pp 93ndash109 2005

[51] J R Thompson J K Bell J Bratt G A Grant and LJ Banaszak ldquoVmax regulation through domain and subunitchanges The active form of phosphoglycerate dehydrogenaserdquoBiochemistry vol 44 no 15 pp 5763ndash5773 2005

[52] V Consalvi R Chiaraluce L Politi A Gambacorta MDe Rosa and R Scandurra ldquoGlutamate dehydrogenase fromthe thermoacidophilic archaebacterium Sulfolobus solfataricusrdquoEuropean Journal of Biochemistry vol 196 no 2 pp 459ndash4671991

[53] A J L Macario M Lange B K Ahring and E Conway DeMacario ldquoStress genes and proteins in the archaeardquo Microbiol-ogy and Molecular Biology Reviews vol 63 no 4 pp 923ndash9671999

[54] J AMaupin-FurlowM A Gil M A Humbard et al ldquoArchaealproteasomes and other regulatory proteasesrdquo Current Opinionin Microbiology vol 8 no 6 pp 720ndash728 2005

[55] N Sun F Beck R Wilhelm Knispel et al ldquoProteomics analysisof Thermoplasma acidophilum with a focus on protein com-plexesrdquoMolecular and Cellular Proteomics vol 6 no 3 pp 492ndash502 2007

[56] L S Madding J K Michel K R Shockley et al ldquoRole of the 1205731subunit in the function and stability of the 20S proteasome inthe hyperthermophilic Archaeon Pyrococcus furiosusrdquo Journalof Bacteriology vol 189 no 2 pp 583ndash590 2007

[57] R Shibata Y Bessho A Shinkai et al ldquoCrystal structureand RNA-binding analysis of the archaeal transcription factorNusArdquo Biochemical and Biophysical Research Communicationsvol 355 no 1 pp 122ndash128 2007

[58] W Bae B Xia M Inouye and K Severinov ldquoEscherichia coliCspA-family RNA chaperones are transcription antitermina-torsrdquo Proceedings of the National Academy of Sciences of theUnited States of America vol 97 no 14 pp 7784ndash7789 2000

[59] R J Ram N C VerBerkmoes M P Thelen et al ldquoMicrobi-ology community proteomics of a natural microbial biofilmrdquoScience vol 308 no 5730 pp 1915ndash1920 2005

[60] I Noel-Georis T Vallaeys R Chauvaux et al ldquoGlobal analysisof the Ralstonia metallidurans proteome prelude for the large-scale study of heavy metal responserdquo Proteomics vol 4 no 1pp 151ndash179 2004

16 Archaea

[61] M TMNovo A CDa Silva RMoreto et al ldquoThiobacillus fer-rooxidans response to copper and other heavy metals growthprotein synthesis and protein phosphorylationrdquo Antonie vanLeeuwenhoek vol 77 no 2 pp 187ndash195 2000

[62] Q She R K Singh F Confalonieri et al ldquoThe complete genomeof the crenarchaeon Sulfolobus solfataricus P2rdquo Proceedings ofthe National Academy of Sciences of the United States of Americavol 98 no 14 pp 7835ndash7840 2001

[63] R P Anderson and J R Roth ldquoTandem genetic duplications inphage and bacteriardquo Annual Review of Microbiology vol 31 pp473ndash505 1977

[64] D Gevers K Vandepoele C Simillion and Y Van De PeerldquoGene duplication and biased functional retention of paralogsin bacterial genomesrdquo Trends in Microbiology vol 12 no 4 pp148ndash154 2004

[65] A B Reams and E L Neidle ldquoSelection for gene clustering bytandemduplicationrdquoAnnual Review ofMicrobiology vol 58 pp119ndash142 2004

[66] L H Orellana and C A Jerez ldquoA genomic island providesAcidithiobacillus ferrooxidans ATCC 53993 additional copperresistance a possible competitive advantagerdquo Applied Microbi-ology and Biotechnology vol 92 no 4 pp 761ndash767 2011

[67] A B Brinkman T J G Ettema W M de Vos and J van derOost ldquoThe Lrp family of transcriptional regulatorsrdquo MolecularMicrobiology vol 48 no 2 pp 287ndash294 2003

[68] C Vollmecke S L Drees J Reimann S V Albers and MLubben ldquoThe ATPases CopA and CopB both contribute tocopper resistance of the thermoacidophilic archaeon SulfolobussolfataricusrdquoMicrobiology vol 158 no 6 pp 1622ndash1633 2012

[69] A Villafane Y Voskoboynik I Ruhl et al ldquoCopR of Sulfolobussolfataricus represents a novel class of archaeal-specific copper-responsive activators of transcriptionrdquo Microbiology vol 157no 10 pp 2808ndash2817 2011

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

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International Journal of

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Zoology

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Microbiology

8 Archaea

Table 2 Proteins upregulated in S metallicus cells exposed to 100mM Cu or 1mM Cd

Spot Molecular weight (kDa) Cu induction levels Putative function Microorganism related Accesion number1dagger 266 infin Proteasome subunit S solfataricus AAK410343dagger 701 infin Ferredoxin oxidoreductase S solfataricus AAK429264dagger 429 infin Alcohol dehydrogenase S solfataricus AAK431545 35 26 Hypothetical protein S acidocaldarius YP 2549766 27 infin Hypothetical protein P aerophilum NP 5598899dagger 57 infin Phosphoglycerate dehydrogenase M thermautotrophicus AAB8546613dagger 27 34 Unknown mdash mdash14 28 infin Unknown mdash mdash15 35 55 Unknown mdash mdash20dagger 461 infin Glutamate dehydrogenase S solfataricus AAK4223021dagger 45 infin Hypothetical protein M acetivorans NP 618380122 49 infin Hypothetical protein P torridus YP 02280723dagger 51 infin ATP synthase subunit B S solfataricus AAK4088024 585 infin HSP60 subunit S shibatae AAG37273130dagger 48 infin Unknown mdash mdash31 16 ND Unknown mdash mdash32 144 ND Putative transcription regulator S solfataricus AAK4041333 138 ND Transcription factor nusA S solfataricus AAK40563Spots refer to those numbered in Figure 2 Proteins in Figure 2 which are not included in this table were not subjected to sequencing due to their low intensitiesin the gelsdaggerSpot up-regulated in cells exposed to Cu and CdND not determinedInfinity symbol indicates that proteins were expressed only in presence of copper or cadmiumM thermoautotrophicus Methanobacterium thermoautotrophicus M acetivorans Methanosarcina acetivorans S shibatae Sulfolobus shibatae P torridusPicrophilus torridus P aerophilum Pyrobaculum aerophilum S acidocaldarius Sulfolobus acidocaldarius

up-regulated in response to Cd (Table 2) Several previ-ous reports have suggested that oxidoreductases contributeto an oxidative protection in both Bacteria and Archaeain response to heavy metals [46ndash48] In the neutrophilicmicroorganisms Escherichia coli and Staphylococcus aureusit has been described that oxidases and dehydrogenasescontribute to oxidative protection Cu homeostasis and stressresponses [46 47] Furthermore some proteins involved inoxidative damage repair such as NADH-dependent oxidasesand thioredoxin reductases were expressed in cells of Facidarmanus exposed to As(III) [16] The expression of thisgroup of proteins has also been observed when the samemicroorganism was exposed to Cu [18] Therefore oxidore-ductases play an important role against oxidative stress andmay eliminate reactive oxygen species which constitute themajor component of the stress caused by transition metals[44 49 50]

The enzymes related to biosynthesis of amino acids werefound as commonly up-regulated either in response to Cu orCdThey corresponded to a phosphoglycerate dehydrogenase(spot 9) and a glutamate dehydrogenase (spot 20) Theformer one catalyzes the NAD+-dependent oxidation of 3-phosphoglycerate into 3-phosphohydroxypyruvate a branchpoint from the glycolytic pathway and the initial reactionin L-serine biosynthesis [51] Glutamate dehydrogenase cat-alyzes the oxidative deamination of glutamate to produce 2-oxoglutarate and ammonia with reduction of NAD+ [52]Proteins involved in amino acids synthesis were expressed

in cells of F acidarmanus exposed to As(III) [16] Moreoverincreased levels of proteins involved in the biosynthesisof sulfur-containing amino acids have been observed inSaccharomyces cerevisiae cells exposed to Cd [24] suggestingthat these proteins might also have a role in the cellularresponse to adverse conditions

Two proteins showed identity to components of the stressresponse mechanism such as one subunit of the HSP60chaperonin (spot 24) and one subunit of the proteasome (spot1) In Archaea the stress protein HSP60 is directly involvedin protein folding processes [53] Proteomic analysis of Facidarmanus cells exposed to As(III) and Cd revealed theexpression of proteins involved in protein folding and DNArepair including HSP60 chaperonin and DnaK heat-shockprotein (HSP70) thereby the authors suggested that thismicroorganismusesmultiplemechanisms to resist high levelsof Cu [16 18] On the other hand proteasomes are describedas nonspecific proteolytic nanomachines associated withprotein catabolism Proteasomes are known to be closelyinvolved in maintaining protein quality control by degradingmiss folded and denatured proteins in response to cell stressin all three domains of life [54] A proteomic analysis inthe thermoacidophilic archaeon Thermoplasma acidophilumshowed that proteasomes and chaperone-related proteinswere highly induced against stress conditions indicatinga high turnover rate of proteins [55] In P furiosus theexpression of the 1205731 subunit of the proteasome was inducedin cells subjected to heat shock stress [56] Additionally the

Archaea 9

150pb

(a)

1016 pb

(b)

2000 pb

(c)

Figure 4 CODEHOP-based PCR for the amplification of the putative genes copA and copM from S metallicus Amplification products(a) using degenerate PCR primers (DOP) copM degF and copM degR designed from amino acid sequence conserved blocks of SulfolobalesCopM proteins (b) using primers copA cdegF and copA cdegR designed by CODEHOP strategy for amplification of a copA-like gene (c)Primers copM degF and copA cdegRwere assesed for the amplification of a copMA-likeDNA regionThose primerswere testedwith genomicS metallicus DNA samples Amplicons expected sizes were as follows ca 150 bp for copM ca 950 bp for copA and ca 2000 bp for copMAPCR products were excised and cloned for later sequencing

proteasome subunit was also found to be up-regulated whenS metallicus was subjected to Cd stress (Table 2)

The transcriptional regulation-related proteins corre-sponded to a putative transcription regulator (spot 32) andthe transcription factor NusA (spot 33) involved in theintrinsic termination of transcription [57] NusA has beenalso reported to display important functions in the cellularresponse to adverse factors [58] The expression of twotranscriptional regulators related to amino acids biosynthesisand metal-dependent genetic repression were found to beinduced in P furiosus cells subjected to oxidative stress[48] Moreover the expression of several genes encodingpredicted transcriptional regulators were induced followingCd exposure in T gammatolerans cells [27]

Spots 5 6 13 14 15 21 22 30 and 31 (Table 2) did notshow significant scores with any protein sequences currentlyavailable in the data bases Interestingly spots 2 4 9 13 21and 30 were found to be commonly upregulated when Smetallicus cells were treated with either Cu or Cd (Figure 3Table 2)

The results presented here are consistent with previ-ous work describing global protein expression profiles inresponse to heavy metals as reported for F acidarmanus [1618] microbial-biofilm communities in an acid mine drainagesite [59] and other prokaryotic and eukaryotic microorgan-isms [24 60]Therefore a coordinated expression of differentgroups of genes suggests the existence of regulatory networkssuch as stress response mechanisms and respiratory chainadjustments to cope with the presence of heavy metal ions[61]

34 Cu-Resistance (Cop) Genes Cluster Is Duplicated in Smetallicus Genome A Cu-resistance (cop) locus has beendescribed to be highly conserved in archaeal genomeswhich consists of genes encoding a new type of archaealtranscriptional regulator (CopT) a metal-binding chaperone(CopM) and a Cu-transporting P-type ATPase (CopA) [19]Since the genome sequence of Smetallicus is not yet availablewe searched for the presence of cop genes by using consen-sus degenerate hybrid oligonucleotide primers-based PCR(CODEHOP-PCR) CopA and CopM amino acid sequencealignments from S acidocaldarius S tokodaii and S solfa-taricus were used to identify conserved sequence blocks andtherefore to design either CODEHOP or DOP (degeneratehybrid oligonucleotide) primers (Figure S1 Table 1) TherebyPCR assays using S metallicus genomic DNA as templateyielded amplicons of ca 150 bp and ca 950 bp for copMand copA CODEHOP pair primers respectively (Figures4(a) 4(b)) As estimated from primer pairrsquos position onamino acid sequence alignments the obtained PCR productscorresponded to the expected sizes suggesting the presenceof copM- and copA-like genes in S metallicus genome Inorder to determine the identity of the amplified DNA frag-ments they were TA-cloned and sequenced Blastx sequenceanalysis showed that the 150 bp DNA fragment coded for anincomplete ORF sharing 63 homology with S solfataricusCopM (SSO10823) whereas the analysis of the 950 pb DNAfragment yielded a partial ORF sharing 54 homology withS solfataricus CopA protein (SSO2651)

The genomic organization of the putative cop genes in Smetallicus was analyzed to find out its similarity with those

10 Archaea

Table 3 Sequence identity comparison of S metallicus Cop proteins to those in other Sulfolobales

S metallicus protein Identity to homologue in other SulfolobalesS solfataricus P2 S acidocaldarius S tokodaii M sedula

CopA1 51 43 44 45CopA2 88 46 49 50CopM1 63 69 71 71CopM2 89 72 70 77CopT1 36 27 30 32CopT2lowast 90 52 52 53Boldface type indicates to which organism the S metallicus homologue shows the highest sequence identitylowastRefers to the analysis obtained using the uncompleted amino acid sequence of CopT2 which represents sim90 in length when compared with S solfataricusCopT sequence

previously described in S solfataricus P2 and F acidarmanuswhere the cop cluster consists of tandem-orientated genesas copTMA [18 19] Thus using copM degF (forward) andcopA cdegR (reverse) primers we attempted to amplify aputative copMA DNA region This PCR yielded a product ofca 2000 pb corresponding to the expected size and resultedin 2 ORFs showing high sequence homology with CopMand CopA from S solfataricus (Figure 4(c)) Interestinglywhen aligning this copMA sequence with the copA and copMsequences obtained previously they were not identical whichstrongly indicated the presence of duplicated putative copgenes encoding for both Cu-ATPases (copA1 and copA2) andmetallochaperones (copM1 and copM2) in the S metallicusgenome

To isolate the entire cop gene sequences the genomewalking method described by Acevedo et al [38] was usedSix different DNA libraries were constructed from S metalli-cus genomic DNA digested with several restriction enzymesincluding 51015840 overhang and blunt end restriction enzymesThe DNA libraries were used as templates in 2 successivePCR reactions From the previously known sequences twospecific sense primers for the 31015840 end amplification and twospecific antisense primers for the 51015840 end amplification weredesigned (abbreviated SP1 and SP2) Therefore the 51015840 and 31015840ends of the putative genes copA1 and copA2 that had beenpartially identified through the genome walking techniquewere amplified

By overlapping the sequence isolated by degenerate PCRand the lateral sequences obtained by genome walking(Figure S2) it was possible to complete the whole nucleotidesequence of the putative gene copA1 Additionally it waspossible to confirm the presence of the upstream copM1 geneanddetermine the occurrence of a partial copT1 gene tandem-orientated upstream of copM1 (Figure 5)Through additionalgenome walking experiments the partial copT1 sequencecould be further completedThe samewas truewhen isolatingthe whole nucleotide sequence of the putative gene copA2as the presence of copM2 and copT2 genes were confirmedupstream of copA2 However it was not possible to completethe whole 51015840 end of the putative gene copT2 as its determinedlength corresponded to only 90 of the full length whencompared with S solfataricus copT gene (Figure 5)

In conclusion degenerate PCR together with genomewalking experiments allowed to describe the occurrence inS metallicus genome of 2 cop genes loci (named as locus cop1and locus cop2) coding for paralogous genes whose productsmay be involved in Cu-resistance (Figure 5) Although somearchaeal genomes exhibit two copies of putative Cu-P-typeATPases as described for S solfataricus [62] the genomicarrangement displayed as a cop gene cluster (copTMA) hasbeen reported to be represented in only one copy for manyarchaeal genomes [10 19 21] Moreover we further updatedthis analysis by searching for duplications of the cop genescluster in all available archaeal genome sequences up to date(October 2012) retrieving only one cop locus in each caseThereby we propose that the discovery of this cop gene clusterduplication in the genome of S metallicus constitutes so faran unprecedented feature for a representative of the Archaeadomain and might contribute to its high metal resistance

In this context it has been widely reported that increasedgene copy number can increase gene expression allowingprokaryotic to thrive under growth limiting conditions [1363ndash68] However alongside the augmented gene copy onecannot exclude that the cop operon duplication in S metal-licus may offer among others a wider repertoire of Cu-ATPases which might differ in terms of metal affinitiesandor specificity efflux rates andor differences in theirabundance regulation

Throughout amino acid sequence analysis it was furtherdetermined that each polypeptide encoded by S metallicuswith the exception of CopT1 showed to be highly homol-ogous to their orthologous counterparts encoded by othersSulfolobales (Table 3) Moreover the 3 ORFs encoded by thelocus cop2 shared about 90 identity with the correspondingS solfataricus orthologous peptides (Table 3) S metallicuscop genes products showed characteristic domains referredto as critical for their respective proposed biological activitiesS metallicus paralogous gene products CopA1 (749 aa1015840)and CopA2 (747 aa1015840) shared 513 of identity These twoputative Cu-ATPases (CopA1 and CopA2) contain the aminoacid sequence motif CPCALGLA which has been proposedto confer Cu-transporting specificity in CPx-ATPases [48]and has been also found in other CopA-like proteins frombiomining Bacteria and Archaea [14]

Archaea 11

HindIII HindIII HindIII

HindIII

CopT1Locus cop1 M1

EcoRIEcoRV

513 bp 219 bp 2250 bp

CopA1

(a)

Locus cop2 M2

EcoRI

CopA2

453 bp 171 bp 2244 bp

CopT2

PstIHindIII HindIII HindIII

(b)

Figure 5 Schematic representation of the two cop gene clusters isolated from S metallicus genome Each cluster codes for anArchaea-specifictranscriptional regulator (copT) a metallochaperone (copM) and a P-type Cu-exporting ATPase (copA) Lengths of each gene are indicatedLocus cop1 corresponds to 4346 sequenced base pairs and shows an intergenic region copT1-M1 of 232 base pairs Locus cop2 correspondsto 3435 base pairs and shows 89 base pairs copT2-M2 intergenic region Genes copM2 and copA2 overlapped in 11 base pairs copT2 was notfully isolated

Control CdSO4 Ag2SO4 NiSO4 ZnSO4 CuSO4

kb3kb2kb1

02 kb

(a)

kb3kb2kb1

02 kb

(b)

(c)

Figure 6 Northern blot analysis to determine the expression of S metallicus copA1 (a) and copA2 (b) putative genes in response to variousmetals S metallicus total RNAwas extracted from cultures grown either in the absence (control) or the presence of CuSO

4(50mM) Ag

2SO4

(008mM) NiSO4(15mM) ZnSO

4(50mM) and CdSO

4(2mM) P32-radioactivelly labelled DNA fragments annealing to copA1 and copA2

sequences respectively were used as probes (c) shows rRNAs as total RNA loading control

On the other hand CopM1 (71 aa) and CopM2 (56 aa)sharing 66 of identity present the proposed metal bindingdomain TRASH (trafficking resistance and sensing of heavymetals) [10] Additionally when analyzing the putative tran-scriptional regulators the presence of a C-terminal TRASHdomain in both CopT1 and CopT2 was also found CopT1sequence also contains an entire N-terminal helix-turn-helix (HTH) motif (not shown) that resembles the DNA-bindingmotifs of prokaryotic transcriptional regulators suchas Lrp-like proteins [67] The HTH motif was only partiallyidentified in CopT2 since the 51015840 region of copT1 gene has notbeen yet fully isolated

35 Transcriptional Analysis of S metallicus Cop Genes Toget insight into the role of the two cop loci the expressionof the corresponding genes was analyzed under variousconditions S metallicus was first grown in presence ofdifferent heavy metals (Cu Zn Cd Ni o Ag) at a givenconcentration that did not affect growth kinetics [30 42]Subsequently total RNA was isolated from late exponentiallygrown cultures and Northern blot experiments were carriedout in order to determine the expression of both copA1 andcopA2 genes As depicted in Figure 6 transcription of bothcopA1 and copA2messengers were specifically induced in thepresence of Cu and Cd ions This gene expression pattern

12 Archaea

954pb

M1 CopA1

(a)

1438 pb

M2 CopA2

(b)

1000 pb

800pb

+ minus

(c)

+ minus

1500 pb

1000 pb

(d)

Figure 7 Cotranscription analysis of copMA1 and copMA2 genes cDNA was synthesized with a reverse primer (dotted arrows) hybridizingtoward the 31015840 end of either copA1 (a) or copA2 (b) S metallicus total RNA was extracted at the late exponential phase from a culture growingin presence of 20mMCu PCR amplifications were carried out with these cDNAs and each corresponding primer pair (black arrows) as listedin Table 1 (c) and (d) show RT-PCR products obtained for the copMA1 and copMA2 intergenic regions respectively Reverse transcriptasereactions with (+) and without (minus) the Improm II reverse transcriptase enzyme were carried out in order to exclude amplification due togenomic DNA contamination Expected sizes (in base pairs) for the corresponding PCR products are given in (a) and (b)

is in good agreement with what has been described for Ssolfataricus where the bicistronic copMA transcript levelswere also found to be up-regulated in response to both Cuand Cd [19]

Northern blot analysis showed that the sizes seen for bothcopA transcripts (sim25 Kb) did not correspond exactly withthe individual genes length (each of about 225Kb) (Figure 6)This strongly suggested that the transcripts could also includethe respective copM gene located upstream of copA in eachcop loci To confirm this assumption a co-transcriptionexperiment was done (Figure 7) in which the cDNAs wereobtained by using RNA extracted from a culture grownin the presence of 20mM Cu and using a reverse primerhybridizing with copA1 (Figure 7(a)) or copA2 (Figure 7(b))gene sequence respectively PCR amplifications were carriedout by using the corresponding cDNAs as templates and eachpair of primers lying in adjacent genes (copM) The presenceof an amplicon of the expected size in each case indicatedthe adjacent genes were part of polycistronic messengers(Figure 7) These results clearly show that in S metallicuseach couple of copMA genes was expressed in the form oftranscriptional units as reported in F acidarmanus and Ssolfataricus [18 19] The coexpression of gene pairrsquos copMAmay suggest a coordinated and dependent function for therespective encoded proteins In the bacterium E hirae themetallochaperone CopZ (CopM in S metallicus) fulfills apivotal role in the mechanism of Cu homeostasis [45] Thusit was shown that this protein interacts directly with the Cu-ATPase CopB (CopA in S metallicus) handing the Cu forsubsequent removal In this context one might expect thatproteins CopM1 and CopM2 from S metallicus have a similarrole to that described for E hirae Furthermore quantitativeRT-PCR experiments were carried out in order to determinedifferential expression of the cop genes in response to differentCu concentrations The expression of copA1 copA2 copT1and copT2 was tested relative to the transcript levels ofrRNA 16S gene since its levels were not significantly affected

in all assessed conditions (data not shown) As shown inFigure 8(a) copA2 and copA1 transcript levels were foundto be concomitantly increased with the increasing Cu con-centrations present in the medium Higher transcripts levelsof copA1 were seen in all tested conditions when comparedwith copA2 levels copA1 transcript levels were found to be325-fold up-regulated when comparing 50mMCu conditionversuscontrol (absence of metal) whereas copA2 transcriptlevels showed an increment of only 175-fold (Figure 8(a))The finding that Cu-ATPases mRNA levels were significantlyincreased in response to Cu ions exposure suggests that thetransport system may operate for Cu efflux in S metallicus

Moreover copA2 and copA1 gene expression was quan-tified when S metallicus was grown using chalcopyrite(CuFeS

2) ore as an oxidizable substrate (Figure 8(a)) Mineral

oxidation mediated by the microorganism generates a pro-gressive increase in Cu ions concentration in the medium Tofind out whether the amount of solubilized Cu induced theexpression of the Cu-ATPases genes total RNAwas extractedfrom a S metallicus culture grown to late exponential phaseand in the presence of 1 CuFeS

2 It was clear that an

up-regulation of the Cu-ATPases encoding genes also tookplace when S metallicus was grown in the presence ofCuFeS

2(Figure 8(a)) copA1 transcript levels increased 14-fold

compared with the control condition in the absence of Cuwhile copA2 gene expression showed an increase of 76 foldAlong with this bymeans of atomic absorption spectrometry(AAS) analysis an overall amount of solubilized Cu ions(Cu2+Cu1+) of 144plusmn21mMwas determined to be present inthemedium indicating that copA2 and copA1 gene expressionwas most likely in response to the Cu present in the culturedue to CuFeS

2microbial-solubilization

The finding that copA1 was highly expressed comparedwith copA2 in all tested conditions may suggest a possiblephysiological hierarchy between the two ATPases whenovercoming either Cu or Cd stress In this regard by meansof a genetic approach it was demonstrated in S solfataricus

Archaea 13

700

600

500

400

300

200

100

00

5 20 50 CuFeS2CuSO4 concentration (mM)

Relativ

egenee

xpression

CopA2CopA1

(a)

8

6

7

5

4

3

2

1

00

5 20 50 CuFeS2CuSO4 concentration (mM)

Relativ

egenee

xpression

CopT1CopT2

(b)

Figure 8 Relative expression levels of S metallicus genes copA1 and copA2 (a) copT1 and copT2 (b) Expression of S metallicus cop geneswasassessed by qRT-PCR and normalized against 16S rRNA gene expression in individual cultures Mean values and standard deviations arefrom analyses of three independent cultures grown in the presence of 0 5 20 50mMCu or in 1 of a chalcopyrite ore (CuFeS

2) concentrate

that while CopA was an effective Cu efflux transporterat low and high Cu concentrations the other Cu-ATPase(CopB) only appeared to be a low-affinity Cu export ATPase[68] Moreover by using a M sedula copA deletion mutantit was demonstrated that this strain compromised metalresistance and consequently abolished chalcopyrite oxidation[22] highlighting the role of Cu detoxification mechanismsduring a given bioleaching process Our attempts to showthe functionality of CopA1 and CopA2 from S metallicusby using E coli as a heterologous host were not successfulApparently the overexpression of these ORFs had a toxiceffect on E coli that compromised its viability Althoughgene disruption tools have not been yet developed for Smetallicus the functional role of both copTMA locimight befurther studied by cross-species complementation of a coppersensitive S solfataricus copR mutant as it was described byMaezato et al [22] It will be of great interest to establishin future studies the possible functionality of the isolatedputative transporters from S metallicus

Likewise quantitative RT-PCR experiments showed thatcopT1 transcript levels increased concomitantly with increas-ing Cu concentrations whereas copT2 showed relativelysimilar transcripts levels most likely indicating a constitutiveexpression profile (Figure 8(b)) Furthermore whereas copT1transcript levels were found to be increased 36 fold inCuFeS

2

grown cultures copT2 levels remained unchanged in com-parison with the control in the absence of Cu (Figure 8(b))The results obtained for S metallicus copT2 gene expressionare consistent with those reported for S solfataricus CopTtranscriptional regulator has been proposed to function as arepressor in S solfataricus showing a constitutive expressionthat in the presence of Cu loses its affinity for the promoterregion of copMA allowing the expression of this polycistron[19] In contrast the increased transcripts levels of both copA1and copT1 concomitant with higher Cu concentrations in the

environment suggest that the putative transcriptional regula-tor CopT1 may act by activating both copMA1 and probablyitself (Figure 8) Regarding this it was recently reported thatCopR (CopT in S metallicus) from S solfataricus strain 982acts as an activator of copT (copM in S metallicus) and copAexpression [69] Nevertheless additional experiments wouldbe required to test this possibility in S metallicus

4 Concluding Remarks

We previously reported that S metallicus resists extremelyhigh Cu concentrations which was mediated to some extentby the use of a possible metal resistance system based oninorganic polyphosphate hydrolysis and consequently Cu-PO4

