Characterization and Expression Analysis of the Cytochrome bd OxidaseOperon from Desulfovibrio gigas

Patr�cia Machado,1 Rute F�lix,1 Rute Rodrigues,1 Solange Oliveira,1,2 Claudina Rodrigues-Pousada1

1Instituto de Tecnologia Qu�mica e Biol)gica, Universidade Nova de Lisboa, Apartado 127, 2780-901, Oeiras, Portugal2Departamento de Biologia, Universidade de 3vora, 3vora, Portugal

Received: 9 June 2005 / Accepted: 14 November 2005

Abstract. Although classified as anaerobic, Desulfovibrio gigas contains a functional canonicalmembrane respiratory chain, including a cytochrome bd quinol oxidase as its terminal element. In thepresent study, we report the identification of the operon cydAB encoding the two subunits of cytochromebd from this bacterium. Two hypothetical promoter regions and sequences resembling transcriptionalregulators-binding sites have been identified. Amino acid sequence analysis revealed a high similarity tocytochrome bd from other organisms, presenting the conserved residues typical from these proteins.Reverse transcription polymerase chain reaction (RT-PCR) and Northern blot analysis confirmed theoperon transcription. Gene expression was assessed by real-time RT-PCR in cells grown in differentmedia and under exposure to oxygen and nitric oxide. mRNA levels were slightly enhanced in thepresence of 150 lM NO. However, in the presence of 10 lM NO, a decrease was observed of the steady-state population of cydAB mRNA. No considerable effect was observed in the presence of fumarate/sulfate medium, 60 lM O2 or 10 lM NO.

The sulfate-reducing bacterium Desulfovibrio gigaswas classified as a strict anaerobe [34]. However,several reports have clearly shown that this organismnot only resists oxygen but also has functional respi-ratory chains with terminal oxidases. A cytoplasmicelectron transfer pathway was first proposed by Chenand co-workers [7, 8]. It consists of three proteins,NADH: rubredoxin oxidoreductase, rubredoxin, andrubredoxin:oxygen oxidoreductase [14], which are ableto couple NADH oxidation to the complete reductionof oxygen to water. This process allows energy con-servation by substrate-level phosphorylation, withNAD+ being regenerated at the expense of polyglucosecarbon reserves [13, 37]. Later, a fully competent andcanonical respiratory chain, allowing the reduction ofdioxygen to water using reducing equivalents fromNADH and succinate, was detected in the membranesfrom D. gigas grown in a fumarate-sulfate medium

[23]. D. gigas membranes were able to consume oxy-gen with a respiratory rate on the same order ofmagnitude of that measured in the membranes of someaerobic bacteria such as Rhodothermus marinus [30].Cytochrome bd, the terminal quinol oxidase of thismembrane chain, was purified and characterized,revealing the ability to completely reduce oxygen [23].Similarly to Escherichia coli cytochrome bd, the en-zyme from D. gigas consists of two subunits, containsthree heme components, one heme D and two hemes B,and is relatively insensitive to cyanide, a characteristicof this type of oxidase [23].

Here we report the characterization of the genesencoding cytochrome bd from D. gigas: cydA andcydB. An adjacent region was also analyzed, revealinga gene encoding a Ca2+/Na+ antiporter and a sigma-54-dependent transcriptional/response regulator. Geneexpression analysis confirmed that cydA and cydB areco-transcribed as a single mRNA. The expression levelof the genes under different growth conditions,including exposure to oxygen and nitric oxide, was alsoexamined.

Correspondence to: Claudina Rodrigues-Pousada; email: claudina@itqb.unl.pt

CURRENT MICROBIOLOGY Vol. 52 (2006), pp. 274–281DOI: 10.1007/s00284-005-0165-0 Current

MicrobiologyAn International Journal

ª Springer Science+Business Media, Inc. 2006

Materials and Methods

Bacterial strains and plasmids. D. gigas ATCC 19364 was grownanaerobically as previously described [16]. E. coli strains P2392 andLE392 were used to screen a D. gigas genomic library and in thepurification of the recombinant positive phages. Competent E. coliXL1-Blue cells (Stratagene) were prepared according to standardprotocols [36] and used to transform the DNA fragments subclonedinto the plasmid pZErO-1 (Invitrogen).

DNA preparation. Genomic DNA was extracted from early-logphase D. gigas cells using the Wizard Genomic DNA Purification Kit(Promega). Plasmid DNA was extracted using the Fast Plasmid MiniKit (Eppendorf) and Phage DNA was extracted with the Lambda MaxiKit (Qiagen).

