Biochemical and Biophysical Research Communications 280, 491–502 (2001)
doi:10.1006/bbrc.2000.4147, available online at http://www.idealibrary.com on
nalysis of the Desulfovibrio gigas Transcriptional Unitontaining Rubredoxin (rd) and Rubredoxin-Oxygenxidoreductase (roo) Genes and Upstream ORFs
abriela Silva,* Solange Oliveira,*,† Jean LeGall,*,‡ntonio V. Xavier,* and Claudina Rodrigues-Pousada*,1
Instituto de Tecnologia Quımica e Biologica, Universidade Nova de Lisboa, 2781-901 Oeiras, Portugal;Departamento de Biologia, Universidade Evora, Evora, Portugal; and ‡Department of Biochemistrynd Molecular Biology, University of Georgia, Athens, Georgia 30602
eceived December 11, 2000
among the flavoprotein family are reported in thiss
o
rtfotetttRtvhdposSteDtSmppcIct
Rubredoxin-oxygen oxidoreductase, an 86-kDa ho-odimeric flavoprotein, is the final component of a sol-ble electron transfer chain that couples NADH oxida-ion with oxygen reduction to water from the sulfate-educing bacterium Desulfovibrio gigas. A 4.2-kbragment of D. gigas chromosomal DNA containing theoo gene and the rubredoxin gene was sequenced. Addi-ional open reading frames designated as ORF-1, ORF-2,nd ORF-3 were also identified in this DNA fragment.RF-1 encodes a protein exhibiting homology to severalroteins of the short-chain dehydrogenase/reductaseamily of enzymes. The N-terminal coenzyme-bindingattern and the active-site pattern characteristic ofhort chain dehydrogenase/reductase proteins are con-erved in ORF-1 product. ORF-2 does not show any sig-ificant homology with any known protein, whereasRF-3 encodes a protein having significant homologiesith the branched-chain amino acid transporter AzlCrotein family. Northern blot hybridization analysisith rd and roo-specific probes identified a common
.5-kb transcript, indicating that these two genes areotranscribed. The transcription start site was identi-ed by primer extension analysis to be a guanidine 87 bppstream the ATG start codon of rubredoxin. The tran-cript size indicates that the rd-roo mRNA terminatesownstream the roo-coding unit. Putative 210 and 235egulator regions of a s70-type promoter, having similar-ty with E. coli s70 promoter elements, are found up-tream the transcription start site. Rubredoxin-oxygenxidoreductase and rubredoxin genes are shown to beonstitutively and abundantly expressed. Using the datavailable from different prokaryotic genomes, theubredoxin genomic organization and the first tenta-ive to understand the phylogenetic relationships
1 To whom correspondence should be addressed at Instituto deecnologia Quımica e Biologica, Apartado-127, 2781-901 Oeiras, Por-ugal. Fax: (351-21) 4433644. E-mail: [email protected].
Although there is growing evidence that sulfate-educing bacteria (SRB) are to some extent oxygenolerant, since many of them even being able to profitrom the presence of oxygen (1–5), Cypionka (6) basedn their phenotypes when exposed to oxygen suggestshat these bacteria are essentially anaerobes. How-ver, they possess several properties that enable themo survive and/or utilize oxygen. Several data revealedhat SRB have protective mechanisms that allow themo cope with reactive oxygen species (ROS). CanonicalOS detoxifying enzymes such as FeSOD and catalase,
ogether with a large array of other iron proteins in-olved in defense mechanisms against oxidative stressave been detected in SRB (7–10). Among the latteresulfoferrodoxins and neelaredoxins were shown toosses superoxide-scavenging activity, either as super-xide dismutases or superoxide reductases (11–13). Be-ides the presence of oxygen detoxifying enzymes, someRB were also shown to have chemosensory pathwayshat trigger physiological responses toward the pres-nce of oxygen (14). Furthermore, some SRB, namelyesulfovibrio (D) species, were shown to reduce oxygen
o water having oxygen-reducing systems (1, 4, 15).uch a process may also be regarded as the safestechanism to undergo the detoxification of ROS. In the
resence of oxygen the sulfate reducer D. gigas is ca-able to generate ATP from the degradation of polyglu-ose reserves by substrate level phosphorylation (16).n this bacterium the reduction of oxygen to water isoupled with NADH oxidation and occurs through ahree-component soluble electron transfer chain (15,
Vol. 280, No. 2, 2001 BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
7–19). In this pathway rubredoxin (Rd), a smallononuclear iron protein (20), transfers electrons
etween a NADH oxidase (NRO) and the terminalxidase rubredoxin-oxygen oxidoreductase (ROO)15, 17, 18).
ROO is an 86 kDa homodimeric protein (15), eachubunit of 402 amino acid residues containing oneMN, two iron atoms, and substoichiometric hemeoieties. The solved 3D structure of ROO at 2.5 Å
esolution revealed that each monomer is built by twotructural domains: one b-lactamase-like, containinghe diiron center, and another flavodoxin-like, harbor-ng the flavin mononucleotide. The enzyme quaternarytructure ensures a direct electron transfer betweenhe flavin moiety and the diiron catalytic reaction cen-er. The close proximity between the diiron site andMN provides the four electrons for full reduction ofxygen to water (19). The work of Gomes et al. (18) haslearly demonstrated the direct reaction of Rd withOO and the direct reduction of O2 to water by aechanism that was further supported by the 3D
tructure data (19).In the present study, it is shown that roo is cotrans-
ribed with rd as a discistronic operon from an up-tream promoter region, which shows similarities withhe 210 and 235 promoter elements recognized by E.oli s70 RNA polymerase. Both roo and rd are shown toe constitutively and abundantly expressed and thed-roo mRNA levels decreased if cells are subjected toigh oxygen concentration for 60 min. However, in thease of hydrogen peroxide treatment no change in theirevels was detected. The sequencing of the region up-tream of rd-roo operon revealed the presence of threeRFs: ORF-1 encoding a short-chain dehydrogenase/
eductase-like protein, ORF-2 product not revealingny homology with any known protein, and ORF-3ncoding a branched-chain amino acid transporterzlC-like protein. Furthermore, it is shown that ROO
s a member of a widespread and conserved proteinamily present in prokaryotes but not in eukaryotes,nd that it seems to be closely related with the M.annaschii flavoproteins.
