APPLIED AND ENVIRONMENTAL MICROBIOLOGY,0099-2240/98/$04.0010

Aug. 1998, p. 2882–2887 Vol. 64, No. 8

Copyright © 1998, American Society for Microbiology. All Rights Reserved.

Deletion of the rbo Gene Increases the Oxygen Sensitivity ofthe Sulfate-Reducing Bacterium Desulfovibrio

vulgaris HildenboroughJOHANNA K. VOORDOUW AND GERRIT VOORDOUW*

Department of Biological Sciences, University of Calgary, Calgary, Alberta T2N 1N4, Canada

Received 10 March 1998/Accepted 24 May 1998

The rbo gene of Desulfovibrio vulgaris Hildenborough encodes rubredoxin oxidoreductase (Rbo), a 14-kDairon sulfur protein; forms an operon with the gene for rubredoxin; and is preceded by the gene for theoxygen-sensing protein DcrA. We have deleted the rbo gene from D. vulgaris with the sacB mutagenesisprocedure developed previously (R. Fu and G. Voordouw, Microbiology 143:1815–1826, 1997). The absence ofthe rbo-gene in the resulting mutant, D. vulgaris L2, was confirmed by PCR and protein blotting withRbo-specific polyclonal antibodies. D. vulgaris L2 grows like the wild type under anaerobic conditions. Expo-sure to air for 24 h caused a 100-fold drop in CFU of L2 relative to the wild type. The lag times of liquid culturesof inocula exposed to air were on average also greater for L2 than for the wild type. These results demonstratethat Rbo, which is not homologous with superoxide dismutase or catalase, acts as an oxygen defense proteinin the anaerobic, sulfate-reducing bacterium D. vulgaris Hildenborough and likely also in other sulfate-reducing bacteria and anaerobic archaea in which it has been found.

Sulfate-reducing bacteria (SRB) can have a considerableimpact on their environment, because their growth is coupledto the production of large amounts of hydrogen sulfide. Thisactivity is important in the removal of acidic, oxidized forms ofsulfur (e.g., SO2) from the environment and in the immobili-zation of toxic metal ions, e.g., as present in acid mine drainageeffluents. Despite these essential, environment-restoring prop-erties of SRB they are considered a nuisance in many environ-ments due to the odor, toxicity, and metal-corroding propertiesof their respiratory end product. Oxygen is one of the best andcheapest agents for controlling the growth and activity of SRBin environments in which they are not wanted (21). The sur-vival of SRB in aerobic environments has, therefore, alreadybeen studied. Hardy and Hamilton credited endogenous su-peroxide dismutase (SOD) and catalase activity for the survivalof Desulfovibrio spp. in oxygenated waters from the North Sea(8). The presence of an Fe-containing SOD in Desulfovibriodesulfuricans had been previously demonstrated by Hatchikianand Henry (9). These enzymes may also repair damage arisingfrom microaerophilic growth (9a). Pianzzola et al. attemptedto clone the sod gene from the SRB Desulfoarculus baarsii byfunctional complementation of a SOD-deficient mutant ofEscherichia coli (19). Sequence analysis of the resulting clonesindicated that these contained the rbo and rub genes of D.boarsii, and further complementation studies indicated thatonly rbo was required for complementing the sod phenotype.The rbo gene is widespread in SRB and anaerobic archaea, andthe amino acid sequence of the Rbo protein has remainedremarkably conserved (Fig. 1). In order to elucidate whetherits function is indeed in the prevention or repair of oxygendamage, as suggested by the heterologous complementationstudies with E. coli, or whether it also plays a role underanaerobic conditions, we have constructed an rbo deletion mu-

tant of Desulfovibrio vulgaris Hildenborough, of which theproperties are reported here.