2minusefflux [15 29 30] Here we have addressed the questionwhether this microorganism coded for other determinantsthat might help to explain its high heavy metal toleranceAs the genomic sequence of this microorganism is notyet available we jointly used CODEHOP-PCR and genomewalking approaches and were able to establish the occurrenceof two nonidentical homologous cop loci into the genome ofS metallicus (cop1 and cop2) Each cop locus codes for anarchaeal transcriptional regulator (CopT) a metal-bindingchaperone (CopM) and a Cu-transporting P-type ATPase(CopA) High levels of the polycistronic mRNAscopMA ofeach cop locus were observed after treatment with either Cuor Cd suggesting that the encoded ATPases efflux heavymetals out in order to detoxify the intracellular environmentAltogether previous reports and the results obtained in thisstudy allow us to suggest that some key elements that mayexplain the high resistance to Cu in S metallicus is the dupli-cation of the Cu resistance cop genes a defensive responseto stress and a polyP-based accumulation mechanism Inthe case of Cd although some similar stress responses were

14 Archaea

observed whether comparable Cu-responsive elements alsoparticipate in Cd responses remains to be seen

Conflict of Interests

The authors declare that the research was done in the absenceof any commercial or financial relationships that could beconstrued as a potential conflict of interests

Acknowledgments

This work was supported by Grant no FONDECYT 1110214and in part by ICM P-05-001-F project

References

[1] A Schippers ldquoMicroorganisms involved in bioleaching andnucleic acid-based molecular methods for their identificationand quantificationrdquo in Microbial Processing of Metal SulfidesR E Donati and W Sand Eds pp 3ndash33 Elsevier BerlinGermany 2007

[2] H R Watling ldquoThe bioleaching of sulphide minerals withemphasis on copper sulphides a reviewrdquo Hydrometallurgy vol84 no 1-2 pp 81ndash108 2006

[3] D E Rawlings D Dew and C Du Plessis ldquoBiomineralizationof metal-containing ores and concentratesrdquo Trends in Biotech-nology vol 21 no 1 pp 38ndash44 2003

[4] L Valenzuela A Chi S Beard et al ldquoGenomics metagenomicsand proteomics in biomining microorganismsrdquo BiotechnologyAdvances vol 24 no 2 pp 197ndash211 2006

[5] C A Jerez ldquoBioleaching and biomining for the industrialrecovery of metalsrdquo in Comprehensive Biotechnology M Moo-Young Ed vol 3 pp 717ndash729 Elsevier Amstaerdam TheNetherlands 2nd edition 2011

[6] P R Norris and J P Owen ldquoMineral sulphide oxidationby enrichment cultures of novel thermoacidophilic bacteriardquoFEMS Microbiology Reviews vol 11 no 1ndash3 pp 51ndash56 1993

[7] D E Rawlings and D B Johnson ldquoThe microbiology ofbiomining development and optimization ofmineral-oxidizingmicrobial consortiardquo Microbiology vol 153 no 2 pp 315ndash3242007

[8] P R Norris N P Burton and N A M Foulis ldquoAcidophiles inbioreactor mineral processingrdquo Extremophiles vol 4 no 2 pp71ndash76 2000

[9] M R Bruins S Kapil and F W Oehme ldquoMicrobial resistancetometals in the environmentrdquo Ecotoxicology and EnvironmentalSafety vol 45 no 3 pp 198ndash207 2000

[10] T J G Ettema M A Huynen W M De Vos and J Van DerOost ldquoTRASH a novel metal-binding domain predicted to beinvolved in heavy-metal sensing trafficking and resistancerdquoTrends in Biochemical Sciences vol 28 no 4 pp 170ndash173 2003

[11] M Dopson C Baker-Austin P R Koppineedi and P L BondldquoGrowth in sulfidic mineral environments metal resistancemechanisms in acidophilic micro-organismsrdquo Microbiologyvol 149 no 8 pp 1959ndash1970 2003

[12] S Franke and C Rensing ldquoAcidophiles Mechanisms to toleratemetal and acid toxicityrdquo in Physiology and Biochemistry ofExtremophiles C Gerday and N Glansdorff Eds pp 271ndash278ASM Press Washington DC USA 2007

[13] C A Navarro L H Orellana C Mauriaca and C A JerezldquoTranscriptional and functional studies of Acidithiobacillusferrooxidans genes related to survival in the presence of copperrdquoApplied and Environmental Microbiology vol 75 no 19 pp6102ndash6109 2009

[14] A Orell C A Navarro R Arancibia J C Mobarec andC A Jerez ldquoLife in blue copper resistance mechanisms ofbacteria and Archaea used in industrial biomining of mineralsrdquoBiotechnology Advances vol 28 no 6 pp 839ndash848 2010

[15] A Orell C A Navarro M Rivero J S Aguilar and C A JerezldquoInorganic polyphosphates in extremophiles and their posiblefunctionsrdquo Extremophiles vol 16 no 4 pp 573ndash583 2012

[16] C Baker-Austin M Dopson M Wexler et al ldquoExtremearsenic resistance by the acidophilic archaeon ldquoFerroplasmaacidarmanusrdquo Fer1rdquo Extremophiles vol 11 no 3 pp 425ndash4342007

[17] E Pedone S Bartolucci and G Fiorentino ldquoSensing andadapting to environmental stress the archaeal tacticrdquo Frontiersin Bioscience vol 9 pp 2909ndash2926 2004

[18] C Baker-Austin M Dopson M Wexler R G Sawers and PL Bond ldquoMolecular insight into extreme copper resistance inthe extremophilic archaeon ldquoFerroplasma acidarmanusrdquo Fer1rdquoMicrobiology vol 151 no 8 pp 2637ndash2646 2005

[19] T J G Ettema A B Brinkman P P Lamers N G Kornet WM de Vos and J van der Oost ldquoMolecular characterization ofa conserved archaeal copper resistance (cop) gene cluster andits copper-responsive regulator in Sulfolobus solfataricus P2rdquoMicrobiology vol 152 no 7 pp 1969ndash1979 2006

[20] A A Villafane Y VoskoboynikM Cuebas I Ruhl and E BinildquoResponse to excess copper in the hyperthermophile Sulfolobussolfataricus strain 982rdquo Biochemical and Biophysical ResearchCommunications vol 385 no 1 pp 67ndash71 2009

[21] K S Auernik Y Maezato P H Blum and R M KellyldquoThe genome sequence of the metal-mobilizing extremelythermoacidophilic archaeon Metallosphaera sedula providesinsights into bioleaching-associated metabolismrdquo Applied andEnvironmental Microbiology vol 74 no 3 pp 682ndash692 2008

[22] Y Maezato T Johnson S McCarthy K Dana and P BlumldquoMetal resistance and lithoautotrophy in the extreme ther-moacidophile Metalosphaera sedulardquo Journal of Bacteriologyvol 194 no 24 pp 6856ndash6863 2012

[23] D H Nies ldquoMicrobial heavy-metal resistancerdquo Applied Micro-biology and Biotechnology vol 51 no 6 pp 730ndash750 1999

[24] K Vido D Spector G Lagniel S Lopez M B Toledano andJ Labarre ldquoA proteome analysis of the cadmium response inSaccharomyces cerevisiaerdquo The Journal of Biological Chemistryvol 276 no 11 pp 8469ndash8474 2001

[25] G Bertin and D Averbeck ldquoCadmium cellular effects mod-ifications of biomolecules modulation of DNA repair andgenotoxic consequences (a review)rdquo Biochimie vol 88 no 11pp 1549ndash1559 2006

[26] M H Joe S W Jung S H Im et al ldquoGenome-wide responseof Deinococcus radiodurans on cadmium toxicityrdquo Journal ofMicrobiology and Biotechnology vol 21 no 4 pp 438ndash447 2011

[27] A Lagorce A Fourcans M Dutertre B Bouyssiere YZivanovic and F Confalonieri ldquoGenome-wide transcriptionalresponse of the archeon Thermococcus gammatolerans to cad-miumrdquo PLOS ONE vol 7 no 7 Article ID e41935 2012

[28] A Kornberg N N Rao and D Ault-Riche ldquoInorganicpolyphosphate a molecule of many functionsrdquo Annual Reviewof Biochemistry vol 68 pp 89ndash125 1999

Archaea 15

[29] J D Keasling ldquoRegulation of intracellular toxic metals andother cations by hydrolysis of polyphosphaterdquoAnnals of theNewYork Academy of Sciences vol 829 pp 242ndash249 1997

[30] F Remonsellez A Orell and C A Jerez ldquoCopper tolerance ofthe thermoacidophilic archaeon Sulfolobus metallicus possiblerole of polyphosphate metabolismrdquoMicrobiology vol 152 no 1pp 59ndash66 2006

[31] V Hatzimanikatis L H Choe and K H Lee ldquoProteomicstheoretical and experimental considerationsrdquo BiotechnologyProgress vol 15 no 3 pp 312ndash318 1999

[32] L H Choe and K H Lee ldquoA comparison of three commerciallyavailable isoelectric focusing units for proteome analysis theMultiphor the IPGphor and the Protean IEF cellrdquo Electrophore-sis vol 21 no 5 pp 993ndash1000 2000

[33] U K Laemmli ldquoCleavage of structural proteins during theassembly of the head of bacteriophage T4rdquo Nature vol 227 no5259 pp 680ndash685 1970

[34] A Shevchenko M Wilm O Vorm and M Mann ldquoMassspectrometric sequencing of proteins from silver-stained poly-acrylamide gelsrdquo Analytical Chemistry vol 68 no 5 pp 850ndash858 1996

[35] P Giavalisco E Nordhoff T Kreitler et al ldquoProteome analysisof Arabidopsis thaliana by two-dimensional gel electrophoresisand matrix-assisted laser desorptionionisation-time of flightmass spectrometryrdquo Proteomics vol 5 no 7 pp 1902ndash19132005

[36] T M Rose E R Schultz J G Henikoff S Pietrokovski CM McCallum and S Henikoff ldquoConsensus-degenerate hybridoligonucleotide primers for amplification of distantly relatedsequencesrdquo Nucleic Acids Research vol 26 no 7 pp 1628ndash16351998

[37] T M Rose J G Henikoff and S Henikoff ldquoCODEHOP(Consensus-Degenerate Hybrid Oligonucleotide Primer) PCRprimer designrdquo Nucleic Acids Research vol 31 no 13 pp 3763ndash3766 2003

[38] J P Acevedo F Reyes L P Parra O Salazar B A Andrews andJ A Asenjo ldquoCloning of complete genes for novel hydrolyticenzymes from Antarctic sea water bacteria by use of animproved genomewalking techniquerdquo Journal of Biotechnologyvol 133 no 3 pp 277ndash286 2008

[39] S Bathe and P R Norris ldquoFerrous iron- and sulfur-inducedgenes in Sulfolobus metallicusrdquo Applied and EnvironmentalMicrobiology vol 73 no 8 pp 2491ndash2497 2007

[40] J Sambrook E F Fritsch and TManiatisMolecular Cloning ALaboratory Manual Cold Spring Harbor Press New York NYUSA 1989

[41] K W Miller S Sass Risanico and J B Risatti ldquoDifferentialtolerance of Sulfolobus strains to transition metalsrdquo FEMSMicrobiology Letters vol 93 no 1 pp 69ndash73 1992

[42] G Huber C Spinnler A Gambacorta and K O Stetter ldquoMet-allosphaera sedula gen and sp nov represents a new genus ofaerobic metal-mobilizing thermoacidophilic archaebacteriardquoSystematic and Applied Microbiology vol 12 pp 38ndash47 1989

[43] J Llanos C Capasso E Parisi D Prieur and C JeanthonldquoSusceptibility to heavy metals and cadmium accumulation inaerobic and anaerobic thermophilic microorganisms isolatedfrom deep-sea hydrothermal ventsrdquo Current Microbiology vol41 no 3 pp 201ndash205 2000

[44] A Kaur M Pan M Meislin M T Facciotti R El-Gewelyand N S Baliga ldquoA systems view of haloarchaeal strategies towithstand stress from transition metalsrdquo Genome Research vol16 no 7 pp 841ndash854 2006

[45] M Solioz and J V Stoyanov ldquoCopper homeostasis in Entero-coccus hiraerdquo FEMS Microbiology Reviews vol 27 no 2-3 pp183ndash195 2003

[46] S Sitthisak K Howieson C Amezola and R K JayaswalldquoCharacterization of a multicopper oxidase gene from Staphy-lococcus aureusrdquo Applied and Environmental Microbiology vol71 no 9 pp 5650ndash5653 2005

[47] L Rodrıguez-Montelongo S I Volentini R N Farıas E MMassa and V A Rapisarda ldquoThe Cu(II)-reductase NADHdehydrogenase-2 of Escherichia coli improves the bacterialgrowth in extreme copper concentrations and increases theresistance to the damage caused by copper and hydroperoxiderdquoArchives of Biochemistry and Biophysics vol 451 no 1 pp 1ndash72006

[48] E Williams T M Lowe J Savas and J DiRuggiero ldquoMicroar-ray analysis of the hyperthermophilic archaeon Pyrococcusfuriosus exposed to gamma irradiationrdquo Extremophiles vol 11no 1 pp 19ndash29 2007

[49] L M Gaetke and C K Chow ldquoCopper toxicity oxidative stressand antioxidant nutrientsrdquo Toxicology vol 189 no 1-2 pp 147ndash163 2003

[50] M J Davies ldquoThe oxidative environment and protein damagerdquoBiochimica et Biophysica Acta vol 1703 no 2 pp 93ndash109 2005

[51] J R Thompson J K Bell J Bratt G A Grant and LJ Banaszak ldquoVmax regulation through domain and subunitchanges The active form of phosphoglycerate dehydrogenaserdquoBiochemistry vol 44 no 15 pp 5763ndash5773 2005

[52] V Consalvi R Chiaraluce L Politi A Gambacorta MDe Rosa and R Scandurra ldquoGlutamate dehydrogenase fromthe thermoacidophilic archaebacterium Sulfolobus solfataricusrdquoEuropean Journal of Biochemistry vol 196 no 2 pp 459ndash4671991

[53] A J L Macario M Lange B K Ahring and E Conway DeMacario ldquoStress genes and proteins in the archaeardquo Microbiol-ogy and Molecular Biology Reviews vol 63 no 4 pp 923ndash9671999

[54] J AMaupin-FurlowM A Gil M A Humbard et al ldquoArchaealproteasomes and other regulatory proteasesrdquo Current Opinionin Microbiology vol 8 no 6 pp 720ndash728 2005

[55] N Sun F Beck R Wilhelm Knispel et al ldquoProteomics analysisof Thermoplasma acidophilum with a focus on protein com-plexesrdquoMolecular and Cellular Proteomics vol 6 no 3 pp 492ndash502 2007

[56] L S Madding J K Michel K R Shockley et al ldquoRole of the 1205731subunit in the function and stability of the 20S proteasome inthe hyperthermophilic Archaeon Pyrococcus furiosusrdquo Journalof Bacteriology vol 189 no 2 pp 583ndash590 2007

[57] R Shibata Y Bessho A Shinkai et al ldquoCrystal structureand RNA-binding analysis of the archaeal transcription factorNusArdquo Biochemical and Biophysical Research Communicationsvol 355 no 1 pp 122ndash128 2007

[58] W Bae B Xia M Inouye and K Severinov ldquoEscherichia coliCspA-family RNA chaperones are transcription antitermina-torsrdquo Proceedings of the National Academy of Sciences of theUnited States of America vol 97 no 14 pp 7784ndash7789 2000

[59] R J Ram N C VerBerkmoes M P Thelen et al ldquoMicrobi-ology community proteomics of a natural microbial biofilmrdquoScience vol 308 no 5730 pp 1915ndash1920 2005

[60] I Noel-Georis T Vallaeys R Chauvaux et al ldquoGlobal analysisof the Ralstonia metallidurans proteome prelude for the large-scale study of heavy metal responserdquo Proteomics vol 4 no 1pp 151ndash179 2004

16 Archaea

[61] M TMNovo A CDa Silva RMoreto et al ldquoThiobacillus fer-rooxidans response to copper and other heavy metals growthprotein synthesis and protein phosphorylationrdquo Antonie vanLeeuwenhoek vol 77 no 2 pp 187ndash195 2000

[62] Q She R K Singh F Confalonieri et al ldquoThe complete genomeof the crenarchaeon Sulfolobus solfataricus P2rdquo Proceedings ofthe National Academy of Sciences of the United States of Americavol 98 no 14 pp 7835ndash7840 2001

[63] R P Anderson and J R Roth ldquoTandem genetic duplications inphage and bacteriardquo Annual Review of Microbiology vol 31 pp473ndash505 1977

[64] D Gevers K Vandepoele C Simillion and Y Van De PeerldquoGene duplication and biased functional retention of paralogsin bacterial genomesrdquo Trends in Microbiology vol 12 no 4 pp148ndash154 2004

[65] A B Reams and E L Neidle ldquoSelection for gene clustering bytandemduplicationrdquoAnnual Review ofMicrobiology vol 58 pp119ndash142 2004

[66] L H Orellana and C A Jerez ldquoA genomic island providesAcidithiobacillus ferrooxidans ATCC 53993 additional copperresistance a possible competitive advantagerdquo Applied Microbi-ology and Biotechnology vol 92 no 4 pp 761ndash767 2011

[67] A B Brinkman T J G Ettema W M de Vos and J van derOost ldquoThe Lrp family of transcriptional regulatorsrdquo MolecularMicrobiology vol 48 no 2 pp 287ndash294 2003

[68] C Vollmecke S L Drees J Reimann S V Albers and MLubben ldquoThe ATPases CopA and CopB both contribute tocopper resistance of the thermoacidophilic archaeon SulfolobussolfataricusrdquoMicrobiology vol 158 no 6 pp 1622ndash1633 2012

[69] A Villafane Y Voskoboynik I Ruhl et al ldquoCopR of Sulfolobussolfataricus represents a novel class of archaeal-specific copper-responsive activators of transcriptionrdquo Microbiology vol 157no 10 pp 2808ndash2817 2011

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Anatomy Research International

PeptidesInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporation httpwwwhindawicom

International Journal of

Volume 2014

Zoology

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Molecular Biology International

GenomicsInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioinformaticsAdvances in

Marine BiologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Signal TransductionJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

Evolutionary BiologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Biochemistry Research International

ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Genetics Research International

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Advances in

Virolog y

Hindawi Publishing Corporationhttpwwwhindawicom

Nucleic AcidsJournal of

Volume 2014

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Enzyme Research

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

International Journal of

Microbiology

Archaea 9

150pb

(a)

1016 pb

(b)

2000 pb

(c)

Figure 4 CODEHOP-based PCR for the amplification of the putative genes copA and copM from S metallicus Amplification products(a) using degenerate PCR primers (DOP) copM degF and copM degR designed from amino acid sequence conserved blocks of SulfolobalesCopM proteins (b) using primers copA cdegF and copA cdegR designed by CODEHOP strategy for amplification of a copA-like gene (c)Primers copM degF and copA cdegRwere assesed for the amplification of a copMA-likeDNA regionThose primerswere testedwith genomicS metallicus DNA samples Amplicons expected sizes were as follows ca 150 bp for copM ca 950 bp for copA and ca 2000 bp for copMAPCR products were excised and cloned for later sequencing

proteasome subunit was also found to be up-regulated whenS metallicus was subjected to Cd stress (Table 2)

The transcriptional regulation-related proteins corre-sponded to a putative transcription regulator (spot 32) andthe transcription factor NusA (spot 33) involved in theintrinsic termination of transcription [57] NusA has beenalso reported to display important functions in the cellularresponse to adverse factors [58] The expression of twotranscriptional regulators related to amino acids biosynthesisand metal-dependent genetic repression were found to beinduced in P furiosus cells subjected to oxidative stress[48] Moreover the expression of several genes encodingpredicted transcriptional regulators were induced followingCd exposure in T gammatolerans cells [27]

Spots 5 6 13 14 15 21 22 30 and 31 (Table 2) did notshow significant scores with any protein sequences currentlyavailable in the data bases Interestingly spots 2 4 9 13 21and 30 were found to be commonly upregulated when Smetallicus cells were treated with either Cu or Cd (Figure 3Table 2)

The results presented here are consistent with previ-ous work describing global protein expression profiles inresponse to heavy metals as reported for F acidarmanus [1618] microbial-biofilm communities in an acid mine drainagesite [59] and other prokaryotic and eukaryotic microorgan-isms [24 60]Therefore a coordinated expression of differentgroups of genes suggests the existence of regulatory networkssuch as stress response mechanisms and respiratory chainadjustments to cope with the presence of heavy metal ions[61]

34 Cu-Resistance (Cop) Genes Cluster Is Duplicated in Smetallicus Genome A Cu-resistance (cop) locus has beendescribed to be highly conserved in archaeal genomeswhich consists of genes encoding a new type of archaealtranscriptional regulator (CopT) a metal-binding chaperone(CopM) and a Cu-transporting P-type ATPase (CopA) [19]Since the genome sequence of Smetallicus is not yet availablewe searched for the presence of cop genes by using consen-sus degenerate hybrid oligonucleotide primers-based PCR(CODEHOP-PCR) CopA and CopM amino acid sequencealignments from S acidocaldarius S tokodaii and S solfa-taricus were used to identify conserved sequence blocks andtherefore to design either CODEHOP or DOP (degeneratehybrid oligonucleotide) primers (Figure S1 Table 1) TherebyPCR assays using S metallicus genomic DNA as templateyielded amplicons of ca 150 bp and ca 950 bp for copMand copA CODEHOP pair primers respectively (Figures4(a) 4(b)) As estimated from primer pairrsquos position onamino acid sequence alignments the obtained PCR productscorresponded to the expected sizes suggesting the presenceof copM- and copA-like genes in S metallicus genome Inorder to determine the identity of the amplified DNA frag-ments they were TA-cloned and sequenced Blastx sequenceanalysis showed that the 150 bp DNA fragment coded for anincomplete ORF sharing 63 homology with S solfataricusCopM (SSO10823) whereas the analysis of the 950 pb DNAfragment yielded a partial ORF sharing 54 homology withS solfataricus CopA protein (SSO2651)

The genomic organization of the putative cop genes in Smetallicus was analyzed to find out its similarity with those

10 Archaea

Table 3 Sequence identity comparison of S metallicus Cop proteins to those in other Sulfolobales

S metallicus protein Identity to homologue in other SulfolobalesS solfataricus P2 S acidocaldarius S tokodaii M sedula

CopA1 51 43 44 45CopA2 88 46 49 50CopM1 63 69 71 71CopM2 89 72 70 77CopT1 36 27 30 32CopT2lowast 90 52 52 53Boldface type indicates to which organism the S metallicus homologue shows the highest sequence identitylowastRefers to the analysis obtained using the uncompleted amino acid sequence of CopT2 which represents sim90 in length when compared with S solfataricusCopT sequence

previously described in S solfataricus P2 and F acidarmanuswhere the cop cluster consists of tandem-orientated genesas copTMA [18 19] Thus using copM degF (forward) andcopA cdegR (reverse) primers we attempted to amplify aputative copMA DNA region This PCR yielded a product ofca 2000 pb corresponding to the expected size and resultedin 2 ORFs showing high sequence homology with CopMand CopA from S solfataricus (Figure 4(c)) Interestinglywhen aligning this copMA sequence with the copA and copMsequences obtained previously they were not identical whichstrongly indicated the presence of duplicated putative copgenes encoding for both Cu-ATPases (copA1 and copA2) andmetallochaperones (copM1 and copM2) in the S metallicusgenome

To isolate the entire cop gene sequences the genomewalking method described by Acevedo et al [38] was usedSix different DNA libraries were constructed from S metalli-cus genomic DNA digested with several restriction enzymesincluding 51015840 overhang and blunt end restriction enzymesThe DNA libraries were used as templates in 2 successivePCR reactions From the previously known sequences twospecific sense primers for the 31015840 end amplification and twospecific antisense primers for the 51015840 end amplification weredesigned (abbreviated SP1 and SP2) Therefore the 51015840 and 31015840ends of the putative genes copA1 and copA2 that had beenpartially identified through the genome walking techniquewere amplified

By overlapping the sequence isolated by degenerate PCRand the lateral sequences obtained by genome walking(Figure S2) it was possible to complete the whole nucleotidesequence of the putative gene copA1 Additionally it waspossible to confirm the presence of the upstream copM1 geneanddetermine the occurrence of a partial copT1 gene tandem-orientated upstream of copM1 (Figure 5)Through additionalgenome walking experiments the partial copT1 sequencecould be further completedThe samewas truewhen isolatingthe whole nucleotide sequence of the putative gene copA2as the presence of copM2 and copT2 genes were confirmedupstream of copA2 However it was not possible to completethe whole 51015840 end of the putative gene copT2 as its determinedlength corresponded to only 90 of the full length whencompared with S solfataricus copT gene (Figure 5)

In conclusion degenerate PCR together with genomewalking experiments allowed to describe the occurrence inS metallicus genome of 2 cop genes loci (named as locus cop1and locus cop2) coding for paralogous genes whose productsmay be involved in Cu-resistance (Figure 5) Although somearchaeal genomes exhibit two copies of putative Cu-P-typeATPases as described for S solfataricus [62] the genomicarrangement displayed as a cop gene cluster (copTMA) hasbeen reported to be represented in only one copy for manyarchaeal genomes [10 19 21] Moreover we further updatedthis analysis by searching for duplications of the cop genescluster in all available archaeal genome sequences up to date(October 2012) retrieving only one cop locus in each caseThereby we propose that the discovery of this cop gene clusterduplication in the genome of S metallicus constitutes so faran unprecedented feature for a representative of the Archaeadomain and might contribute to its high metal resistance

In this context it has been widely reported that increasedgene copy number can increase gene expression allowingprokaryotic to thrive under growth limiting conditions [1363ndash68] However alongside the augmented gene copy onecannot exclude that the cop operon duplication in S metal-licus may offer among others a wider repertoire of Cu-ATPases which might differ in terms of metal affinitiesandor specificity efflux rates andor differences in theirabundance regulation

Throughout amino acid sequence analysis it was furtherdetermined that each polypeptide encoded by S metallicuswith the exception of CopT1 showed to be highly homol-ogous to their orthologous counterparts encoded by othersSulfolobales (Table 3) Moreover the 3 ORFs encoded by thelocus cop2 shared about 90 identity with the correspondingS solfataricus orthologous peptides (Table 3) S metallicuscop genes products showed characteristic domains referredto as critical for their respective proposed biological activitiesS metallicus paralogous gene products CopA1 (749 aa1015840)and CopA2 (747 aa1015840) shared 513 of identity These twoputative Cu-ATPases (CopA1 and CopA2) contain the aminoacid sequence motif CPCALGLA which has been proposedto confer Cu-transporting specificity in CPx-ATPases [48]and has been also found in other CopA-like proteins frombiomining Bacteria and Archaea [14]

Archaea 11

HindIII HindIII HindIII

HindIII

CopT1Locus cop1 M1

EcoRIEcoRV

513 bp 219 bp 2250 bp

CopA1

(a)

Locus cop2 M2

EcoRI

CopA2

453 bp 171 bp 2244 bp

CopT2

PstIHindIII HindIII HindIII

(b)

Figure 5 Schematic representation of the two cop gene clusters isolated from S metallicus genome Each cluster codes for anArchaea-specifictranscriptional regulator (copT) a metallochaperone (copM) and a P-type Cu-exporting ATPase (copA) Lengths of each gene are indicatedLocus cop1 corresponds to 4346 sequenced base pairs and shows an intergenic region copT1-M1 of 232 base pairs Locus cop2 correspondsto 3435 base pairs and shows 89 base pairs copT2-M2 intergenic region Genes copM2 and copA2 overlapped in 11 base pairs copT2 was notfully isolated

Control CdSO4 Ag2SO4 NiSO4 ZnSO4 CuSO4

kb3kb2kb1

02 kb

(a)

kb3kb2kb1

02 kb

(b)

(c)

