Genomic insights into microbial iron oxidation and iron uptake

Minireview

Genomic insights into microbial iron oxidation andiron uptake strategies in extremely acidicenvironmentsemi_2626 1..15

Violaine Bonnefoy1,2* and David S. Holmes3

1Laboratoire de Chimie Bactérienne, UPR-CNRS 9043,Institut de Microbiologie de la Méditerranée, Marseille,France.2Aix-Marseille Université, Marseille, France.3Center for Bioinformatics and Genome Biology,Fundación Ciencia para la Vida and Depto. de CienciasBiologicas, Facultad de Ciencias Biologicas,Universidad Andres Bello, Santiago, Chile.

Summary

This minireview presents recent advances in ourunderstanding of iron oxidation and homeostasis inacidophilic Bacteria and Archaea. These processesinfluence the flux of metals and nutrients in pristineand man-made acidic environments such as acid minedrainage and industrial bioleaching operations.Acido-philes are also being studied to understand life inextreme conditions and their role in the generation ofbiomarkers used in the search for evidence of existingor past extra-terrestrial life. Iron oxidation in acido-philes is best understood in the model organismAcidithiobacillus ferrooxidans. However, recent func-tional genomic analysis of acidophiles is leading to adeeper appreciation of the diversity of acidophilic iron-oxidizing pathways. Although it is too early to paint adetailed picture of the role played by lateral genetransfer in the evolution of iron oxidation, emergingevidence tends to support the view that iron oxidationarose independently more than once in evolution.Acidic environments are generally rich in soluble ironand extreme acidophiles (e.g. the Leptospirillumgenus) have considerably fewer iron uptake systemscompared with neutrophiles. However, some acido-philes have been shown to grow as high as pH 6 and, inthe case of the Acidithiobacillus genus, to have mul-

tiple iron uptake systems. This could be an adaptionallowing them to respond to different iron con-centrations via the use of a multiplicity of differentsiderophores. Both Leptospirillum spp. and Acidithio-bacillus spp. are predicted to synthesize the acidstable citrate siderophore for Fe(III) uptake. In addi-tion, both groups have predicted receptors forsiderophores produced by other microorganisms,suggesting that competition for iron occurs influenc-ing the ecophysiology of acidic environments. Little isknown about the genetic regulation of iron oxidationand iron uptake in acidophiles, especially how the useof iron as an energy source is balanced with its need totake up iron for metabolism. It is anticipated that inte-grated and complex regulatory networks sensing dif-ferent environmental signals, such as the energysource and/or the redox state of the cell as well as theoxygen availability, are involved.

Introduction

Scope of this minireview

This minireview focuses on iron-oxidizing microorganismsthat inhabit acidophilic environments. There are a numberof other reviews and papers that discuss issues related tolife in extremely acidic conditions that will not be coveredhere, including the occurrence and composition of acido-philic communities (Gonzalez-Toril et al., 2003; Rawlingsand Johnson, 2007; Johnson, 2008; Demergasso et al.,2010), resistance to low pH and metal homeostasis(Baker-Austin and Dopson, 2007; Franke and Rensing,2007; Dopson, 2010), reduced inorganic sulfur com-pounds (RISCs) energetics (Holmes and Bonnefoy, 2007;Quatrini et al., 2009; Bonnefoy, 2010) and genomics andmetabolic reconstruction (Tyson et al., 2004; Valenzuelaet al., 2006; Holmes and Bonnefoy, 2007; Jerez, 2007;2008; Quatrini et al., 2007a; 2009; Valdes et al., 2008;Siezen and Wilson, 2009; Bonnefoy, 2010; Denef et al.,2010) including genomics of over 50 acidophiles (Carde-nas et al., 2010).

Received 17 June, 2011; accepted 16 September, 2011. *For corre-spondence. E-mail [email protected]; Tel. (+33) 4 91 16 4146; Fax (+33) 4 91 71 89 14.

Environmental Microbiology (2011) doi:10.1111/j.1462-2920.2011.02626.x

© 2011 Society for Applied Microbiology and Blackwell Publishing Ltd

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Acidophiles and their environments

We define ‘extreme acidophiles’ as those organismswhose growth optimum is lower than pH 3. Most acido-philes belong to the eubacterial or archaeal kingdoms(prokaryotes) (Johnson, 1998; 2007; 2009; Hallberg andJohnson, 2001; Johnson and Hallberg, 2003), althoughunicellular eukaryotes have also been detected in someacidic environments (e.g. Lopez-Archilla et al., 2001;Amaral Zettler et al., 2002; Baker et al., 2009; Johnson,2009; Cid et al., 2010).

Acidophilic microorganisms inhabit pristine environ-ments such as volcanic, geothermal areas and acid rockdrainage (ARD) (Lopez-Archilla et al., 2001; Gonzalez-Toril et al., 2003). They are also found in acidic environ-ments of anthropogenic origin such as bioleaching heapsand acid mine drainage (AMD) (Rawlings, 2002; 2007;Baker and Banfield, 2003; Johnson and Hallberg, 2003;Johnson, 2007; 2009; Norris, 2007; Schippers, 2007;Schippers et al., 2010). Acidophiles are found over a widerange of temperatures and include psychrotolerants (butnot psychrophiles) that can grow down to 4°C (Harrison,1982; Johnson et al., 2001; Dopson et al., 2007; Hallberget al., 2010), mesophiles (up to 40°C), moderate thermo-philes (between 40°C and 55°C) and extreme thermo-philes (above 55°C) (Hallberg and Johnson, 2001; Norris,2007; Schippers, 2007; Johnson, 2009). Many acidophilicprokaryotes are obligate chemolithoautotrophs obtainingtheir energy and electrons from the oxidation of ferrousiron [Fe(II)] and RISCs (Hallberg and Johnson, 2001;Rawlings, 2005; 2007; Johnson and Hallberg, 2008;Johnson, 2009) and their carbon by fixing CO2 from theatmosphere (Rawlings, 2007; Johnson and Hallberg,2008). Some also fix atmospheric N2 (Johnson, 2007;Rawlings, 2007). Heterotrophs have also been detectedthat scavenge organic carbon compounds produced bythe autotrophs (Hallberg and Johnson, 2001; Rawlings,2002; Johnson and Hallberg, 2003; Johnson, 2007; 2009;Norris, 2007; Schippers, 2007; Nancucheo and Johnson,2010; Schippers et al., 2010).