MATERIALS AND METHODS

Materials. Restriction and DNA modification enzymes and bacteriophage lDNA were obtained from Pharmacia. [a-32P]dCTP (10 mCi/ml; 3,000 Ci/mmol)was from ICN. Mixed gas (85% [vol/vol] N2, 10% [vol/vol] CO2, and 5% [vol/vol]H2) was from Praxair Products Inc. Reagents for the construction and purifica-tion of a MalE-Rbo fusion protein (expression vector pMALc2, factor Xa pro-tease, and amylose resin), as well as anti-mouse immunoglobulin G alkalinephosphatase-linked antibody, were from New England Biolabs. Other immuno-blotting reagents (nitroblue tetrazolium and 5-bromo-4-chloro-3-indolylphos-phate) were from Promega, whereas Hybond-N membrane filters were obtainedfrom Amersham. Reagent grade chemicals were from either BDH, Fisher, orSigma. Deoxyoligonucleotide primers were obtained from University Core DNAServices of the University of Calgary.

Bacterial strains, plasmids, and growth conditions. Strains, plasmids, andprimers used or constructed in this study are listed in Table 1. E. coli and D.vulgaris strains were grown as described elsewhere (5, 21, 22, 30).

Construction of an rbo deletion mutant. The strategy used for gene replace-ment mutagenesis was similar to that described previously (5, 10). PlasmidpNotDRboCmMOB was transferred from E. coli S17-1 to D. vulgaris by conju-gation by a filter mating method (5, 22). Aliquots (50 to 100 ml) of the resus-pended mating mixture were plated onto medium E with kanamycin and chlor-amphenicol (CAM). The plates were incubated at 35°C for 5 to 7 days to selectCmr Kmr transconjugal integrants. D. vulgaris R1, in which pNotDRboCmMOBhad been integrated downstream from the rbo-rub operon (see Fig. 2), waspurified from contaminating E. coli by plating on the same medium. D. vulgarisR1 was next grown in medium C with CAM and sucrose. Growth was monitoredwith a Klett meter and was slow relative to that of D. vulgaris F100, a strain thatis both Sucr and Cmr, in the same medium. At midsaturation, 200-ml aliquots ofthis culture, diluted either 102- or 104-fold, were plated on medium E with CAM.Fifty isolated colonies were picked and grown in 1 ml of medium C with CAM.Aliquots (0.5 ml) of these cultures were used to inoculate 5 ml of medium C withCAM and 5 ml of medium C with CAM and sucrose. Observation of similar Klettreadings for the two cultures after growth to saturation was considered evidencethat the picked colony was Cmr and Sucr. The two cultures were then combined;0.5 ml was inoculated into 20 ml of medium B with kanamycin and CAM, grownto saturation, and stored at 4°C. The remainder was used for DNA isolationaccording to the method of Marmur (17).

Southern blot and PCR analyses for identification of the rbo deletion mutant.The DNAs from Cmr and Sucr cultures were digested with PstI, separated byagarose gel electrophoresis, and transferred to Hybond-N membrane filters. Theblots were then hybridized with a sacB probe, obtained as a 2.4-kb XbaI fragmentfrom plasmid pMOB2, and 32P labeled by extension of random hexamers asdescribed previously (31). After washing and drying, the radioactive images ofthe blots were displayed with a Fuji BAS 1000 Bioimaging Analyzer. DNAs thatdid not hybridize with sacB were further tested by PCR with primers P122-rbo-f

* Corresponding author. Mailing address: Department of BiologicalSciences, University of Calgary, 2500 University Dr. N.W., Calgary,Alberta T2N 1N4, Canada. Phone: (403) 220-6388. Fax: (403) 289-9311. E-mail: voordouw@acs.ucalgary.ca.

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and P123-rbo-r. PCR amplification was done with a Perkin-Elmer Gene Amp2400 PCR system using TaqI DNA polymerase and reagents, as indicated else-where (28). Reaction was for 30 cycles of 94°C (30 s), 60°C (30 s), and 72°C (90s). This allowed efficient amplification of PCR products in the 2-kb range. Thereaction time at 72°C was shortened for primer combinations which yielded onlysmaller PCR products (0.4 to 0.7 kb). D. vulgaris L2 was selected as the desiredreplacement mutant.