Figure 6 Northern blot analysis to determine the expression of S metallicus copA1 (a) and copA2 (b) putative genes in response to variousmetals S metallicus total RNAwas extracted from cultures grown either in the absence (control) or the presence of CuSO

4(50mM) Ag

2SO4

(008mM) NiSO4(15mM) ZnSO

4(50mM) and CdSO

4(2mM) P32-radioactivelly labelled DNA fragments annealing to copA1 and copA2

sequences respectively were used as probes (c) shows rRNAs as total RNA loading control

On the other hand CopM1 (71 aa) and CopM2 (56 aa)sharing 66 of identity present the proposed metal bindingdomain TRASH (trafficking resistance and sensing of heavymetals) [10] Additionally when analyzing the putative tran-scriptional regulators the presence of a C-terminal TRASHdomain in both CopT1 and CopT2 was also found CopT1sequence also contains an entire N-terminal helix-turn-helix (HTH) motif (not shown) that resembles the DNA-bindingmotifs of prokaryotic transcriptional regulators suchas Lrp-like proteins [67] The HTH motif was only partiallyidentified in CopT2 since the 51015840 region of copT1 gene has notbeen yet fully isolated

35 Transcriptional Analysis of S metallicus Cop Genes Toget insight into the role of the two cop loci the expressionof the corresponding genes was analyzed under variousconditions S metallicus was first grown in presence ofdifferent heavy metals (Cu Zn Cd Ni o Ag) at a givenconcentration that did not affect growth kinetics [30 42]Subsequently total RNA was isolated from late exponentiallygrown cultures and Northern blot experiments were carriedout in order to determine the expression of both copA1 andcopA2 genes As depicted in Figure 6 transcription of bothcopA1 and copA2messengers were specifically induced in thepresence of Cu and Cd ions This gene expression pattern

12 Archaea

954pb

M1 CopA1

(a)

1438 pb

M2 CopA2

(b)

1000 pb

800pb

+ minus

(c)

+ minus

1500 pb

1000 pb

(d)

Figure 7 Cotranscription analysis of copMA1 and copMA2 genes cDNA was synthesized with a reverse primer (dotted arrows) hybridizingtoward the 31015840 end of either copA1 (a) or copA2 (b) S metallicus total RNA was extracted at the late exponential phase from a culture growingin presence of 20mMCu PCR amplifications were carried out with these cDNAs and each corresponding primer pair (black arrows) as listedin Table 1 (c) and (d) show RT-PCR products obtained for the copMA1 and copMA2 intergenic regions respectively Reverse transcriptasereactions with (+) and without (minus) the Improm II reverse transcriptase enzyme were carried out in order to exclude amplification due togenomic DNA contamination Expected sizes (in base pairs) for the corresponding PCR products are given in (a) and (b)

is in good agreement with what has been described for Ssolfataricus where the bicistronic copMA transcript levelswere also found to be up-regulated in response to both Cuand Cd [19]

Northern blot analysis showed that the sizes seen for bothcopA transcripts (sim25 Kb) did not correspond exactly withthe individual genes length (each of about 225Kb) (Figure 6)This strongly suggested that the transcripts could also includethe respective copM gene located upstream of copA in eachcop loci To confirm this assumption a co-transcriptionexperiment was done (Figure 7) in which the cDNAs wereobtained by using RNA extracted from a culture grownin the presence of 20mM Cu and using a reverse primerhybridizing with copA1 (Figure 7(a)) or copA2 (Figure 7(b))gene sequence respectively PCR amplifications were carriedout by using the corresponding cDNAs as templates and eachpair of primers lying in adjacent genes (copM) The presenceof an amplicon of the expected size in each case indicatedthe adjacent genes were part of polycistronic messengers(Figure 7) These results clearly show that in S metallicuseach couple of copMA genes was expressed in the form oftranscriptional units as reported in F acidarmanus and Ssolfataricus [18 19] The coexpression of gene pairrsquos copMAmay suggest a coordinated and dependent function for therespective encoded proteins In the bacterium E hirae themetallochaperone CopZ (CopM in S metallicus) fulfills apivotal role in the mechanism of Cu homeostasis [45] Thusit was shown that this protein interacts directly with the Cu-ATPase CopB (CopA in S metallicus) handing the Cu forsubsequent removal In this context one might expect thatproteins CopM1 and CopM2 from S metallicus have a similarrole to that described for E hirae Furthermore quantitativeRT-PCR experiments were carried out in order to determinedifferential expression of the cop genes in response to differentCu concentrations The expression of copA1 copA2 copT1and copT2 was tested relative to the transcript levels ofrRNA 16S gene since its levels were not significantly affected

in all assessed conditions (data not shown) As shown inFigure 8(a) copA2 and copA1 transcript levels were foundto be concomitantly increased with the increasing Cu con-centrations present in the medium Higher transcripts levelsof copA1 were seen in all tested conditions when comparedwith copA2 levels copA1 transcript levels were found to be325-fold up-regulated when comparing 50mMCu conditionversuscontrol (absence of metal) whereas copA2 transcriptlevels showed an increment of only 175-fold (Figure 8(a))The finding that Cu-ATPases mRNA levels were significantlyincreased in response to Cu ions exposure suggests that thetransport system may operate for Cu efflux in S metallicus

Moreover copA2 and copA1 gene expression was quan-tified when S metallicus was grown using chalcopyrite(CuFeS

2) ore as an oxidizable substrate (Figure 8(a)) Mineral

oxidation mediated by the microorganism generates a pro-gressive increase in Cu ions concentration in the medium Tofind out whether the amount of solubilized Cu induced theexpression of the Cu-ATPases genes total RNAwas extractedfrom a S metallicus culture grown to late exponential phaseand in the presence of 1 CuFeS

2 It was clear that an

up-regulation of the Cu-ATPases encoding genes also tookplace when S metallicus was grown in the presence ofCuFeS

2(Figure 8(a)) copA1 transcript levels increased 14-fold

compared with the control condition in the absence of Cuwhile copA2 gene expression showed an increase of 76 foldAlong with this bymeans of atomic absorption spectrometry(AAS) analysis an overall amount of solubilized Cu ions(Cu2+Cu1+) of 144plusmn21mMwas determined to be present inthemedium indicating that copA2 and copA1 gene expressionwas most likely in response to the Cu present in the culturedue to CuFeS

2microbial-solubilization

The finding that copA1 was highly expressed comparedwith copA2 in all tested conditions may suggest a possiblephysiological hierarchy between the two ATPases whenovercoming either Cu or Cd stress In this regard by meansof a genetic approach it was demonstrated in S solfataricus

Archaea 13

700

600

500

400

300

200

100

00

5 20 50 CuFeS2CuSO4 concentration (mM)

Relativ

egenee

xpression

CopA2CopA1

(a)

8

6

7

5

4

3

2

1

00

5 20 50 CuFeS2CuSO4 concentration (mM)

Relativ

egenee

xpression

CopT1CopT2

(b)

Figure 8 Relative expression levels of S metallicus genes copA1 and copA2 (a) copT1 and copT2 (b) Expression of S metallicus cop geneswasassessed by qRT-PCR and normalized against 16S rRNA gene expression in individual cultures Mean values and standard deviations arefrom analyses of three independent cultures grown in the presence of 0 5 20 50mMCu or in 1 of a chalcopyrite ore (CuFeS

2) concentrate

that while CopA was an effective Cu efflux transporterat low and high Cu concentrations the other Cu-ATPase(CopB) only appeared to be a low-affinity Cu export ATPase[68] Moreover by using a M sedula copA deletion mutantit was demonstrated that this strain compromised metalresistance and consequently abolished chalcopyrite oxidation[22] highlighting the role of Cu detoxification mechanismsduring a given bioleaching process Our attempts to showthe functionality of CopA1 and CopA2 from S metallicusby using E coli as a heterologous host were not successfulApparently the overexpression of these ORFs had a toxiceffect on E coli that compromised its viability Althoughgene disruption tools have not been yet developed for Smetallicus the functional role of both copTMA locimight befurther studied by cross-species complementation of a coppersensitive S solfataricus copR mutant as it was described byMaezato et al [22] It will be of great interest to establishin future studies the possible functionality of the isolatedputative transporters from S metallicus

Likewise quantitative RT-PCR experiments showed thatcopT1 transcript levels increased concomitantly with increas-ing Cu concentrations whereas copT2 showed relativelysimilar transcripts levels most likely indicating a constitutiveexpression profile (Figure 8(b)) Furthermore whereas copT1transcript levels were found to be increased 36 fold inCuFeS

2

grown cultures copT2 levels remained unchanged in com-parison with the control in the absence of Cu (Figure 8(b))The results obtained for S metallicus copT2 gene expressionare consistent with those reported for S solfataricus CopTtranscriptional regulator has been proposed to function as arepressor in S solfataricus showing a constitutive expressionthat in the presence of Cu loses its affinity for the promoterregion of copMA allowing the expression of this polycistron[19] In contrast the increased transcripts levels of both copA1and copT1 concomitant with higher Cu concentrations in the

environment suggest that the putative transcriptional regula-tor CopT1 may act by activating both copMA1 and probablyitself (Figure 8) Regarding this it was recently reported thatCopR (CopT in S metallicus) from S solfataricus strain 982acts as an activator of copT (copM in S metallicus) and copAexpression [69] Nevertheless additional experiments wouldbe required to test this possibility in S metallicus

4 Concluding Remarks

We previously reported that S metallicus resists extremelyhigh Cu concentrations which was mediated to some extentby the use of a possible metal resistance system based oninorganic polyphosphate hydrolysis and consequently Cu-PO4

2minusefflux [15 29 30] Here we have addressed the questionwhether this microorganism coded for other determinantsthat might help to explain its high heavy metal toleranceAs the genomic sequence of this microorganism is notyet available we jointly used CODEHOP-PCR and genomewalking approaches and were able to establish the occurrenceof two nonidentical homologous cop loci into the genome ofS metallicus (cop1 and cop2) Each cop locus codes for anarchaeal transcriptional regulator (CopT) a metal-bindingchaperone (CopM) and a Cu-transporting P-type ATPase(CopA) High levels of the polycistronic mRNAscopMA ofeach cop locus were observed after treatment with either Cuor Cd suggesting that the encoded ATPases efflux heavymetals out in order to detoxify the intracellular environmentAltogether previous reports and the results obtained in thisstudy allow us to suggest that some key elements that mayexplain the high resistance to Cu in S metallicus is the dupli-cation of the Cu resistance cop genes a defensive responseto stress and a polyP-based accumulation mechanism Inthe case of Cd although some similar stress responses were

14 Archaea

observed whether comparable Cu-responsive elements alsoparticipate in Cd responses remains to be seen

Conflict of Interests

The authors declare that the research was done in the absenceof any commercial or financial relationships that could beconstrued as a potential conflict of interests

Acknowledgments

This work was supported by Grant no FONDECYT 1110214and in part by ICM P-05-001-F project

References

[1] A Schippers ldquoMicroorganisms involved in bioleaching andnucleic acid-based molecular methods for their identificationand quantificationrdquo in Microbial Processing of Metal SulfidesR E Donati and W Sand Eds pp 3ndash33 Elsevier BerlinGermany 2007

[2] H R Watling ldquoThe bioleaching of sulphide minerals withemphasis on copper sulphides a reviewrdquo Hydrometallurgy vol84 no 1-2 pp 81ndash108 2006

[3] D E Rawlings D Dew and C Du Plessis ldquoBiomineralizationof metal-containing ores and concentratesrdquo Trends in Biotech-nology vol 21 no 1 pp 38ndash44 2003

[4] L Valenzuela A Chi S Beard et al ldquoGenomics metagenomicsand proteomics in biomining microorganismsrdquo BiotechnologyAdvances vol 24 no 2 pp 197ndash211 2006

[5] C A Jerez ldquoBioleaching and biomining for the industrialrecovery of metalsrdquo in Comprehensive Biotechnology M Moo-Young Ed vol 3 pp 717ndash729 Elsevier Amstaerdam TheNetherlands 2nd edition 2011

[6] P R Norris and J P Owen ldquoMineral sulphide oxidationby enrichment cultures of novel thermoacidophilic bacteriardquoFEMS Microbiology Reviews vol 11 no 1ndash3 pp 51ndash56 1993

[7] D E Rawlings and D B Johnson ldquoThe microbiology ofbiomining development and optimization ofmineral-oxidizingmicrobial consortiardquo Microbiology vol 153 no 2 pp 315ndash3242007

[8] P R Norris N P Burton and N A M Foulis ldquoAcidophiles inbioreactor mineral processingrdquo Extremophiles vol 4 no 2 pp71ndash76 2000

[9] M R Bruins S Kapil and F W Oehme ldquoMicrobial resistancetometals in the environmentrdquo Ecotoxicology and EnvironmentalSafety vol 45 no 3 pp 198ndash207 2000

[10] T J G Ettema M A Huynen W M De Vos and J Van DerOost ldquoTRASH a novel metal-binding domain predicted to beinvolved in heavy-metal sensing trafficking and resistancerdquoTrends in Biochemical Sciences vol 28 no 4 pp 170ndash173 2003

[11] M Dopson C Baker-Austin P R Koppineedi and P L BondldquoGrowth in sulfidic mineral environments metal resistancemechanisms in acidophilic micro-organismsrdquo Microbiologyvol 149 no 8 pp 1959ndash1970 2003

[12] S Franke and C Rensing ldquoAcidophiles Mechanisms to toleratemetal and acid toxicityrdquo in Physiology and Biochemistry ofExtremophiles C Gerday and N Glansdorff Eds pp 271ndash278ASM Press Washington DC USA 2007

[13] C A Navarro L H Orellana C Mauriaca and C A JerezldquoTranscriptional and functional studies of Acidithiobacillusferrooxidans genes related to survival in the presence of copperrdquoApplied and Environmental Microbiology vol 75 no 19 pp6102ndash6109 2009

[14] A Orell C A Navarro R Arancibia J C Mobarec andC A Jerez ldquoLife in blue copper resistance mechanisms ofbacteria and Archaea used in industrial biomining of mineralsrdquoBiotechnology Advances vol 28 no 6 pp 839ndash848 2010

[15] A Orell C A Navarro M Rivero J S Aguilar and C A JerezldquoInorganic polyphosphates in extremophiles and their posiblefunctionsrdquo Extremophiles vol 16 no 4 pp 573ndash583 2012

[16] C Baker-Austin M Dopson M Wexler et al ldquoExtremearsenic resistance by the acidophilic archaeon ldquoFerroplasmaacidarmanusrdquo Fer1rdquo Extremophiles vol 11 no 3 pp 425ndash4342007

[17] E Pedone S Bartolucci and G Fiorentino ldquoSensing andadapting to environmental stress the archaeal tacticrdquo Frontiersin Bioscience vol 9 pp 2909ndash2926 2004

[18] C Baker-Austin M Dopson M Wexler R G Sawers and PL Bond ldquoMolecular insight into extreme copper resistance inthe extremophilic archaeon ldquoFerroplasma acidarmanusrdquo Fer1rdquoMicrobiology vol 151 no 8 pp 2637ndash2646 2005

[19] T J G Ettema A B Brinkman P P Lamers N G Kornet WM de Vos and J van der Oost ldquoMolecular characterization ofa conserved archaeal copper resistance (cop) gene cluster andits copper-responsive regulator in Sulfolobus solfataricus P2rdquoMicrobiology vol 152 no 7 pp 1969ndash1979 2006

[20] A A Villafane Y VoskoboynikM Cuebas I Ruhl and E BinildquoResponse to excess copper in the hyperthermophile Sulfolobussolfataricus strain 982rdquo Biochemical and Biophysical ResearchCommunications vol 385 no 1 pp 67ndash71 2009

[21] K S Auernik Y Maezato P H Blum and R M KellyldquoThe genome sequence of the metal-mobilizing extremelythermoacidophilic archaeon Metallosphaera sedula providesinsights into bioleaching-associated metabolismrdquo Applied andEnvironmental Microbiology vol 74 no 3 pp 682ndash692 2008

[22] Y Maezato T Johnson S McCarthy K Dana and P BlumldquoMetal resistance and lithoautotrophy in the extreme ther-moacidophile Metalosphaera sedulardquo Journal of Bacteriologyvol 194 no 24 pp 6856ndash6863 2012

[23] D H Nies ldquoMicrobial heavy-metal resistancerdquo Applied Micro-biology and Biotechnology vol 51 no 6 pp 730ndash750 1999

[24] K Vido D Spector G Lagniel S Lopez M B Toledano andJ Labarre ldquoA proteome analysis of the cadmium response inSaccharomyces cerevisiaerdquo The Journal of Biological Chemistryvol 276 no 11 pp 8469ndash8474 2001

[25] G Bertin and D Averbeck ldquoCadmium cellular effects mod-ifications of biomolecules modulation of DNA repair andgenotoxic consequences (a review)rdquo Biochimie vol 88 no 11pp 1549ndash1559 2006

[26] M H Joe S W Jung S H Im et al ldquoGenome-wide responseof Deinococcus radiodurans on cadmium toxicityrdquo Journal ofMicrobiology and Biotechnology vol 21 no 4 pp 438ndash447 2011

[27] A Lagorce A Fourcans M Dutertre B Bouyssiere YZivanovic and F Confalonieri ldquoGenome-wide transcriptionalresponse of the archeon Thermococcus gammatolerans to cad-miumrdquo PLOS ONE vol 7 no 7 Article ID e41935 2012

[28] A Kornberg N N Rao and D Ault-Riche ldquoInorganicpolyphosphate a molecule of many functionsrdquo Annual Reviewof Biochemistry vol 68 pp 89ndash125 1999

Archaea 15

[29] J D Keasling ldquoRegulation of intracellular toxic metals andother cations by hydrolysis of polyphosphaterdquoAnnals of theNewYork Academy of Sciences vol 829 pp 242ndash249 1997

[30] F Remonsellez A Orell and C A Jerez ldquoCopper tolerance ofthe thermoacidophilic archaeon Sulfolobus metallicus possiblerole of polyphosphate metabolismrdquoMicrobiology vol 152 no 1pp 59ndash66 2006

[31] V Hatzimanikatis L H Choe and K H Lee ldquoProteomicstheoretical and experimental considerationsrdquo BiotechnologyProgress vol 15 no 3 pp 312ndash318 1999

[32] L H Choe and K H Lee ldquoA comparison of three commerciallyavailable isoelectric focusing units for proteome analysis theMultiphor the IPGphor and the Protean IEF cellrdquo Electrophore-sis vol 21 no 5 pp 993ndash1000 2000

[33] U K Laemmli ldquoCleavage of structural proteins during theassembly of the head of bacteriophage T4rdquo Nature vol 227 no5259 pp 680ndash685 1970

[34] A Shevchenko M Wilm O Vorm and M Mann ldquoMassspectrometric sequencing of proteins from silver-stained poly-acrylamide gelsrdquo Analytical Chemistry vol 68 no 5 pp 850ndash858 1996

[35] P Giavalisco E Nordhoff T Kreitler et al ldquoProteome analysisof Arabidopsis thaliana by two-dimensional gel electrophoresisand matrix-assisted laser desorptionionisation-time of flightmass spectrometryrdquo Proteomics vol 5 no 7 pp 1902ndash19132005

[36] T M Rose E R Schultz J G Henikoff S Pietrokovski CM McCallum and S Henikoff ldquoConsensus-degenerate hybridoligonucleotide primers for amplification of distantly relatedsequencesrdquo Nucleic Acids Research vol 26 no 7 pp 1628ndash16351998

[37] T M Rose J G Henikoff and S Henikoff ldquoCODEHOP(Consensus-Degenerate Hybrid Oligonucleotide Primer) PCRprimer designrdquo Nucleic Acids Research vol 31 no 13 pp 3763ndash3766 2003

[38] J P Acevedo F Reyes L P Parra O Salazar B A Andrews andJ A Asenjo ldquoCloning of complete genes for novel hydrolyticenzymes from Antarctic sea water bacteria by use of animproved genomewalking techniquerdquo Journal of Biotechnologyvol 133 no 3 pp 277ndash286 2008

[39] S Bathe and P R Norris ldquoFerrous iron- and sulfur-inducedgenes in Sulfolobus metallicusrdquo Applied and EnvironmentalMicrobiology vol 73 no 8 pp 2491ndash2497 2007

[40] J Sambrook E F Fritsch and TManiatisMolecular Cloning ALaboratory Manual Cold Spring Harbor Press New York NYUSA 1989

[41] K W Miller S Sass Risanico and J B Risatti ldquoDifferentialtolerance of Sulfolobus strains to transition metalsrdquo FEMSMicrobiology Letters vol 93 no 1 pp 69ndash73 1992

[42] G Huber C Spinnler A Gambacorta and K O Stetter ldquoMet-allosphaera sedula gen and sp nov represents a new genus ofaerobic metal-mobilizing thermoacidophilic archaebacteriardquoSystematic and Applied Microbiology vol 12 pp 38ndash47 1989

[43] J Llanos C Capasso E Parisi D Prieur and C JeanthonldquoSusceptibility to heavy metals and cadmium accumulation inaerobic and anaerobic thermophilic microorganisms isolatedfrom deep-sea hydrothermal ventsrdquo Current Microbiology vol41 no 3 pp 201ndash205 2000

[44] A Kaur M Pan M Meislin M T Facciotti R El-Gewelyand N S Baliga ldquoA systems view of haloarchaeal strategies towithstand stress from transition metalsrdquo Genome Research vol16 no 7 pp 841ndash854 2006

[45] M Solioz and J V Stoyanov ldquoCopper homeostasis in Entero-coccus hiraerdquo FEMS Microbiology Reviews vol 27 no 2-3 pp183ndash195 2003

[46] S Sitthisak K Howieson C Amezola and R K JayaswalldquoCharacterization of a multicopper oxidase gene from Staphy-lococcus aureusrdquo Applied and Environmental Microbiology vol71 no 9 pp 5650ndash5653 2005

[47] L Rodrıguez-Montelongo S I Volentini R N Farıas E MMassa and V A Rapisarda ldquoThe Cu(II)-reductase NADHdehydrogenase-2 of Escherichia coli improves the bacterialgrowth in extreme copper concentrations and increases theresistance to the damage caused by copper and hydroperoxiderdquoArchives of Biochemistry and Biophysics vol 451 no 1 pp 1ndash72006

[48] E Williams T M Lowe J Savas and J DiRuggiero ldquoMicroar-ray analysis of the hyperthermophilic archaeon Pyrococcusfuriosus exposed to gamma irradiationrdquo Extremophiles vol 11no 1 pp 19ndash29 2007

[49] L M Gaetke and C K Chow ldquoCopper toxicity oxidative stressand antioxidant nutrientsrdquo Toxicology vol 189 no 1-2 pp 147ndash163 2003

[50] M J Davies ldquoThe oxidative environment and protein damagerdquoBiochimica et Biophysica Acta vol 1703 no 2 pp 93ndash109 2005

[51] J R Thompson J K Bell J Bratt G A Grant and LJ Banaszak ldquoVmax regulation through domain and subunitchanges The active form of phosphoglycerate dehydrogenaserdquoBiochemistry vol 44 no 15 pp 5763ndash5773 2005

[52] V Consalvi R Chiaraluce L Politi A Gambacorta MDe Rosa and R Scandurra ldquoGlutamate dehydrogenase fromthe thermoacidophilic archaebacterium Sulfolobus solfataricusrdquoEuropean Journal of Biochemistry vol 196 no 2 pp 459ndash4671991

[53] A J L Macario M Lange B K Ahring and E Conway DeMacario ldquoStress genes and proteins in the archaeardquo Microbiol-ogy and Molecular Biology Reviews vol 63 no 4 pp 923ndash9671999

[54] J AMaupin-FurlowM A Gil M A Humbard et al ldquoArchaealproteasomes and other regulatory proteasesrdquo Current Opinionin Microbiology vol 8 no 6 pp 720ndash728 2005

[55] N Sun F Beck R Wilhelm Knispel et al ldquoProteomics analysisof Thermoplasma acidophilum with a focus on protein com-plexesrdquoMolecular and Cellular Proteomics vol 6 no 3 pp 492ndash502 2007

[56] L S Madding J K Michel K R Shockley et al ldquoRole of the 1205731subunit in the function and stability of the 20S proteasome inthe hyperthermophilic Archaeon Pyrococcus furiosusrdquo Journalof Bacteriology vol 189 no 2 pp 583ndash590 2007

[57] R Shibata Y Bessho A Shinkai et al ldquoCrystal structureand RNA-binding analysis of the archaeal transcription factorNusArdquo Biochemical and Biophysical Research Communicationsvol 355 no 1 pp 122ndash128 2007

[58] W Bae B Xia M Inouye and K Severinov ldquoEscherichia coliCspA-family RNA chaperones are transcription antitermina-torsrdquo Proceedings of the National Academy of Sciences of theUnited States of America vol 97 no 14 pp 7784ndash7789 2000

[59] R J Ram N C VerBerkmoes M P Thelen et al ldquoMicrobi-ology community proteomics of a natural microbial biofilmrdquoScience vol 308 no 5730 pp 1915ndash1920 2005

[60] I Noel-Georis T Vallaeys R Chauvaux et al ldquoGlobal analysisof the Ralstonia metallidurans proteome prelude for the large-scale study of heavy metal responserdquo Proteomics vol 4 no 1pp 151ndash179 2004

16 Archaea

[61] M TMNovo A CDa Silva RMoreto et al ldquoThiobacillus fer-rooxidans response to copper and other heavy metals growthprotein synthesis and protein phosphorylationrdquo Antonie vanLeeuwenhoek vol 77 no 2 pp 187ndash195 2000

[62] Q She R K Singh F Confalonieri et al ldquoThe complete genomeof the crenarchaeon Sulfolobus solfataricus P2rdquo Proceedings ofthe National Academy of Sciences of the United States of Americavol 98 no 14 pp 7835ndash7840 2001

[63] R P Anderson and J R Roth ldquoTandem genetic duplications inphage and bacteriardquo Annual Review of Microbiology vol 31 pp473ndash505 1977

[64] D Gevers K Vandepoele C Simillion and Y Van De PeerldquoGene duplication and biased functional retention of paralogsin bacterial genomesrdquo Trends in Microbiology vol 12 no 4 pp148ndash154 2004

[65] A B Reams and E L Neidle ldquoSelection for gene clustering bytandemduplicationrdquoAnnual Review ofMicrobiology vol 58 pp119ndash142 2004

[66] L H Orellana and C A Jerez ldquoA genomic island providesAcidithiobacillus ferrooxidans ATCC 53993 additional copperresistance a possible competitive advantagerdquo Applied Microbi-ology and Biotechnology vol 92 no 4 pp 761ndash767 2011

[67] A B Brinkman T J G Ettema W M de Vos and J van derOost ldquoThe Lrp family of transcriptional regulatorsrdquo MolecularMicrobiology vol 48 no 2 pp 287ndash294 2003

[68] C Vollmecke S L Drees J Reimann S V Albers and MLubben ldquoThe ATPases CopA and CopB both contribute tocopper resistance of the thermoacidophilic archaeon SulfolobussolfataricusrdquoMicrobiology vol 158 no 6 pp 1622ndash1633 2012

[69] A Villafane Y Voskoboynik I Ruhl et al ldquoCopR of Sulfolobussolfataricus represents a novel class of archaeal-specific copper-responsive activators of transcriptionrdquo Microbiology vol 157no 10 pp 2808ndash2817 2011

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Anatomy Research International

PeptidesInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporation httpwwwhindawicom

International Journal of

Volume 2014

Zoology

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Molecular Biology International

GenomicsInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioinformaticsAdvances in

Marine BiologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Signal TransductionJournal of

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BioMed Research International

Evolutionary BiologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Biochemistry Research International

ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Genetics Research International

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Advances in

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Nucleic AcidsJournal of

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Enzyme Research

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

International Journal of

Microbiology

10 Archaea

Table 3 Sequence identity comparison of S metallicus Cop proteins to those in other Sulfolobales

S metallicus protein Identity to homologue in other SulfolobalesS solfataricus P2 S acidocaldarius S tokodaii M sedula