Cloning and sequencing. Degenerate oligonucleotide primers(5¢-TTCTGGGGCAAGCT(G/C)TT(C/T)(A/C)TCATC-3¢ and 5¢-CATGC(G/C)GAAAC(A/G)CCCAGCAC(A/G)AAG-3¢) (Thermo)were designed based on highly conserved regions within thecytochrome bd subunit I (CydA) polypeptide sequence from severalbacteria. The primers were used to amplify D. gigas genomic DNA byPCR (94�C, 3 min; (30 cycles of 94�C, 45 s; 55�C, 30 s; 72�C, 1 min30 s); 72�C, 10 min). The product, with 449 bp, was cloned in pZErO-1and subjected to DNA sequencing, showing consistent homology withcydA genes. The 449-bp fragment was radiolabeled with [a-32P] dATPusing the Megaprime DNA Labelling Systems (Amersham) and usedas a probe in a Southern blot analysis of genomic DNA and to screen aD. gigas genomic library constructed in the vector (k-Dash II(Stratagene). One positive phage was purified, its DNA digestedwith Apa I (Promega), and two positive fragments were identified bySouthern blotting. The fragments were subcloned in pZErO-1 andsequenced in both strands with ABI Prism 373A Automatic Sequencer(Perkin-Elmer) using ABI Prism DyeDeoxy Terminator CycleSequencing Kit. Protein identification and amino acid sequenceanalysis was performed with Blast 2.0 [1] and Expasy serveranalysis tools [2]. Hydropathy profiles were determined usingTMpred [21]. Multiple sequence alignments were performed withClustalW [20] and GeneDoc [27].

RNA isolation, Northern blotting, and RT-PCR analysis. TotalRNA was extracted from early-log phase D. gigas cells grownanaerobically at 37�C in lactate/sulfate medium, according to Malkiet al. [26], and treated with DNase I RNase-free (Gibco). RNA wasseparated on 1.2% agarose in MOPS buffer and 6% formaldehyde (v/v)gel using a 0.24–9.5 kb RNA Ladder (Gibco) as a size marker.Northern blotting and membrane hybridization with the [a-32P]-labeledcydA probe were carried out as described by Silva et al. [38].

Reverse transcription polymerase chain reaction (RT-PCR)experiments were performed using Superscript First-Strand SynthesisSystem for RT-PCR (Gibco), as described by the manufacturer.Primers specific for a 232-bp region in cydA (5¢-GATGGCGG-ACAGGTTGGTGG-3¢ and 5¢-GGGCGTGGTGACGGGCATTA-3¢)were used in PCR assays with cDNA as template. The RNA used wastreated with DNAse I RNase-free (Gibco) and a negative control wasperformed: a RNA sample was incubated under the conditions used forreverse transcriptase but without the enzyme and then used as templatein PCR with cydA specific primers.

RNA isolation and real-time RT- PCR analysis. In order to analyzethe effect of different growth media on the expression levels of cydAB,cells were grown anaerobically at 37�C in lactate/sulfate medium(control) and in fumarate/sulfate medium, as previously described [26].Total RNA was extracted from early-log phase D. gigas cellsaccording to Ausubel et al. [3].

In order to analyze the expression of cytochrome bd operon underoxygen and nitric oxide exposure, cells were grown anaerobically at37�C in lactate/sulfate medium until early-log phase and then 60 lMO2, 10 lM NO, or 150 lM NO was added to the cultures. Cells wereallowed to grow for 1 h after the addition, and total RNA was extractedas previously described [3].

RNAs were treated with Dnase I Rnase-free (Roche). Tran-scriptor Reverse Transcriptase (Roche) was used to synthetize cDNAwith the random primer p(dN)6, according to the manufacturer�sinstructions. A Light Cycler and the DNA Master SYBR Green I Kit(both from Roche) performed the amplification and quantification ofcDNA, using specific primers for cydA amplification (the same used inRT-PCR, presented above). Relative standard curves were constructedfor the cydA gene and for the housekeeping gene 16S rRNA, usingduplicate serial dilutions of cDNA. The expression of cydA gene fromcells in different growth conditions was normalized against the one of16S rRNA.

Results and Discussion

D. Gigas cytochrome bd operon sequence analysis. Inthe present work, we have identified the operonencoding the two subunits of cytochrome bd fromD. gigas. Nucleotide sequence analysis revealed that thegenes encoding subunit I (CydA) and subunit II (CydB)are separated by 12 bp. This short distance between thegenes, which have the same transcription direction,without any promoter region, suggests that cydA andcydB are co-transcribed (Fig. 1).