Protein blotting. Expression of rbo was monitored by protein blotting withpolyclonal antibodies generated in mice against Rbo, overexpressed in E. coli asa MalE-Rbo fusion protein, and purified by affinity chromatography on amyloseresin, according to procedures suggested by the manufacturer. The MalE andRbo portions of this fusion protein could be separated by proteolysis with factorXa protease. Cells of D. vulgaris wild type, F100, and L2 were grown in 5 ml ofmedium C. The cells were suspended in 300 to 350 ml of water (depending on themeasured cell density), an equal volume of sodium dodecyl sulfate (SDS)-con-taining incubation buffer was added, and the samples were placed immediately ina boiling water bath. The samples were then subjected to SDS-polyacrylamide gelelectrophoresis with 15% (wt/vol) polyacrylamide gels, according to the methodof Laemmli (13). Separated proteins were electroblotted onto nitrocellulose(29), and the blots were incubated sequentially with gelatin-containing blockingbuffer and the Rbo-recognizing primary antibody. Bound primary antibodieswere detected with an alkaline phosphatase-conjugated anti-mouse secondaryantibody and immunoblot staining reagents (20).

Survival in air. Cultures (5 ml) of D. vulgaris L2 and the wild type were grownanaerobically in medium C overnight. The cell densities were verified with a Klettmeter. For growth under anaerobic conditions, identical inocula (ca. 50 ml of 109

CFU/ml) were diluted into 5 ml of medium C in a 13- by 100-mm tube, afterwhich growth was monitored with the Klett meter. For exposure to air, identicalinocula (ca. 5 ml, depending on the measured cell density) were diluted into 500ml of medium C stirred continuously in air with a magnetic stirrer. Samples of 5ml of these aerobically incubated cells were pipetted periodically into 13- by100-mm tubes. These were transferred to anaerobic conditions, after whichgrowth was monitored with the Klett meter. D. vulgaris wild type and L2 do notgrow under aerobic conditions. Also, 100-ml aliquots, as well as 100-ml aliquotsof 102 and 104 dilutions, of several of these samples were plated immediatelyafter transfer to anaerobic conditions onto medium E plates. The number ofsurviving CFU per milliliter was determined from these plates by countingcolonies after 1 week of incubation at 35°C under anaerobic conditions.

RESULTS

Construction and application of the suicide vector.pNotRboI, consisting of a modified pUC vector of 2.6 kb anda 1.1-kb SalI fragment containing the rbo-rub operon, was usedas the starting point for directed mutagenesis. The rbo-rub

operon is located downstream from the 39 end of the dcrA gene(4) on this 1.1-kb fragment (Fig. 2, WT). PCR of pNotRboI(3.7 kb) with primers P121-Drbo-f and P120-Drbo-r gave a3.6-kb product. In plasmid pNotDRbo, obtained following li-gation and transformation of this PCR product, 90 bp from therbo coding region (Fig. 2; WT D) was replaced by a BamHIlinker. Insertion of the 1.4-kb cat gene and 4.8-kb oriT sacBcassettes gave pNotDRboCmMOB, a plasmid of 9.8 kb. Theidentity of this plasmid was confirmed as follows. (i) PCRamplification with P122-rbo-f and P123-rbo-r gave a 1.7-kbproduct, 1.3 kb larger than the 0.4-kb product obtained withpJK29 or wild-type chromosomal DNA. (ii) Digestion withBamHI released a 1.4-kb cat-gene-containing insert (Fig. 2, R1and L2 I). (iii) Digestion with NotI gave two similar-sizedfragments of 5.0 and 4.8 kb. (iv) The sacB gene could bereleased by PstI digestion as a 2.6-kb fragment (6). In the mapof D. vulgaris R1 (Fig. 2), pNotDRboCmMOB extends fromnucleotides (nt) 2800 to 12600.