CopA1 51 43 44 45CopA2 88 46 49 50CopM1 63 69 71 71CopM2 89 72 70 77CopT1 36 27 30 32CopT2lowast 90 52 52 53Boldface type indicates to which organism the S metallicus homologue shows the highest sequence identitylowastRefers to the analysis obtained using the uncompleted amino acid sequence of CopT2 which represents sim90 in length when compared with S solfataricusCopT sequence

previously described in S solfataricus P2 and F acidarmanuswhere the cop cluster consists of tandem-orientated genesas copTMA [18 19] Thus using copM degF (forward) andcopA cdegR (reverse) primers we attempted to amplify aputative copMA DNA region This PCR yielded a product ofca 2000 pb corresponding to the expected size and resultedin 2 ORFs showing high sequence homology with CopMand CopA from S solfataricus (Figure 4(c)) Interestinglywhen aligning this copMA sequence with the copA and copMsequences obtained previously they were not identical whichstrongly indicated the presence of duplicated putative copgenes encoding for both Cu-ATPases (copA1 and copA2) andmetallochaperones (copM1 and copM2) in the S metallicusgenome

To isolate the entire cop gene sequences the genomewalking method described by Acevedo et al [38] was usedSix different DNA libraries were constructed from S metalli-cus genomic DNA digested with several restriction enzymesincluding 51015840 overhang and blunt end restriction enzymesThe DNA libraries were used as templates in 2 successivePCR reactions From the previously known sequences twospecific sense primers for the 31015840 end amplification and twospecific antisense primers for the 51015840 end amplification weredesigned (abbreviated SP1 and SP2) Therefore the 51015840 and 31015840ends of the putative genes copA1 and copA2 that had beenpartially identified through the genome walking techniquewere amplified

By overlapping the sequence isolated by degenerate PCRand the lateral sequences obtained by genome walking(Figure S2) it was possible to complete the whole nucleotidesequence of the putative gene copA1 Additionally it waspossible to confirm the presence of the upstream copM1 geneanddetermine the occurrence of a partial copT1 gene tandem-orientated upstream of copM1 (Figure 5)Through additionalgenome walking experiments the partial copT1 sequencecould be further completedThe samewas truewhen isolatingthe whole nucleotide sequence of the putative gene copA2as the presence of copM2 and copT2 genes were confirmedupstream of copA2 However it was not possible to completethe whole 51015840 end of the putative gene copT2 as its determinedlength corresponded to only 90 of the full length whencompared with S solfataricus copT gene (Figure 5)

In conclusion degenerate PCR together with genomewalking experiments allowed to describe the occurrence inS metallicus genome of 2 cop genes loci (named as locus cop1and locus cop2) coding for paralogous genes whose productsmay be involved in Cu-resistance (Figure 5) Although somearchaeal genomes exhibit two copies of putative Cu-P-typeATPases as described for S solfataricus [62] the genomicarrangement displayed as a cop gene cluster (copTMA) hasbeen reported to be represented in only one copy for manyarchaeal genomes [10 19 21] Moreover we further updatedthis analysis by searching for duplications of the cop genescluster in all available archaeal genome sequences up to date(October 2012) retrieving only one cop locus in each caseThereby we propose that the discovery of this cop gene clusterduplication in the genome of S metallicus constitutes so faran unprecedented feature for a representative of the Archaeadomain and might contribute to its high metal resistance

In this context it has been widely reported that increasedgene copy number can increase gene expression allowingprokaryotic to thrive under growth limiting conditions [1363ndash68] However alongside the augmented gene copy onecannot exclude that the cop operon duplication in S metal-licus may offer among others a wider repertoire of Cu-ATPases which might differ in terms of metal affinitiesandor specificity efflux rates andor differences in theirabundance regulation

Throughout amino acid sequence analysis it was furtherdetermined that each polypeptide encoded by S metallicuswith the exception of CopT1 showed to be highly homol-ogous to their orthologous counterparts encoded by othersSulfolobales (Table 3) Moreover the 3 ORFs encoded by thelocus cop2 shared about 90 identity with the correspondingS solfataricus orthologous peptides (Table 3) S metallicuscop genes products showed characteristic domains referredto as critical for their respective proposed biological activitiesS metallicus paralogous gene products CopA1 (749 aa1015840)and CopA2 (747 aa1015840) shared 513 of identity These twoputative Cu-ATPases (CopA1 and CopA2) contain the aminoacid sequence motif CPCALGLA which has been proposedto confer Cu-transporting specificity in CPx-ATPases [48]and has been also found in other CopA-like proteins frombiomining Bacteria and Archaea [14]

Archaea 11

HindIII HindIII HindIII

HindIII

CopT1Locus cop1 M1

EcoRIEcoRV

513 bp 219 bp 2250 bp

CopA1

(a)

Locus cop2 M2

EcoRI

CopA2

453 bp 171 bp 2244 bp

CopT2

PstIHindIII HindIII HindIII

(b)

Figure 5 Schematic representation of the two cop gene clusters isolated from S metallicus genome Each cluster codes for anArchaea-specifictranscriptional regulator (copT) a metallochaperone (copM) and a P-type Cu-exporting ATPase (copA) Lengths of each gene are indicatedLocus cop1 corresponds to 4346 sequenced base pairs and shows an intergenic region copT1-M1 of 232 base pairs Locus cop2 correspondsto 3435 base pairs and shows 89 base pairs copT2-M2 intergenic region Genes copM2 and copA2 overlapped in 11 base pairs copT2 was notfully isolated

Control CdSO4 Ag2SO4 NiSO4 ZnSO4 CuSO4

kb3kb2kb1

02 kb

(a)

kb3kb2kb1

02 kb

(b)

(c)

Figure 6 Northern blot analysis to determine the expression of S metallicus copA1 (a) and copA2 (b) putative genes in response to variousmetals S metallicus total RNAwas extracted from cultures grown either in the absence (control) or the presence of CuSO

4(50mM) Ag

2SO4

(008mM) NiSO4(15mM) ZnSO

4(50mM) and CdSO

4(2mM) P32-radioactivelly labelled DNA fragments annealing to copA1 and copA2

sequences respectively were used as probes (c) shows rRNAs as total RNA loading control

On the other hand CopM1 (71 aa) and CopM2 (56 aa)sharing 66 of identity present the proposed metal bindingdomain TRASH (trafficking resistance and sensing of heavymetals) [10] Additionally when analyzing the putative tran-scriptional regulators the presence of a C-terminal TRASHdomain in both CopT1 and CopT2 was also found CopT1sequence also contains an entire N-terminal helix-turn-helix (HTH) motif (not shown) that resembles the DNA-bindingmotifs of prokaryotic transcriptional regulators suchas Lrp-like proteins [67] The HTH motif was only partiallyidentified in CopT2 since the 51015840 region of copT1 gene has notbeen yet fully isolated

35 Transcriptional Analysis of S metallicus Cop Genes Toget insight into the role of the two cop loci the expressionof the corresponding genes was analyzed under variousconditions S metallicus was first grown in presence ofdifferent heavy metals (Cu Zn Cd Ni o Ag) at a givenconcentration that did not affect growth kinetics [30 42]Subsequently total RNA was isolated from late exponentiallygrown cultures and Northern blot experiments were carriedout in order to determine the expression of both copA1 andcopA2 genes As depicted in Figure 6 transcription of bothcopA1 and copA2messengers were specifically induced in thepresence of Cu and Cd ions This gene expression pattern

12 Archaea

954pb

M1 CopA1

(a)

1438 pb

M2 CopA2

(b)

1000 pb

800pb

+ minus

(c)

+ minus

1500 pb

1000 pb

(d)

Figure 7 Cotranscription analysis of copMA1 and copMA2 genes cDNA was synthesized with a reverse primer (dotted arrows) hybridizingtoward the 31015840 end of either copA1 (a) or copA2 (b) S metallicus total RNA was extracted at the late exponential phase from a culture growingin presence of 20mMCu PCR amplifications were carried out with these cDNAs and each corresponding primer pair (black arrows) as listedin Table 1 (c) and (d) show RT-PCR products obtained for the copMA1 and copMA2 intergenic regions respectively Reverse transcriptasereactions with (+) and without (minus) the Improm II reverse transcriptase enzyme were carried out in order to exclude amplification due togenomic DNA contamination Expected sizes (in base pairs) for the corresponding PCR products are given in (a) and (b)

is in good agreement with what has been described for Ssolfataricus where the bicistronic copMA transcript levelswere also found to be up-regulated in response to both Cuand Cd [19]

Northern blot analysis showed that the sizes seen for bothcopA transcripts (sim25 Kb) did not correspond exactly withthe individual genes length (each of about 225Kb) (Figure 6)This strongly suggested that the transcripts could also includethe respective copM gene located upstream of copA in eachcop loci To confirm this assumption a co-transcriptionexperiment was done (Figure 7) in which the cDNAs wereobtained by using RNA extracted from a culture grownin the presence of 20mM Cu and using a reverse primerhybridizing with copA1 (Figure 7(a)) or copA2 (Figure 7(b))gene sequence respectively PCR amplifications were carriedout by using the corresponding cDNAs as templates and eachpair of primers lying in adjacent genes (copM) The presenceof an amplicon of the expected size in each case indicatedthe adjacent genes were part of polycistronic messengers(Figure 7) These results clearly show that in S metallicuseach couple of copMA genes was expressed in the form oftranscriptional units as reported in F acidarmanus and Ssolfataricus [18 19] The coexpression of gene pairrsquos copMAmay suggest a coordinated and dependent function for therespective encoded proteins In the bacterium E hirae themetallochaperone CopZ (CopM in S metallicus) fulfills apivotal role in the mechanism of Cu homeostasis [45] Thusit was shown that this protein interacts directly with the Cu-ATPase CopB (CopA in S metallicus) handing the Cu forsubsequent removal In this context one might expect thatproteins CopM1 and CopM2 from S metallicus have a similarrole to that described for E hirae Furthermore quantitativeRT-PCR experiments were carried out in order to determinedifferential expression of the cop genes in response to differentCu concentrations The expression of copA1 copA2 copT1and copT2 was tested relative to the transcript levels ofrRNA 16S gene since its levels were not significantly affected

in all assessed conditions (data not shown) As shown inFigure 8(a) copA2 and copA1 transcript levels were foundto be concomitantly increased with the increasing Cu con-centrations present in the medium Higher transcripts levelsof copA1 were seen in all tested conditions when comparedwith copA2 levels copA1 transcript levels were found to be325-fold up-regulated when comparing 50mMCu conditionversuscontrol (absence of metal) whereas copA2 transcriptlevels showed an increment of only 175-fold (Figure 8(a))The finding that Cu-ATPases mRNA levels were significantlyincreased in response to Cu ions exposure suggests that thetransport system may operate for Cu efflux in S metallicus

Moreover copA2 and copA1 gene expression was quan-tified when S metallicus was grown using chalcopyrite(CuFeS

2) ore as an oxidizable substrate (Figure 8(a)) Mineral

oxidation mediated by the microorganism generates a pro-gressive increase in Cu ions concentration in the medium Tofind out whether the amount of solubilized Cu induced theexpression of the Cu-ATPases genes total RNAwas extractedfrom a S metallicus culture grown to late exponential phaseand in the presence of 1 CuFeS

2 It was clear that an

up-regulation of the Cu-ATPases encoding genes also tookplace when S metallicus was grown in the presence ofCuFeS

2(Figure 8(a)) copA1 transcript levels increased 14-fold

compared with the control condition in the absence of Cuwhile copA2 gene expression showed an increase of 76 foldAlong with this bymeans of atomic absorption spectrometry(AAS) analysis an overall amount of solubilized Cu ions(Cu2+Cu1+) of 144plusmn21mMwas determined to be present inthemedium indicating that copA2 and copA1 gene expressionwas most likely in response to the Cu present in the culturedue to CuFeS

2microbial-solubilization

The finding that copA1 was highly expressed comparedwith copA2 in all tested conditions may suggest a possiblephysiological hierarchy between the two ATPases whenovercoming either Cu or Cd stress In this regard by meansof a genetic approach it was demonstrated in S solfataricus

Archaea 13

700

600

500

400

300

200

100

00

5 20 50 CuFeS2CuSO4 concentration (mM)

Relativ

egenee

xpression

CopA2CopA1

(a)

8

6

7

5

4

3

2

1

00

5 20 50 CuFeS2CuSO4 concentration (mM)

Relativ

egenee

xpression

CopT1CopT2

(b)

Figure 8 Relative expression levels of S metallicus genes copA1 and copA2 (a) copT1 and copT2 (b) Expression of S metallicus cop geneswasassessed by qRT-PCR and normalized against 16S rRNA gene expression in individual cultures Mean values and standard deviations arefrom analyses of three independent cultures grown in the presence of 0 5 20 50mMCu or in 1 of a chalcopyrite ore (CuFeS

2) concentrate

that while CopA was an effective Cu efflux transporterat low and high Cu concentrations the other Cu-ATPase(CopB) only appeared to be a low-affinity Cu export ATPase[68] Moreover by using a M sedula copA deletion mutantit was demonstrated that this strain compromised metalresistance and consequently abolished chalcopyrite oxidation[22] highlighting the role of Cu detoxification mechanismsduring a given bioleaching process Our attempts to showthe functionality of CopA1 and CopA2 from S metallicusby using E coli as a heterologous host were not successfulApparently the overexpression of these ORFs had a toxiceffect on E coli that compromised its viability Althoughgene disruption tools have not been yet developed for Smetallicus the functional role of both copTMA locimight befurther studied by cross-species complementation of a coppersensitive S solfataricus copR mutant as it was described byMaezato et al [22] It will be of great interest to establishin future studies the possible functionality of the isolatedputative transporters from S metallicus

Likewise quantitative RT-PCR experiments showed thatcopT1 transcript levels increased concomitantly with increas-ing Cu concentrations whereas copT2 showed relativelysimilar transcripts levels most likely indicating a constitutiveexpression profile (Figure 8(b)) Furthermore whereas copT1transcript levels were found to be increased 36 fold inCuFeS

2

grown cultures copT2 levels remained unchanged in com-parison with the control in the absence of Cu (Figure 8(b))The results obtained for S metallicus copT2 gene expressionare consistent with those reported for S solfataricus CopTtranscriptional regulator has been proposed to function as arepressor in S solfataricus showing a constitutive expressionthat in the presence of Cu loses its affinity for the promoterregion of copMA allowing the expression of this polycistron[19] In contrast the increased transcripts levels of both copA1and copT1 concomitant with higher Cu concentrations in the

environment suggest that the putative transcriptional regula-tor CopT1 may act by activating both copMA1 and probablyitself (Figure 8) Regarding this it was recently reported thatCopR (CopT in S metallicus) from S solfataricus strain 982acts as an activator of copT (copM in S metallicus) and copAexpression [69] Nevertheless additional experiments wouldbe required to test this possibility in S metallicus

4 Concluding Remarks

We previously reported that S metallicus resists extremelyhigh Cu concentrations which was mediated to some extentby the use of a possible metal resistance system based oninorganic polyphosphate hydrolysis and consequently Cu-PO4

2minusefflux [15 29 30] Here we have addressed the questionwhether this microorganism coded for other determinantsthat might help to explain its high heavy metal toleranceAs the genomic sequence of this microorganism is notyet available we jointly used CODEHOP-PCR and genomewalking approaches and were able to establish the occurrenceof two nonidentical homologous cop loci into the genome ofS metallicus (cop1 and cop2) Each cop locus codes for anarchaeal transcriptional regulator (CopT) a metal-bindingchaperone (CopM) and a Cu-transporting P-type ATPase(CopA) High levels of the polycistronic mRNAscopMA ofeach cop locus were observed after treatment with either Cuor Cd suggesting that the encoded ATPases efflux heavymetals out in order to detoxify the intracellular environmentAltogether previous reports and the results obtained in thisstudy allow us to suggest that some key elements that mayexplain the high resistance to Cu in S metallicus is the dupli-cation of the Cu resistance cop genes a defensive responseto stress and a polyP-based accumulation mechanism Inthe case of Cd although some similar stress responses were

14 Archaea

observed whether comparable Cu-responsive elements alsoparticipate in Cd responses remains to be seen

Conflict of Interests

The authors declare that the research was done in the absenceof any commercial or financial relationships that could beconstrued as a potential conflict of interests

Acknowledgments

This work was supported by Grant no FONDECYT 1110214and in part by ICM P-05-001-F project

References

[1] A Schippers ldquoMicroorganisms involved in bioleaching andnucleic acid-based molecular methods for their identificationand quantificationrdquo in Microbial Processing of Metal SulfidesR E Donati and W Sand Eds pp 3ndash33 Elsevier BerlinGermany 2007

[2] H R Watling ldquoThe bioleaching of sulphide minerals withemphasis on copper sulphides a reviewrdquo Hydrometallurgy vol84 no 1-2 pp 81ndash108 2006

[3] D E Rawlings D Dew and C Du Plessis ldquoBiomineralizationof metal-containing ores and concentratesrdquo Trends in Biotech-nology vol 21 no 1 pp 38ndash44 2003

[4] L Valenzuela A Chi S Beard et al ldquoGenomics metagenomicsand proteomics in biomining microorganismsrdquo BiotechnologyAdvances vol 24 no 2 pp 197ndash211 2006

[5] C A Jerez ldquoBioleaching and biomining for the industrialrecovery of metalsrdquo in Comprehensive Biotechnology M Moo-Young Ed vol 3 pp 717ndash729 Elsevier Amstaerdam TheNetherlands 2nd edition 2011

[6] P R Norris and J P Owen ldquoMineral sulphide oxidationby enrichment cultures of novel thermoacidophilic bacteriardquoFEMS Microbiology Reviews vol 11 no 1ndash3 pp 51ndash56 1993

[7] D E Rawlings and D B Johnson ldquoThe microbiology ofbiomining development and optimization ofmineral-oxidizingmicrobial consortiardquo Microbiology vol 153 no 2 pp 315ndash3242007

[8] P R Norris N P Burton and N A M Foulis ldquoAcidophiles inbioreactor mineral processingrdquo Extremophiles vol 4 no 2 pp71ndash76 2000

[9] M R Bruins S Kapil and F W Oehme ldquoMicrobial resistancetometals in the environmentrdquo Ecotoxicology and EnvironmentalSafety vol 45 no 3 pp 198ndash207 2000

[10] T J G Ettema M A Huynen W M De Vos and J Van DerOost ldquoTRASH a novel metal-binding domain predicted to beinvolved in heavy-metal sensing trafficking and resistancerdquoTrends in Biochemical Sciences vol 28 no 4 pp 170ndash173 2003

[11] M Dopson C Baker-Austin P R Koppineedi and P L BondldquoGrowth in sulfidic mineral environments metal resistancemechanisms in acidophilic micro-organismsrdquo Microbiologyvol 149 no 8 pp 1959ndash1970 2003

[12] S Franke and C Rensing ldquoAcidophiles Mechanisms to toleratemetal and acid toxicityrdquo in Physiology and Biochemistry ofExtremophiles C Gerday and N Glansdorff Eds pp 271ndash278ASM Press Washington DC USA 2007

[13] C A Navarro L H Orellana C Mauriaca and C A JerezldquoTranscriptional and functional studies of Acidithiobacillusferrooxidans genes related to survival in the presence of copperrdquoApplied and Environmental Microbiology vol 75 no 19 pp6102ndash6109 2009

[14] A Orell C A Navarro R Arancibia J C Mobarec andC A Jerez ldquoLife in blue copper resistance mechanisms ofbacteria and Archaea used in industrial biomining of mineralsrdquoBiotechnology Advances vol 28 no 6 pp 839ndash848 2010

[15] A Orell C A Navarro M Rivero J S Aguilar and C A JerezldquoInorganic polyphosphates in extremophiles and their posiblefunctionsrdquo Extremophiles vol 16 no 4 pp 573ndash583 2012

[16] C Baker-Austin M Dopson M Wexler et al ldquoExtremearsenic resistance by the acidophilic archaeon ldquoFerroplasmaacidarmanusrdquo Fer1rdquo Extremophiles vol 11 no 3 pp 425ndash4342007

[17] E Pedone S Bartolucci and G Fiorentino ldquoSensing andadapting to environmental stress the archaeal tacticrdquo Frontiersin Bioscience vol 9 pp 2909ndash2926 2004

[18] C Baker-Austin M Dopson M Wexler R G Sawers and PL Bond ldquoMolecular insight into extreme copper resistance inthe extremophilic archaeon ldquoFerroplasma acidarmanusrdquo Fer1rdquoMicrobiology vol 151 no 8 pp 2637ndash2646 2005

[19] T J G Ettema A B Brinkman P P Lamers N G Kornet WM de Vos and J van der Oost ldquoMolecular characterization ofa conserved archaeal copper resistance (cop) gene cluster andits copper-responsive regulator in Sulfolobus solfataricus P2rdquoMicrobiology vol 152 no 7 pp 1969ndash1979 2006

[20] A A Villafane Y VoskoboynikM Cuebas I Ruhl and E BinildquoResponse to excess copper in the hyperthermophile Sulfolobussolfataricus strain 982rdquo Biochemical and Biophysical ResearchCommunications vol 385 no 1 pp 67ndash71 2009

[21] K S Auernik Y Maezato P H Blum and R M KellyldquoThe genome sequence of the metal-mobilizing extremelythermoacidophilic archaeon Metallosphaera sedula providesinsights into bioleaching-associated metabolismrdquo Applied andEnvironmental Microbiology vol 74 no 3 pp 682ndash692 2008

[22] Y Maezato T Johnson S McCarthy K Dana and P BlumldquoMetal resistance and lithoautotrophy in the extreme ther-moacidophile Metalosphaera sedulardquo Journal of Bacteriologyvol 194 no 24 pp 6856ndash6863 2012

[23] D H Nies ldquoMicrobial heavy-metal resistancerdquo Applied Micro-biology and Biotechnology vol 51 no 6 pp 730ndash750 1999

[24] K Vido D Spector G Lagniel S Lopez M B Toledano andJ Labarre ldquoA proteome analysis of the cadmium response inSaccharomyces cerevisiaerdquo The Journal of Biological Chemistryvol 276 no 11 pp 8469ndash8474 2001

[25] G Bertin and D Averbeck ldquoCadmium cellular effects mod-ifications of biomolecules modulation of DNA repair andgenotoxic consequences (a review)rdquo Biochimie vol 88 no 11pp 1549ndash1559 2006

[26] M H Joe S W Jung S H Im et al ldquoGenome-wide responseof Deinococcus radiodurans on cadmium toxicityrdquo Journal ofMicrobiology and Biotechnology vol 21 no 4 pp 438ndash447 2011

[27] A Lagorce A Fourcans M Dutertre B Bouyssiere YZivanovic and F Confalonieri ldquoGenome-wide transcriptionalresponse of the archeon Thermococcus gammatolerans to cad-miumrdquo PLOS ONE vol 7 no 7 Article ID e41935 2012

[28] A Kornberg N N Rao and D Ault-Riche ldquoInorganicpolyphosphate a molecule of many functionsrdquo Annual Reviewof Biochemistry vol 68 pp 89ndash125 1999

Archaea 15

[29] J D Keasling ldquoRegulation of intracellular toxic metals andother cations by hydrolysis of polyphosphaterdquoAnnals of theNewYork Academy of Sciences vol 829 pp 242ndash249 1997

[30] F Remonsellez A Orell and C A Jerez ldquoCopper tolerance ofthe thermoacidophilic archaeon Sulfolobus metallicus possiblerole of polyphosphate metabolismrdquoMicrobiology vol 152 no 1pp 59ndash66 2006

[31] V Hatzimanikatis L H Choe and K H Lee ldquoProteomicstheoretical and experimental considerationsrdquo BiotechnologyProgress vol 15 no 3 pp 312ndash318 1999

[32] L H Choe and K H Lee ldquoA comparison of three commerciallyavailable isoelectric focusing units for proteome analysis theMultiphor the IPGphor and the Protean IEF cellrdquo Electrophore-sis vol 21 no 5 pp 993ndash1000 2000

[33] U K Laemmli ldquoCleavage of structural proteins during theassembly of the head of bacteriophage T4rdquo Nature vol 227 no5259 pp 680ndash685 1970

[34] A Shevchenko M Wilm O Vorm and M Mann ldquoMassspectrometric sequencing of proteins from silver-stained poly-acrylamide gelsrdquo Analytical Chemistry vol 68 no 5 pp 850ndash858 1996

[35] P Giavalisco E Nordhoff T Kreitler et al ldquoProteome analysisof Arabidopsis thaliana by two-dimensional gel electrophoresisand matrix-assisted laser desorptionionisation-time of flightmass spectrometryrdquo Proteomics vol 5 no 7 pp 1902ndash19132005

[36] T M Rose E R Schultz J G Henikoff S Pietrokovski CM McCallum and S Henikoff ldquoConsensus-degenerate hybridoligonucleotide primers for amplification of distantly relatedsequencesrdquo Nucleic Acids Research vol 26 no 7 pp 1628ndash16351998

[37] T M Rose J G Henikoff and S Henikoff ldquoCODEHOP(Consensus-Degenerate Hybrid Oligonucleotide Primer) PCRprimer designrdquo Nucleic Acids Research vol 31 no 13 pp 3763ndash3766 2003

[38] J P Acevedo F Reyes L P Parra O Salazar B A Andrews andJ A Asenjo ldquoCloning of complete genes for novel hydrolyticenzymes from Antarctic sea water bacteria by use of animproved genomewalking techniquerdquo Journal of Biotechnologyvol 133 no 3 pp 277ndash286 2008

[39] S Bathe and P R Norris ldquoFerrous iron- and sulfur-inducedgenes in Sulfolobus metallicusrdquo Applied and EnvironmentalMicrobiology vol 73 no 8 pp 2491ndash2497 2007

[40] J Sambrook E F Fritsch and TManiatisMolecular Cloning ALaboratory Manual Cold Spring Harbor Press New York NYUSA 1989

[41] K W Miller S Sass Risanico and J B Risatti ldquoDifferentialtolerance of Sulfolobus strains to transition metalsrdquo FEMSMicrobiology Letters vol 93 no 1 pp 69ndash73 1992

[42] G Huber C Spinnler A Gambacorta and K O Stetter ldquoMet-allosphaera sedula gen and sp nov represents a new genus ofaerobic metal-mobilizing thermoacidophilic archaebacteriardquoSystematic and Applied Microbiology vol 12 pp 38ndash47 1989

[43] J Llanos C Capasso E Parisi D Prieur and C JeanthonldquoSusceptibility to heavy metals and cadmium accumulation inaerobic and anaerobic thermophilic microorganisms isolatedfrom deep-sea hydrothermal ventsrdquo Current Microbiology vol41 no 3 pp 201ndash205 2000

[44] A Kaur M Pan M Meislin M T Facciotti R El-Gewelyand N S Baliga ldquoA systems view of haloarchaeal strategies towithstand stress from transition metalsrdquo Genome Research vol16 no 7 pp 841ndash854 2006

[45] M Solioz and J V Stoyanov ldquoCopper homeostasis in Entero-coccus hiraerdquo FEMS Microbiology Reviews vol 27 no 2-3 pp183ndash195 2003

[46] S Sitthisak K Howieson C Amezola and R K JayaswalldquoCharacterization of a multicopper oxidase gene from Staphy-lococcus aureusrdquo Applied and Environmental Microbiology vol71 no 9 pp 5650ndash5653 2005

[47] L Rodrıguez-Montelongo S I Volentini R N Farıas E MMassa and V A Rapisarda ldquoThe Cu(II)-reductase NADHdehydrogenase-2 of Escherichia coli improves the bacterialgrowth in extreme copper concentrations and increases theresistance to the damage caused by copper and hydroperoxiderdquoArchives of Biochemistry and Biophysics vol 451 no 1 pp 1ndash72006

[48] E Williams T M Lowe J Savas and J DiRuggiero ldquoMicroar-ray analysis of the hyperthermophilic archaeon Pyrococcusfuriosus exposed to gamma irradiationrdquo Extremophiles vol 11no 1 pp 19ndash29 2007

[49] L M Gaetke and C K Chow ldquoCopper toxicity oxidative stressand antioxidant nutrientsrdquo Toxicology vol 189 no 1-2 pp 147ndash163 2003

[50] M J Davies ldquoThe oxidative environment and protein damagerdquoBiochimica et Biophysica Acta vol 1703 no 2 pp 93ndash109 2005