The ATG start codon of each gene is immediatelypreceded by a sequence corresponding to a hypotheticalribosome-binding site. Upstream of the cydA, twohypothetical promoter regions were identified, based onthe similarity to the E. coli d70 promoter consensus se-quence [19]: 5¢-CTTGAAA-N17-TAAAAT-3¢ and 5¢-GTTCAAA-N17-ATTTTT-3¢, separated by 24 bp(Fig. 1). In E. coli, five promoter regions were described[17], but in Azotobacter vinelandii only a single pro-moter was detected [42]. Some other sequences resem-bling –35 regions, such as TTGTCT and TTGCCC, arepresent upstream of the D. gigas cydA, but the biologicalmeaning of these regions needs further studies (Fig. 1).

In the cydAB promoter region of E. coli, severaloxygen-responsive transcriptional regulator-bindingsites were identified: three ArcA-P-binding sites [25]and two Fnr-binding sites [10]. In D. gigas, sequencesresembling these sites are also present upstream of thecydAB, as diagrammed in Fig. 1. It is plausible to sug-gest that the expression of D. gigas cydAB is controlledby Arc and Fnr homologous regulator proteins. Con-sistent with this prediction, transcriptional regulatorgenes homologous to fnr and arcA were found in thegenomes of D. vulgaris and D. desulfuricans. (accessionnr. YP_012322 and accession nr. ZP_00129138,respectively).

P. Machado et al.: Cytochrome bd Operon from Desulfovibrio gigas 275

Genomic Southern analysis using a cydA probe re-vealed only one positive fragment in each digestion(data not shown), suggesting that there is only one copyof the cydAB operon in D. gigas, as it happens in theclosely related D. vulgaris and D. desulfuricans. How-ever, more than one cytochrome bd was found in severalbacteria [29].

The total region sequenced in this work, includingthe cydAB operon and its vicinity, was submitted toGenbank (accession nr. DQ088033).

CYdAB expression analysis. RT-PCR analysis ofD. gigas total RNA extracted from cells grown inlactate/sulfate medium using a cydA probe revealed thatcydA is expressed under standard growth conditions. APCR product was obtained with a size of 282 bp aspredicted (Fig. 2A). Northern blot analysis showed asignal corresponding to a band with an approximate sizeof 2.5 kb, which is expected from the nucleotidesequence (Fig. 2B).

The expression of the cydAB operon under differentgrowth conditions was analyzed by real-time reversetranscription PCR, using D. gigas RNA extracted fromcells growing under standard conditions and in fumarate/sulfate medium or in the presence of either O2 or NO.Similar levels of cydAB mRNA were detected whencells were grown in the standard lactate/sulfate mediumor in fumarate/sulfate medium (data not shown), indi-cating that there is no induction of the cydAB operonexpression in fumarate/sulfate medium. This result

suggests that the high amount of cytochrome bd inD. gigas grown in fumarate/sulfate [23] is not due to anincrease of the cydAB mRNA levels. It seems, therefore,that the cytochrome bd synthesis in response to fumarateis not primarily controlled at the cydAB transcriptionlevel.

Analysis of the cydAB expression in D. gigas grownunder exposure to 60 lM O2 revealed no considerablechanges in cydAB mRNA levels (Fig. 2C). It is possiblethat the O2 concentration is not adequate in order toinduce the transcription of the operon. Indeed, in thefacultative anaerobe E. coli, the cydAB expression pat-tern is achieved through the combined action of Fnr andthe sensor kinase-response regulator pair ArcB-ArcA[18, 25]. ArcAB is responsible for the microaerobicactivation of the operon, with its transcription beingmaximal at ca. 7% air saturation [40] (1.47% oxygentension, equivalent to 3.7 lM, approximately, consider-ing the oxygen solubility in the medium of 8 mg/mL).When oxygen is further decreased, Fnr becomes active,repressing the cydAB transcription [40]. Sequencesresembling Arc-P and Fnr-binding sites were identifiedin the promotor region of the cydAB operon fromD. gigas and therefore, it is possible that a similarcontrol mechanism occurs in this organism.