We planned to use PCR to monitor the formation of newDNA junctions by homologous recombination of plasmidpNotDRboCmMOB with the D. vulgaris chromosome. PrimersP128-cat and P129-cat, hybridizing with the cat gene insert, andP127-f and P130-r, designed to hybridize immediately outsidethe 1.1-kb SalI fragment (Fig. 2), were synthesized for thispurpose. Synthesis of P130-r required additional sequencing,because the available sequence information did not extendbeyond the rightmost SalI site. Sequencing of plasmid pJK34,which contained a 2.6-kb EcoRI insert extending rightwardfrom nt 3067 in the map of the wild type in Fig. 2, gave therequired information. Chromosomal DNA from a putativeplasmid integrant, D. vulgaris R1, was subjected to PCR withvarious primer pairs. Only the use of P128-cat and P130-r gavethe expected 590-bp PCR product (not shown), indicating thatintegration had occurred through homologous recombinationof the downstream regions (Fig. 2, R1). The 590-bp productwas not formed when wild-type chromosomal DNA was usedfor PCR.

Verification of the D. vulgaris L2 genotype. The desired L2genotype can, in theory, be distinguished from wild-type, R1,and R1SR strains by the formation of a characteristic 650-bpPCR product with primers P127-f and P129-cat (Fig. 2). Cul-tures of D. vulgaris R1 in medium C with CAM and sucrosewere, therefore, plated on medium E with CAM, and 30 col-onies were toothpicked directly into PCR mix containing thisprimer pair. However, following amplification, formation ofthe 650-bp PCR product was not clearly shown for any ofthese. DNAs isolated from 37 Cmr and Sucr colonies weretherefore digested with PstI and analyzed by Southern blotting,with the radiolabeled sacB gene as the probe. The results for11 colonies are shown in Fig. 3. The amounts of digested DNAloaded were identical for all 11 samples, as indicated byethidium bromide staining prior to blotting (not shown). Onlysamples L2 and L6 lacked sacB hybridization, indicating thatthese were candidates for the desired homologous recombina-tion through the upstream regions. Samples L4, L5, L9, andL11 showed hybridization of a 2.6-kb PstI fragment with thesacB probe, similar to that observed for D. vulgaris R1 (notshown). Samples L3, L7, L8, and L10 had a 3.8-kb hybridizingPstI fragment, indicating ISD1 insertion (6), whereas L1showed both hybridizing bands. PCR analysis of chromosomalDNA from D. vulgaris L2 with primers P122-rbo-f and P123-rbo-r indicated exclusively a 1.7-kb product, whereas wild-typeDNA gave exclusively a 0.4-kb product when amplified underthe same conditions (Fig. 4). Chromosomal DNA from D.vulgaris R1 gave both the 0.4- and 1.7-kb products (Fig. 4).These results are in agreement with the maps shown in Fig. 2.

FIG. 1. Comparison of amino acid sequences of Rbo from D. vulgaris Hilden-borough (Rbodvh), D. vulgaris Miyazaki F (Rbomya), D. desulfuricans (Rbodde),Desulfoarculus baarsii (Rbodab), Archaeoglobus fulgidus (Rboarf), and Meth-anobacterium thermoautotrophicum (Rbomta), as reported in references 2, 3, 11,12, 19 and 27. Residues identical to or deleted from the D. vulgaris sequence areindicated by dots and tildes, respectively. Residues that are conserved in all 6sequences are indicated in the consensus sequence. DNA encoding amino acids49 to 78 (underlined) was deleted from the rbo gene in the construction ofpNotDRbo.

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PCR amplification of purified chromosomal DNA from D.vulgaris L2 with primers P127-f and P129-cat gave a weak650-bp band that was not seen when DNA from the wild typeor D. vulgaris R1 was used. This product was not obtainedwhen toothpicked colonies of D. vulgaris L2 were used as atemplate for PCR, explaining the failure of our earlier at-tempts at PCR screening of Cmr and Sucr colonies.