[51] J R Thompson J K Bell J Bratt G A Grant and LJ Banaszak ldquoVmax regulation through domain and subunitchanges The active form of phosphoglycerate dehydrogenaserdquoBiochemistry vol 44 no 15 pp 5763ndash5773 2005

[52] V Consalvi R Chiaraluce L Politi A Gambacorta MDe Rosa and R Scandurra ldquoGlutamate dehydrogenase fromthe thermoacidophilic archaebacterium Sulfolobus solfataricusrdquoEuropean Journal of Biochemistry vol 196 no 2 pp 459ndash4671991

[53] A J L Macario M Lange B K Ahring and E Conway DeMacario ldquoStress genes and proteins in the archaeardquo Microbiol-ogy and Molecular Biology Reviews vol 63 no 4 pp 923ndash9671999

[54] J AMaupin-FurlowM A Gil M A Humbard et al ldquoArchaealproteasomes and other regulatory proteasesrdquo Current Opinionin Microbiology vol 8 no 6 pp 720ndash728 2005

[55] N Sun F Beck R Wilhelm Knispel et al ldquoProteomics analysisof Thermoplasma acidophilum with a focus on protein com-plexesrdquoMolecular and Cellular Proteomics vol 6 no 3 pp 492ndash502 2007

[56] L S Madding J K Michel K R Shockley et al ldquoRole of the 1205731subunit in the function and stability of the 20S proteasome inthe hyperthermophilic Archaeon Pyrococcus furiosusrdquo Journalof Bacteriology vol 189 no 2 pp 583ndash590 2007

[57] R Shibata Y Bessho A Shinkai et al ldquoCrystal structureand RNA-binding analysis of the archaeal transcription factorNusArdquo Biochemical and Biophysical Research Communicationsvol 355 no 1 pp 122ndash128 2007

[58] W Bae B Xia M Inouye and K Severinov ldquoEscherichia coliCspA-family RNA chaperones are transcription antitermina-torsrdquo Proceedings of the National Academy of Sciences of theUnited States of America vol 97 no 14 pp 7784ndash7789 2000

[59] R J Ram N C VerBerkmoes M P Thelen et al ldquoMicrobi-ology community proteomics of a natural microbial biofilmrdquoScience vol 308 no 5730 pp 1915ndash1920 2005

[60] I Noel-Georis T Vallaeys R Chauvaux et al ldquoGlobal analysisof the Ralstonia metallidurans proteome prelude for the large-scale study of heavy metal responserdquo Proteomics vol 4 no 1pp 151ndash179 2004

16 Archaea

[61] M TMNovo A CDa Silva RMoreto et al ldquoThiobacillus fer-rooxidans response to copper and other heavy metals growthprotein synthesis and protein phosphorylationrdquo Antonie vanLeeuwenhoek vol 77 no 2 pp 187ndash195 2000

[62] Q She R K Singh F Confalonieri et al ldquoThe complete genomeof the crenarchaeon Sulfolobus solfataricus P2rdquo Proceedings ofthe National Academy of Sciences of the United States of Americavol 98 no 14 pp 7835ndash7840 2001

[63] R P Anderson and J R Roth ldquoTandem genetic duplications inphage and bacteriardquo Annual Review of Microbiology vol 31 pp473ndash505 1977

[64] D Gevers K Vandepoele C Simillion and Y Van De PeerldquoGene duplication and biased functional retention of paralogsin bacterial genomesrdquo Trends in Microbiology vol 12 no 4 pp148ndash154 2004

[65] A B Reams and E L Neidle ldquoSelection for gene clustering bytandemduplicationrdquoAnnual Review ofMicrobiology vol 58 pp119ndash142 2004

[66] L H Orellana and C A Jerez ldquoA genomic island providesAcidithiobacillus ferrooxidans ATCC 53993 additional copperresistance a possible competitive advantagerdquo Applied Microbi-ology and Biotechnology vol 92 no 4 pp 761ndash767 2011

[67] A B Brinkman T J G Ettema W M de Vos and J van derOost ldquoThe Lrp family of transcriptional regulatorsrdquo MolecularMicrobiology vol 48 no 2 pp 287ndash294 2003

[68] C Vollmecke S L Drees J Reimann S V Albers and MLubben ldquoThe ATPases CopA and CopB both contribute tocopper resistance of the thermoacidophilic archaeon SulfolobussolfataricusrdquoMicrobiology vol 158 no 6 pp 1622ndash1633 2012

[69] A Villafane Y Voskoboynik I Ruhl et al ldquoCopR of Sulfolobussolfataricus represents a novel class of archaeal-specific copper-responsive activators of transcriptionrdquo Microbiology vol 157no 10 pp 2808ndash2817 2011

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Anatomy Research International

PeptidesInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporation httpwwwhindawicom

International Journal of

Volume 2014

Zoology

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Molecular Biology International

GenomicsInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioinformaticsAdvances in

Marine BiologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Signal TransductionJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

Evolutionary BiologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Biochemistry Research International

ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Genetics Research International

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Advances in

Virolog y

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Nucleic AcidsJournal of

Volume 2014

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

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Enzyme Research

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

International Journal of

Microbiology

Archaea 11

HindIII HindIII HindIII

HindIII

CopT1Locus cop1 M1

EcoRIEcoRV

513 bp 219 bp 2250 bp

CopA1

(a)

Locus cop2 M2

EcoRI

CopA2

453 bp 171 bp 2244 bp

CopT2

PstIHindIII HindIII HindIII

(b)

Figure 5 Schematic representation of the two cop gene clusters isolated from S metallicus genome Each cluster codes for anArchaea-specifictranscriptional regulator (copT) a metallochaperone (copM) and a P-type Cu-exporting ATPase (copA) Lengths of each gene are indicatedLocus cop1 corresponds to 4346 sequenced base pairs and shows an intergenic region copT1-M1 of 232 base pairs Locus cop2 correspondsto 3435 base pairs and shows 89 base pairs copT2-M2 intergenic region Genes copM2 and copA2 overlapped in 11 base pairs copT2 was notfully isolated

Control CdSO4 Ag2SO4 NiSO4 ZnSO4 CuSO4

kb3kb2kb1

02 kb

(a)

kb3kb2kb1

02 kb

(b)

(c)

Figure 6 Northern blot analysis to determine the expression of S metallicus copA1 (a) and copA2 (b) putative genes in response to variousmetals S metallicus total RNAwas extracted from cultures grown either in the absence (control) or the presence of CuSO

4(50mM) Ag

2SO4

(008mM) NiSO4(15mM) ZnSO

4(50mM) and CdSO

4(2mM) P32-radioactivelly labelled DNA fragments annealing to copA1 and copA2

sequences respectively were used as probes (c) shows rRNAs as total RNA loading control

On the other hand CopM1 (71 aa) and CopM2 (56 aa)sharing 66 of identity present the proposed metal bindingdomain TRASH (trafficking resistance and sensing of heavymetals) [10] Additionally when analyzing the putative tran-scriptional regulators the presence of a C-terminal TRASHdomain in both CopT1 and CopT2 was also found CopT1sequence also contains an entire N-terminal helix-turn-helix (HTH) motif (not shown) that resembles the DNA-bindingmotifs of prokaryotic transcriptional regulators suchas Lrp-like proteins [67] The HTH motif was only partiallyidentified in CopT2 since the 51015840 region of copT1 gene has notbeen yet fully isolated

35 Transcriptional Analysis of S metallicus Cop Genes Toget insight into the role of the two cop loci the expressionof the corresponding genes was analyzed under variousconditions S metallicus was first grown in presence ofdifferent heavy metals (Cu Zn Cd Ni o Ag) at a givenconcentration that did not affect growth kinetics [30 42]Subsequently total RNA was isolated from late exponentiallygrown cultures and Northern blot experiments were carriedout in order to determine the expression of both copA1 andcopA2 genes As depicted in Figure 6 transcription of bothcopA1 and copA2messengers were specifically induced in thepresence of Cu and Cd ions This gene expression pattern

12 Archaea

954pb

M1 CopA1

(a)

1438 pb

M2 CopA2

(b)

1000 pb

800pb

+ minus

(c)

+ minus

1500 pb

1000 pb

(d)

Figure 7 Cotranscription analysis of copMA1 and copMA2 genes cDNA was synthesized with a reverse primer (dotted arrows) hybridizingtoward the 31015840 end of either copA1 (a) or copA2 (b) S metallicus total RNA was extracted at the late exponential phase from a culture growingin presence of 20mMCu PCR amplifications were carried out with these cDNAs and each corresponding primer pair (black arrows) as listedin Table 1 (c) and (d) show RT-PCR products obtained for the copMA1 and copMA2 intergenic regions respectively Reverse transcriptasereactions with (+) and without (minus) the Improm II reverse transcriptase enzyme were carried out in order to exclude amplification due togenomic DNA contamination Expected sizes (in base pairs) for the corresponding PCR products are given in (a) and (b)

is in good agreement with what has been described for Ssolfataricus where the bicistronic copMA transcript levelswere also found to be up-regulated in response to both Cuand Cd [19]

Northern blot analysis showed that the sizes seen for bothcopA transcripts (sim25 Kb) did not correspond exactly withthe individual genes length (each of about 225Kb) (Figure 6)This strongly suggested that the transcripts could also includethe respective copM gene located upstream of copA in eachcop loci To confirm this assumption a co-transcriptionexperiment was done (Figure 7) in which the cDNAs wereobtained by using RNA extracted from a culture grownin the presence of 20mM Cu and using a reverse primerhybridizing with copA1 (Figure 7(a)) or copA2 (Figure 7(b))gene sequence respectively PCR amplifications were carriedout by using the corresponding cDNAs as templates and eachpair of primers lying in adjacent genes (copM) The presenceof an amplicon of the expected size in each case indicatedthe adjacent genes were part of polycistronic messengers(Figure 7) These results clearly show that in S metallicuseach couple of copMA genes was expressed in the form oftranscriptional units as reported in F acidarmanus and Ssolfataricus [18 19] The coexpression of gene pairrsquos copMAmay suggest a coordinated and dependent function for therespective encoded proteins In the bacterium E hirae themetallochaperone CopZ (CopM in S metallicus) fulfills apivotal role in the mechanism of Cu homeostasis [45] Thusit was shown that this protein interacts directly with the Cu-ATPase CopB (CopA in S metallicus) handing the Cu forsubsequent removal In this context one might expect thatproteins CopM1 and CopM2 from S metallicus have a similarrole to that described for E hirae Furthermore quantitativeRT-PCR experiments were carried out in order to determinedifferential expression of the cop genes in response to differentCu concentrations The expression of copA1 copA2 copT1and copT2 was tested relative to the transcript levels ofrRNA 16S gene since its levels were not significantly affected

in all assessed conditions (data not shown) As shown inFigure 8(a) copA2 and copA1 transcript levels were foundto be concomitantly increased with the increasing Cu con-centrations present in the medium Higher transcripts levelsof copA1 were seen in all tested conditions when comparedwith copA2 levels copA1 transcript levels were found to be325-fold up-regulated when comparing 50mMCu conditionversuscontrol (absence of metal) whereas copA2 transcriptlevels showed an increment of only 175-fold (Figure 8(a))The finding that Cu-ATPases mRNA levels were significantlyincreased in response to Cu ions exposure suggests that thetransport system may operate for Cu efflux in S metallicus

Moreover copA2 and copA1 gene expression was quan-tified when S metallicus was grown using chalcopyrite(CuFeS

2) ore as an oxidizable substrate (Figure 8(a)) Mineral

oxidation mediated by the microorganism generates a pro-gressive increase in Cu ions concentration in the medium Tofind out whether the amount of solubilized Cu induced theexpression of the Cu-ATPases genes total RNAwas extractedfrom a S metallicus culture grown to late exponential phaseand in the presence of 1 CuFeS

2 It was clear that an

up-regulation of the Cu-ATPases encoding genes also tookplace when S metallicus was grown in the presence ofCuFeS

2(Figure 8(a)) copA1 transcript levels increased 14-fold

compared with the control condition in the absence of Cuwhile copA2 gene expression showed an increase of 76 foldAlong with this bymeans of atomic absorption spectrometry(AAS) analysis an overall amount of solubilized Cu ions(Cu2+Cu1+) of 144plusmn21mMwas determined to be present inthemedium indicating that copA2 and copA1 gene expressionwas most likely in response to the Cu present in the culturedue to CuFeS

2microbial-solubilization

The finding that copA1 was highly expressed comparedwith copA2 in all tested conditions may suggest a possiblephysiological hierarchy between the two ATPases whenovercoming either Cu or Cd stress In this regard by meansof a genetic approach it was demonstrated in S solfataricus

Archaea 13

700

600

500

400

300

200

100

00

5 20 50 CuFeS2CuSO4 concentration (mM)

Relativ

egenee

xpression

CopA2CopA1

(a)

8

6

7

5

4

3

2

1

00

5 20 50 CuFeS2CuSO4 concentration (mM)

Relativ

egenee

xpression

CopT1CopT2

(b)

Figure 8 Relative expression levels of S metallicus genes copA1 and copA2 (a) copT1 and copT2 (b) Expression of S metallicus cop geneswasassessed by qRT-PCR and normalized against 16S rRNA gene expression in individual cultures Mean values and standard deviations arefrom analyses of three independent cultures grown in the presence of 0 5 20 50mMCu or in 1 of a chalcopyrite ore (CuFeS

2) concentrate

that while CopA was an effective Cu efflux transporterat low and high Cu concentrations the other Cu-ATPase(CopB) only appeared to be a low-affinity Cu export ATPase[68] Moreover by using a M sedula copA deletion mutantit was demonstrated that this strain compromised metalresistance and consequently abolished chalcopyrite oxidation[22] highlighting the role of Cu detoxification mechanismsduring a given bioleaching process Our attempts to showthe functionality of CopA1 and CopA2 from S metallicusby using E coli as a heterologous host were not successfulApparently the overexpression of these ORFs had a toxiceffect on E coli that compromised its viability Althoughgene disruption tools have not been yet developed for Smetallicus the functional role of both copTMA locimight befurther studied by cross-species complementation of a coppersensitive S solfataricus copR mutant as it was described byMaezato et al [22] It will be of great interest to establishin future studies the possible functionality of the isolatedputative transporters from S metallicus

Likewise quantitative RT-PCR experiments showed thatcopT1 transcript levels increased concomitantly with increas-ing Cu concentrations whereas copT2 showed relativelysimilar transcripts levels most likely indicating a constitutiveexpression profile (Figure 8(b)) Furthermore whereas copT1transcript levels were found to be increased 36 fold inCuFeS

2

grown cultures copT2 levels remained unchanged in com-parison with the control in the absence of Cu (Figure 8(b))The results obtained for S metallicus copT2 gene expressionare consistent with those reported for S solfataricus CopTtranscriptional regulator has been proposed to function as arepressor in S solfataricus showing a constitutive expressionthat in the presence of Cu loses its affinity for the promoterregion of copMA allowing the expression of this polycistron[19] In contrast the increased transcripts levels of both copA1and copT1 concomitant with higher Cu concentrations in the

environment suggest that the putative transcriptional regula-tor CopT1 may act by activating both copMA1 and probablyitself (Figure 8) Regarding this it was recently reported thatCopR (CopT in S metallicus) from S solfataricus strain 982acts as an activator of copT (copM in S metallicus) and copAexpression [69] Nevertheless additional experiments wouldbe required to test this possibility in S metallicus

4 Concluding Remarks

We previously reported that S metallicus resists extremelyhigh Cu concentrations which was mediated to some extentby the use of a possible metal resistance system based oninorganic polyphosphate hydrolysis and consequently Cu-PO4

2minusefflux [15 29 30] Here we have addressed the questionwhether this microorganism coded for other determinantsthat might help to explain its high heavy metal toleranceAs the genomic sequence of this microorganism is notyet available we jointly used CODEHOP-PCR and genomewalking approaches and were able to establish the occurrenceof two nonidentical homologous cop loci into the genome ofS metallicus (cop1 and cop2) Each cop locus codes for anarchaeal transcriptional regulator (CopT) a metal-bindingchaperone (CopM) and a Cu-transporting P-type ATPase(CopA) High levels of the polycistronic mRNAscopMA ofeach cop locus were observed after treatment with either Cuor Cd suggesting that the encoded ATPases efflux heavymetals out in order to detoxify the intracellular environmentAltogether previous reports and the results obtained in thisstudy allow us to suggest that some key elements that mayexplain the high resistance to Cu in S metallicus is the dupli-cation of the Cu resistance cop genes a defensive responseto stress and a polyP-based accumulation mechanism Inthe case of Cd although some similar stress responses were

14 Archaea

observed whether comparable Cu-responsive elements alsoparticipate in Cd responses remains to be seen

Conflict of Interests

The authors declare that the research was done in the absenceof any commercial or financial relationships that could beconstrued as a potential conflict of interests

Acknowledgments

This work was supported by Grant no FONDECYT 1110214and in part by ICM P-05-001-F project

References

[1] A Schippers ldquoMicroorganisms involved in bioleaching andnucleic acid-based molecular methods for their identificationand quantificationrdquo in Microbial Processing of Metal SulfidesR E Donati and W Sand Eds pp 3ndash33 Elsevier BerlinGermany 2007

[2] H R Watling ldquoThe bioleaching of sulphide minerals withemphasis on copper sulphides a reviewrdquo Hydrometallurgy vol84 no 1-2 pp 81ndash108 2006

[3] D E Rawlings D Dew and C Du Plessis ldquoBiomineralizationof metal-containing ores and concentratesrdquo Trends in Biotech-nology vol 21 no 1 pp 38ndash44 2003

[4] L Valenzuela A Chi S Beard et al ldquoGenomics metagenomicsand proteomics in biomining microorganismsrdquo BiotechnologyAdvances vol 24 no 2 pp 197ndash211 2006

[5] C A Jerez ldquoBioleaching and biomining for the industrialrecovery of metalsrdquo in Comprehensive Biotechnology M Moo-Young Ed vol 3 pp 717ndash729 Elsevier Amstaerdam TheNetherlands 2nd edition 2011

[6] P R Norris and J P Owen ldquoMineral sulphide oxidationby enrichment cultures of novel thermoacidophilic bacteriardquoFEMS Microbiology Reviews vol 11 no 1ndash3 pp 51ndash56 1993

[7] D E Rawlings and D B Johnson ldquoThe microbiology ofbiomining development and optimization ofmineral-oxidizingmicrobial consortiardquo Microbiology vol 153 no 2 pp 315ndash3242007

[8] P R Norris N P Burton and N A M Foulis ldquoAcidophiles inbioreactor mineral processingrdquo Extremophiles vol 4 no 2 pp71ndash76 2000

[9] M R Bruins S Kapil and F W Oehme ldquoMicrobial resistancetometals in the environmentrdquo Ecotoxicology and EnvironmentalSafety vol 45 no 3 pp 198ndash207 2000

[10] T J G Ettema M A Huynen W M De Vos and J Van DerOost ldquoTRASH a novel metal-binding domain predicted to beinvolved in heavy-metal sensing trafficking and resistancerdquoTrends in Biochemical Sciences vol 28 no 4 pp 170ndash173 2003

[11] M Dopson C Baker-Austin P R Koppineedi and P L BondldquoGrowth in sulfidic mineral environments metal resistancemechanisms in acidophilic micro-organismsrdquo Microbiologyvol 149 no 8 pp 1959ndash1970 2003

[12] S Franke and C Rensing ldquoAcidophiles Mechanisms to toleratemetal and acid toxicityrdquo in Physiology and Biochemistry ofExtremophiles C Gerday and N Glansdorff Eds pp 271ndash278ASM Press Washington DC USA 2007

[13] C A Navarro L H Orellana C Mauriaca and C A JerezldquoTranscriptional and functional studies of Acidithiobacillusferrooxidans genes related to survival in the presence of copperrdquoApplied and Environmental Microbiology vol 75 no 19 pp6102ndash6109 2009

[14] A Orell C A Navarro R Arancibia J C Mobarec andC A Jerez ldquoLife in blue copper resistance mechanisms ofbacteria and Archaea used in industrial biomining of mineralsrdquoBiotechnology Advances vol 28 no 6 pp 839ndash848 2010

[15] A Orell C A Navarro M Rivero J S Aguilar and C A JerezldquoInorganic polyphosphates in extremophiles and their posiblefunctionsrdquo Extremophiles vol 16 no 4 pp 573ndash583 2012

[16] C Baker-Austin M Dopson M Wexler et al ldquoExtremearsenic resistance by the acidophilic archaeon ldquoFerroplasmaacidarmanusrdquo Fer1rdquo Extremophiles vol 11 no 3 pp 425ndash4342007

[17] E Pedone S Bartolucci and G Fiorentino ldquoSensing andadapting to environmental stress the archaeal tacticrdquo Frontiersin Bioscience vol 9 pp 2909ndash2926 2004

[18] C Baker-Austin M Dopson M Wexler R G Sawers and PL Bond ldquoMolecular insight into extreme copper resistance inthe extremophilic archaeon ldquoFerroplasma acidarmanusrdquo Fer1rdquoMicrobiology vol 151 no 8 pp 2637ndash2646 2005

[19] T J G Ettema A B Brinkman P P Lamers N G Kornet WM de Vos and J van der Oost ldquoMolecular characterization ofa conserved archaeal copper resistance (cop) gene cluster andits copper-responsive regulator in Sulfolobus solfataricus P2rdquoMicrobiology vol 152 no 7 pp 1969ndash1979 2006

[20] A A Villafane Y VoskoboynikM Cuebas I Ruhl and E BinildquoResponse to excess copper in the hyperthermophile Sulfolobussolfataricus strain 982rdquo Biochemical and Biophysical ResearchCommunications vol 385 no 1 pp 67ndash71 2009

[21] K S Auernik Y Maezato P H Blum and R M KellyldquoThe genome sequence of the metal-mobilizing extremelythermoacidophilic archaeon Metallosphaera sedula providesinsights into bioleaching-associated metabolismrdquo Applied andEnvironmental Microbiology vol 74 no 3 pp 682ndash692 2008

[22] Y Maezato T Johnson S McCarthy K Dana and P BlumldquoMetal resistance and lithoautotrophy in the extreme ther-moacidophile Metalosphaera sedulardquo Journal of Bacteriologyvol 194 no 24 pp 6856ndash6863 2012

[23] D H Nies ldquoMicrobial heavy-metal resistancerdquo Applied Micro-biology and Biotechnology vol 51 no 6 pp 730ndash750 1999

[24] K Vido D Spector G Lagniel S Lopez M B Toledano andJ Labarre ldquoA proteome analysis of the cadmium response inSaccharomyces cerevisiaerdquo The Journal of Biological Chemistryvol 276 no 11 pp 8469ndash8474 2001

[25] G Bertin and D Averbeck ldquoCadmium cellular effects mod-ifications of biomolecules modulation of DNA repair andgenotoxic consequences (a review)rdquo Biochimie vol 88 no 11pp 1549ndash1559 2006

[26] M H Joe S W Jung S H Im et al ldquoGenome-wide responseof Deinococcus radiodurans on cadmium toxicityrdquo Journal ofMicrobiology and Biotechnology vol 21 no 4 pp 438ndash447 2011

[27] A Lagorce A Fourcans M Dutertre B Bouyssiere YZivanovic and F Confalonieri ldquoGenome-wide transcriptionalresponse of the archeon Thermococcus gammatolerans to cad-miumrdquo PLOS ONE vol 7 no 7 Article ID e41935 2012

[28] A Kornberg N N Rao and D Ault-Riche ldquoInorganicpolyphosphate a molecule of many functionsrdquo Annual Reviewof Biochemistry vol 68 pp 89ndash125 1999

Archaea 15

[29] J D Keasling ldquoRegulation of intracellular toxic metals andother cations by hydrolysis of polyphosphaterdquoAnnals of theNewYork Academy of Sciences vol 829 pp 242ndash249 1997

[30] F Remonsellez A Orell and C A Jerez ldquoCopper tolerance ofthe thermoacidophilic archaeon Sulfolobus metallicus possiblerole of polyphosphate metabolismrdquoMicrobiology vol 152 no 1pp 59ndash66 2006

[31] V Hatzimanikatis L H Choe and K H Lee ldquoProteomicstheoretical and experimental considerationsrdquo BiotechnologyProgress vol 15 no 3 pp 312ndash318 1999

[32] L H Choe and K H Lee ldquoA comparison of three commerciallyavailable isoelectric focusing units for proteome analysis theMultiphor the IPGphor and the Protean IEF cellrdquo Electrophore-sis vol 21 no 5 pp 993ndash1000 2000

[33] U K Laemmli ldquoCleavage of structural proteins during theassembly of the head of bacteriophage T4rdquo Nature vol 227 no5259 pp 680ndash685 1970

[34] A Shevchenko M Wilm O Vorm and M Mann ldquoMassspectrometric sequencing of proteins from silver-stained poly-acrylamide gelsrdquo Analytical Chemistry vol 68 no 5 pp 850ndash858 1996

[35] P Giavalisco E Nordhoff T Kreitler et al ldquoProteome analysisof Arabidopsis thaliana by two-dimensional gel electrophoresisand matrix-assisted laser desorptionionisation-time of flightmass spectrometryrdquo Proteomics vol 5 no 7 pp 1902ndash19132005

[36] T M Rose E R Schultz J G Henikoff S Pietrokovski CM McCallum and S Henikoff ldquoConsensus-degenerate hybridoligonucleotide primers for amplification of distantly relatedsequencesrdquo Nucleic Acids Research vol 26 no 7 pp 1628ndash16351998

[37] T M Rose J G Henikoff and S Henikoff ldquoCODEHOP(Consensus-Degenerate Hybrid Oligonucleotide Primer) PCRprimer designrdquo Nucleic Acids Research vol 31 no 13 pp 3763ndash3766 2003

[38] J P Acevedo F Reyes L P Parra O Salazar B A Andrews andJ A Asenjo ldquoCloning of complete genes for novel hydrolyticenzymes from Antarctic sea water bacteria by use of animproved genomewalking techniquerdquo Journal of Biotechnologyvol 133 no 3 pp 277ndash286 2008

[39] S Bathe and P R Norris ldquoFerrous iron- and sulfur-inducedgenes in Sulfolobus metallicusrdquo Applied and EnvironmentalMicrobiology vol 73 no 8 pp 2491ndash2497 2007

[40] J Sambrook E F Fritsch and TManiatisMolecular Cloning ALaboratory Manual Cold Spring Harbor Press New York NYUSA 1989

[41] K W Miller S Sass Risanico and J B Risatti ldquoDifferentialtolerance of Sulfolobus strains to transition metalsrdquo FEMSMicrobiology Letters vol 93 no 1 pp 69ndash73 1992

[42] G Huber C Spinnler A Gambacorta and K O Stetter ldquoMet-allosphaera sedula gen and sp nov represents a new genus ofaerobic metal-mobilizing thermoacidophilic archaebacteriardquoSystematic and Applied Microbiology vol 12 pp 38ndash47 1989

[43] J Llanos C Capasso E Parisi D Prieur and C JeanthonldquoSusceptibility to heavy metals and cadmium accumulation inaerobic and anaerobic thermophilic microorganisms isolatedfrom deep-sea hydrothermal ventsrdquo Current Microbiology vol41 no 3 pp 201ndash205 2000

[44] A Kaur M Pan M Meislin M T Facciotti R El-Gewelyand N S Baliga ldquoA systems view of haloarchaeal strategies towithstand stress from transition metalsrdquo Genome Research vol16 no 7 pp 841ndash854 2006

[45] M Solioz and J V Stoyanov ldquoCopper homeostasis in Entero-coccus hiraerdquo FEMS Microbiology Reviews vol 27 no 2-3 pp183ndash195 2003

[46] S Sitthisak K Howieson C Amezola and R K JayaswalldquoCharacterization of a multicopper oxidase gene from Staphy-lococcus aureusrdquo Applied and Environmental Microbiology vol71 no 9 pp 5650ndash5653 2005