FIG. 1. Genomic organization of D. gigas roo. Partial restrictionositions of roo and rd genes together with other putative ORFs locatrrows indicate the promoter (P) of rd-roo operon and a putative rhrrows indicate the direction of transcription.
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ATERIALS AND METHODS
Bacterial strains and growth conditions. D. gigas (ATCC 19364)as grown as previously described (21). The E. coli strains E. coliOP 10F9 (Invitrogen) and XL2-Blue (Stratagene, La Jolla, CA)arrying the pZErO–1 (Invitrogen, San Diego, CA) and pZErO-1-erived vectors were grown in LB broth (containing, per litter, Tryp-one (Difco), 10g; yeast extract (Difco), 5 g; and sodium chloride, 5 g)ith 20 mg zeocin ml21 or with 20 mg zeocin ml21 and 100 mg IPTGl21. Competent E. coli TOP 10F9 and XL2-Blue cells were prepared
s described in Invitrogen.
Preparation of DNA. Genomic DNA isolated from D. gigas wasxtracted as described in (22). Plasmid DNA was prepared using thetandards protocols or the plasmid purification kit Qiagen (Diagen).ligonucleotides used were synthesized by Gibco BRL.
Cloning and sequencing of a DNA fragment containing the rooene. During the screening of a Sau3A1 genomic library from D.igas constructed in the vector l-Dash II (Stratagene) with a rooomologous probe two overlapping phages carrying the roo geneere identified. One with a 14.4 kb chromosomal DNA fragment
rom D. gigas and the other with a 12 kb DNA fragment overlappinghe first in an 11.4 kb. These DNA fragments were identified, clonednd sequenced as described in (18) using the Thermo Sequenaseycle sequencing kit from Amersham Life Science and [a-35S]dATProm ICN. The identification of ORFs was performed using theodepreference program of Wisconsin Package Version 10.0, Genet-
cs Computer Group (GCG), based on the codon preference used by D.igas. Putative rho-independent terminators were found using theCG terminator search program. Homology searches for putativeene products were performed using BLAST 2.1 of GenBank data-ase (http://www.ncbi.nlm.nih.gov/). Sequence alignments wereade using the GCG or ClustalW (23). Phylogenetic studies were
erformed using the Phylip program version 3.5c; distances analysissed the program Protdist with a PAM 250 substitution matrix andrees calculated by neighbor-joining algorithms. Bootstrap analysessed 500 replicates of a single round of random addition each. Theonsensus tree by the majority-rule consensus tree method was dis-layed using the TreeView program (24).
RNA isolation and Northern blot analysis. Total RNA was iso-ated from exponentially and stationary anaerobically grown D.igas cells. RNA extraction was performed as follows: the cells wereapidly cooled by adding 1/20 vol of ice cooled stop solution (22),arvested by centrifugation, and the cell pellets were quickly dis-olved in 7 ml lysis buffer (1% SDS, 20 mM sodium acetate pH 5.5,0 mM EDTA, 30 mM Tris–HCl pH 7.5, 10 mM VRC) containing 100g proteinase K ml21. Equal volume of hot-buffered phenol (pH.5–5.0) was added to the lysate, followed by 20 min incubation at0°C. After centrifugation the supernatant was extracted 3 timesith phenol:chloroform:isoamyl alcohol (25:24:1), and the nucleiccids precipitated with 0.2 M LiCl. The pellet was then dissolved in
p of a 4.2-kb DNA fragment. The restriction sites and the relativepstream the rd gene (ORF-1, ORF-2, and ORF-3) are indicated. The
ndependent terminator (T) located downstream roo gene. The gray
maed uo-i
Ooipu
FIG. 2. Partial nucleotide sequence of 4.2-kb DNA fragment from D. gigas. The DNA sequence containing the coding regions of ORF-1,RF-2, ORF-3, and rd is given in the 59 to 39 direction, and numbering corresponds to the database entry. The predicted amino acid sequencesf ORF-1, ORF-2, and rd are given in single-letter amino acid code above the nucleotide sequence, and for ORF-3 below the sequence. Asteriskndicates translation stop codons. Putative ribosome-binding sites (rbs) preceding each gene are shown. The determined transcription startoints of rd-roo mRNA are indicated by bent arrows. Potential 210 and 235 promoter elements of a s70 upstream P1 are indicated. The P4pstream region discussed in the text is underlined by a dotted line.
DTaafTooggod
mtpCawhptpAl
Brah
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EPC-treated water and subjected to a proteinase K treatment.he samples were again extracted with phenol:chloroform:isoamyllcohol, and the total RNA precipitated with 0.3 M potassiumcetate. The RNA was further purified by treatment with RNase-ree DNase I in the presence of RNase-out (Gibco BRL, Lifeechnologies) followed by reading the absorbance at 260 nm inrder to determine its concentration. The RNA was then separatedn a 1.5–2% agarose in 13 Mops buffer and 6% formaldehyde (v/v)el using a 0.24-9.5 kb RNA Ladder (Gibco BRL, Life Technolo-ies) as a size marker. Northern blotting was essentially carriedut as in (22), and filters were prehybridized and hybridized asescribed in (12) at 53–58°C. 32P-labeled oligonucleotides comple-
FIG. 3. Comparison of ORF-1 encoding a SDR-like protein with. halodurans 3-oxoacyl-reductase; Pa1537, P. aeruginosa probable
eductase; PfadhA, P. furiosus short-chain alcohol dehydrogenase;lcohol dehydrogenase. The NAD-/NADP-binding and the active sitesistidine residues of ORF-1-encoded protein.