Protein blotting confirmed the absence of the rbo gene fromD. vulgaris L2. Identical amounts of cells were loaded for D.vulgaris F100, a dcrA deletion mutant that overexpresses rbo(5), for the wild type, and for D. vulgaris L2. Incubation of theblot containing the SDS-polyacrylamide gel electrophoresis-separated proteins with the Rbo-specific antibody indicatedreaction with purified 14-kDa Rbo (Fig. 5, lanes 7 to 10), aswell as with Rbo in D. vulgaris F100 and the wild type (Fig. 5,lanes 1 and 2 and lanes 3 and 4, respectively). D. vulgaris L2clearly lacked an immunoreactive 14-kDa protein (Fig. 5, lanes5 and 6).

Phenotype of D. vulgaris L2. The growth curves of D. vulgariswild type and L2 under anaerobic conditions were very similar(Fig. 6A), indicating a similar doubling time and final celldensity. Growth of liquid cultures that had been exposed to air

proceeded only after a lag time, defined as the time requiredfor the aerated culture to reach midsaturation minus 18 h,which was the time required for the anaerobic culture to reachmidsaturation (Fig. 6A). For 2 h of air exposure, lag times of 14and 53 h were observed for D. vulgaris wild type and L2,respectively (Fig. 6B). Lag times were compared for 25 pairs of5-ml cultures of D. vulgaris wild type and L2, which wereexposed to air for 1 to 36 h prior to transfer to the anaerobichood. These received inocula of ca. 107 cells/ml and grewidentically without exposure to air (as in Fig. 6A). The lagtimes for D. vulgaris L2 exceeded those for the wild type in 19of the 25 experiments. This included 13 cases in which D.vulgaris L2 failed to regrow. The lag times for D. vulgaris wildtype exceeded those for L2 in 2 of the 25 experiments, includ-ing one case in which the wild type failed to regrow. No con-clusions could be drawn for 4 of the 25 experiments becauseboth the wild type and D. vulgaris L2 failed to regrow. Thesedata indicated a significantly increased sensitivity of D. vulgarisL2 to inactivation by oxygen compared to the wild type.

The survival of the two strains following exposure to air wasalso compared in plating experiments. Plating after air expo-sure led to a wider range of colony sizes than observed for

TABLE 1. Bacterial strains, plasmids, and primers

Strain, plasmid, orprimer Relevant characteristics Reference

or source

D. vulgarisHildenborough NCIMB 8303; wild type; Kmr Cms Sucr 21F100 dcrA gene replaced with a cat gene cassette from pUC19Cm; Kmr Cmr Sucr 5R1 pNotDRboCmMOB integrated into the chromosome; Kmr Cmr Sucs This studyR1SR R1 derivative with mutated sacB gene: Kmr Cmr Sucr This studyL2 rbo gene replaced with a cat gene cassette from pUC19Cm; Kmr Cmr Sucr This study

E. coliTG2 D(lac-pro) supE thi hsdM hsdR recA F9 (traD36 proAB1 lacZDM15Iq); used for general

molecular biological work24

S17-1 thi pro hsdR recA with RP4-2[Tc::Mu,Km::Tn7] in the chromosome; mobilizer strain 26

PlasmidspJK29 rbo-rub operon on a 1.1-kb SalI fragment in pUC8; Apr 2pJK34 2.6-kb EcoRI fragment starting at nt 3067a; cloned in pUC8 This studypMOB2 Containing an oriT-sacBR cassette on a 4.8-kb NotI fragment; Kmr 25pNOT19 pUC19 with 10-bp NdeI-NotI adapter in NdeI site; Apr 5pSUP104 Broad-host-range vector; Cmr 23pUC8, pUC19 Cloning vector, pMB1 origin of replication; Apr 24pUC19Cm pUC19 containing a 1.4-kb SacII-TthIII fragment from pSUP104 with the cat gene in its BamHI

site; Apr Cmr5

pNotRboI 1.1-kb SalI fragment from pJK29 in SalI site of pNOT19; Apr This studypNotRboI9 pNotRboI with BamHI site deleted from the polylinker; Apr This studypNotDRbo pNotRboI9 with part of the rbo gene replaced by a BamHI-containing sequence; Apr This studypNotDRboCm cat gene of pUC19Cm inserted in the BamHI site of pNotDRbo; Apr Cmr This studypNotDRboCmMOB oriT and sacBR containing NotI fragment of pMOB2 cloned into NotI site of pNotDRboCm;