[47] L Rodrıguez-Montelongo S I Volentini R N Farıas E MMassa and V A Rapisarda ldquoThe Cu(II)-reductase NADHdehydrogenase-2 of Escherichia coli improves the bacterialgrowth in extreme copper concentrations and increases theresistance to the damage caused by copper and hydroperoxiderdquoArchives of Biochemistry and Biophysics vol 451 no 1 pp 1ndash72006

[48] E Williams T M Lowe J Savas and J DiRuggiero ldquoMicroar-ray analysis of the hyperthermophilic archaeon Pyrococcusfuriosus exposed to gamma irradiationrdquo Extremophiles vol 11no 1 pp 19ndash29 2007

[49] L M Gaetke and C K Chow ldquoCopper toxicity oxidative stressand antioxidant nutrientsrdquo Toxicology vol 189 no 1-2 pp 147ndash163 2003

[50] M J Davies ldquoThe oxidative environment and protein damagerdquoBiochimica et Biophysica Acta vol 1703 no 2 pp 93ndash109 2005

[51] J R Thompson J K Bell J Bratt G A Grant and LJ Banaszak ldquoVmax regulation through domain and subunitchanges The active form of phosphoglycerate dehydrogenaserdquoBiochemistry vol 44 no 15 pp 5763ndash5773 2005

[52] V Consalvi R Chiaraluce L Politi A Gambacorta MDe Rosa and R Scandurra ldquoGlutamate dehydrogenase fromthe thermoacidophilic archaebacterium Sulfolobus solfataricusrdquoEuropean Journal of Biochemistry vol 196 no 2 pp 459ndash4671991

[53] A J L Macario M Lange B K Ahring and E Conway DeMacario ldquoStress genes and proteins in the archaeardquo Microbiol-ogy and Molecular Biology Reviews vol 63 no 4 pp 923ndash9671999

[54] J AMaupin-FurlowM A Gil M A Humbard et al ldquoArchaealproteasomes and other regulatory proteasesrdquo Current Opinionin Microbiology vol 8 no 6 pp 720ndash728 2005

[55] N Sun F Beck R Wilhelm Knispel et al ldquoProteomics analysisof Thermoplasma acidophilum with a focus on protein com-plexesrdquoMolecular and Cellular Proteomics vol 6 no 3 pp 492ndash502 2007

[56] L S Madding J K Michel K R Shockley et al ldquoRole of the 1205731subunit in the function and stability of the 20S proteasome inthe hyperthermophilic Archaeon Pyrococcus furiosusrdquo Journalof Bacteriology vol 189 no 2 pp 583ndash590 2007

[57] R Shibata Y Bessho A Shinkai et al ldquoCrystal structureand RNA-binding analysis of the archaeal transcription factorNusArdquo Biochemical and Biophysical Research Communicationsvol 355 no 1 pp 122ndash128 2007

[58] W Bae B Xia M Inouye and K Severinov ldquoEscherichia coliCspA-family RNA chaperones are transcription antitermina-torsrdquo Proceedings of the National Academy of Sciences of theUnited States of America vol 97 no 14 pp 7784ndash7789 2000

[59] R J Ram N C VerBerkmoes M P Thelen et al ldquoMicrobi-ology community proteomics of a natural microbial biofilmrdquoScience vol 308 no 5730 pp 1915ndash1920 2005

[60] I Noel-Georis T Vallaeys R Chauvaux et al ldquoGlobal analysisof the Ralstonia metallidurans proteome prelude for the large-scale study of heavy metal responserdquo Proteomics vol 4 no 1pp 151ndash179 2004

16 Archaea

[61] M TMNovo A CDa Silva RMoreto et al ldquoThiobacillus fer-rooxidans response to copper and other heavy metals growthprotein synthesis and protein phosphorylationrdquo Antonie vanLeeuwenhoek vol 77 no 2 pp 187ndash195 2000

[62] Q She R K Singh F Confalonieri et al ldquoThe complete genomeof the crenarchaeon Sulfolobus solfataricus P2rdquo Proceedings ofthe National Academy of Sciences of the United States of Americavol 98 no 14 pp 7835ndash7840 2001

[63] R P Anderson and J R Roth ldquoTandem genetic duplications inphage and bacteriardquo Annual Review of Microbiology vol 31 pp473ndash505 1977

[64] D Gevers K Vandepoele C Simillion and Y Van De PeerldquoGene duplication and biased functional retention of paralogsin bacterial genomesrdquo Trends in Microbiology vol 12 no 4 pp148ndash154 2004

[65] A B Reams and E L Neidle ldquoSelection for gene clustering bytandemduplicationrdquoAnnual Review ofMicrobiology vol 58 pp119ndash142 2004

[66] L H Orellana and C A Jerez ldquoA genomic island providesAcidithiobacillus ferrooxidans ATCC 53993 additional copperresistance a possible competitive advantagerdquo Applied Microbi-ology and Biotechnology vol 92 no 4 pp 761ndash767 2011

[67] A B Brinkman T J G Ettema W M de Vos and J van derOost ldquoThe Lrp family of transcriptional regulatorsrdquo MolecularMicrobiology vol 48 no 2 pp 287ndash294 2003

[68] C Vollmecke S L Drees J Reimann S V Albers and MLubben ldquoThe ATPases CopA and CopB both contribute tocopper resistance of the thermoacidophilic archaeon SulfolobussolfataricusrdquoMicrobiology vol 158 no 6 pp 1622ndash1633 2012

[69] A Villafane Y Voskoboynik I Ruhl et al ldquoCopR of Sulfolobussolfataricus represents a novel class of archaeal-specific copper-responsive activators of transcriptionrdquo Microbiology vol 157no 10 pp 2808ndash2817 2011

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Anatomy Research International

PeptidesInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporation httpwwwhindawicom

International Journal of

Volume 2014

Zoology

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Molecular Biology International

GenomicsInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioinformaticsAdvances in

Marine BiologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Signal TransductionJournal of

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BioMed Research International

Evolutionary BiologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Biochemistry Research International

ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Genetics Research International

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Advances in

Virolog y

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Nucleic AcidsJournal of

Volume 2014

Stem CellsInternational

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Enzyme Research

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

International Journal of

Microbiology

12 Archaea

954pb

M1 CopA1

(a)

1438 pb

M2 CopA2

(b)

1000 pb

800pb

+ minus

(c)

+ minus

1500 pb

1000 pb

(d)

Figure 7 Cotranscription analysis of copMA1 and copMA2 genes cDNA was synthesized with a reverse primer (dotted arrows) hybridizingtoward the 31015840 end of either copA1 (a) or copA2 (b) S metallicus total RNA was extracted at the late exponential phase from a culture growingin presence of 20mMCu PCR amplifications were carried out with these cDNAs and each corresponding primer pair (black arrows) as listedin Table 1 (c) and (d) show RT-PCR products obtained for the copMA1 and copMA2 intergenic regions respectively Reverse transcriptasereactions with (+) and without (minus) the Improm II reverse transcriptase enzyme were carried out in order to exclude amplification due togenomic DNA contamination Expected sizes (in base pairs) for the corresponding PCR products are given in (a) and (b)

is in good agreement with what has been described for Ssolfataricus where the bicistronic copMA transcript levelswere also found to be up-regulated in response to both Cuand Cd [19]

Northern blot analysis showed that the sizes seen for bothcopA transcripts (sim25 Kb) did not correspond exactly withthe individual genes length (each of about 225Kb) (Figure 6)This strongly suggested that the transcripts could also includethe respective copM gene located upstream of copA in eachcop loci To confirm this assumption a co-transcriptionexperiment was done (Figure 7) in which the cDNAs wereobtained by using RNA extracted from a culture grownin the presence of 20mM Cu and using a reverse primerhybridizing with copA1 (Figure 7(a)) or copA2 (Figure 7(b))gene sequence respectively PCR amplifications were carriedout by using the corresponding cDNAs as templates and eachpair of primers lying in adjacent genes (copM) The presenceof an amplicon of the expected size in each case indicatedthe adjacent genes were part of polycistronic messengers(Figure 7) These results clearly show that in S metallicuseach couple of copMA genes was expressed in the form oftranscriptional units as reported in F acidarmanus and Ssolfataricus [18 19] The coexpression of gene pairrsquos copMAmay suggest a coordinated and dependent function for therespective encoded proteins In the bacterium E hirae themetallochaperone CopZ (CopM in S metallicus) fulfills apivotal role in the mechanism of Cu homeostasis [45] Thusit was shown that this protein interacts directly with the Cu-ATPase CopB (CopA in S metallicus) handing the Cu forsubsequent removal In this context one might expect thatproteins CopM1 and CopM2 from S metallicus have a similarrole to that described for E hirae Furthermore quantitativeRT-PCR experiments were carried out in order to determinedifferential expression of the cop genes in response to differentCu concentrations The expression of copA1 copA2 copT1and copT2 was tested relative to the transcript levels ofrRNA 16S gene since its levels were not significantly affected

in all assessed conditions (data not shown) As shown inFigure 8(a) copA2 and copA1 transcript levels were foundto be concomitantly increased with the increasing Cu con-centrations present in the medium Higher transcripts levelsof copA1 were seen in all tested conditions when comparedwith copA2 levels copA1 transcript levels were found to be325-fold up-regulated when comparing 50mMCu conditionversuscontrol (absence of metal) whereas copA2 transcriptlevels showed an increment of only 175-fold (Figure 8(a))The finding that Cu-ATPases mRNA levels were significantlyincreased in response to Cu ions exposure suggests that thetransport system may operate for Cu efflux in S metallicus

Moreover copA2 and copA1 gene expression was quan-tified when S metallicus was grown using chalcopyrite(CuFeS

2) ore as an oxidizable substrate (Figure 8(a)) Mineral

oxidation mediated by the microorganism generates a pro-gressive increase in Cu ions concentration in the medium Tofind out whether the amount of solubilized Cu induced theexpression of the Cu-ATPases genes total RNAwas extractedfrom a S metallicus culture grown to late exponential phaseand in the presence of 1 CuFeS

2 It was clear that an

up-regulation of the Cu-ATPases encoding genes also tookplace when S metallicus was grown in the presence ofCuFeS

2(Figure 8(a)) copA1 transcript levels increased 14-fold

compared with the control condition in the absence of Cuwhile copA2 gene expression showed an increase of 76 foldAlong with this bymeans of atomic absorption spectrometry(AAS) analysis an overall amount of solubilized Cu ions(Cu2+Cu1+) of 144plusmn21mMwas determined to be present inthemedium indicating that copA2 and copA1 gene expressionwas most likely in response to the Cu present in the culturedue to CuFeS

2microbial-solubilization

The finding that copA1 was highly expressed comparedwith copA2 in all tested conditions may suggest a possiblephysiological hierarchy between the two ATPases whenovercoming either Cu or Cd stress In this regard by meansof a genetic approach it was demonstrated in S solfataricus

Archaea 13

700

600

500

400

300

200

100

00

5 20 50 CuFeS2CuSO4 concentration (mM)

Relativ

egenee

xpression

CopA2CopA1

(a)

8

6

7

5

4

3

2

1

00

5 20 50 CuFeS2CuSO4 concentration (mM)

Relativ

egenee

xpression

CopT1CopT2

(b)

Figure 8 Relative expression levels of S metallicus genes copA1 and copA2 (a) copT1 and copT2 (b) Expression of S metallicus cop geneswasassessed by qRT-PCR and normalized against 16S rRNA gene expression in individual cultures Mean values and standard deviations arefrom analyses of three independent cultures grown in the presence of 0 5 20 50mMCu or in 1 of a chalcopyrite ore (CuFeS

2) concentrate

that while CopA was an effective Cu efflux transporterat low and high Cu concentrations the other Cu-ATPase(CopB) only appeared to be a low-affinity Cu export ATPase[68] Moreover by using a M sedula copA deletion mutantit was demonstrated that this strain compromised metalresistance and consequently abolished chalcopyrite oxidation[22] highlighting the role of Cu detoxification mechanismsduring a given bioleaching process Our attempts to showthe functionality of CopA1 and CopA2 from S metallicusby using E coli as a heterologous host were not successfulApparently the overexpression of these ORFs had a toxiceffect on E coli that compromised its viability Althoughgene disruption tools have not been yet developed for Smetallicus the functional role of both copTMA locimight befurther studied by cross-species complementation of a coppersensitive S solfataricus copR mutant as it was described byMaezato et al [22] It will be of great interest to establishin future studies the possible functionality of the isolatedputative transporters from S metallicus

Likewise quantitative RT-PCR experiments showed thatcopT1 transcript levels increased concomitantly with increas-ing Cu concentrations whereas copT2 showed relativelysimilar transcripts levels most likely indicating a constitutiveexpression profile (Figure 8(b)) Furthermore whereas copT1transcript levels were found to be increased 36 fold inCuFeS

2

grown cultures copT2 levels remained unchanged in com-parison with the control in the absence of Cu (Figure 8(b))The results obtained for S metallicus copT2 gene expressionare consistent with those reported for S solfataricus CopTtranscriptional regulator has been proposed to function as arepressor in S solfataricus showing a constitutive expressionthat in the presence of Cu loses its affinity for the promoterregion of copMA allowing the expression of this polycistron[19] In contrast the increased transcripts levels of both copA1and copT1 concomitant with higher Cu concentrations in the

environment suggest that the putative transcriptional regula-tor CopT1 may act by activating both copMA1 and probablyitself (Figure 8) Regarding this it was recently reported thatCopR (CopT in S metallicus) from S solfataricus strain 982acts as an activator of copT (copM in S metallicus) and copAexpression [69] Nevertheless additional experiments wouldbe required to test this possibility in S metallicus

4 Concluding Remarks

We previously reported that S metallicus resists extremelyhigh Cu concentrations which was mediated to some extentby the use of a possible metal resistance system based oninorganic polyphosphate hydrolysis and consequently Cu-PO4

2minusefflux [15 29 30] Here we have addressed the questionwhether this microorganism coded for other determinantsthat might help to explain its high heavy metal toleranceAs the genomic sequence of this microorganism is notyet available we jointly used CODEHOP-PCR and genomewalking approaches and were able to establish the occurrenceof two nonidentical homologous cop loci into the genome ofS metallicus (cop1 and cop2) Each cop locus codes for anarchaeal transcriptional regulator (CopT) a metal-bindingchaperone (CopM) and a Cu-transporting P-type ATPase(CopA) High levels of the polycistronic mRNAscopMA ofeach cop locus were observed after treatment with either Cuor Cd suggesting that the encoded ATPases efflux heavymetals out in order to detoxify the intracellular environmentAltogether previous reports and the results obtained in thisstudy allow us to suggest that some key elements that mayexplain the high resistance to Cu in S metallicus is the dupli-cation of the Cu resistance cop genes a defensive responseto stress and a polyP-based accumulation mechanism Inthe case of Cd although some similar stress responses were

14 Archaea

observed whether comparable Cu-responsive elements alsoparticipate in Cd responses remains to be seen

Conflict of Interests

The authors declare that the research was done in the absenceof any commercial or financial relationships that could beconstrued as a potential conflict of interests

Acknowledgments

This work was supported by Grant no FONDECYT 1110214and in part by ICM P-05-001-F project

References

[1] A Schippers ldquoMicroorganisms involved in bioleaching andnucleic acid-based molecular methods for their identificationand quantificationrdquo in Microbial Processing of Metal SulfidesR E Donati and W Sand Eds pp 3ndash33 Elsevier BerlinGermany 2007

[2] H R Watling ldquoThe bioleaching of sulphide minerals withemphasis on copper sulphides a reviewrdquo Hydrometallurgy vol84 no 1-2 pp 81ndash108 2006

[3] D E Rawlings D Dew and C Du Plessis ldquoBiomineralizationof metal-containing ores and concentratesrdquo Trends in Biotech-nology vol 21 no 1 pp 38ndash44 2003

[4] L Valenzuela A Chi S Beard et al ldquoGenomics metagenomicsand proteomics in biomining microorganismsrdquo BiotechnologyAdvances vol 24 no 2 pp 197ndash211 2006

[5] C A Jerez ldquoBioleaching and biomining for the industrialrecovery of metalsrdquo in Comprehensive Biotechnology M Moo-Young Ed vol 3 pp 717ndash729 Elsevier Amstaerdam TheNetherlands 2nd edition 2011

[6] P R Norris and J P Owen ldquoMineral sulphide oxidationby enrichment cultures of novel thermoacidophilic bacteriardquoFEMS Microbiology Reviews vol 11 no 1ndash3 pp 51ndash56 1993

[7] D E Rawlings and D B Johnson ldquoThe microbiology ofbiomining development and optimization ofmineral-oxidizingmicrobial consortiardquo Microbiology vol 153 no 2 pp 315ndash3242007

[8] P R Norris N P Burton and N A M Foulis ldquoAcidophiles inbioreactor mineral processingrdquo Extremophiles vol 4 no 2 pp71ndash76 2000

[9] M R Bruins S Kapil and F W Oehme ldquoMicrobial resistancetometals in the environmentrdquo Ecotoxicology and EnvironmentalSafety vol 45 no 3 pp 198ndash207 2000

[10] T J G Ettema M A Huynen W M De Vos and J Van DerOost ldquoTRASH a novel metal-binding domain predicted to beinvolved in heavy-metal sensing trafficking and resistancerdquoTrends in Biochemical Sciences vol 28 no 4 pp 170ndash173 2003

[11] M Dopson C Baker-Austin P R Koppineedi and P L BondldquoGrowth in sulfidic mineral environments metal resistancemechanisms in acidophilic micro-organismsrdquo Microbiologyvol 149 no 8 pp 1959ndash1970 2003

[12] S Franke and C Rensing ldquoAcidophiles Mechanisms to toleratemetal and acid toxicityrdquo in Physiology and Biochemistry ofExtremophiles C Gerday and N Glansdorff Eds pp 271ndash278ASM Press Washington DC USA 2007

[13] C A Navarro L H Orellana C Mauriaca and C A JerezldquoTranscriptional and functional studies of Acidithiobacillusferrooxidans genes related to survival in the presence of copperrdquoApplied and Environmental Microbiology vol 75 no 19 pp6102ndash6109 2009

[14] A Orell C A Navarro R Arancibia J C Mobarec andC A Jerez ldquoLife in blue copper resistance mechanisms ofbacteria and Archaea used in industrial biomining of mineralsrdquoBiotechnology Advances vol 28 no 6 pp 839ndash848 2010

[15] A Orell C A Navarro M Rivero J S Aguilar and C A JerezldquoInorganic polyphosphates in extremophiles and their posiblefunctionsrdquo Extremophiles vol 16 no 4 pp 573ndash583 2012

[16] C Baker-Austin M Dopson M Wexler et al ldquoExtremearsenic resistance by the acidophilic archaeon ldquoFerroplasmaacidarmanusrdquo Fer1rdquo Extremophiles vol 11 no 3 pp 425ndash4342007

[17] E Pedone S Bartolucci and G Fiorentino ldquoSensing andadapting to environmental stress the archaeal tacticrdquo Frontiersin Bioscience vol 9 pp 2909ndash2926 2004

[18] C Baker-Austin M Dopson M Wexler R G Sawers and PL Bond ldquoMolecular insight into extreme copper resistance inthe extremophilic archaeon ldquoFerroplasma acidarmanusrdquo Fer1rdquoMicrobiology vol 151 no 8 pp 2637ndash2646 2005

[19] T J G Ettema A B Brinkman P P Lamers N G Kornet WM de Vos and J van der Oost ldquoMolecular characterization ofa conserved archaeal copper resistance (cop) gene cluster andits copper-responsive regulator in Sulfolobus solfataricus P2rdquoMicrobiology vol 152 no 7 pp 1969ndash1979 2006

[20] A A Villafane Y VoskoboynikM Cuebas I Ruhl and E BinildquoResponse to excess copper in the hyperthermophile Sulfolobussolfataricus strain 982rdquo Biochemical and Biophysical ResearchCommunications vol 385 no 1 pp 67ndash71 2009

[21] K S Auernik Y Maezato P H Blum and R M KellyldquoThe genome sequence of the metal-mobilizing extremelythermoacidophilic archaeon Metallosphaera sedula providesinsights into bioleaching-associated metabolismrdquo Applied andEnvironmental Microbiology vol 74 no 3 pp 682ndash692 2008

[22] Y Maezato T Johnson S McCarthy K Dana and P BlumldquoMetal resistance and lithoautotrophy in the extreme ther-moacidophile Metalosphaera sedulardquo Journal of Bacteriologyvol 194 no 24 pp 6856ndash6863 2012

[23] D H Nies ldquoMicrobial heavy-metal resistancerdquo Applied Micro-biology and Biotechnology vol 51 no 6 pp 730ndash750 1999

[24] K Vido D Spector G Lagniel S Lopez M B Toledano andJ Labarre ldquoA proteome analysis of the cadmium response inSaccharomyces cerevisiaerdquo The Journal of Biological Chemistryvol 276 no 11 pp 8469ndash8474 2001

[25] G Bertin and D Averbeck ldquoCadmium cellular effects mod-ifications of biomolecules modulation of DNA repair andgenotoxic consequences (a review)rdquo Biochimie vol 88 no 11pp 1549ndash1559 2006

[26] M H Joe S W Jung S H Im et al ldquoGenome-wide responseof Deinococcus radiodurans on cadmium toxicityrdquo Journal ofMicrobiology and Biotechnology vol 21 no 4 pp 438ndash447 2011

[27] A Lagorce A Fourcans M Dutertre B Bouyssiere YZivanovic and F Confalonieri ldquoGenome-wide transcriptionalresponse of the archeon Thermococcus gammatolerans to cad-miumrdquo PLOS ONE vol 7 no 7 Article ID e41935 2012

[28] A Kornberg N N Rao and D Ault-Riche ldquoInorganicpolyphosphate a molecule of many functionsrdquo Annual Reviewof Biochemistry vol 68 pp 89ndash125 1999

Archaea 15

[29] J D Keasling ldquoRegulation of intracellular toxic metals andother cations by hydrolysis of polyphosphaterdquoAnnals of theNewYork Academy of Sciences vol 829 pp 242ndash249 1997

[30] F Remonsellez A Orell and C A Jerez ldquoCopper tolerance ofthe thermoacidophilic archaeon Sulfolobus metallicus possiblerole of polyphosphate metabolismrdquoMicrobiology vol 152 no 1pp 59ndash66 2006

[31] V Hatzimanikatis L H Choe and K H Lee ldquoProteomicstheoretical and experimental considerationsrdquo BiotechnologyProgress vol 15 no 3 pp 312ndash318 1999

[32] L H Choe and K H Lee ldquoA comparison of three commerciallyavailable isoelectric focusing units for proteome analysis theMultiphor the IPGphor and the Protean IEF cellrdquo Electrophore-sis vol 21 no 5 pp 993ndash1000 2000

[33] U K Laemmli ldquoCleavage of structural proteins during theassembly of the head of bacteriophage T4rdquo Nature vol 227 no5259 pp 680ndash685 1970

[34] A Shevchenko M Wilm O Vorm and M Mann ldquoMassspectrometric sequencing of proteins from silver-stained poly-acrylamide gelsrdquo Analytical Chemistry vol 68 no 5 pp 850ndash858 1996

[35] P Giavalisco E Nordhoff T Kreitler et al ldquoProteome analysisof Arabidopsis thaliana by two-dimensional gel electrophoresisand matrix-assisted laser desorptionionisation-time of flightmass spectrometryrdquo Proteomics vol 5 no 7 pp 1902ndash19132005

[36] T M Rose E R Schultz J G Henikoff S Pietrokovski CM McCallum and S Henikoff ldquoConsensus-degenerate hybridoligonucleotide primers for amplification of distantly relatedsequencesrdquo Nucleic Acids Research vol 26 no 7 pp 1628ndash16351998

[37] T M Rose J G Henikoff and S Henikoff ldquoCODEHOP(Consensus-Degenerate Hybrid Oligonucleotide Primer) PCRprimer designrdquo Nucleic Acids Research vol 31 no 13 pp 3763ndash3766 2003

[38] J P Acevedo F Reyes L P Parra O Salazar B A Andrews andJ A Asenjo ldquoCloning of complete genes for novel hydrolyticenzymes from Antarctic sea water bacteria by use of animproved genomewalking techniquerdquo Journal of Biotechnologyvol 133 no 3 pp 277ndash286 2008

[39] S Bathe and P R Norris ldquoFerrous iron- and sulfur-inducedgenes in Sulfolobus metallicusrdquo Applied and EnvironmentalMicrobiology vol 73 no 8 pp 2491ndash2497 2007

[40] J Sambrook E F Fritsch and TManiatisMolecular Cloning ALaboratory Manual Cold Spring Harbor Press New York NYUSA 1989

[41] K W Miller S Sass Risanico and J B Risatti ldquoDifferentialtolerance of Sulfolobus strains to transition metalsrdquo FEMSMicrobiology Letters vol 93 no 1 pp 69ndash73 1992

[42] G Huber C Spinnler A Gambacorta and K O Stetter ldquoMet-allosphaera sedula gen and sp nov represents a new genus ofaerobic metal-mobilizing thermoacidophilic archaebacteriardquoSystematic and Applied Microbiology vol 12 pp 38ndash47 1989

[43] J Llanos C Capasso E Parisi D Prieur and C JeanthonldquoSusceptibility to heavy metals and cadmium accumulation inaerobic and anaerobic thermophilic microorganisms isolatedfrom deep-sea hydrothermal ventsrdquo Current Microbiology vol41 no 3 pp 201ndash205 2000

[44] A Kaur M Pan M Meislin M T Facciotti R El-Gewelyand N S Baliga ldquoA systems view of haloarchaeal strategies towithstand stress from transition metalsrdquo Genome Research vol16 no 7 pp 841ndash854 2006

[45] M Solioz and J V Stoyanov ldquoCopper homeostasis in Entero-coccus hiraerdquo FEMS Microbiology Reviews vol 27 no 2-3 pp183ndash195 2003

[46] S Sitthisak K Howieson C Amezola and R K JayaswalldquoCharacterization of a multicopper oxidase gene from Staphy-lococcus aureusrdquo Applied and Environmental Microbiology vol71 no 9 pp 5650ndash5653 2005

[47] L Rodrıguez-Montelongo S I Volentini R N Farıas E MMassa and V A Rapisarda ldquoThe Cu(II)-reductase NADHdehydrogenase-2 of Escherichia coli improves the bacterialgrowth in extreme copper concentrations and increases theresistance to the damage caused by copper and hydroperoxiderdquoArchives of Biochemistry and Biophysics vol 451 no 1 pp 1ndash72006

[48] E Williams T M Lowe J Savas and J DiRuggiero ldquoMicroar-ray analysis of the hyperthermophilic archaeon Pyrococcusfuriosus exposed to gamma irradiationrdquo Extremophiles vol 11no 1 pp 19ndash29 2007

[49] L M Gaetke and C K Chow ldquoCopper toxicity oxidative stressand antioxidant nutrientsrdquo Toxicology vol 189 no 1-2 pp 147ndash163 2003

[50] M J Davies ldquoThe oxidative environment and protein damagerdquoBiochimica et Biophysica Acta vol 1703 no 2 pp 93ndash109 2005

[51] J R Thompson J K Bell J Bratt G A Grant and LJ Banaszak ldquoVmax regulation through domain and subunitchanges The active form of phosphoglycerate dehydrogenaserdquoBiochemistry vol 44 no 15 pp 5763ndash5773 2005

[52] V Consalvi R Chiaraluce L Politi A Gambacorta MDe Rosa and R Scandurra ldquoGlutamate dehydrogenase fromthe thermoacidophilic archaebacterium Sulfolobus solfataricusrdquoEuropean Journal of Biochemistry vol 196 no 2 pp 459ndash4671991

[53] A J L Macario M Lange B K Ahring and E Conway DeMacario ldquoStress genes and proteins in the archaeardquo Microbiol-ogy and Molecular Biology Reviews vol 63 no 4 pp 923ndash9671999

[54] J AMaupin-FurlowM A Gil M A Humbard et al ldquoArchaealproteasomes and other regulatory proteasesrdquo Current Opinionin Microbiology vol 8 no 6 pp 720ndash728 2005

[55] N Sun F Beck R Wilhelm Knispel et al ldquoProteomics analysisof Thermoplasma acidophilum with a focus on protein com-plexesrdquoMolecular and Cellular Proteomics vol 6 no 3 pp 492ndash502 2007