494
entary to roo (59-CTAGGCGGCCAGCTTGGCCTTCAG-39) ando rd (59-CTACTGCTTTTCGAAGGCGTCCTTG-39) were used asrobes. A 32P-labeled 23SrRNA primer (59-CTTTTCRCCTTTC-CTCACGGTAC-39) was used as an internal control of loadingnd hybridization conditions. After hybridization, filters wereashed in 23 SSC/0.1% SDS and 0.23 SSC/0.1% SDS underomologous hybridization temperatures. To reuse the filters therobe was removed by pouring onto the membrane a boiling solu-ion of 0.1% SDS. Quantification analysis of Northern blots waserformed using ImageQuant version 3.1 (Molecular Dynamics).ll solutions used were prepared in ultra pure water MilliQ (Mil-
ipore Corp.) or in DEPC treated ultra pure water.
orthologues. Hsinb, Halobacterium sp. NRC-1 Inb protein; BhfabG,rt-chain dehydrogenase; Dr1620, D. radiodurans probable ketoacylcpA, E. coli UCPA oxidoreductase; PaSDR1, P. abies short-chainSDR proteins are indicated. The asterisks indicate the cysteine and
itsshoEcuof
oaltaG
ofs
haa
aAOFp
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Primer extension analysis. The transcriptional start of rd-rooperon was determined by using 50 mg of total RNA extracted fromnaerobically grown D. gigas cells as described in (22). Two 32P-abeled oligonucleotides complementary to the rd sequence from 127o 148-primer ROOOP2 (59-GGCAGGATCGTATTCGTAGCCG-39)nd from 191 to 1111-primer ROOOP3 (59-CCAGTCGTCG-GCAGGTCTTC-39) were used. The extended primers were loaded
FIG. 4. Sequence alignment of ORF-3-encoded polypeptide witheruginosa PA01 hypothetical protein (Accession AAG05427); VcA1zlC (Accession G75494); RsORF1, R. sphaeroides putative membra07942); Af1755, A. fulgidus hypothetical protein AF1755 (Accessio71831); NmB0892, N. meningitidis MC58 AzlC-related protein (Acrotein are indicated.
495
nto sequencing gel together with the sequencing reactions per-ormed using the same primers and the Thermo Sequenase cycleequencing kit (Amersham).Nucleotide sequence accession numbers. The coding region of roo
as been deposited in GenBank with the Accession No. AF218053,nd the 2640 nucleotide sequence containing ORF-1, ORF-2, ORF-3,nd rd is deposited in GenBank with the Accession No. AF325447.
mologous proteins. DgazlC, D. gigas AzlC-like protein; Pa2039, P., V. cholerae AzlC (Accession AAF96898); Dr0633, D. radioduransprotein (Accession CAB89200); BsazlC, B. subtilis AzlC (Accession28519); Hp1251, H. pylori hypothetical protein jhp1251 (Accessionion AAF41301). The hydrophobic regions of D. gigas AzlC putative
ho002nen Ocess
R
ilcmTDgiFdssi
tdCqng(dt
typical pairwise residue identity of 15–30% for SDR en-zso(AdNrasnAP(fiwPspNGdc
NhcR(LDwf
cctr(c
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ESULTS AND DISCUSSION
Organization of ORFs upstream the roo gene. Dur-ng the screening of roo gene from D. gigas two over-apping phages were obtained: one containing a 14.4 kbhromosomal DNA and the other containing a frag-ent of 12 kb, with a 0.6 kb nonoverlapping region.he complete sequence of the left 4.2 kb region of theseNA fragments besides containing the rd and rooenes, contains three additional complete ORFs, des-gnated by ORF-1, ORF-2 and ORF-3 as illustrated inig. 1. ORF-1 and ORF-2 are transcribed in the sameirection as rd and roo genes, whereas ORF-3 is tran-cribed in the opposite direction (Fig. 1). Nucleotideequences that may act as ribosome-binding sites aredentified upstream of each ORF (Fig. 2).
Analysis of ORF-1 sequence encoding a SDR-like pro-ein. ORF-1 encodes a protein of 270 amino acid resi-ues, with a predicted molecular mass of 28,620 Da.omparison of the predicted amino acid sequence to se-uences deposited in database revealed that it has sig-ificant homology with the short-chain dehydro-enase/reductase family of enzymes (SDR) (EC 1.1.1.-)25), most of which are known to be NAD- or NADP-ependent oxidoreductases. The overall amino acid iden-ity ranges from 29 to 34%, which is consistent with the
FIG. 5. Transcription analysis of the D. gigas rd-roo operon. (A)orthern blot analysis of D. gigas total RNA using rd and rooomologous probe. M represents molecular mass standards withorresponding sizes marked on the left; lane 1 contains 20 mg totalNA from D. gigas. The arrow indicates the size of the rd-roo mRNA.
B) Determination of the transcription start site of rd-roo mRNA.ane 59, primer extension product. Lanes A, C, G, and T show theNA sequencing reaction from the corresponding region carried outith the same primer. P1, P2, P3, and P4 indicate the position of the
our primer extension products.