Problems of high iron load in acidic conditions

Acidic environments provide a special opportunity and atthe same time an unusual challenge for life. Naturalgeomicrobiological processes and industrial operations,such as bioleaching, convert insoluble metal sulfides intowater-soluble metal sulfates that include extraordinarilyhigh concentrations of soluble iron that can reach valuesas high as 160 g l-1, a concentration about 1016 higherthan typically found in circum-neutral environments. Inoxygen (O2)-saturated environments at neutral pH, Fe(II)is rapidly oxidized to ferric iron [Fe(III)]. Thus, iron pre-dominantly occurs in the ferric form as poorly soluble iron

hydroxides (as low as 10-18 M at pH 7.0), rendering itdifficult to access by biological systems. In contrast, underacidic conditions Fe(II) is stable even in the presence ofatmospheric oxygen. This provides an opportunity formicroorganisms to use Fe(II) as an electron donor and asa source of energy that is not readily available to micro-organisms living in circum-neutral environments.However, extremely acidic environments can also chal-lenge microorganisms with the highest levels of solubleiron and the threat that this imposes on life via the reactionof Fe(II) with oxygen generating free radicals (Fentonreaction) that damage macromolecules and cause celldeath (Touati, 2000).

Phylogeny of iron oxidizers

The ability to oxidize iron is widely distributed in acido-philic Bacteria and Archaea (Fig. 1) (Weber et al., 2006;Emerson et al., 2010; Hedrich et al., 2011). Until now, themajority of known acidophilic iron oxidizers are found inthe Nitrospira class as well as in Gram-positive (Firmicuteand Actinobacteria) and archaeal (Crenarchaeotaand Euryarchaeota) phyla. In Proteobacteria, only fiveacidophiles are encountered (‘Ferrovum myxofaciens’,Acidiferrobacter thiooxydans, ‘Thiobacillus prosperus’,Acidithiobacillus ferrivorans and At. ferrooxidans)(Hedrich et al., 2011). It is not yet known whether thisdistribution reflects real evolutionary trends or merelyresults from an incomplete analysis due to biases ofsampling that have relied mainly on culture-basedapproaches.

Widespread distribution of iron oxidation capabilitieshas also been observed in microorganisms from circum-neutral pH environments. However, as opposed to theacidophiles, there seems to be an enrichment of ironoxidizers in the Proteobacteria (Emerson et al., 2010).This minireview will focus mainly on iron oxidation inacidic environments. Microbial iron oxidation in circum-neutral environments has been reviewed recently(Emerson et al., 2010; Bird et al., 2011) and will not bediscussed here, except for comparison with acidophilicpathways.

Fe(II) iron oxidation

Bioenergetic problems of iron oxidation

When growing on Fe(II), autotrophs are confronted witha crucial bioenergetic problem. The reduction potential ofthe Fe(II)/Fe(III) couple is +0.78 V which is just belowthat of O2/H2O (1.12 V) at the pH of the external medium(pH 2), consequently, there is little energy available fromthe oxidation of Fe(II) (Ingledew, 1982; Bird et al., 2011).Also, since the reduction potential of NAD+ (-0.32 V at

2 V. Bonnefoy and D. S. Holmes

© 2011 Society for Applied Microbiology and Blackwell Publishing Ltd, Environmental Microbiology

Fig. 1. An inferred phylogenetic tree of acidophilic iron-oxidizing Bacteria and Archaea. The tree was produced by a neighbour-joining methodbased on 16S rRNA gene sequences (Kumar et al., 2008). Numbers at nodes represent bootstrap values. The scale bars represent theaverage number of substitutions per site. Whereas At. ferrooxidans clusters with the Gammaproteobacteria in this tree, it has recently beenproposed to have arisen before the split between Gamma- and Betaproteobacteria based upon a phylogeny using protein concatenation(Williams et al., 2010). Strain designations (when specified) and 16 rRNA gene accession numbers are: 1 = embl|HM044161|, 2 = DSM 2392and embl |AF387301|, 3 = DSM 5130 and ENA|EU653291|, 4 = DSM 22755 and ENA|AF376020|, 5 = ATCC 23270 and gi|28194033|,6 = ATCC BAA-1645 and ENA|AY140237|, 7 = ENA|GQ225721|, 8 = ENA|AF251436|, 9 = DSM 10331 and ENA|CP001631|,10 = gi|157649846|, 11 = gi|10998845|, 12 = ATCC 51911 and ENA|AB089843|, 13 = DSM 16297 and ENA|AB222265|, 14 = ATCC 700253 andENA|EF088287|, 15 = DSM 9293 and ENA|AB089844|, 16 = DSM 17362 and ENA |AM502928|, 17 = gi|225380152|, 18 = DSM 17363T andENA|AY079150|, 19 = ATCC BAA-1181 and ENA|ACNP01000064|, 20 = DSM 16297/ENA and AF356832|, 21 = ENA|AAWO01000056|,22 = DSM 14647 and ENA|AF356830|, 23 = DSM 18409T and gi|261599790|, 24 = DSM 16651 and gi|254971292|,25 = ENA|AABC05000010|, 26 = DSM 6482 and ENA|AJ224936|, 27 = DSM 16993 and ENA|BA000023|, 28 = DSM 6482 and ENA|D85519|,29 = DSM 3191 and ENA|D85505|, 30 = DSM 1651 and ENA|D26489|, 31 = DSM 5348 and ENA|CP00682|.

Iron oxidation and iron homeostasis in acidophiles 3


pH 6.5, the pH of the cytoplasm) is much more negativethan that of the Fe(II)/Fe(III) couple, electrons must bepushed ‘uphill’ or ‘in reverse’ from Fe(II) to NAD+ againstthe redox potential gradient. The energy to accomplishthis comes from the proton motive force across the cellmembrane that results from the high concentration ofprotons outside the cell (pH 2) compared with inside (pH6.5). On the other hand, electrons extracted from Fe(II)can pass ‘downhill’ via a thermodynamically favourablegradient to reduce O2 to water, thus neutralizing protonsthat have entered the cell via the ATPase complex.These up- and downhill pathways are intimately con-nected at the level of electron and proton fluxes and arelikely to be homeostatically regulated in order to balanceATP production with the reconstitution of reducing power(NADH) as been demonstrated for Acidithiobacilluscaldus (Dopson et al., 2002).