A 1.6-fold induction of the mRNA levels was ob-tained when cells were exposed to 150 lM NO. Thisincrease can be due to a partial inactivation of a cydABrepressor D. gigas Fnr homologue, as described inA. vinelandii, where CydR, the Fnr counterpart, is

1050 bp1332 bp 1011 bp 1023 bp

350 aa (incomplete)

443 aa 336 aa 340 aa

49859 Da 37334 Da 35402 Da

54-sigmaregulator

1 kb

CydA CydBP

Ca2+/ Na+ antiporter P1 P2

Fig. 1. Genetic organization of the D. gigas cytochrome bd oxidase operon (cydAB) and adjacent region. The number of amino acids and molecularmass are indicated. The direction of transcription is from left to right, except for the 54-sigma transcriptional/response regulator gene. Sequencefrom cydAB upstream region is shown. Hypothetical )10 and )35 boxes for each promoter (P1 and P2), a putative ribosome binding site (rbs), andthe start codon of cydA are indicated. Sequences resembling the transcriptional regulatory elements consensus sequences, Fnr-binding site (5¢-TTGATN4ATCAA-3¢), and Arc-P box (5¢-[A/T]GTTAATTA[A/T]-3¢) are underlined and labeled Fnr and Arc-P, respectively. P, P1, and P2,hypothetical promoter sequences; aa, amino acid. )343 corresponds to the position of the last nucleotide presented relatively to the ATG (+1).

276 CURRENT MICROBIOLOGY Vol. 52 (2006)

inactivated by NO [42]. However, the steady-statepopulation of cydAB mRNA is reduced in 50% of thecontrol cells when grown upon exposure to 10 lM NO. Itis plausible that at low NO concentrations the Fnrhomologue is exerting its repressing role.

Analysis of the predicted polypeptide sequence ofcytochrome bd. The deduced amino acid sequencesrevealed a high similarity to subunits I and II ofcytochrome bd quinol oxidase from D. desulfuricans(77% and 71.6%, respectively) and D. vulgaris (79.9%and 68.9%, respectively) and to CydA and CydB fromE. coli (52.6% and 48.5%, respectively). Subunit I

sequence alignment clearly indicates that the axialligands of heme b558 (His186 [12] and Met 393 [22] inE. coli) and of heme b595 (His19 in E. coli [39]) areconserved in D. gigas as His183, Met331, and His18,respectively. The highly conserved motifGWXXXEXGRQPW is also located in D. gigas(Fig. 3), in which the glutamate seems to be requiredto stabilize the binding of heme b595 [45]. Thecounterparts of the Glu99 and Glu107 residues ofE. coli CydA, proposed to form part of a protonchannel lying from the cytoplasmic side of themembrane to the oxygen reactive site [29, 44], arealso conserved (Fig. 3).

0

1

2

LS LS+ LS+ LS+

Exposure conditions

Cyd

A /

16S

rR

NA

(a

rbit

rary

un

its)

1 2 3 4

200 bp

300 bp

(A) (B)

2

(C)

3

4

65

1.51

0.5

kb

2.5

Fig. 2. CydAB expression analysis.(A) RT-PCR analysis. 1, 4,molecular marker (1Kb Plus,Invitrogen); 2, PCR product fromthe cydA gene; 3, negative control.(B) Northern blot of total RNAisolated from D. gigas grownanaerobically in lactate/sulfatestandard medium after hybridizationwith a [a-32P]-labeled cydA probeRNA molecular markers areindicated in the right side of thefigures. (C) Expression level ofcydA gene under exposure to oxygenand nitric oxide assessed by Real-Time Reverse Transcription PCRanalysis. LS, mRNA from cellsgrown anaerobically in lactate/sulfate medium not exposed to anycompound (control); mRNA fromcells grown anaerobically in lactate/sulfate and exposed for 1 h to 60 lM

O2 (LS + O2 60 lM), to 10 lM NO(LS + NO 10 lM) or to 150 lM NO(LS+NO 150 lM). The RNAexpression levels were obtained asdescribed in Materials and Methods.The ratio between the valuesobtained for the different treatmentsand the control (LS) are indicated inthe Y-axis.