496
ymes, and is most significant at the NAD/NADP-bindingite. Among other proteins, it shows homology with axidoreductase homolog from Halobacterium sp. NRC-134% identity and 38.3% similarity) (Accession No.AG19050), the 3-oxoacyl-reductase from Bacillus halo-urans (32% identity and 42.4% similarity) (Accessiono. BAB06210), a probable ketoacyl reductase from the
adioresistant Deinococcus radiodurans (31% identitynd 38.6% similarity) (Accession No. F75374), a probablehort-chain dehydrogenase from Pseudomonas aerugi-osa (31.6% identity and 38.3% similarity) (Accession No.AG04926), a short-chain alcohol dehydrogenase fromyrococcus furiosus (31% identity and 38.1% similarity)
Accession No. AAC25556), the oxidoreductase UCPArom Escherichia coli (30.2% identity and 39.5% similar-ty) (Accession No. P37440). It also shows similaritiesith a short-chain type dehydrogenase/reductase fromicea abies (29.3% identity and 38.3% similarity) (Acces-ion No. Q08632). The alignment of the predictedolypeptide with its orthologues (Fig. 3) reveals the-terminal coenzyme-binding pattern of typicallyXXXGXG, and the active-site pattern of YXXXK for theehydrogenase/reductase proteins, whereas the midhain pattern NNAG is partially present (25, 26). The low
FIG. 6. Analysis of rd-roo transcription in O2 saturated D. gigasells. (A) Northern blot analysis of 20 mg total RNA from D. gigasells saturated with O2 for 0, 10, 30, and 60 min (lanes 1–4, respec-ively). As loading and hybridization control it was used the 23SRNA. The arrows indicate the rd-roo mRNA (rd-roo) and 23S rRNA23S). (B) Densitometric analysis of Northern blots of A. Valuesorrespond to the average of three independent experiments.
swticalmfd
ba
smapt2stklmmsbtI(t
Aqcgbtcbtc
vDcwRcgvbttndiwspsato
rodb
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imilarity found at the C-terminal region is consistentith variations observed in the C-terminus of SDR pro-
ein family, which is related with the individual functionsn each case. Moreover, the putative D. gigas SDR proteinontains 8 histidine and 6 cysteine residues (marked byn asterisk in Fig. 3), not conserved among its homo-ogues, some of which may be involved in the binding of
etal ions such as Zn and Fe similarly to other proteinsrom the SDR family, namely the short-chain alcoholehydrogenases (27, 28).The predicted polypeptide of 63 amino acids encoded
y ORF-2 does not show any significant homology withny known protein.
Analysis of ORF-3 sequence. An ORF occurring up-tream rd coding unit and encoded by the comple-entary DNA strand, designated by ORF-3, encodesputative protein of 235 residues. Comparison of theredicted amino acid sequence to other sequences inhe GenBank-EMBL/Swiss-Prot databases revealed5 to 39% sequence identity (37 to 50% sequenceimilarity) with the branched-chain amino acidransporter AzlC family protein from several pro-aryotes. The predicted polypeptide, with a molecu-
ar mass of 25 kDa, has a length within the range ofost AzlC proteins. Its hydropatic profile, deter-ined according to the method of Kyte-Doolittle,
hows the presence of VII hydrophobic regions capa-le of spanning the membrane (Fig. 4), indicatinghat it is likely to be an integral membrane protein.n B. subtilis AzlC protein, 5 out of 7 of these regionsregions I, II, IV, V, and VII) were identified to beransmembrane regions (29). Alignment of D. gigas
FIG. 7. Rubredoxin gene localization among prokaryotes. Coocceducers and in potential operons of several available prokaryotic gexidoreductase; rbo, rubredoxin oxidoreductase; rbr and rr, rubrerytfx, desulfoferrodoxin; fprA and fprA3, flavoproteins; ahp, alkyl hydoxes indicate genes located in the same operon.
497
zlC-like protein with orthologues revealed that se-uence similarity among the predicted polypeptidesompared is almost limited to the hydrophobic re-ions, and to a region located between the hydropho-ic regions III and IV (see Fig. 4). Although no func-ion was yet attributed to this region, it probablyorresponds to the region that interacts with theranched chain amino acids or with other Azl pro-eins, such as AzlD and AzlE (29), as it is the mostonserved region among the compared polypeptides.
Characterization of rd-roo operon of D. gigas. Pre-ious sequencing analysis of a 3.6 kb BamHI/BamHINA fragment suggested that rd and roo genes are
lustered in the same operon (18). To investigatehether or not these genes are cotranscribed, totalNA extracted from mid-log-phase (OD450nm ' 0.3–0.4)
ells of D. gigas was probed with rd and roo homolo-ous probes. Northern blot hybridization analysis re-ealed a transcript of approximately 1.5 kb hybridizingoth with the rd and roo probes (Fig. 5A), indicatinghat rd and roo genes are cotranscribed. Moreover, theranscript size indicates that the rd-roo mRNA termi-ates in the putative rho-independent terminatorownstream ROO coding unit. Primer extension exper-ments to determine rd-roo transcription start siteere performed using two different primers. The re-
ults presented in Fig. 5B show 4 primer extensionroducts designated P1, P2, P3, and P4. These tran-cription start sites correspond to positions 87, 56, 47nd 34 for P1, P2, P3, and P4 respectively, upstreamhe translation initiation codon (Fig. 2). Examinationf the upstream region of P1 transcription start site
ence of rubredoxin gene with others in the same operon in sulfatees. The genes shown are of: rd, rubredoxin; roo, rubredoxin-oxygen; rdl, rubredoxin-like; sor, superoxide reductase; nlr, neelaredoxin;
Vol. 280, No. 2, 2001 BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
showed sequences TTGAAG and TACATC at 235 and2r2a2qqsPsstsi2tdpTht2tidTsu
toOp
orAt1Fgtc
indicating that this regulator proteins are not involvedi
eTogOpevdac6sFmFgatrscscott(umleahipcnfii
PNMANQAS(t
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10, optimally separated by 17 bp, resembling thoseecognized by E. coli RNA polymerase (RNAP) s70 (Fig.). These regions match four of the six bases (boldface)t the 235 (TTGACA) and three of the six bases at the10 (TATAAT) E. coli s70 consensus promoter se-uences. On the other hand, no promoter-like se-uences having homology with the 235 and 210 of the70-dependent promoter were detected upstream P2,3 and P4 primer extension products. This observationuggests that P2, P3 and P4 products probably corre-pond to premature termination of reverse transcrip-ase caused by the high GC content in the region up-tream the P4. A search for s54–dependent promoters,ncluding GG and GC doublets at positions 224 and12, respectively, could suggest the existence of a po-
ential s54 promoter upstream the P4 (underlined by aoted line in Fig. 2). Furthermore, despite the fact thatromoter sequences recognized by s54 (TGGCAC-N5-TGC) are well conserved (30), the sequences of theypothetical s54 promoter located upstream the P4 al-hough containing the GG and GC doublets at positions24 and 212, do not show relevant similarities with
he s54 consensus promoter. This fact corroborates thedea that P2, P3 and P4 primer extension products areue to premature termination of reverse transcriptase.hese data indicate that the real transcriptional startite of the rd-roo operon is the guanidine in position 87pstream the rd ATG.In conclusion it seems that rd-roo operon, in contrast
o operons containing genes encoding for ROO homol-gous proteins, namely the product of R. capsulatusRF14 (31), is under the control of a s70-dependentromoter.