Best-studied case: At. ferrooxidans

The most detailed account of electron transport pathwaysand the molecular complexes used during Fe(II) oxidationis available for the Gram-negative bacterium At. ferrooxi-dans that was considered until recently to be a member ofthe Gammaproteobacteria but now is thought to havearisen before the split between Gamma- and Betaproteo-bacteria (Williams et al., 2010). These studies were initi-ated almost 30 years ago in a pioneering paper byIngledew who not only provided a theoretical frameworkfor the electrochemistry of electron and proton fluxes butalso proposed the bifurcating (uphill and downhill) elec-tron transport pathways and suggested which molecularcomplexes might be involved (Ingledew, 1982). The Ingle-dew model has proved to be essentially correct and, in theintervening years, has received validation from numerousexperimental and bioinformatics studies (see Fig. 2)(reviewed in Holmes and Bonnefoy, 2007; Quatrini et al.,2009; Bonnefoy, 2010; Bird et al., 2011 and additionalreferences therein).

Iron oxidation in other acidophiles

In addition to At. ferrooxidans, other known acidophilicprokaryotes whose iron oxidation pathways have beenstudied, include the bacteria At. ferrivorans, ‘T. prospe-rus’, Leptospirillum ferrooxidans, ‘L. rubarum’ and ‘L. fer-rodiazotrophum’ and the archaea Metallosphaera sedula,‘Ferroplasma acidarmanus’, Ferroplasma Types I and II,Sulfolobus metallicus and S. tokodaii.

It has been known since the 1990s that At. ferrooxi-dans, L. ferrooxidans, M. sedula and S. metallicuscontain spectrally distinct redox proteins when grown onFe(II) (Barr et al., 1990; Blake et al., 1992; 1993) suggest-ing that several different Fe(II) oxidation pathways have

evolved in prokaryotes. This hypothesis has sincereceived additional bioinformatic and proteomic/transcriptomic support (see reviews Holmes and Bonne-foy, 2007; Bonnefoy, 2010).

Acidithiobacillus ferrivorans. Acidithiobacillus ferrivoranshas been recently characterized as an Fe(II)- and RISCs-oxidizing acidophile (Hallberg et al., 2010; Amouric et al.,2011). Although it is phylogenetically closely related toAt. ferrooxidans at the 16S rRNA gene level (see Fig. 1),unlike this microorganism it is motile and psychrotolerant.It exhibits iro (Hallberg et al., 2009; Amouric et al., 2011)encoding a high potential iron-sulfur protein (Table 1) thathas been proposed to be the iron oxidase (Fukumoriet al., 1988; Kusano et al., 1992; Cavazza et al., 1995). Inaddition, it has not only the archetypal rusA encoding theblue copper protein rusticyanin (Liljeqvist et al., 2011) asfound in the At. ferrooxidans type strain and others (Ben-grine et al., 1998; Valdes et al., 2008) but also rusB (Hall-berg et al., 2010; Liljeqvist et al., 2011; Amouric et al.,2011) as described in some other At. ferrooxidans strains(Ida et al., 2003; Sasaki et al., 2003).

The iron-oxidizing Acidithiobacillus spp. have recentlybeen shown to be composed of at least four groups likelyrepresenting four species (Amouric et al., 2011). In two ofthese groups, including the At. ferrooxidans type strain,only rusA was detected, while the two other taxons, whichinclude At. ferrivorans, encode iro and have either rusAand rusB or no rus (Liljeqvist et al., 2011; Amouric et al.,2011). This suggests that at least two different pathwaysfor Fe(II) oxidation exist in the Acidithiobacillus spp., thefirst via rusA and the second possibly through the HiPIPencoded by iro.

‘Thiobacillus prosperus’. This Gammaproteobacterium isan Fe(II) and S°-oxidizing, salt-tolerant acidophile. Acluster of genes with similarity to a substantial part of therus operon of At. ferrooxidans was identified including: apredicted outer membrane cytochrome c (37% identitywith Cyc2); a multicopper oxidase whose function issubject to controversy (46% identity with Cup) (Quatriniet al., 2009; Castelle et al., 2010); four subunits of a puta-tive aa3-type cytochrome oxidase (49%, 54%, 31% and32% identity with CoxB, A, C and D respectively) and apredicted blue copper protein showing 50% identity withrusticyanin A and 49% with rusticyanin B (Table 1) (Nicolleet al., 2009). However, cyc1 encoding the membrane-bound cytochrome c proposed to transfer the electronsduring uphill flow from rusticyanin to the terminal aa3

oxidase in At. ferrooxidans was not detected in this locus(see Fig. 2). In ‘T. prosperus’, it is possible that this func-tion is assumed by another electron transfer protein, suchas the multicopper oxidase Cup. Alternatively, electrontransfer between rusticyanin and the terminal oxidase is



made directly without the need for an intermediaryprotein. Also, it cannot be excluded that cyc1 exists insome other part of the genome and is not associated withthe rus operon as in At. ferrooxidans.

Another difference is that rus is transcribed indepen-dently from the upstream genes in ‘T. prosperus’ whereasit is either co-transcribed with them or transcribed from aninternal promoter in At. ferrooxidans (Bengrine et al.,1998). In addition, it has been reported that in ‘T. prospe-rus’ transcription of cyc2 and rus is higher in S° thanin Fe(II)-medium while the coxBACD cluster is moreexpressed during growth on Fe(II) (Nicolle et al., 2009).This contrasts with the situation in At. ferrooxidans whererus, cyc2 and coxBACD are upregulated in mediumcontaining Fe(II) (Yarzabal et al., 2004; Quatrini et al.,

2009). However, based on the sequence similaritybetween the redox proteins and the organization of thecorresponding genes, it has been proposed that the com-position of the electron transfer chain from Fe(II) to O2 in‘T. prosperus’ might be similar to that of At. ferrooxidans(Nicolle et al., 2009).