[56] L S Madding J K Michel K R Shockley et al ldquoRole of the 1205731subunit in the function and stability of the 20S proteasome inthe hyperthermophilic Archaeon Pyrococcus furiosusrdquo Journalof Bacteriology vol 189 no 2 pp 583ndash590 2007

[57] R Shibata Y Bessho A Shinkai et al ldquoCrystal structureand RNA-binding analysis of the archaeal transcription factorNusArdquo Biochemical and Biophysical Research Communicationsvol 355 no 1 pp 122ndash128 2007

[58] W Bae B Xia M Inouye and K Severinov ldquoEscherichia coliCspA-family RNA chaperones are transcription antitermina-torsrdquo Proceedings of the National Academy of Sciences of theUnited States of America vol 97 no 14 pp 7784ndash7789 2000

[59] R J Ram N C VerBerkmoes M P Thelen et al ldquoMicrobi-ology community proteomics of a natural microbial biofilmrdquoScience vol 308 no 5730 pp 1915ndash1920 2005

[60] I Noel-Georis T Vallaeys R Chauvaux et al ldquoGlobal analysisof the Ralstonia metallidurans proteome prelude for the large-scale study of heavy metal responserdquo Proteomics vol 4 no 1pp 151ndash179 2004

16 Archaea

[61] M TMNovo A CDa Silva RMoreto et al ldquoThiobacillus fer-rooxidans response to copper and other heavy metals growthprotein synthesis and protein phosphorylationrdquo Antonie vanLeeuwenhoek vol 77 no 2 pp 187ndash195 2000

[62] Q She R K Singh F Confalonieri et al ldquoThe complete genomeof the crenarchaeon Sulfolobus solfataricus P2rdquo Proceedings ofthe National Academy of Sciences of the United States of Americavol 98 no 14 pp 7835ndash7840 2001

[63] R P Anderson and J R Roth ldquoTandem genetic duplications inphage and bacteriardquo Annual Review of Microbiology vol 31 pp473ndash505 1977

[64] D Gevers K Vandepoele C Simillion and Y Van De PeerldquoGene duplication and biased functional retention of paralogsin bacterial genomesrdquo Trends in Microbiology vol 12 no 4 pp148ndash154 2004

[65] A B Reams and E L Neidle ldquoSelection for gene clustering bytandemduplicationrdquoAnnual Review ofMicrobiology vol 58 pp119ndash142 2004

[66] L H Orellana and C A Jerez ldquoA genomic island providesAcidithiobacillus ferrooxidans ATCC 53993 additional copperresistance a possible competitive advantagerdquo Applied Microbi-ology and Biotechnology vol 92 no 4 pp 761ndash767 2011

[67] A B Brinkman T J G Ettema W M de Vos and J van derOost ldquoThe Lrp family of transcriptional regulatorsrdquo MolecularMicrobiology vol 48 no 2 pp 287ndash294 2003

[68] C Vollmecke S L Drees J Reimann S V Albers and MLubben ldquoThe ATPases CopA and CopB both contribute tocopper resistance of the thermoacidophilic archaeon SulfolobussolfataricusrdquoMicrobiology vol 158 no 6 pp 1622ndash1633 2012

[69] A Villafane Y Voskoboynik I Ruhl et al ldquoCopR of Sulfolobussolfataricus represents a novel class of archaeal-specific copper-responsive activators of transcriptionrdquo Microbiology vol 157no 10 pp 2808ndash2817 2011

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Anatomy Research International

PeptidesInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporation httpwwwhindawicom

International Journal of

Volume 2014

Zoology

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Molecular Biology International

GenomicsInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioinformaticsAdvances in

Marine BiologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Signal TransductionJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

Evolutionary BiologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Biochemistry Research International

ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Genetics Research International

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Advances in

Virolog y

Hindawi Publishing Corporationhttpwwwhindawicom

Nucleic AcidsJournal of

Volume 2014

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Enzyme Research

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

International Journal of

Microbiology

Archaea 13

700

600

500

400

300

200

100

00

5 20 50 CuFeS2CuSO4 concentration (mM)

Relativ

egenee

xpression

CopA2CopA1

(a)

8

6

7

5

4

3

2

1

00

5 20 50 CuFeS2CuSO4 concentration (mM)

Relativ

egenee

xpression

CopT1CopT2

(b)

Figure 8 Relative expression levels of S metallicus genes copA1 and copA2 (a) copT1 and copT2 (b) Expression of S metallicus cop geneswasassessed by qRT-PCR and normalized against 16S rRNA gene expression in individual cultures Mean values and standard deviations arefrom analyses of three independent cultures grown in the presence of 0 5 20 50mMCu or in 1 of a chalcopyrite ore (CuFeS

2) concentrate

that while CopA was an effective Cu efflux transporterat low and high Cu concentrations the other Cu-ATPase(CopB) only appeared to be a low-affinity Cu export ATPase[68] Moreover by using a M sedula copA deletion mutantit was demonstrated that this strain compromised metalresistance and consequently abolished chalcopyrite oxidation[22] highlighting the role of Cu detoxification mechanismsduring a given bioleaching process Our attempts to showthe functionality of CopA1 and CopA2 from S metallicusby using E coli as a heterologous host were not successfulApparently the overexpression of these ORFs had a toxiceffect on E coli that compromised its viability Althoughgene disruption tools have not been yet developed for Smetallicus the functional role of both copTMA locimight befurther studied by cross-species complementation of a coppersensitive S solfataricus copR mutant as it was described byMaezato et al [22] It will be of great interest to establishin future studies the possible functionality of the isolatedputative transporters from S metallicus

Likewise quantitative RT-PCR experiments showed thatcopT1 transcript levels increased concomitantly with increas-ing Cu concentrations whereas copT2 showed relativelysimilar transcripts levels most likely indicating a constitutiveexpression profile (Figure 8(b)) Furthermore whereas copT1transcript levels were found to be increased 36 fold inCuFeS

2

grown cultures copT2 levels remained unchanged in com-parison with the control in the absence of Cu (Figure 8(b))The results obtained for S metallicus copT2 gene expressionare consistent with those reported for S solfataricus CopTtranscriptional regulator has been proposed to function as arepressor in S solfataricus showing a constitutive expressionthat in the presence of Cu loses its affinity for the promoterregion of copMA allowing the expression of this polycistron[19] In contrast the increased transcripts levels of both copA1and copT1 concomitant with higher Cu concentrations in the

environment suggest that the putative transcriptional regula-tor CopT1 may act by activating both copMA1 and probablyitself (Figure 8) Regarding this it was recently reported thatCopR (CopT in S metallicus) from S solfataricus strain 982acts as an activator of copT (copM in S metallicus) and copAexpression [69] Nevertheless additional experiments wouldbe required to test this possibility in S metallicus

4 Concluding Remarks

We previously reported that S metallicus resists extremelyhigh Cu concentrations which was mediated to some extentby the use of a possible metal resistance system based oninorganic polyphosphate hydrolysis and consequently Cu-PO4

2minusefflux [15 29 30] Here we have addressed the questionwhether this microorganism coded for other determinantsthat might help to explain its high heavy metal toleranceAs the genomic sequence of this microorganism is notyet available we jointly used CODEHOP-PCR and genomewalking approaches and were able to establish the occurrenceof two nonidentical homologous cop loci into the genome ofS metallicus (cop1 and cop2) Each cop locus codes for anarchaeal transcriptional regulator (CopT) a metal-bindingchaperone (CopM) and a Cu-transporting P-type ATPase(CopA) High levels of the polycistronic mRNAscopMA ofeach cop locus were observed after treatment with either Cuor Cd suggesting that the encoded ATPases efflux heavymetals out in order to detoxify the intracellular environmentAltogether previous reports and the results obtained in thisstudy allow us to suggest that some key elements that mayexplain the high resistance to Cu in S metallicus is the dupli-cation of the Cu resistance cop genes a defensive responseto stress and a polyP-based accumulation mechanism Inthe case of Cd although some similar stress responses were

14 Archaea

observed whether comparable Cu-responsive elements alsoparticipate in Cd responses remains to be seen

Conflict of Interests

The authors declare that the research was done in the absenceof any commercial or financial relationships that could beconstrued as a potential conflict of interests

Acknowledgments

This work was supported by Grant no FONDECYT 1110214and in part by ICM P-05-001-F project

References

[1] A Schippers ldquoMicroorganisms involved in bioleaching andnucleic acid-based molecular methods for their identificationand quantificationrdquo in Microbial Processing of Metal SulfidesR E Donati and W Sand Eds pp 3ndash33 Elsevier BerlinGermany 2007

[2] H R Watling ldquoThe bioleaching of sulphide minerals withemphasis on copper sulphides a reviewrdquo Hydrometallurgy vol84 no 1-2 pp 81ndash108 2006

[3] D E Rawlings D Dew and C Du Plessis ldquoBiomineralizationof metal-containing ores and concentratesrdquo Trends in Biotech-nology vol 21 no 1 pp 38ndash44 2003

[4] L Valenzuela A Chi S Beard et al ldquoGenomics metagenomicsand proteomics in biomining microorganismsrdquo BiotechnologyAdvances vol 24 no 2 pp 197ndash211 2006

[5] C A Jerez ldquoBioleaching and biomining for the industrialrecovery of metalsrdquo in Comprehensive Biotechnology M Moo-Young Ed vol 3 pp 717ndash729 Elsevier Amstaerdam TheNetherlands 2nd edition 2011

[6] P R Norris and J P Owen ldquoMineral sulphide oxidationby enrichment cultures of novel thermoacidophilic bacteriardquoFEMS Microbiology Reviews vol 11 no 1ndash3 pp 51ndash56 1993

[7] D E Rawlings and D B Johnson ldquoThe microbiology ofbiomining development and optimization ofmineral-oxidizingmicrobial consortiardquo Microbiology vol 153 no 2 pp 315ndash3242007

[8] P R Norris N P Burton and N A M Foulis ldquoAcidophiles inbioreactor mineral processingrdquo Extremophiles vol 4 no 2 pp71ndash76 2000

[9] M R Bruins S Kapil and F W Oehme ldquoMicrobial resistancetometals in the environmentrdquo Ecotoxicology and EnvironmentalSafety vol 45 no 3 pp 198ndash207 2000

[10] T J G Ettema M A Huynen W M De Vos and J Van DerOost ldquoTRASH a novel metal-binding domain predicted to beinvolved in heavy-metal sensing trafficking and resistancerdquoTrends in Biochemical Sciences vol 28 no 4 pp 170ndash173 2003

[11] M Dopson C Baker-Austin P R Koppineedi and P L BondldquoGrowth in sulfidic mineral environments metal resistancemechanisms in acidophilic micro-organismsrdquo Microbiologyvol 149 no 8 pp 1959ndash1970 2003

[12] S Franke and C Rensing ldquoAcidophiles Mechanisms to toleratemetal and acid toxicityrdquo in Physiology and Biochemistry ofExtremophiles C Gerday and N Glansdorff Eds pp 271ndash278ASM Press Washington DC USA 2007

[13] C A Navarro L H Orellana C Mauriaca and C A JerezldquoTranscriptional and functional studies of Acidithiobacillusferrooxidans genes related to survival in the presence of copperrdquoApplied and Environmental Microbiology vol 75 no 19 pp6102ndash6109 2009

[14] A Orell C A Navarro R Arancibia J C Mobarec andC A Jerez ldquoLife in blue copper resistance mechanisms ofbacteria and Archaea used in industrial biomining of mineralsrdquoBiotechnology Advances vol 28 no 6 pp 839ndash848 2010

[15] A Orell C A Navarro M Rivero J S Aguilar and C A JerezldquoInorganic polyphosphates in extremophiles and their posiblefunctionsrdquo Extremophiles vol 16 no 4 pp 573ndash583 2012

[16] C Baker-Austin M Dopson M Wexler et al ldquoExtremearsenic resistance by the acidophilic archaeon ldquoFerroplasmaacidarmanusrdquo Fer1rdquo Extremophiles vol 11 no 3 pp 425ndash4342007

[17] E Pedone S Bartolucci and G Fiorentino ldquoSensing andadapting to environmental stress the archaeal tacticrdquo Frontiersin Bioscience vol 9 pp 2909ndash2926 2004

[18] C Baker-Austin M Dopson M Wexler R G Sawers and PL Bond ldquoMolecular insight into extreme copper resistance inthe extremophilic archaeon ldquoFerroplasma acidarmanusrdquo Fer1rdquoMicrobiology vol 151 no 8 pp 2637ndash2646 2005

[19] T J G Ettema A B Brinkman P P Lamers N G Kornet WM de Vos and J van der Oost ldquoMolecular characterization ofa conserved archaeal copper resistance (cop) gene cluster andits copper-responsive regulator in Sulfolobus solfataricus P2rdquoMicrobiology vol 152 no 7 pp 1969ndash1979 2006

[20] A A Villafane Y VoskoboynikM Cuebas I Ruhl and E BinildquoResponse to excess copper in the hyperthermophile Sulfolobussolfataricus strain 982rdquo Biochemical and Biophysical ResearchCommunications vol 385 no 1 pp 67ndash71 2009

[21] K S Auernik Y Maezato P H Blum and R M KellyldquoThe genome sequence of the metal-mobilizing extremelythermoacidophilic archaeon Metallosphaera sedula providesinsights into bioleaching-associated metabolismrdquo Applied andEnvironmental Microbiology vol 74 no 3 pp 682ndash692 2008

[22] Y Maezato T Johnson S McCarthy K Dana and P BlumldquoMetal resistance and lithoautotrophy in the extreme ther-moacidophile Metalosphaera sedulardquo Journal of Bacteriologyvol 194 no 24 pp 6856ndash6863 2012

[23] D H Nies ldquoMicrobial heavy-metal resistancerdquo Applied Micro-biology and Biotechnology vol 51 no 6 pp 730ndash750 1999

[24] K Vido D Spector G Lagniel S Lopez M B Toledano andJ Labarre ldquoA proteome analysis of the cadmium response inSaccharomyces cerevisiaerdquo The Journal of Biological Chemistryvol 276 no 11 pp 8469ndash8474 2001

[25] G Bertin and D Averbeck ldquoCadmium cellular effects mod-ifications of biomolecules modulation of DNA repair andgenotoxic consequences (a review)rdquo Biochimie vol 88 no 11pp 1549ndash1559 2006

[26] M H Joe S W Jung S H Im et al ldquoGenome-wide responseof Deinococcus radiodurans on cadmium toxicityrdquo Journal ofMicrobiology and Biotechnology vol 21 no 4 pp 438ndash447 2011

[27] A Lagorce A Fourcans M Dutertre B Bouyssiere YZivanovic and F Confalonieri ldquoGenome-wide transcriptionalresponse of the archeon Thermococcus gammatolerans to cad-miumrdquo PLOS ONE vol 7 no 7 Article ID e41935 2012

[28] A Kornberg N N Rao and D Ault-Riche ldquoInorganicpolyphosphate a molecule of many functionsrdquo Annual Reviewof Biochemistry vol 68 pp 89ndash125 1999

Archaea 15

[29] J D Keasling ldquoRegulation of intracellular toxic metals andother cations by hydrolysis of polyphosphaterdquoAnnals of theNewYork Academy of Sciences vol 829 pp 242ndash249 1997

[30] F Remonsellez A Orell and C A Jerez ldquoCopper tolerance ofthe thermoacidophilic archaeon Sulfolobus metallicus possiblerole of polyphosphate metabolismrdquoMicrobiology vol 152 no 1pp 59ndash66 2006

[31] V Hatzimanikatis L H Choe and K H Lee ldquoProteomicstheoretical and experimental considerationsrdquo BiotechnologyProgress vol 15 no 3 pp 312ndash318 1999

[32] L H Choe and K H Lee ldquoA comparison of three commerciallyavailable isoelectric focusing units for proteome analysis theMultiphor the IPGphor and the Protean IEF cellrdquo Electrophore-sis vol 21 no 5 pp 993ndash1000 2000

[33] U K Laemmli ldquoCleavage of structural proteins during theassembly of the head of bacteriophage T4rdquo Nature vol 227 no5259 pp 680ndash685 1970

[34] A Shevchenko M Wilm O Vorm and M Mann ldquoMassspectrometric sequencing of proteins from silver-stained poly-acrylamide gelsrdquo Analytical Chemistry vol 68 no 5 pp 850ndash858 1996

[35] P Giavalisco E Nordhoff T Kreitler et al ldquoProteome analysisof Arabidopsis thaliana by two-dimensional gel electrophoresisand matrix-assisted laser desorptionionisation-time of flightmass spectrometryrdquo Proteomics vol 5 no 7 pp 1902ndash19132005

[36] T M Rose E R Schultz J G Henikoff S Pietrokovski CM McCallum and S Henikoff ldquoConsensus-degenerate hybridoligonucleotide primers for amplification of distantly relatedsequencesrdquo Nucleic Acids Research vol 26 no 7 pp 1628ndash16351998

[37] T M Rose J G Henikoff and S Henikoff ldquoCODEHOP(Consensus-Degenerate Hybrid Oligonucleotide Primer) PCRprimer designrdquo Nucleic Acids Research vol 31 no 13 pp 3763ndash3766 2003

[38] J P Acevedo F Reyes L P Parra O Salazar B A Andrews andJ A Asenjo ldquoCloning of complete genes for novel hydrolyticenzymes from Antarctic sea water bacteria by use of animproved genomewalking techniquerdquo Journal of Biotechnologyvol 133 no 3 pp 277ndash286 2008

[39] S Bathe and P R Norris ldquoFerrous iron- and sulfur-inducedgenes in Sulfolobus metallicusrdquo Applied and EnvironmentalMicrobiology vol 73 no 8 pp 2491ndash2497 2007

[40] J Sambrook E F Fritsch and TManiatisMolecular Cloning ALaboratory Manual Cold Spring Harbor Press New York NYUSA 1989

[41] K W Miller S Sass Risanico and J B Risatti ldquoDifferentialtolerance of Sulfolobus strains to transition metalsrdquo FEMSMicrobiology Letters vol 93 no 1 pp 69ndash73 1992

[42] G Huber C Spinnler A Gambacorta and K O Stetter ldquoMet-allosphaera sedula gen and sp nov represents a new genus ofaerobic metal-mobilizing thermoacidophilic archaebacteriardquoSystematic and Applied Microbiology vol 12 pp 38ndash47 1989

[43] J Llanos C Capasso E Parisi D Prieur and C JeanthonldquoSusceptibility to heavy metals and cadmium accumulation inaerobic and anaerobic thermophilic microorganisms isolatedfrom deep-sea hydrothermal ventsrdquo Current Microbiology vol41 no 3 pp 201ndash205 2000

[44] A Kaur M Pan M Meislin M T Facciotti R El-Gewelyand N S Baliga ldquoA systems view of haloarchaeal strategies towithstand stress from transition metalsrdquo Genome Research vol16 no 7 pp 841ndash854 2006

[45] M Solioz and J V Stoyanov ldquoCopper homeostasis in Entero-coccus hiraerdquo FEMS Microbiology Reviews vol 27 no 2-3 pp183ndash195 2003

[46] S Sitthisak K Howieson C Amezola and R K JayaswalldquoCharacterization of a multicopper oxidase gene from Staphy-lococcus aureusrdquo Applied and Environmental Microbiology vol71 no 9 pp 5650ndash5653 2005

[47] L Rodrıguez-Montelongo S I Volentini R N Farıas E MMassa and V A Rapisarda ldquoThe Cu(II)-reductase NADHdehydrogenase-2 of Escherichia coli improves the bacterialgrowth in extreme copper concentrations and increases theresistance to the damage caused by copper and hydroperoxiderdquoArchives of Biochemistry and Biophysics vol 451 no 1 pp 1ndash72006

[48] E Williams T M Lowe J Savas and J DiRuggiero ldquoMicroar-ray analysis of the hyperthermophilic archaeon Pyrococcusfuriosus exposed to gamma irradiationrdquo Extremophiles vol 11no 1 pp 19ndash29 2007

[49] L M Gaetke and C K Chow ldquoCopper toxicity oxidative stressand antioxidant nutrientsrdquo Toxicology vol 189 no 1-2 pp 147ndash163 2003

[50] M J Davies ldquoThe oxidative environment and protein damagerdquoBiochimica et Biophysica Acta vol 1703 no 2 pp 93ndash109 2005

[51] J R Thompson J K Bell J Bratt G A Grant and LJ Banaszak ldquoVmax regulation through domain and subunitchanges The active form of phosphoglycerate dehydrogenaserdquoBiochemistry vol 44 no 15 pp 5763ndash5773 2005

[52] V Consalvi R Chiaraluce L Politi A Gambacorta MDe Rosa and R Scandurra ldquoGlutamate dehydrogenase fromthe thermoacidophilic archaebacterium Sulfolobus solfataricusrdquoEuropean Journal of Biochemistry vol 196 no 2 pp 459ndash4671991

[53] A J L Macario M Lange B K Ahring and E Conway DeMacario ldquoStress genes and proteins in the archaeardquo Microbiol-ogy and Molecular Biology Reviews vol 63 no 4 pp 923ndash9671999

[54] J AMaupin-FurlowM A Gil M A Humbard et al ldquoArchaealproteasomes and other regulatory proteasesrdquo Current Opinionin Microbiology vol 8 no 6 pp 720ndash728 2005

[55] N Sun F Beck R Wilhelm Knispel et al ldquoProteomics analysisof Thermoplasma acidophilum with a focus on protein com-plexesrdquoMolecular and Cellular Proteomics vol 6 no 3 pp 492ndash502 2007

[56] L S Madding J K Michel K R Shockley et al ldquoRole of the 1205731subunit in the function and stability of the 20S proteasome inthe hyperthermophilic Archaeon Pyrococcus furiosusrdquo Journalof Bacteriology vol 189 no 2 pp 583ndash590 2007

[57] R Shibata Y Bessho A Shinkai et al ldquoCrystal structureand RNA-binding analysis of the archaeal transcription factorNusArdquo Biochemical and Biophysical Research Communicationsvol 355 no 1 pp 122ndash128 2007

[58] W Bae B Xia M Inouye and K Severinov ldquoEscherichia coliCspA-family RNA chaperones are transcription antitermina-torsrdquo Proceedings of the National Academy of Sciences of theUnited States of America vol 97 no 14 pp 7784ndash7789 2000

[59] R J Ram N C VerBerkmoes M P Thelen et al ldquoMicrobi-ology community proteomics of a natural microbial biofilmrdquoScience vol 308 no 5730 pp 1915ndash1920 2005

[60] I Noel-Georis T Vallaeys R Chauvaux et al ldquoGlobal analysisof the Ralstonia metallidurans proteome prelude for the large-scale study of heavy metal responserdquo Proteomics vol 4 no 1pp 151ndash179 2004

16 Archaea

[61] M TMNovo A CDa Silva RMoreto et al ldquoThiobacillus fer-rooxidans response to copper and other heavy metals growthprotein synthesis and protein phosphorylationrdquo Antonie vanLeeuwenhoek vol 77 no 2 pp 187ndash195 2000

[62] Q She R K Singh F Confalonieri et al ldquoThe complete genomeof the crenarchaeon Sulfolobus solfataricus P2rdquo Proceedings ofthe National Academy of Sciences of the United States of Americavol 98 no 14 pp 7835ndash7840 2001

[63] R P Anderson and J R Roth ldquoTandem genetic duplications inphage and bacteriardquo Annual Review of Microbiology vol 31 pp473ndash505 1977

[64] D Gevers K Vandepoele C Simillion and Y Van De PeerldquoGene duplication and biased functional retention of paralogsin bacterial genomesrdquo Trends in Microbiology vol 12 no 4 pp148ndash154 2004

[65] A B Reams and E L Neidle ldquoSelection for gene clustering bytandemduplicationrdquoAnnual Review ofMicrobiology vol 58 pp119ndash142 2004

[66] L H Orellana and C A Jerez ldquoA genomic island providesAcidithiobacillus ferrooxidans ATCC 53993 additional copperresistance a possible competitive advantagerdquo Applied Microbi-ology and Biotechnology vol 92 no 4 pp 761ndash767 2011

[67] A B Brinkman T J G Ettema W M de Vos and J van derOost ldquoThe Lrp family of transcriptional regulatorsrdquo MolecularMicrobiology vol 48 no 2 pp 287ndash294 2003

[68] C Vollmecke S L Drees J Reimann S V Albers and MLubben ldquoThe ATPases CopA and CopB both contribute tocopper resistance of the thermoacidophilic archaeon SulfolobussolfataricusrdquoMicrobiology vol 158 no 6 pp 1622ndash1633 2012

[69] A Villafane Y Voskoboynik I Ruhl et al ldquoCopR of Sulfolobussolfataricus represents a novel class of archaeal-specific copper-responsive activators of transcriptionrdquo Microbiology vol 157no 10 pp 2808ndash2817 2011

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Anatomy Research International

PeptidesInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporation httpwwwhindawicom

International Journal of

Volume 2014

Zoology

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Molecular Biology International

GenomicsInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioinformaticsAdvances in

Marine BiologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Signal TransductionJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

Evolutionary BiologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Biochemistry Research International

ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Genetics Research International

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Advances in

Virolog y

Hindawi Publishing Corporationhttpwwwhindawicom

Nucleic AcidsJournal of

Volume 2014

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Enzyme Research

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

International Journal of

Microbiology

14 Archaea

observed whether comparable Cu-responsive elements alsoparticipate in Cd responses remains to be seen

Conflict of Interests

The authors declare that the research was done in the absenceof any commercial or financial relationships that could beconstrued as a potential conflict of interests

Acknowledgments

This work was supported by Grant no FONDECYT 1110214and in part by ICM P-05-001-F project

References

[1] A Schippers ldquoMicroorganisms involved in bioleaching andnucleic acid-based molecular methods for their identificationand quantificationrdquo in Microbial Processing of Metal SulfidesR E Donati and W Sand Eds pp 3ndash33 Elsevier BerlinGermany 2007

[2] H R Watling ldquoThe bioleaching of sulphide minerals withemphasis on copper sulphides a reviewrdquo Hydrometallurgy vol84 no 1-2 pp 81ndash108 2006

[3] D E Rawlings D Dew and C Du Plessis ldquoBiomineralizationof metal-containing ores and concentratesrdquo Trends in Biotech-nology vol 21 no 1 pp 38ndash44 2003

[4] L Valenzuela A Chi S Beard et al ldquoGenomics metagenomicsand proteomics in biomining microorganismsrdquo BiotechnologyAdvances vol 24 no 2 pp 197ndash211 2006

[5] C A Jerez ldquoBioleaching and biomining for the industrialrecovery of metalsrdquo in Comprehensive Biotechnology M Moo-Young Ed vol 3 pp 717ndash729 Elsevier Amstaerdam TheNetherlands 2nd edition 2011

[6] P R Norris and J P Owen ldquoMineral sulphide oxidationby enrichment cultures of novel thermoacidophilic bacteriardquoFEMS Microbiology Reviews vol 11 no 1ndash3 pp 51ndash56 1993

[7] D E Rawlings and D B Johnson ldquoThe microbiology ofbiomining development and optimization ofmineral-oxidizingmicrobial consortiardquo Microbiology vol 153 no 2 pp 315ndash3242007

[8] P R Norris N P Burton and N A M Foulis ldquoAcidophiles inbioreactor mineral processingrdquo Extremophiles vol 4 no 2 pp71ndash76 2000

[9] M R Bruins S Kapil and F W Oehme ldquoMicrobial resistancetometals in the environmentrdquo Ecotoxicology and EnvironmentalSafety vol 45 no 3 pp 198ndash207 2000

[10] T J G Ettema M A Huynen W M De Vos and J Van DerOost ldquoTRASH a novel metal-binding domain predicted to beinvolved in heavy-metal sensing trafficking and resistancerdquoTrends in Biochemical Sciences vol 28 no 4 pp 170ndash173 2003

[11] M Dopson C Baker-Austin P R Koppineedi and P L BondldquoGrowth in sulfidic mineral environments metal resistancemechanisms in acidophilic micro-organismsrdquo Microbiologyvol 149 no 8 pp 1959ndash1970 2003