Expression of rd-roo genes. Expression of terminalxidoreductases in E. coli is shown to be regulated inesponse to oxygen by the regulator proteins FNR andrcAB (32, 33). Since ROO is a terminal oxidoreduc-
ase that reduces oxygen to water in D. gigas (15,7–19), as a first approach, a search for binding sites ofNR and ArcAB regulators in the rd-roo promoter re-ion was performed. No consensus sequences similar tohose recognized by ArcAB or Fnr regulator proteinsould however be found in the rd-roo promoter region,
FIG. 8. Complete amino acid alignment of flavoproteins. The multipileup program. DgROO, D. gigas rubredoxin-oxygen oxidoreductase (tho. A72338); PAB0596, P. abyssi flavoprotein (Accession No. A75136TH157, MTH1350 and MTH220, M. thermoautotrophicum flavoproteAB85827), and flavoprotein A homolog (II) (Accession No. C69127); Affpo. G69270) and FprA-2 (Accession No. G69439); MJ0748, MJ0534 and58158), MJ0534 (Accession No. Q57954) and MJ0732 (AccessionAG00802); EcORF-o479, E. coli flavoprotein (Accession No. B65051); R39896) and potential FMN-protein (Accession No. BAA02789); Ss1102
Accession Nos. S74578, S75748, S74576 and S76209, respectively). Theristic of this protein family are indicated by #, and the ROO iron liga
499
n the control of rd-roo expression.In contrast to other terminal oxidoreductases rd-roo
xpression may not be regulated in response to oxygen.o evaluate rd-roo gene expression in the presence ofxygen, total RNA was extracted from anaerobicallyrown mid-log-phase cells of D. gigas saturated with2 (;1.27 mM O2) during 0, 10, 30, and 60 min androbed both with rd and roo homologous probes. North-rn blot hybridization and densitometric analyses re-ealed no significant change in the rd-roo mRNA levelsuring the first 30 min in O2 saturated cells, but anpproximate 59% decrease in rd-roo mRNA levels inultures exposed to O2 for 60 min was measured (Fig.). The observation that no decrease in rd-roo expres-ion occurs during the first minutes of O2 exposure (seeig. 6) suggests that the decrease at 60 min O2 treat-ent is probably due to rd-roo mRNA degradation.urther experiments with anaerobically grown D. gi-as cells subjected to O2 for higher periods of time (90nd 120 min) showed that the rd-roo mRNA was par-ially degraded which is accompanied by a similar deg-adation of the highly stable 23S rRNA (data nothown). These findings support the idea that the de-rease of rd-roo mRNA levels observed at 60 min O2-aturated cells is due to its degradation under theseonditions. As high numbers of SRB are found in thexic zone of microbial mats, several authors suggesthat these bacteria can deal with temporal exposureso elevated oxygen concentrations of even up to 1.5 mM3, 5). However, it seems that the O2 concentration heresed, near to the one used by Dilling et al. (2) (1.17M), is too high for pure cultures of SRB to tolerate for
arge periods of time. Indeed, in the highly aeratednvironments, besides forming aggregates as a mech-nism to protect themselves against the toxic effect ofigh O2 concentration levels, SRB are shown to occur
n association with facultative aerobic bacteria, whichrobably maintain O2 concentration at levels that SRBan tolerate (5, 34). Nevertheless, the observation thato decrease in rd-roo mRNA is observed during therst 30 min of O2 exposure indicates that rd-roo mRNA
s to some extent resistant to high O2 concentrations.
lignment between ROO and ROO homologues was made with the GCGork); TM0755, T. maritima conserved hypothetical protein (AccessionH1085, P. horikoshii probable flavoprotein (Accession No. B71103);homolog (III) (Accession No. E69076), flavoprotein AI (Accession No.
-1 and AffprA-2, A. fulgidus flavoprotein homologues FprA-1 (Accession0732, M. jannaschii hypothetical flavoproteins MJ0748 (Accession No.D64391); MtfprA, M. thermoacetica flavoprotein A (Accession No.F14 and RcORFU1, R. capsulatus flavoprotein homolog (Accession No.
, Ss1105550, Ss110219, and Ss111521, Synechocystis sp. flavoproteinsmino acid residues corresponding to the flavodoxin signature charac-are marked by asterisks.
le ais w); Pin ArAMJ
No.cOR17e ands
Expression of rd-roo genes was also analyzed in D.giwas(i
tRttt
oscljlccfonm(3sDieebwwttaapivflgd
Rnrwikvp
tasmfras
afiaitasgtl9b(Rjet
ajept1oi
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igas cells treated with 100 mM H2O2 hydrogen perox-de and no significant change of rd-roo mRNA levelsas detected in these cultures (data not shown). Undernaerobic conditions, the levels of rd-roo expressionlightly decrease upon entry in stationary phaseOD450nm ' 0.8), a common feature among microorgan-sms.
All these data indicate that rd-roo genes are consti-utively and abundantly expressed in the presence ofOS, in agreement with the functions performed by
he proteins encoded by these genes (18, 19) and withhe observation that SRB can occur in constant orransiently oxygenated environments.