Leptospirillum spp. Leptospirillum spp. are bacteriabelonging to the deep branching class Nitrospira. Basedon metagenomic and metaproteomic information derivedfrom a study of biofilms in the acid mine drainage of IronMountain (USA), a model has been proposed for Fe(II)oxidation for Leptospirillum ferriphilum, ‘L. rubarum’ and‘L. ferrodiazotrophum’ (Table 1) (Tyson et al., 2004; Ramet al., 2005; Lo et al., 2007; Simmons et al., 2008; Golts-

Fig. 2. Model of the oxidation of Fe(II) in At. ferrooxidans based on biochemical, molecular genetics, bioenergetic, bioinformatic and functionalgenomics evidence. A redox tower has been placed below the model to facilitate comparison of the redox potentials of some of the reactions.Electrons extracted from the oxidation of Fe(II) by the outer membrane embedded cytochrome c Cyc2 are passed to the periplasmic copperprotein rusticyanin (R). From rusticyanin, the electrons can take a download pathway to reduce O2 to water (solid arrow) passing through thecytochrome c4 Cyc1 and the aa3 type cytochrome oxidase complex or an uphill pathway (dotted arrow) to the NADH1 complex via thecytochrome c4 CycA1, the bc1 complex and membrane-associated quinones. The energy to push electrons uphill against thisthermodynamically unfavourable gradient is postulated to come from the influx of protons (solid arrows) generated by the proton motive forceacross the inner membrane resulting from the difference in proton concentration inside the cell (pH 6.5) and outside (pH 2). This uphillpathway is similar in many respects to the pathways taken by electrons and protons in mitochondrial and prokaryotic oxidativephosphorylation, only in reverse. The figure also shows the influx of protons through the ATP synthetase complex (ATPase) driving thebiosynthesis of ATP. These protons and also those that enter the cell to drive electrons uphill are postulated to be consumed, at least in part,by the reduction of O2 to water using electrons derived from the oxidation of Fe(II) via the downhill pathway. Abbreviations used: R,rusticyanin; OM, outer membrane; IM, inner membrane. * = values of E•

0 for the Fe(III)/Fe(II) and 1/2 O2/H2O couples for pH 2 (Ferguson andIngledew, 2008). Figure reproduced with modifications from Quatrini and colleagues (2009).



man et al., 2009; Singer et al., 2010). In this model, elec-trons are extracted from Fe(II) by an outer membranecytochrome c termed Cyc572. Cyc572 passes these elec-trons to a periplasmic cytochrome c termed Cyt579, whichsubsequently transfers them via periplasmic cytochromesc either downhill to a cytoplasmic membrane embeddedcbb3 terminal oxidase that reduces O2 or uphill first to abc1 complex and then to the NADH dehydrogenasecomplex via the quinone pool. The postulated route andprotein complexes for both downhill and uphill flow inLeptospirillum has many features similar to those pro-posed for At. ferrooxidans with the notable difference thatLeptospirillum has no equivalent to rusticyanin and thebranch point at which electrons from Fe(II) oxidation arechannelled either uphill or downhill was proposed to beCyt579.

Ferroplasma spp. These acidophilic archaea are aerobicFe(II) oxidizers that grow mixotrophically or chemohet-erotrophically. An analysis of the metagenome of the IronMountain biofilm (Tyson et al., 2004; Allen et al., 2007)and biochemical, genomic and proteomic analysis(Dopson et al., 2005), allowed a model to be built forFe(II) oxidation in three Ferroplasma spp. (Table 1). Thepresence of the blue copper sulfocyanin, a member of afamily of proteins to which rusticyanin also belongs, isproposed to transfer electrons to a cbb3 terminal oxidase,but little is known about the protein(s) involved in theinitial extraction of electrons from Fe(II) nor in otherpotential intermediate steps. Unlike At. ferrooxidans,cytochrome c has not been detected in Ferroplasma spp.The presence of sulfocyanin raises the possibility that itserves as branch point like rusticyanin between uphilland downhill electron flow.

No electron carriers for an uphill pathway of Fe(II) oxi-dation have been identified in Ferroplasma spp., but it ispossible that this pathway is not required since theorganisms may be able to generate NADH using elec-trons and energy derived from the oxidation of carbon.

Sulfolobus spp. These archaea are acidophilic andextremely thermophilic. Among them, two species havebeen shown to oxidize Fe(II): S. metallicus and S. toko-daii. As for all thermoacidophilic archaea studied so far,there is no evidence for genes encoding cytochromes c. ACbsA-like cytochrome b and a haem-copper terminaloxidase encoded by the fox [for Fe(II) oxidation] clusterhave been proposed to be involved in Fe(II) oxidationin S. metallicus based on a subtractive hybridizationapproach (Table 1) (Bathe and Norris, 2007). In this samecluster, are found genes encoding electron transporters,such as putative ferredoxins and other iron-sulfur pro-teins. In agreement with the hypothesis that this cluster isinvolved in Fe(II) oxidation, these genes have also beendetected in the genome sequence of S. tokodaii, but not inother Sulfolobus species that do not oxidize Fe(II) (Batheand Norris, 2007).

The fox cluster may encode components involved inboth downhill and uphill electron flow since it has genespotentially encoding a haem terminal oxidase, presum-ably involved in O2 reduction, and also cytochrome b andiron-sulfur proteins that could be components of a bc1

complex.

Metallosphaera sedula. This obligate thermoacidophilicarchaeon is a facultative chemolithoautotroph, being ableto oxidize Fe(II) or RISCs in aerobic conditions, and togrow on complex organic substrates. Genomic and tran-scriptomic analyses have allowed the prediction ofcomponents involved in Fe(II) oxidation (Auernik andKelly, 2008; Auernik et al., 2008). A fox cluster has beendetected in M. sedula and has been determined to bemore expressed in the presence of Fe(II) than S°. It hasbeen suggested that this cluster could be involved in the‘uphill’ pathway (Auernik and Kelly, 2008). However,given the possible connection between the fox clusterand terminal electron transfer in S. metallicus, wesuggest that it is more likely that the fox cluster alsoplays a similar role in downhill electron flow in M. sedula.