P. Machado et al.: Cytochrome bd Operon from Desulfovibrio gigas 277

Hydropathy plots of E. coli [44] and D. gigas (notshown) subunit I amino acid sequences are very similarto each other, both showing nine membrane-spanning

regions with similar locations. However, D. gigas CydAlacks the C-terminal half of the long periplasmic loopbetween the putative transmembrane segments VI and

Fig. 3. Alignment of the deduced sequence ofcytochrome bd subunit I in D. gigas and otherknown sequences. Highly conserved residues areindicated, including: the heme-binding ligands(H19, H186, and M393, in E. coli), the aminoacids proposed to be part of a proton channel(E99 and E107, in E. coli) and the highlyconserved region (GWXXXEXGRQPW). TheQ-loop region is located between the residues236 and 323 of D. gigas. Dg, D. gigas; Dd,D. desulfuricans (accession nr. ZP_00128927);Dv, D. vulgaris (accession nr. YP_012481); Mt,M. Thermoacetica (accession nr. ZP_00330517);Ma, M. acetivorans (accession nr. NP_615957);Ec, E. coli (accession nr. P11026); Av, A.vinelandii (accession nr. Q09049).

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VII (the Q-loop). This region is also missing in CydAsof several different bacteria such as the closely relatedD. desulfuricans and D. vulgaris, the gram-positiveMoorella thermoacetica, and the archaea Methanosar-cina acetivorans (Fig. 3). Although the Q-loop wassuggested to be important for quinol oxidation [11, 43],the missing part does not seem to be essential for thefunction [29].

Analysis of the CydAB adjacent region. The cydABdownstream region was also sequenced. This 2288-bpregion included two genes whose products revealedhomology with Ca2+/Na+ antiporters and sigma-54dependent transcriptional/response regulators (Fig. 1).

Ca2+/Na+ antiporters are sodium/calcium exchangerintegral membrane proteins that regulate intracellularCa2+ concentrations [28]. The exchanger is primarily aCa2+ extrusion mechanism and as such, requires theenergy of the Na+ gradient produced by a Na+ pump[31]. Interestingly, there are several studies reportingthat cytochrome bd seems to operate as a Na+ pump inthe Na+-coupled energetics alternative to the H+ cycle[4–6]. Regarding its amino acid sequence, Ca2+/Na+

exchangers have an internal repeat in their membranedomain that presumably has arisen from a primordialgene duplication event [35]. This repeat was identifiedin the predicted polypeptide from D. gigas.

The second gene downstream cydAB, transcribed inan opposite direction, was incompletely sequenced.Only the C-terminal region was determined, where itwas identified as a sigma-54 interaction domain signa-ture, WPGNVRELgN (consensus sequence [FYW]-P-[GS]-N-[LIVM]-R-[EQ]-L-x-[NHAT]) (accession nr.PS00688), suggesting that the gene encodes a sigma-54dependent transcriptional/response regulator. Twoputative ATP-binding sites (GETGVGKE and AH-GGVLFLDE) (accession nr. PS50045) were also rec-ognized. Sigma-54 factor is required for expression of awide variety of genes involved in many diverse func-tions such as energy metabolism [24], RNA modifica-tion [15], and flagellation [32].

No cydC or cydD genes, encoding ABC-transporterproteins, were found downstream the D. gigas cydABoperon. These proteins are required for assembly of thecytochrome bd complex [9, 33], and there are studiesshowing that no functional cytochrome bd is made ifCydCD is not present [41]. Although cydC and cydD arefrequently contiguous to the cydAB genes, such as inB. subtilis, in which they form the cydABCD operon[41], their absence in the cydAB adjacent region is notsurprising because there are several bacteria showing thesame gene organization, e.g., E. coli (accession nr.U00096), Vibrio cholerae (accession nr. NC_002505),

D. vulgaris (accession nr. NC_002937), and D. desul-furicans (accession nr. NZ_AABN02000001).

In spite of being an anaerobe, D. gigas contains acytochrome bd, which may exert a role in the transportof electron towards oxygen. Indeed, Lemos et al. [23]have shown that membranes from D. gigas grown in afumarate-sulfate medium are able to reduce dioxygen towater. Although D. gigas contains the required equip-ment to respire oxygen, it is not yet ruled out whetherthis bacterium requires this cytochrome chain to surviveonly to the presence of oxygen. This reveals that theanaerobic bacteria may present several flexible respira-tory systems to cope with the modifications of theenvironment. Consequently, gene expression is repro-grammed as in the case of the sensing of oxygen, nitrate,or nitric oxide.

ACKNOWLEDGMENTS

This work was supported by Fundażo para a CiÞncia e Tecnologia(POCTI/BME/37480/2001 to C.R.-P. and fellowships 018/BIC/2004 toP.M., 031/BIC/2003 to R.F., SFRH/BD/5219/2001 to R.R.). STABGen)mica is also acknowledged for running the sequencing reactions.

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