Rubredoxin genomic organization. A search for rdrthologues in database revealed that Rd proteins ineveral of the available prokaryotic genomes are en-oded by genes adjacent to other encoding ROO homo-ogues as in D. gigas (Fig. 7). This is the case of M.annaschii locus U67520, and M. thermoautotrophicumocus AE000804. However, besides being in the sameluster with flavoprotein genes, rd is also found inlusters with genes encoding proteins for which theunction has been related to oxidative stress in anaer-bic bacteria (Fig. 7), namely desulfoferrodoxins (Dfx),eelaredoxins (Nlr), superoxide reductases and dis-utases (SOR and SOD, respectively), rubrerythrins
Rbr/Rr) and alkyl hydroperoxide reductases (Ahp) (8,5–37). The same neighborhood is also found in theulfate reducers, D. vulgaris Hildenborough (8, 38) and. baarsii (35), where rd is located in operons contain-
ng genes encoding for desulfoferrodoxin and rubr-rythrin proteins. The co-occurrence of rd with genesncoding these proteins (Fig. 7) suggests that Rd maye involved in the transfer of electrons to them. Thisas shown to be the case of D. gigas rd and roo genes,here Rd transfers electrons to ROO during the reduc-
ion of oxygen to water (18). It should be emphasizedhat until now the electron donors for Dfx, Nlr, SORnd Rr/Rbr proteins have not yet been determined,lthough rubredoxin of P. furiosus was proposed as ahysiological donor in a SOR assay (36). Interestingly,n E. coli both rd and ROO are contained in the fla-oprotein encoded by ORF-o479 (39, 40), where theavodomain of this protein, which is highly homolo-ous to D. gigas ROO, is fused to a rubredoxin-likeomain.
D. gigas ROO orthologues. Comparison studies ofOO with homologous proteins reveal that it has sig-ificant homology with flavoproteins both from bacte-ia and archaea domains. However, no homologiesere found with eukaryotic proteins, a fact that may
ndicate that these proteins are present only in pro-aryotes. The overall sequence similarity among fla-oproteins varies substantially between individualairs of sequences, from 100% to lower than 20% iden-
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ity (23 to 100% similarity). Despite this diversity, thelignment of these flavoproteins reveals several con-erved regions, with the N- and C-terminal regionsore highly variable than the internal ones (Fig. 8). In
act, the 3D structure of ROO at 2.5 Å resolution (19)eveals that its structural and functional importantmino acid residues are located in these internal con-erved regions.The alignment of all flavoprotein sequences was an-
lyzed by neighbor-joining method with statistical con-dence measured by bootstrap analysis. All sets ofnalysis define three major groups in this protein fam-ly (Fig. 9), suggesting that there were at one timehree ancestral genes. However, the branching ordermong these three ancestral genes is not clearly re-olved by these analyses. Therefore, these analysesive strong statistical confidence that two of the ances-ral genes, corresponding to groups II and III, areikely to share a more recent common origin (Fig. 9,5% bootstrap). Within these groups, the protein mem-ers of group III are more likely to be closely relatedFig. 9, 96% bootstrap). Furthermore, from this treeOO is likely to be more closely related with the M.
annaschii flavoproteins MJ0534 and MJ0732. How-ver, due to the low confidence level (36% bootstrap)hey seem to share a very ancient common origin.
FIG. 9. Phylogenetic tree reflecting the relationships among thenalyzed flavoproteins. The tree shown was derived by neighbor-oining distance analysis between roo and its orthologues from sev-ral prokaryotes. Bootstrap values for each branch are given as aercentage of 500 resamplings. The three major groups of flavopro-eins identified in this tree are indicated. The scale bar represents0% difference in amino acid sequence determined by taking the sumf the length of the horizontal line segments connecting two organ-sms. For abbreviations, see legend to Fig. 8.
Examination of this tree reveals that group I is them
fAttRadORapcclg(tcFsr(Fiiotvflvttlttaipb
A
CcC
R
3. Canfield, D. E., and Des Marais, D. J. (1991) Aerobic sulfate
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ost heterogeneous one (36–100% bootstrap).In a first attempt to characterize the flavoprotein
amily based on cofactor and sequencing analysis of six-type flavoproteins, Wasserfallen et al. (39) suggested
he existence of four subfamilies: (1) simple flavopro-eins binding only FMN (found in methanogens and in. capsulatus); (2) diflavin flavoproteins binding FMNnd FAD (present in Synechocystis sp); (3) flavorubre-oxin binding FMN and mononuclear iron (E. coliRF-o479 product); and (4) hemoflavoprotein (D. gigasOO). Except for D. gigas ROO no functions were yetttributed to the other flavoproteins, although it wasroposed that by analogy to ROO these flavoproteinsould be involved in keeping low intracellular oxygenoncentration (39). This could be the case of R. capsu-atus flavoprotein gene, which is cotranscribed withenes encoding proteins involved in nitrogen fixation31, 41). As already mentioned, in the methanogens M.hermoautotrophicum and M. jannaschii the genes en-oding the flavoproteins FprA3 (locus AE000804) andprA (locus U67520), respectively, are located in theame transcriptional unit with genes encodingubredoxin-like proteins, similarly to D. gigas ROOFig. 7). The same gene organization occurs with thepaA gene of M. thermoautotrophicum (42). However,
n most prokaryotes flavoprotein genes are containedn operons with genes encoding hypothetical proteinsr other proteins different from rubredoxins. This ishe case of A. fulgidus locus AE000997 where the fla-oprotein gene is clustered with the genes of iron sulfuravoproteins and DNA/pantothenate metabolism fla-oprotein. Thus, based on their different cofactor con-ents and gene environments, the different members ofhe flavoprotein family may be involved in other cellu-ar mechanisms besides keeping low oxygen concentra-ion within the cell. Moreover, the observation thathese proteins exist in strict anaerobic prokaryotes butlso in facultative ones, suggests that not only they arenvolved in O2 reactions but they might also play otherhysiological roles possessing therefore a very generaliological relevance.