Table 1. Comparison of the redox proteins demonstrated, or proposed, to be involved in Fe(II) oxidation in acidophiles.

Bacteria Outer membrane Periplasm Inner membrane peripheral Inner membrane

Acidithiobacillus ferrooxidans Cyc2 Rus A Cyc1 aa3 oxidaseCycA1 bc1 complex

Acidithiobacillus ferrivorans IroRus A or B?

‘Thiobacillus prosperus’ Cyc2-like Rus-like aa3 oxidaseLeptospirillum spp. Cyc572 Cyc579 Cytc? cbb3 oxidase

Cytc? bc1 complex

Archaea Membrane peripheral Inner membrane

Ferroplasma spp. Sulfocyanin cbb3 oxidaseSulfolobus metallicus and S. tokodaii cyt b, [Fe-S], haem-copper terminal oxidaseMetallosphaera sedula cyt b, [Fe-S], haem-copper terminal oxidase



In addition, a cytochrome ba complex encoded by thesoxN-soxL-cbsABA locus and proposed recently to beanalogous to the bc1 complex of bacteria and mitochon-dria (Bandeiras et al., 2009) seems to be also involved inthe oxidation of Fe(II) and could therefore represent theuphill pathways (Table 1). However, these genes are alsopresent in Acidianus ambivalens, Caldivirga maquilin-gensis, Sulfolobus acidocaldarius and Sulfolobus solfa-taricus (Bandeiras et al., 2009).

Summary of Fe(II) oxidation pathways in acidophiles

A pathway for Fe(II) oxidation is known in detail onlyin At. ferrooxidans. However, information is emergingfrom other acidophiles and Table 1 summarizes ourcurrent knowledge. A comparison of known Fe(II)oxidation pathways in acidophilic bacteria suggeststhat they all accomplish the preliminary removal of elec-trons from their Fe(II) substrates using an outer mem-brane cytochrome c. Most likely this is to avoid theproduction of damaging free radicals and precipitates offerric oxyhydroxides within the cell in the case of solublesubstrates such FeSO4. Cyc2, which accomplishes thisrole in At. ferrooxidans, has 47% and 48% sequencesimilarity within a stretch of 489 and 65 amino acidsincluding the haem C binding site with the predictedouter membrane cytochromes of ‘T. prosperus’ and Lep-tospirillum spp., respectively, suggesting that they mayhave arisen from a common ancestor. However, sincethe identity is very low they either diverged very earlierin evolution or else have been subjected to rapidselection.

A commonality between acidophilic Bacteria andArchaea is the use of a terminal oxidase located in theinner membrane that uses electrons derived from theoxidation of Fe(II) (downhill pathway) to consume orextrude protons that enter the cell via the ATP synthetasein order to maintain intracellular neutrality.

A third feature that appears to be shared is the abilityto push electrons uphill against a thermodynamicallyunfavourable gradient using PMF in order to reduceNAD+. This appears to be accomplished by a bc1

complex.However, what is striking, is the dissimilarity in the com-

ponents involved in Fe(II) oxidation in many acidophiles,and we speculate that the ability to oxidize Fe(II) aroseindependently more than once in evolution. For example,whereas At. ferrooxidans and Ferroplasma spp. usemembers of the small copper protein family as electroncarriers (rusticyanin and sulfocyanin respectively), thisrole may be accomplished by Fe-S proteins in Sulfolobusspp. and in M. sedula. In addition, the terminal oxidaseused can be of the aa3 (e.g. At. ferrooxidans) or cbb3 type(e.g. Leptospirillum sp.).

A comparison of Fe(II) oxidation between acidophilesand neutrophiles

Whereas information regarding iron oxidation for severalacidophiles is beginning to emerge after its first charac-terization in At. ferrooxidans, data are scarce for neutro-philes with only two operons described (Bird et al., 2011).In Rhodobacter capsulatus SB1003, a cytochrome c, apyrroloquinoline quinone containing protein and an innermembrane protein encoded by the foxEYZ operon (Croalet al., 2007) while a decahaem periplasmic cytochrome c,an outer membrane protein and a high potential iron sulfurprotein (HiPIP) encoded by the pioABC operon inRhodopseudomonas palustris TIE-1 (Jiao and Newman,2007) have been suggested to be involved in iron oxida-tion. Interestingly, PioA and PioB present some similaritiesto the soluble decahaem cytochrome c MtrA and to theouter membrane MtrB of Shewanella spp. and Geobacterspp. In these latter Fe(III)-reducing neutrophilic bacteria,MtrB was proposed to serve as a sheath within which theperiplasmic MtrA receives the electrons from the outermembrane MtrC cytochrome c which reduces Fe(III)(Hartshorne et al., 2009). By homology, PioB could pos-sibly allow the transfer of the electrons from Fe(II) to thecytochrome c PioA or to the HiPIP PioC which are pre-dicted to be periplasmic. Therefore, the Fe(II) oxidationsystem of R. palustris TIE-1 shares more similarities tothe ferric-reducing system of Shewanella spp. and Geo-bacter spp. than to the Fe(II) oxidation system of R. cap-sulatus SB1003, which is phylogenetically closer. ApioABC operon was also detected in the genome of theFe(II) oxidizer Gallionella capsiferriformans suggestingthat this Betaproteobacterium oxidizes Fe(II) in a similarway to R. palustris TIE-1. In most acidophilic bacteria,while an outer membrane cytochrome c has been pre-dicted, and in some cases characterized (see above), noequivalent to pioB/mtrB was detected. Even if a HiPIP wasproposed to be the iron oxidase in the iron-oxidizingAcidithiobacillus spp. of Groups III and IV (Kusano et al.,1992; Amouric et al., 2011) (see above), the percentage ofidentity/similarity with PioC is rather low (about 36/50%)and the genetic context completely different (Kusanoet al., 1992; Jiao and Newman, 2007; Amouric et al.,2011) suggesting that the model proposed for R. palustrisTIE-1 is unlikely in these acidophiles. Therefore, fromwhat we know nowadays on the iron oxidation pathways,no consensus model in neutrophiles or acidophiles can bededuced and each prokaryote seems to have evolved itsown system.