[12] S Franke and C Rensing ldquoAcidophiles Mechanisms to toleratemetal and acid toxicityrdquo in Physiology and Biochemistry ofExtremophiles C Gerday and N Glansdorff Eds pp 271ndash278ASM Press Washington DC USA 2007

[13] C A Navarro L H Orellana C Mauriaca and C A JerezldquoTranscriptional and functional studies of Acidithiobacillusferrooxidans genes related to survival in the presence of copperrdquoApplied and Environmental Microbiology vol 75 no 19 pp6102ndash6109 2009

[14] A Orell C A Navarro R Arancibia J C Mobarec andC A Jerez ldquoLife in blue copper resistance mechanisms ofbacteria and Archaea used in industrial biomining of mineralsrdquoBiotechnology Advances vol 28 no 6 pp 839ndash848 2010

[15] A Orell C A Navarro M Rivero J S Aguilar and C A JerezldquoInorganic polyphosphates in extremophiles and their posiblefunctionsrdquo Extremophiles vol 16 no 4 pp 573ndash583 2012

[16] C Baker-Austin M Dopson M Wexler et al ldquoExtremearsenic resistance by the acidophilic archaeon ldquoFerroplasmaacidarmanusrdquo Fer1rdquo Extremophiles vol 11 no 3 pp 425ndash4342007

[17] E Pedone S Bartolucci and G Fiorentino ldquoSensing andadapting to environmental stress the archaeal tacticrdquo Frontiersin Bioscience vol 9 pp 2909ndash2926 2004

[18] C Baker-Austin M Dopson M Wexler R G Sawers and PL Bond ldquoMolecular insight into extreme copper resistance inthe extremophilic archaeon ldquoFerroplasma acidarmanusrdquo Fer1rdquoMicrobiology vol 151 no 8 pp 2637ndash2646 2005

[19] T J G Ettema A B Brinkman P P Lamers N G Kornet WM de Vos and J van der Oost ldquoMolecular characterization ofa conserved archaeal copper resistance (cop) gene cluster andits copper-responsive regulator in Sulfolobus solfataricus P2rdquoMicrobiology vol 152 no 7 pp 1969ndash1979 2006

[20] A A Villafane Y VoskoboynikM Cuebas I Ruhl and E BinildquoResponse to excess copper in the hyperthermophile Sulfolobussolfataricus strain 982rdquo Biochemical and Biophysical ResearchCommunications vol 385 no 1 pp 67ndash71 2009

[21] K S Auernik Y Maezato P H Blum and R M KellyldquoThe genome sequence of the metal-mobilizing extremelythermoacidophilic archaeon Metallosphaera sedula providesinsights into bioleaching-associated metabolismrdquo Applied andEnvironmental Microbiology vol 74 no 3 pp 682ndash692 2008

[22] Y Maezato T Johnson S McCarthy K Dana and P BlumldquoMetal resistance and lithoautotrophy in the extreme ther-moacidophile Metalosphaera sedulardquo Journal of Bacteriologyvol 194 no 24 pp 6856ndash6863 2012

[23] D H Nies ldquoMicrobial heavy-metal resistancerdquo Applied Micro-biology and Biotechnology vol 51 no 6 pp 730ndash750 1999

[24] K Vido D Spector G Lagniel S Lopez M B Toledano andJ Labarre ldquoA proteome analysis of the cadmium response inSaccharomyces cerevisiaerdquo The Journal of Biological Chemistryvol 276 no 11 pp 8469ndash8474 2001

[25] G Bertin and D Averbeck ldquoCadmium cellular effects mod-ifications of biomolecules modulation of DNA repair andgenotoxic consequences (a review)rdquo Biochimie vol 88 no 11pp 1549ndash1559 2006

[26] M H Joe S W Jung S H Im et al ldquoGenome-wide responseof Deinococcus radiodurans on cadmium toxicityrdquo Journal ofMicrobiology and Biotechnology vol 21 no 4 pp 438ndash447 2011

[27] A Lagorce A Fourcans M Dutertre B Bouyssiere YZivanovic and F Confalonieri ldquoGenome-wide transcriptionalresponse of the archeon Thermococcus gammatolerans to cad-miumrdquo PLOS ONE vol 7 no 7 Article ID e41935 2012

[28] A Kornberg N N Rao and D Ault-Riche ldquoInorganicpolyphosphate a molecule of many functionsrdquo Annual Reviewof Biochemistry vol 68 pp 89ndash125 1999

Archaea 15

[29] J D Keasling ldquoRegulation of intracellular toxic metals andother cations by hydrolysis of polyphosphaterdquoAnnals of theNewYork Academy of Sciences vol 829 pp 242ndash249 1997

[30] F Remonsellez A Orell and C A Jerez ldquoCopper tolerance ofthe thermoacidophilic archaeon Sulfolobus metallicus possiblerole of polyphosphate metabolismrdquoMicrobiology vol 152 no 1pp 59ndash66 2006

[31] V Hatzimanikatis L H Choe and K H Lee ldquoProteomicstheoretical and experimental considerationsrdquo BiotechnologyProgress vol 15 no 3 pp 312ndash318 1999

[32] L H Choe and K H Lee ldquoA comparison of three commerciallyavailable isoelectric focusing units for proteome analysis theMultiphor the IPGphor and the Protean IEF cellrdquo Electrophore-sis vol 21 no 5 pp 993ndash1000 2000

[33] U K Laemmli ldquoCleavage of structural proteins during theassembly of the head of bacteriophage T4rdquo Nature vol 227 no5259 pp 680ndash685 1970

[34] A Shevchenko M Wilm O Vorm and M Mann ldquoMassspectrometric sequencing of proteins from silver-stained poly-acrylamide gelsrdquo Analytical Chemistry vol 68 no 5 pp 850ndash858 1996

[35] P Giavalisco E Nordhoff T Kreitler et al ldquoProteome analysisof Arabidopsis thaliana by two-dimensional gel electrophoresisand matrix-assisted laser desorptionionisation-time of flightmass spectrometryrdquo Proteomics vol 5 no 7 pp 1902ndash19132005

[36] T M Rose E R Schultz J G Henikoff S Pietrokovski CM McCallum and S Henikoff ldquoConsensus-degenerate hybridoligonucleotide primers for amplification of distantly relatedsequencesrdquo Nucleic Acids Research vol 26 no 7 pp 1628ndash16351998

[37] T M Rose J G Henikoff and S Henikoff ldquoCODEHOP(Consensus-Degenerate Hybrid Oligonucleotide Primer) PCRprimer designrdquo Nucleic Acids Research vol 31 no 13 pp 3763ndash3766 2003

[38] J P Acevedo F Reyes L P Parra O Salazar B A Andrews andJ A Asenjo ldquoCloning of complete genes for novel hydrolyticenzymes from Antarctic sea water bacteria by use of animproved genomewalking techniquerdquo Journal of Biotechnologyvol 133 no 3 pp 277ndash286 2008

[39] S Bathe and P R Norris ldquoFerrous iron- and sulfur-inducedgenes in Sulfolobus metallicusrdquo Applied and EnvironmentalMicrobiology vol 73 no 8 pp 2491ndash2497 2007

[40] J Sambrook E F Fritsch and TManiatisMolecular Cloning ALaboratory Manual Cold Spring Harbor Press New York NYUSA 1989

[41] K W Miller S Sass Risanico and J B Risatti ldquoDifferentialtolerance of Sulfolobus strains to transition metalsrdquo FEMSMicrobiology Letters vol 93 no 1 pp 69ndash73 1992

[42] G Huber C Spinnler A Gambacorta and K O Stetter ldquoMet-allosphaera sedula gen and sp nov represents a new genus ofaerobic metal-mobilizing thermoacidophilic archaebacteriardquoSystematic and Applied Microbiology vol 12 pp 38ndash47 1989

[43] J Llanos C Capasso E Parisi D Prieur and C JeanthonldquoSusceptibility to heavy metals and cadmium accumulation inaerobic and anaerobic thermophilic microorganisms isolatedfrom deep-sea hydrothermal ventsrdquo Current Microbiology vol41 no 3 pp 201ndash205 2000

[44] A Kaur M Pan M Meislin M T Facciotti R El-Gewelyand N S Baliga ldquoA systems view of haloarchaeal strategies towithstand stress from transition metalsrdquo Genome Research vol16 no 7 pp 841ndash854 2006

[45] M Solioz and J V Stoyanov ldquoCopper homeostasis in Entero-coccus hiraerdquo FEMS Microbiology Reviews vol 27 no 2-3 pp183ndash195 2003

[46] S Sitthisak K Howieson C Amezola and R K JayaswalldquoCharacterization of a multicopper oxidase gene from Staphy-lococcus aureusrdquo Applied and Environmental Microbiology vol71 no 9 pp 5650ndash5653 2005

[47] L Rodrıguez-Montelongo S I Volentini R N Farıas E MMassa and V A Rapisarda ldquoThe Cu(II)-reductase NADHdehydrogenase-2 of Escherichia coli improves the bacterialgrowth in extreme copper concentrations and increases theresistance to the damage caused by copper and hydroperoxiderdquoArchives of Biochemistry and Biophysics vol 451 no 1 pp 1ndash72006

[48] E Williams T M Lowe J Savas and J DiRuggiero ldquoMicroar-ray analysis of the hyperthermophilic archaeon Pyrococcusfuriosus exposed to gamma irradiationrdquo Extremophiles vol 11no 1 pp 19ndash29 2007

[49] L M Gaetke and C K Chow ldquoCopper toxicity oxidative stressand antioxidant nutrientsrdquo Toxicology vol 189 no 1-2 pp 147ndash163 2003

[50] M J Davies ldquoThe oxidative environment and protein damagerdquoBiochimica et Biophysica Acta vol 1703 no 2 pp 93ndash109 2005

[51] J R Thompson J K Bell J Bratt G A Grant and LJ Banaszak ldquoVmax regulation through domain and subunitchanges The active form of phosphoglycerate dehydrogenaserdquoBiochemistry vol 44 no 15 pp 5763ndash5773 2005

[52] V Consalvi R Chiaraluce L Politi A Gambacorta MDe Rosa and R Scandurra ldquoGlutamate dehydrogenase fromthe thermoacidophilic archaebacterium Sulfolobus solfataricusrdquoEuropean Journal of Biochemistry vol 196 no 2 pp 459ndash4671991

[53] A J L Macario M Lange B K Ahring and E Conway DeMacario ldquoStress genes and proteins in the archaeardquo Microbiol-ogy and Molecular Biology Reviews vol 63 no 4 pp 923ndash9671999

[54] J AMaupin-FurlowM A Gil M A Humbard et al ldquoArchaealproteasomes and other regulatory proteasesrdquo Current Opinionin Microbiology vol 8 no 6 pp 720ndash728 2005

[55] N Sun F Beck R Wilhelm Knispel et al ldquoProteomics analysisof Thermoplasma acidophilum with a focus on protein com-plexesrdquoMolecular and Cellular Proteomics vol 6 no 3 pp 492ndash502 2007

[56] L S Madding J K Michel K R Shockley et al ldquoRole of the 1205731subunit in the function and stability of the 20S proteasome inthe hyperthermophilic Archaeon Pyrococcus furiosusrdquo Journalof Bacteriology vol 189 no 2 pp 583ndash590 2007

[57] R Shibata Y Bessho A Shinkai et al ldquoCrystal structureand RNA-binding analysis of the archaeal transcription factorNusArdquo Biochemical and Biophysical Research Communicationsvol 355 no 1 pp 122ndash128 2007

[58] W Bae B Xia M Inouye and K Severinov ldquoEscherichia coliCspA-family RNA chaperones are transcription antitermina-torsrdquo Proceedings of the National Academy of Sciences of theUnited States of America vol 97 no 14 pp 7784ndash7789 2000

[59] R J Ram N C VerBerkmoes M P Thelen et al ldquoMicrobi-ology community proteomics of a natural microbial biofilmrdquoScience vol 308 no 5730 pp 1915ndash1920 2005

[60] I Noel-Georis T Vallaeys R Chauvaux et al ldquoGlobal analysisof the Ralstonia metallidurans proteome prelude for the large-scale study of heavy metal responserdquo Proteomics vol 4 no 1pp 151ndash179 2004

16 Archaea

[61] M TMNovo A CDa Silva RMoreto et al ldquoThiobacillus fer-rooxidans response to copper and other heavy metals growthprotein synthesis and protein phosphorylationrdquo Antonie vanLeeuwenhoek vol 77 no 2 pp 187ndash195 2000

[62] Q She R K Singh F Confalonieri et al ldquoThe complete genomeof the crenarchaeon Sulfolobus solfataricus P2rdquo Proceedings ofthe National Academy of Sciences of the United States of Americavol 98 no 14 pp 7835ndash7840 2001

[63] R P Anderson and J R Roth ldquoTandem genetic duplications inphage and bacteriardquo Annual Review of Microbiology vol 31 pp473ndash505 1977

[64] D Gevers K Vandepoele C Simillion and Y Van De PeerldquoGene duplication and biased functional retention of paralogsin bacterial genomesrdquo Trends in Microbiology vol 12 no 4 pp148ndash154 2004

[65] A B Reams and E L Neidle ldquoSelection for gene clustering bytandemduplicationrdquoAnnual Review ofMicrobiology vol 58 pp119ndash142 2004

[66] L H Orellana and C A Jerez ldquoA genomic island providesAcidithiobacillus ferrooxidans ATCC 53993 additional copperresistance a possible competitive advantagerdquo Applied Microbi-ology and Biotechnology vol 92 no 4 pp 761ndash767 2011

[67] A B Brinkman T J G Ettema W M de Vos and J van derOost ldquoThe Lrp family of transcriptional regulatorsrdquo MolecularMicrobiology vol 48 no 2 pp 287ndash294 2003

[68] C Vollmecke S L Drees J Reimann S V Albers and MLubben ldquoThe ATPases CopA and CopB both contribute tocopper resistance of the thermoacidophilic archaeon SulfolobussolfataricusrdquoMicrobiology vol 158 no 6 pp 1622ndash1633 2012

[69] A Villafane Y Voskoboynik I Ruhl et al ldquoCopR of Sulfolobussolfataricus represents a novel class of archaeal-specific copper-responsive activators of transcriptionrdquo Microbiology vol 157no 10 pp 2808ndash2817 2011

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Anatomy Research International

PeptidesInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporation httpwwwhindawicom

International Journal of

Volume 2014

Zoology

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Molecular Biology International

GenomicsInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioinformaticsAdvances in

Marine BiologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Signal TransductionJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

Evolutionary BiologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Biochemistry Research International

ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Genetics Research International

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Advances in

Virolog y

Hindawi Publishing Corporationhttpwwwhindawicom

Nucleic AcidsJournal of

Volume 2014

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Enzyme Research

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

International Journal of

Microbiology

Archaea 15

[29] J D Keasling ldquoRegulation of intracellular toxic metals andother cations by hydrolysis of polyphosphaterdquoAnnals of theNewYork Academy of Sciences vol 829 pp 242ndash249 1997

[30] F Remonsellez A Orell and C A Jerez ldquoCopper tolerance ofthe thermoacidophilic archaeon Sulfolobus metallicus possiblerole of polyphosphate metabolismrdquoMicrobiology vol 152 no 1pp 59ndash66 2006

[31] V Hatzimanikatis L H Choe and K H Lee ldquoProteomicstheoretical and experimental considerationsrdquo BiotechnologyProgress vol 15 no 3 pp 312ndash318 1999

[32] L H Choe and K H Lee ldquoA comparison of three commerciallyavailable isoelectric focusing units for proteome analysis theMultiphor the IPGphor and the Protean IEF cellrdquo Electrophore-sis vol 21 no 5 pp 993ndash1000 2000

[33] U K Laemmli ldquoCleavage of structural proteins during theassembly of the head of bacteriophage T4rdquo Nature vol 227 no5259 pp 680ndash685 1970

[34] A Shevchenko M Wilm O Vorm and M Mann ldquoMassspectrometric sequencing of proteins from silver-stained poly-acrylamide gelsrdquo Analytical Chemistry vol 68 no 5 pp 850ndash858 1996

[35] P Giavalisco E Nordhoff T Kreitler et al ldquoProteome analysisof Arabidopsis thaliana by two-dimensional gel electrophoresisand matrix-assisted laser desorptionionisation-time of flightmass spectrometryrdquo Proteomics vol 5 no 7 pp 1902ndash19132005

[36] T M Rose E R Schultz J G Henikoff S Pietrokovski CM McCallum and S Henikoff ldquoConsensus-degenerate hybridoligonucleotide primers for amplification of distantly relatedsequencesrdquo Nucleic Acids Research vol 26 no 7 pp 1628ndash16351998

[37] T M Rose J G Henikoff and S Henikoff ldquoCODEHOP(Consensus-Degenerate Hybrid Oligonucleotide Primer) PCRprimer designrdquo Nucleic Acids Research vol 31 no 13 pp 3763ndash3766 2003

[38] J P Acevedo F Reyes L P Parra O Salazar B A Andrews andJ A Asenjo ldquoCloning of complete genes for novel hydrolyticenzymes from Antarctic sea water bacteria by use of animproved genomewalking techniquerdquo Journal of Biotechnologyvol 133 no 3 pp 277ndash286 2008

[39] S Bathe and P R Norris ldquoFerrous iron- and sulfur-inducedgenes in Sulfolobus metallicusrdquo Applied and EnvironmentalMicrobiology vol 73 no 8 pp 2491ndash2497 2007

[40] J Sambrook E F Fritsch and TManiatisMolecular Cloning ALaboratory Manual Cold Spring Harbor Press New York NYUSA 1989

[41] K W Miller S Sass Risanico and J B Risatti ldquoDifferentialtolerance of Sulfolobus strains to transition metalsrdquo FEMSMicrobiology Letters vol 93 no 1 pp 69ndash73 1992

[42] G Huber C Spinnler A Gambacorta and K O Stetter ldquoMet-allosphaera sedula gen and sp nov represents a new genus ofaerobic metal-mobilizing thermoacidophilic archaebacteriardquoSystematic and Applied Microbiology vol 12 pp 38ndash47 1989

[43] J Llanos C Capasso E Parisi D Prieur and C JeanthonldquoSusceptibility to heavy metals and cadmium accumulation inaerobic and anaerobic thermophilic microorganisms isolatedfrom deep-sea hydrothermal ventsrdquo Current Microbiology vol41 no 3 pp 201ndash205 2000

[44] A Kaur M Pan M Meislin M T Facciotti R El-Gewelyand N S Baliga ldquoA systems view of haloarchaeal strategies towithstand stress from transition metalsrdquo Genome Research vol16 no 7 pp 841ndash854 2006

[45] M Solioz and J V Stoyanov ldquoCopper homeostasis in Entero-coccus hiraerdquo FEMS Microbiology Reviews vol 27 no 2-3 pp183ndash195 2003

[46] S Sitthisak K Howieson C Amezola and R K JayaswalldquoCharacterization of a multicopper oxidase gene from Staphy-lococcus aureusrdquo Applied and Environmental Microbiology vol71 no 9 pp 5650ndash5653 2005

[47] L Rodrıguez-Montelongo S I Volentini R N Farıas E MMassa and V A Rapisarda ldquoThe Cu(II)-reductase NADHdehydrogenase-2 of Escherichia coli improves the bacterialgrowth in extreme copper concentrations and increases theresistance to the damage caused by copper and hydroperoxiderdquoArchives of Biochemistry and Biophysics vol 451 no 1 pp 1ndash72006

[48] E Williams T M Lowe J Savas and J DiRuggiero ldquoMicroar-ray analysis of the hyperthermophilic archaeon Pyrococcusfuriosus exposed to gamma irradiationrdquo Extremophiles vol 11no 1 pp 19ndash29 2007

[49] L M Gaetke and C K Chow ldquoCopper toxicity oxidative stressand antioxidant nutrientsrdquo Toxicology vol 189 no 1-2 pp 147ndash163 2003

[50] M J Davies ldquoThe oxidative environment and protein damagerdquoBiochimica et Biophysica Acta vol 1703 no 2 pp 93ndash109 2005

[51] J R Thompson J K Bell J Bratt G A Grant and LJ Banaszak ldquoVmax regulation through domain and subunitchanges The active form of phosphoglycerate dehydrogenaserdquoBiochemistry vol 44 no 15 pp 5763ndash5773 2005

[52] V Consalvi R Chiaraluce L Politi A Gambacorta MDe Rosa and R Scandurra ldquoGlutamate dehydrogenase fromthe thermoacidophilic archaebacterium Sulfolobus solfataricusrdquoEuropean Journal of Biochemistry vol 196 no 2 pp 459ndash4671991

[53] A J L Macario M Lange B K Ahring and E Conway DeMacario ldquoStress genes and proteins in the archaeardquo Microbiol-ogy and Molecular Biology Reviews vol 63 no 4 pp 923ndash9671999

[54] J AMaupin-FurlowM A Gil M A Humbard et al ldquoArchaealproteasomes and other regulatory proteasesrdquo Current Opinionin Microbiology vol 8 no 6 pp 720ndash728 2005

[55] N Sun F Beck R Wilhelm Knispel et al ldquoProteomics analysisof Thermoplasma acidophilum with a focus on protein com-plexesrdquoMolecular and Cellular Proteomics vol 6 no 3 pp 492ndash502 2007

[56] L S Madding J K Michel K R Shockley et al ldquoRole of the 1205731subunit in the function and stability of the 20S proteasome inthe hyperthermophilic Archaeon Pyrococcus furiosusrdquo Journalof Bacteriology vol 189 no 2 pp 583ndash590 2007

[57] R Shibata Y Bessho A Shinkai et al ldquoCrystal structureand RNA-binding analysis of the archaeal transcription factorNusArdquo Biochemical and Biophysical Research Communicationsvol 355 no 1 pp 122ndash128 2007

[58] W Bae B Xia M Inouye and K Severinov ldquoEscherichia coliCspA-family RNA chaperones are transcription antitermina-torsrdquo Proceedings of the National Academy of Sciences of theUnited States of America vol 97 no 14 pp 7784ndash7789 2000

[59] R J Ram N C VerBerkmoes M P Thelen et al ldquoMicrobi-ology community proteomics of a natural microbial biofilmrdquoScience vol 308 no 5730 pp 1915ndash1920 2005

[60] I Noel-Georis T Vallaeys R Chauvaux et al ldquoGlobal analysisof the Ralstonia metallidurans proteome prelude for the large-scale study of heavy metal responserdquo Proteomics vol 4 no 1pp 151ndash179 2004

16 Archaea

[61] M TMNovo A CDa Silva RMoreto et al ldquoThiobacillus fer-rooxidans response to copper and other heavy metals growthprotein synthesis and protein phosphorylationrdquo Antonie vanLeeuwenhoek vol 77 no 2 pp 187ndash195 2000

[62] Q She R K Singh F Confalonieri et al ldquoThe complete genomeof the crenarchaeon Sulfolobus solfataricus P2rdquo Proceedings ofthe National Academy of Sciences of the United States of Americavol 98 no 14 pp 7835ndash7840 2001

[63] R P Anderson and J R Roth ldquoTandem genetic duplications inphage and bacteriardquo Annual Review of Microbiology vol 31 pp473ndash505 1977

[64] D Gevers K Vandepoele C Simillion and Y Van De PeerldquoGene duplication and biased functional retention of paralogsin bacterial genomesrdquo Trends in Microbiology vol 12 no 4 pp148ndash154 2004

[65] A B Reams and E L Neidle ldquoSelection for gene clustering bytandemduplicationrdquoAnnual Review ofMicrobiology vol 58 pp119ndash142 2004

[66] L H Orellana and C A Jerez ldquoA genomic island providesAcidithiobacillus ferrooxidans ATCC 53993 additional copperresistance a possible competitive advantagerdquo Applied Microbi-ology and Biotechnology vol 92 no 4 pp 761ndash767 2011

[67] A B Brinkman T J G Ettema W M de Vos and J van derOost ldquoThe Lrp family of transcriptional regulatorsrdquo MolecularMicrobiology vol 48 no 2 pp 287ndash294 2003

[68] C Vollmecke S L Drees J Reimann S V Albers and MLubben ldquoThe ATPases CopA and CopB both contribute tocopper resistance of the thermoacidophilic archaeon SulfolobussolfataricusrdquoMicrobiology vol 158 no 6 pp 1622ndash1633 2012

[69] A Villafane Y Voskoboynik I Ruhl et al ldquoCopR of Sulfolobussolfataricus represents a novel class of archaeal-specific copper-responsive activators of transcriptionrdquo Microbiology vol 157no 10 pp 2808ndash2817 2011

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Anatomy Research International

PeptidesInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporation httpwwwhindawicom

International Journal of

Volume 2014

Zoology

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Molecular Biology International

GenomicsInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioinformaticsAdvances in

Marine BiologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Signal TransductionJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

Evolutionary BiologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Biochemistry Research International

ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Genetics Research International

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Advances in

Virolog y

Hindawi Publishing Corporationhttpwwwhindawicom

Nucleic AcidsJournal of

Volume 2014

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Enzyme Research

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

International Journal of

Microbiology

16 Archaea

[61] M TMNovo A CDa Silva RMoreto et al ldquoThiobacillus fer-rooxidans response to copper and other heavy metals growthprotein synthesis and protein phosphorylationrdquo Antonie vanLeeuwenhoek vol 77 no 2 pp 187ndash195 2000

[62] Q She R K Singh F Confalonieri et al ldquoThe complete genomeof the crenarchaeon Sulfolobus solfataricus P2rdquo Proceedings ofthe National Academy of Sciences of the United States of Americavol 98 no 14 pp 7835ndash7840 2001

[63] R P Anderson and J R Roth ldquoTandem genetic duplications inphage and bacteriardquo Annual Review of Microbiology vol 31 pp473ndash505 1977

[64] D Gevers K Vandepoele C Simillion and Y Van De PeerldquoGene duplication and biased functional retention of paralogsin bacterial genomesrdquo Trends in Microbiology vol 12 no 4 pp148ndash154 2004

[65] A B Reams and E L Neidle ldquoSelection for gene clustering bytandemduplicationrdquoAnnual Review ofMicrobiology vol 58 pp119ndash142 2004

[66] L H Orellana and C A Jerez ldquoA genomic island providesAcidithiobacillus ferrooxidans ATCC 53993 additional copperresistance a possible competitive advantagerdquo Applied Microbi-ology and Biotechnology vol 92 no 4 pp 761ndash767 2011

[67] A B Brinkman T J G Ettema W M de Vos and J van derOost ldquoThe Lrp family of transcriptional regulatorsrdquo MolecularMicrobiology vol 48 no 2 pp 287ndash294 2003

[68] C Vollmecke S L Drees J Reimann S V Albers and MLubben ldquoThe ATPases CopA and CopB both contribute tocopper resistance of the thermoacidophilic archaeon SulfolobussolfataricusrdquoMicrobiology vol 158 no 6 pp 1622ndash1633 2012

[69] A Villafane Y Voskoboynik I Ruhl et al ldquoCopR of Sulfolobussolfataricus represents a novel class of archaeal-specific copper-responsive activators of transcriptionrdquo Microbiology vol 157no 10 pp 2808ndash2817 2011

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Anatomy Research International

PeptidesInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporation httpwwwhindawicom

International Journal of

Volume 2014

Zoology

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Molecular Biology International

GenomicsInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioinformaticsAdvances in

Marine BiologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Signal TransductionJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

Evolutionary BiologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Biochemistry Research International

ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Genetics Research International

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Advances in

Virolog y

Hindawi Publishing Corporationhttpwwwhindawicom

Nucleic AcidsJournal of

Volume 2014

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Enzyme Research

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

International Journal of

Microbiology

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Anatomy Research International

PeptidesInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporation httpwwwhindawicom

International Journal of

Volume 2014

Zoology

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Molecular Biology International

GenomicsInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioinformaticsAdvances in

Marine BiologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Signal TransductionJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

Evolutionary BiologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Biochemistry Research International

ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Genetics Research International

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Advances in

Virolog y

Hindawi Publishing Corporationhttpwwwhindawicom

Nucleic AcidsJournal of

Volume 2014

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Enzyme Research

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

International Journal of

Microbiology