CKNOWLEDGMENTS
This work was supported by PRAXIS XXI 32/96 and 11074/98 toRP and PRAXIS XXI/BD/9016 to G.S., from Fundacao para a Cien-ia e Tecnologia. We thank Isabel Marques (Instituto Gulbenkian deiencia) for helping with the Phylip program.
EFERENCES
1. Abdollahy, H., and Wimpenny, J. W. T. (1990) Effects of oxygenon the growth of Desulfovibrio desulfuricans. J. Gen. Microbiol.136, 1025–1030.
2. Dilling, W., and Cypionka, H. (1990) Aerobic respiration insulfate-reducing bacteria. FEMS Microbiol. Lett. 71, 123–128.
501
reduction in microbial mats. Science 251, 1471–1473.4. van Niel, E. W. J., and Gottschal, J. (1998) Oxygen consumption
by Desulfovibrio strains with and without polyglucose. Appl.Environ Microbiol. 64, 1034–1039.
5. Sigalevich, P., Baev, M. V., Teske, A., and Cohen, Y. (2000)Sulphate reduction and possible aerobic metabolism of thesulfate-reducing bacterium Desulfovibrio oxyclinae in a chemo-stat coculture with Marinobacter sp. strain MB under exposureto increasing oxygen concentrations. Appl. Environ. Microbiol.66, 5013–5018.
6. Cypionka, H. (2000) Oxygen respiration by Desulfovibrio species.Arch. Microbiol. 54, 827–848.
7. Hatchikian, E. C., LeGall, J., and Bell, G. R. (1977) Significanceof superoxide dismutase and catalase activities in the strictanaerobes, sulfate-reducing bacteria. In Superoxide and Super-oxide Dismutase A. M. (Michelson, A. M., McCord, J. M., andFridovich, I., Ed.),. pp. 159–172. Academic Press, London, UK.
8. Lumppio, H. L., Shenvi, N. V., Garg, R. P., Summers, A. O., andKurtz, D. M., Jr. (1997) A rubrerythrin operon and nigerythringene in Desulfovibrio vulgaris (Hildenborough). J. Bacteriol.179, 4607–4615.
9. Voordouw, J. K., and Voordouw, G. (1998) Deletion of the rbogene increases the oxygen sensitivity of the sulfate-reducingbacterium Desulfovibrio vulgaris Hildenborough. Appl. Environ.Microbiol. 64, 2882–2887.
0. Dos Santos, W. G., Pacheco, I., Liu, M.-Y., Teixeira, M., Xavier,A. V., and LeGall, J. (2000) Purification and characterization ofan iron superoxide dismutase and a catalase from the sulfate-reducing bacterium Desulfovibrio gigas. J. Bacteriol. 182, 796–804.
1. Romao, C. V., Liu, M.-Y., LeGall, J., Gomes, C. M., Braga, V.,Pacheco, I., Xavier, A. V., and Teixeira, M. (1999) The super-oxide dismutase activity of desulfoferrodoxin from Desulfovibriodesulfuricans (ATCC 27774). Eur. J. Biochem. 261, 438–443.
2. Silva, G., Oliveira, S., Gomes, C. M., Pacheco, I., Liu, M.-Y.,Xavier, A. V., Teixeira, M., LeGall, J., and Rodrigues-Pousada,C. (1999) Desulfovibrio gigas Neelaredoxin – A novel superoxidedismutase integrated in a putative oxygen sensory operon of ananaerobe. Eur. J. Biochem. 259, 235–243.
3. Abreu, I. A., Saraiva, L. M., Carita, J., Huber, H., Stetter, K. O.,Cabelli, D., and Teixeira, M. (2000) Oxygen detoxification in thestrict anaerobic archaeon Archaeoglobus fulgidus: Superoxidescavenging by Neelaredoxin. Mol. Microbiol. 38, 322–334.
4. Fu, R., Wall, J. D., and Voordouw, G. (1994) DcrA, a c-typeheme-containing methyl-accepting protein from Desulfovibriovulgaris Hildenborough, senses the oxygen concentration or re-dox potential of the environment. J. Bacteriol. 176, 344–350.
5. Chen, L., Liu, M.-Y., LeGall, J., Fareleira, P., Santos, H., andXavier, A. V. (1993) Rubredoxin oxidase, a new flavo-hemo-protein, is the site of oxygen reduction to water by the “strictanaerobe” Desulfovibrio gigas. Biochem Biophys. Res. Commun.193, 100–105.
6. Santos, H., Fareleira, P., Xavier, A. V., Chen, L., Liu, M.-Y., andLeGall, J. (1993) Aerobic metabolism of carbon reserves by the“obligate anaerobe” Desulfovibrio gigas. Biochem. Biophys. Res.Commun. 195, 551–557.
7. Chen, L., Liu, M.-Y., LeGall, J., Fareleira, P., Santos, H., andXavier, A. V. (1993) Purification and characterization of anNADH-rubredoxin oxidoreductase involved in the utilization ofoxygen by Desulfovibrio gigas. Eur. J. Biochem. 216, 443–448.
8. Gomes, C. M., Silva, G., Oliveira, S., LeGall, J., Liu, M.-Y.,Xavier, A. V., Rodrigues-Pousada, C., and Teixeira, M. (1997)Studies on the redox centers of the terminal oxidase from Desul-
fovibrio gigas and evidence for its interaction with rubredoxin.
1
2
2
2
2
2
2
2
2
2
2
3
3
in electron transport to nitrogenase. Mol. Gen. Genet. 241, 602–
3
3
3
3
3
3
3
3
4
4
4
Vol. 280, No. 2, 2001 BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
J. Biol. Chem. 272, 22502–22508.9. Frazao, C., Silva, G., Gomes, C. M., Matias, P., Coelho, R.,
Sieker, L., Macedo, S., Liu, M.-Y., Oliveira, S., Teixeira, M.,Xavier, A. V., Rodrigues-Pousada, C., Carrondo, M. A., LeGall, J.(2000) Structure of a dioxygen reduction enzyme from Desulfo-vibrio gigas. Nat. Struct. Biol. 7, 1041–10445.