However, there are six species that do not follow thesplit between acidophilic and neutrophilic iron oxidizersaccording to the 16S rRNA gene phylogenetic tree(Fig. 1): namely the neutrophile Crenarchaeota Ferro-globus placidus and the acidophilic Proteobacteria



‘Ferrovum myxofaciens’, Acidiferrobacter thiooxydans,‘T. prosperus’, At. ferrivorans and At. ferrooxidans. Atleast four of these acidophiles, the Groups I (At. ferrooxi-dans), II and III (At. ferrivorans) of the iron-oxidizingAcidithiobacillus spp. as well as the ‘T. prosperus’, share arus locus presenting similarities (see above) while theiron-oxidizing Acidithiobacillus spp. from Groups III andIV, which are phylogenetically and physiologically close toGroups I and II, have in addition the rusB gene or nodetectable rus gene at all (Liljeqvist et al., 2011; Amouricet al., 2011). One possibility is that lateral gene transferscould have occurred.

Neutrophilic Fe(III)-reducing and Fe(II)-oxidizing micro-organisms have been shown to use external electroncarriers (reviewed in Gralnick and Newman, 2007) suchas redox-active antibiotics (Hernandez et al., 2004; Wangtand Newman, 2008), humic substances (Lovley et al.,1999) and quinones (Newman and Kolter, 2000) to shuttleelectrons from insoluble iron (such as ferrihydrite atneutral pH) to cellular electron carriers such as multihaemcytochromes c and conductive pili (nanowires).

However, few investigations have explored the possibleuse of external electron carriers in acidophilic Fe(II) oxi-dation. It was suggested that extracellular polymers withembedded ferric iron molecules could play a role in metalsulfide ores oxidation in biofilms of At. ferrooxidans (Ingle-dew, 1986; Sand and Gehrke, 2006). The aporusticyaninof At. ferrooxidans has been claimed to be excreted andto potentially shuttle electrons from mineral surfaces tothe cell (Ohmura and Blake, 1997); however these dataare controversial.

Iron uptake strategies

Introduction

A bioinformatic analysis of the conservation, organizationand distribution of iron homeostasis functions in acido-philic microorganisms is beginning to identify the mole-

cular adaptations that underpin the ability of these micro-organisms to cope with high concentrations of solubleiron. Attention has focused to date on Bacteria of theLeptospirillum and Acidithiobacillus genera and Archaeaof the Ferroplasma genera.

Iron homeostasis in the Leptospirillum spp. andAcidithiobacillus spp.

The apparently obligatory Fe(II)-oxidizing Leptospirillumspp. have only a few predicted iron uptake mechanismsthat include EfeU (FtrI-Fet3P-like permease) for Fe(II)and only three TonB-dependent ferri-citrate siderophorereceptor systems for Fe(III) (Tyson et al., 2004; Parroet al., 2007; Osorio et al., 2008). Citrate siderophoreuptake systems are among the most stable siderophore–Fe(III) complexes at low pH (Harrington and Crumbliss,2009; Hider and Kong, 2010) and therefore would be bestsuited to the extremely low and restricted pH environmentof the Leptospirillum spp. The paucity of Fe(III) uptakesystems in the Leptospirillum spp. might also beexplained by their occupation of this low-pH environmentwhere high concentrations of soluble Fe(II) would nearlyalways be available, rendering it unnecessary to have anextensive suite of Fe(III) uptake systems as is typicallyfound in neutrophiles (Andrews et al., 2003).

In contrast, Acidithiobacillus spp. exhibit a surprisinglylarge number of predicted iron transporters (Quatriniet al., 2005a; Osorio et al., 2008; Valdes et al., 2008)including predicted FeoB-like Fe(II) and Nramp-like Fe(II)-Mn(II) transporters and 14 different TonB-dependentferri-siderophore transporters of diverse siderophorespecificity. The latter include siderophores of the citrate,linear and cyclic catecholate and hydroxamate familiescompared with just the citrate family of the Leptospirillumspp. (Fig. 3).

It was hypothesized that the abundance of differenttypes of siderophore receptors might provide versatility

Fig. 3. Predicted Fe(III) siderophoretransporters in Acidithiobacillus spp. andLeptospirillum spp. The Venn diagram showsspecies-specific and shared TonB-dependentouter membrane receptors: Fecfamily = citrate; Cir family = linear catecholate;Fep family = cyclic catecholate; Fhufamily = hydroxamate. Figure after Osorio andcolleagues (2008).



for growth of Acidithiobacillus spp. in higher-pH environ-ments with less abundant sources of iron and where ironco-precipitation with phosphates or sulfates during ironand sulfur biooxidation might compromise their ability toscavenge iron (Osorio et al., 2008). On the other hand,Leptospirillum spp. live in more acidic and narrow pHrange habitats than Acidithiobacillus spp. and exhibit onlycitrate siderophore uptake systems which are the moststable siderophore–Fe(III) complexes at low pH (Har-rington and Crumbliss, 2009; Hider and Kong, 2010).

Neither the Acidithiobacillus spp. nor the Leptospiril-lum spp. have predicted genes that encode classical sid-erophore biosynthesis pathways, including an absenceof pathways for the linear and cyclic catecholates andhydroxamate siderophores for which the Acidithiobacillusspp. have receptors. Instead, they have predicted path-ways for citrate synthesis and possibly for phosphate-chelation-mediated iron uptake (see Fig. 4) (Osorioet al., 2008). The presence of the citrate biosynthesispathways is consistent with the aforementioned acidstability of citrate siderophores and the presence ofcognate receptors in both the Acidithiobacillus spp. andLeptospirillum spp. The absence of siderophore biosyn-thesis pathways for the three other types of siderophorereceptor complexes suggests that the Acidithiobacillus

spp. may be capable of scavenging siderophores pro-duced by other microorganisms, influencing the ecologyof the community.

Acidithiobacillus ferrooxidans, and the acidophilicsulfur-oxidizers At. thiooxidans and At. caldus, havecandidate genes for the iron storage protein bacterio-ferritin, although its predicted amino acid sequencein all three cases suggests that it could exhibit differ-ences in its ability to bind haem groups compared withthe classical bacterioferritins found in neutrophiles(Osorio et al., 2008). The Acidithiobacillus spp. may alsostore iron in polyphosphate inclusion bodies (Osorioet al., 2008). No genes potentially encoding bacteriofer-ritin or polyphosphate inclusion bodies have beendetected in the Leptospirillum spp. Perhaps theseextreme acidophiles, living in an environment with abun-dant soluble Fe(II), do not have a requirement to storeiron.

Iron storage proteins have also been implicated as away of relieving oxidative stress by serving as a sink ofpotentially dangerous excess iron. Thus, the apparentabsence of conventional iron storage capabilities in Lep-tospirillum spp. might present it with a special challengefor avoiding oxidative stress. One possibility is that Fe(II)oxidation might serve not only as an energy source but

Fig. 4. Predicted iron acquisition modules in Acidithiobacillus spp. Fe(II) uptake probably occurs primarily through an FeoABCP system,although a secondary MtnH system has also been implicated. Fourteen different siderophore systems for the uptake of Fe(III) have beenpredicted based on the TonB/ABC outer membrane receptor system, although only one potential siderophore Fe(III) biosynthesis system hasbeen detected that has similarity to the citrate system. A potential iron/phosphonate biosynthesis and uptake system may also be involved inFe(III) acquisition. Figure after Osorio and colleagues (2008).



also as a way to evade stress by converting the potentiallydangerous soluble Fe(II) to the less soluble Fe(III) (Osorioet al., 2008).

Iron homeostasis in Ferroplasma spp.

Ferroplasma acidiphilum, a member of the Euryarchaea,lives at an extremely acidic pH (around pH 1) with highconcentrations of ferrous iron. A proteomic analysis hasshown that it exhibits the unusual property of having alarge number of iron containing proteins. It has beensuggested that the presence of iron aids in the stabili-zation of these proteins at the relatively low pH (pH 5.6)of the cytoplasm (Ferrer et al., 2007; 2008). The highiron load per cell could increase the potential for oxida-tive damage. A further combined bioinformatics, tran-script profiling and proteomic study of iron uptake andhomeostasis in ‘F. acidarmanus’ Fer1 and F. acidiphilumYT has been carried out (Potrykus et al., 2011). Potentialimporters for Fe(II) via an MtnH-like NRAMP andfor Fe(III) via a Fhu-hydroxamate-like family sidero-phore receptor were detected and their genes seem tobe regulated in an Fe(II)/Fe(III)-dependent manner. TheFerroplasma spp. also exhibit three candidate genesfor isochorismatase potentially involved in the produc-tion of siderophores of the enterobactin family thatare upregulated under iron-limiting conditions. Theenterobactin-like siderophores have a high affinity forFe(III) but are less stable at low pH than thehydroxamate-like siderophores (Hider and Kong, 2010).The presence of enterobactin-like siderophores is unex-pected since Ferroplasma spp. lives at very low pH andmight be expected to use the more acid stable citrate-type siderophores.

Regulation of Fe(II) oxidation and iron homeostasis

Introduction

All microorganisms use iron as a cofactor of manyenzymes involved in essential biological systems andthey must balance the uptake and storage of this metabo-lite with its potential for exacerbating oxidative stressthrough the Fenton reaction. Microorganisms that alsouse Fe(II) as a source of energy and electrons face theadditional problem of balancing iron uptake for metabo-lism versus its use for energy and electron transfer. Verylittle is known about how this balance is achieved and wefocus on initial investigations that are beginning to shedlight on some of the regulatory processes that At. ferrooxi-dans may be using to modulate its use of iron. Hopefully,some of this information will subsequently prove to berelevant for understanding iron function regulation in otheriron oxidizers.

Regulation of iron oxidation in At. ferrooxidans

Acidithiobacillus ferrooxidans is confronted with the chal-lenge of regulating the electron flux derived from Fe(II)oxidation either uphill towards the reduction of NAD+ foranabolic activities such as CO2 and N2 fixation or down-hill towards the reduction of O2 to water to aid in theneutralization of protons that entered the cell via the ATPsynthetase complex. It was suggested about a decadeago that the cellular ATP/ADP ratio regulates thebalance of reducing equivalents from Fe(II), favouringeither the activation of the aa3 cytochrome oxidase andthus promote the downhill pathway or, conversely, therepression of the aa3 cytochrome oxidase promoting theuse of the uphill pathway (Elbehti et al., 2000). In addi-tion to regulatory decisions regarding the flux of elec-trons uphill or downhill, At. ferrooxidans also regulatesenzymes and electron carriers depending on whether itsenergetic substrate is Fe(II) or RISCs (Yarzabal et al.,2004; Bruscella et al., 2007; Amouric et al., 2009;Quatrini et al., 2009).

Regulator circuits potentially involved in making deci-sions about electron flux might therefore exist. Recently,candidate regulator systems have emerged that might beinvolved in these functions. Adjacent to the rus operonencoding components of the downhill pathway is a sixgene operon (ctaABTRUS) that contains genes predictedto be involved in the biogenesis of the aa3 cytochromeoxidase. The expression of this operon is upregulated inthe presence of Fe(II) as an energy source in a mannersimilar to the rus operon (Amouric et al., 2009; Quatriniet al., 2009). CtaR is predicted to encode an iron respon-sive regulator of the Rrf2 family of transcriptional regula-tors that, in many organisms, contains an [Fe-S] clusterand responds indirectly to iron concentration and oxida-tive stress as measured by the availability of intracellular[Fe-S] clusters (Johnston et al., 2007). CtaR may functionas a regulator of its cognate operon as observed inother organisms. Since it may also be a regulator of therus operon, this suggests a model in which the expres-sion of the cta and rus operons could be coordinatelyregulated.