0. Moura, I., Moura, J. J. G., Santos, M. H., Xavier, A. V., and LeGall, J. (1979) Redox studies on rubredoxins from sulphate andsulphur reducing bacteria. FEBS Lett. 107, 419–421.
1. Chen, L., Sharma, P., LeGall, J., Mariano, A. M., Teixeira, M.,and Xavier, A. V. (1994) A blue non-heme iron protein fromDesulfovibrio gigas. Eur. J. Biochem. 226, 613–618.
2. Ausubel, F. M., Brent, R., Kingston, R. E., Moore, D. D., Seid-man, J. G., Smith, J. A., and Struhl, K. (1995) Current Protocolsin Molecular Biology, Greene Publishing Associates and Wiley-Interscience, New York.
3. Thompson, J. D., Higgins. D. G., and Gibson, T. J. (1994)CLUSTAL W: Improving the sensitivity of progressive multiplesequence alignment trough sequence weighting, position-specificgap penalties and weight matrix choice. Nucleic Acids Res. 22,4673–4680.
4. Page, R. D. M. (1996) Treeview: An application to display phy-logenetic trees on personal computers. Comput. Appl. Biosci. 12,357–358.
5. Jornvall, H., Persson, B., Krook, M., Atrian, S., Gonzalez-Duarte, R., Jeffery, J., and Ghosh, D. (1995) Short-chaindehydrogenases/reductases (SDR). Biochemistry 34, 6003–6013.
6. Jornvall, H., Hoog, J.-O., and Persson, B. (1999) SDR and MDR:Completed genome sequences show these protein families to belarge, of old origin, and of complex nature. FEBS Lett. 445,261–264.
7. Scopes, R. K. (1983) An-iron activated alcohol dehydrogenase.FEBS Lett. 156, 303–306.
8. Ma, K., and Adams, M. W. W. (1999) An unusual oxygen-sensitive, iron- and zinc-containing alcohol dehydrogenase fromthe hyperthermophilic archaeon Pyrococcus furiosus. J. Bacte-riol. 181,1163–1170.
9. Belitsky, B. R., Gustafsson, M. C. U., Sonenshein, A. L., and vonWachenfeldt, C. (1997) An lrp-like gene of Bacillus subtilis in-volved in branched-chain amino acid transport. J. Bacteriol. 179,5448–5457.
0. Merrick, M. J. (1993) In a class of its own—The RNA polymerasesigma factor s54 (sN). Mol. Microbiol. 10, 903–909.
1. Schmehl, M., Jahn, A., zu Vilsendorf, A. M., Hennecke, S., Mase-pohl, B., Schuppler, M., Marxer, M., Oelze, J., and Klipp, W.(1993) Identification of a new class of nitrogen fixation genes inRhodobacter capsulatus: A putative membrane complex involved
502
615.2. Gunsalus, R. P. (1992) Control of electron flow in Escherichia
coli: Coordinated transcription of respiratory pathway genes. J.Bacteriol. 174, 7069–7074.
3. Govantes, F., Albrecht, J. A., and Gunsalus, R. P. (2000) Oxygenregulation of the Escherichia coli cytochrome d oxidase (cydAB)operon: roles of multiple promoters and the Fnr-1 and Fnr-2binding sites. Mol. Microbiol. 37, 1456–1469.
4. Krekeler, D., Teske, A., and Cypionka, H. (1997) Strategies ofsulfate-reducing bacteria to escape oxygen stress in a cyanobac-terial mat. FEMS Microbiol. Ecol. 25, 89–96.
5. Pianzzola, M. J., Soubes, M., and Touati, D. (1996) Overproduc-tion of the rbo gene product from Desulfovibrio species sup-presses all deleterious effects of lack of superoxide dismutase inEscherichia coli. J. Bacteriol. 178, 6736–6742.
6. Jenney, F. E. Jr., Verhagen, M. F. J. M., Cui, X., and Adams,M. W. W. (1999) Anaerobic microbes: oxygen detoxification with-out superoxide dismutase. Science 286, 306–309.
7. Rocha, E. R., and Smith, C. J. (1999) Role of the alkyl hydroper-oxide reductase (ahpCF) gene in oxidative stress defense of theobligate anaerobe Bacteroides fragilis. J. Bacteriol. 181, 5701–5710.
8. Brumlik, M. J., and Voordouw, G. (1989) Analysis of the tran-scriptional unit encoding the genes for rubredoxin (rub) and aputative rubredoxin oxidoreductase (rbo) in Desulfovibrio vul-garis Hildenborough. J. Bacteriol. 17, 4996–5004.
9. Wasserfallen, A., Ragettli, S., Jouanneau, Y., and Leisinger, T.(1998) A family of flavoproteins in the domains Archaea andBacteria. Eur. J. Biochem. 254, 325–332.
0. Gomes, C. M., Vicente, J. B., Wasserfallen, A., and Teixeira, M.(2000) Spectroscopic studies and characterisation of a novel elec-tron transfer chain from Escherichia coli involving a flavorubre-doxin and its flavoprotein reductase partner. Biochemistry, inpress.
1. Saeki, K., Tokuda, K., Fujiwara, T., and Matsubara, H. (1993)Nucleotide sequence and genetic analysis of the region essentialfor functional expression of the gene for ferrodoxin I, fdxN, inRhodobacter capsulatus: Sharing of one upstream activator se-quence in opposite directions by two operons related to nitrogenfixation. Plant Cell Physiol. 34, 185–199.
2. Nolling, J., Ishii, M., Koch, J., Pihl, T. D., Reeve, J. N., Thauer,R. K., and Hedderich, R. (1995) Characterization of a 45-kDaflavoprotein and evidence for a rubredoxin, two proteins thatcould participate in electron transport from H2 to CO2 in metha-nogenesis in Methanobacterium thermoautotrophicum. Eur.J. Biochem. 231, 628–638.
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