Genes potentially encoding a sensor/regulator two-component signal transducing system of the RegB/RegAfamily involved in redox sensing in other microorganisms(Wu and Bauer, 2008; Bauer et al., 2009) have also beendetected further downstream of the rus operon (Amouricet al., 2009; Quatrini et al., 2009). This system has beenshown to measure the redox state of the quinone pool inmany microorganisms via a quinone bound to a trans-membrane domain and also by the redox state of a cys-teine located in the cytoplasmic transmitter domain (Wuand Bauer, 2008; Bauer et al., 2009). Both redox-sensingdomains are conserved in RegB in At. ferrooxidans



(Quatrini et al., 2009). The regBA gene pair is upregu-lated in the presence of Fe(II) similarly to the rus operon(Amouric et al., 2009; Quatrini et al., 2009) and couldpotentially activate/repress other genes involved in Fe(II)oxidation.

Other candidate genes potentially involved in the regu-lation of Fe(II) oxidation are Fur (Lefimil et al., 2009) andFNR (Osorio et al., 2009), master regulators of iron homeo-stasis and anaerobiosis respectively in At. ferrooxidans.

Regulation of iron uptake and homeostasis

Genomic and experimental evidence demonstrate thatAt. ferrooxidans has a functional transcriptional regulatorof the Fur family involved in the regulation of iron respon-sive functions such as Fe(II) and Fe(III) uptake and ironstorage (Quatrini et al., 2005b; 2007b; Osorio et al.,2008). Also, bioinformatic predictions suggest thatAt. thiooxidans and At. caldus have a suite of Fur regu-lated genes similar to those identified in At. ferrooxidans(Osorio et al., 2008). It is predicted that these threeAcidithiobacillus spp. have two other members of the Furfamily: a haem responsive (Irr-type) regulator responsiblefor the control of haem biosynthesis in response to ironavailability and a peroxide responsive (PerR-type) regu-lator responsible for the control of a variety of basic physi-ological processes in response to peroxide stress (Osorioet al., 2008).

From a combined metagenomic and proteomic studies,‘L. rubarum’, L. ferriphilum and ‘L. ferrodiazotrophum’ arepredicted to encode three members of the Fur family: aniron responsive Fur-type regulator, a peroxide-sensitivePerR-type regulator and a zinc responsive Zur typeregulator. Gene context analysis supports a role for thePerR-like regulator in alkylperoxide stress response inLeptospirillum sp. group II, where a cytochrome c peroxi-dase and a peroxiredoxin of the AhpC/Tsa family aredivergently transcribed. Partial conservation of thiscontext also occurs in the Acidithiobacillus spp. suggest-ing a similar function of the PerR-like regulator in thisgroup (Osorio et al., 2008).

Several of the iron responsive pathways predicted tobe under the regulation of Fur in the Acidithiobacillusspp. and Leptospirillum spp. exhibit functional redun-dancy that could provide an advantage in changing envi-ronmental conditions such as might be found in naturallyacidic conditions and in industrial copper bioleachingheaps in which differences in iron availability couldarise due to variations in the environmental pH, directlyaffecting iron solubility (Osorio et al., 2008). Understand-ing how regulatory factors other than Fur control expres-sion of iron uptake genes in acidophiles is still limited,yet the picture is growing increasingly complex with therecent findings of superimposed positive regulation by

several different transcriptional regulators (Osorio et al.,2008).

Concluding remarks

• Whereas substantial progress has been made in ourunderstanding of Fe(II) oxidation and iron homeostasisin the model acidophile At. ferrooxidans, knowledge ofthese processes in other acidophiles lags far behind.The recent advent of sequence information for over50 genomes and metagenomes of acidophiles willadvance our understanding of the diversity of pathwaysinvolved in Fe(II) and iron homeostasis and provideinsight into their evolution.

There are a number of different pathways for oxidizingiron that are phylogenetically widespread in Bacteria andArchaea and it seems plausible that the capacity tooxidize iron was invented independently more than onceduring evolution perhaps followed by later diversificationand lateral gene transfer. Major diversification andexploitation of new opportunities could have taken placeduring the ‘great oxygenation event’ about 2.2–2.4 billionyears when acidophiles would have been presented withnovel opportunities to use Fe(II) as an energy sourceusing oxygen as a terminal electron acceptor. However,they would also have been challenged by the significantincrease in soluble Fe(III) in their environment, poten-tially exacerbating problems of oxidative stress.

• Most likely, neither redox nor Fe(II) sensors and theirsignals operate in isolation to regulate the expression ofFe(II) oxidation. It is more probable that integrated andcomplex regulatory networks are involved, providingoptimal responses to changing levels of oxygen and asdifferent energy sources become available. It is hopedthat the availability of sequence information from manynew genomes will provide improved predictive modelsnot only for At. ferrooxidans but for other Fe(II) oxidizersas well.

• Although preliminary data suggest that genes involvedin iron metabolism in At. ferrooxidans are regulated bytranscription factors that sense iron and/or the redoxstate as well as oxygen availability, much remains to bedone to understand how acidophiles discriminatebetween iron as a micronutrient and as an energysource and how the coupling of electron flow betweenthe NAD+ uphill pathway and the O2 reduction downhillpathway reduction is regulated. The molecular mecha-nisms and network connections that underlie these keyregulatory decisions are most likely far more complexthan the preliminary evidence suggests and a majoreffort will be required to unravel the suspected dynamicconnections that underlie the proposed versatility ofpathway connections.



• The paucity of investigations into the possible use ofextracellular electron carriers in acidophilic Fe(II) oxida-tion represents a substantial lacuna in our knowledgeand merits further attention. Since many naturallyoccurring substrates such as pyrite and chalcopyrite areinsoluble, the more widespread use of excreted electroncarriers as aids in initial oxidation of such substratesmight be expected, as has been observed in cases ofneutrophilic iron oxidation and iron reduction.

Acknowledgements

Fondecyt 1090451, DI-UNAB 34-06, Conicyt Basal CCTEPFB16 and an ECOS-Conicyt award. Part of this work wasfinanced by the EU framework 6 project ‘BioMinE’ (N°NM2.ct,2005.500329), and we thank our various partners on theproject for their contributions to the work reported in thisarticle. We thank René Sepúlvida for his help in constructingthe phylogenetic tree. The G. capsiferriformans data wereproduced by the US Department of Energy Joint GenomeInstitute (http://www.jgi.doe.gov/) in collaboration with theuser community.

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Genomic insights into microbial iron oxidation and iron uptake