Apr Cmr SucsThis study

PrimersP120-Drbo-r p-cccggatccCTTTTCCTTGGCCCCGTCAGA (uppercase is not 2672–2652a; lowercase is a

BamHI linker)This study

P121-Drbo-f p-TGGATTGAGCTTGTCGCAGACGGT (nt 2763 to 2786a) This studyP122-rbo-f p-ATGCCCAACCAGTACGAAAT (nt 2529 to 2549a) This studyP123-rbo-r p-CATCGTGGATTCCTCGGGGTT (nt 2928 to 2908a) This studyP130-r p-GAAGTCGCGGCTGTTGTGGTCGAC (nt 3282 to 3259b) This studyP127-f p-GAGGGCATGGCCCAGAGGCTTGAGGCCCT (nt 2138 to 2166a) This studyP129-cat p-CAGGAAGATACTTAACAGGGAAGT (nt 582 to 605c) This studyP128-cat p-GAGTGGCAGGGCGGGGCGTAATTTT (nt 639 to 663c) This study

a Numbers are for the sequence of the dcrA rbo rub region (nt 1 to 3264) of accession no. M81168.b Sequence determined by extension of M81168.c Relative to start codon of the cat gene.

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plating of cells that were not exposed to air. New small coloniesemerged on plates containing air-exposed cells even after 4 to5 days, whereas anaerobically kept cells grew to a uniformcolony size in 2 to 3 days. This made evaluation of remainingCFU per milliliter more difficult than we had anticipated. Theresults of an experiment in which all colonies visible after 1week of incubation without magnification were counted areshown in Fig. 7. Following 24 h of air exposure, the number ofL2 survivors was 100-fold smaller than that of the wild type. Intwo other experiments, the numbers of colonies formed by D.vulgaris L2 after 24 h of exposure to air were 60- and 100-foldlower than those formed by the wild type.

DISCUSSION

The rbo gene of D. vulgaris Hildenborough was discoveredby Brumlik and Voordouw (2) through research aimed at elu-cidating the physiological function of rubredoxin, a 6-kDa re-dox protein with a single iron atom coordinated to four cystei-nyl residues (FeS4 center; E0, 250 to 0 mV). Rubredoxinresides in the cytoplasm of Desulfovibrio spp., where the resi-dent redox potential is generally much lower, causing it to bepresent in the reduced form. Analysis of the rub gene indicatedthat it forms an operon with a gene for a 14-kDa redox protein(2) which was named rubredoxin oxidoreductase because it waslikely to function in oxidation-reduction reactions with rubre-doxin as a redox partner. Since then, the rbo gene has beenfound to be closely associated with rub in other SRB, e.g., in D.vulgaris Miyazaki F (11) and in the more distantly relatedDesulfoarculus baarsii (19).

The sequence of Rbo indicated that it was a redox proteinbecause its N terminus was highly homologous to desulfore-doxin (1, 2), a redox protein of only 36 amino acids fromDesulfovibrio gigas, which, like rubredoxin, has a single FeS4center. Chemical and spectroscopic analysis of purified Rboindicated the presence of a second bound iron atom, in addi-tion to an FeS4 site similar to that present in desulforedoxin.The additional iron atom represented a high spin site thatremained in the ferrous state even under aerobic conditionsand appeared to be coordinated primarily with oxygen andnitrogen ligands (18). Indeed, Rbo has only a single conservedcysteine residue (C-117 [Fig. 1]) outside those in the desul-foredoxin domain (C-10, C-13, C-29, and C-30) [Fig. 1]).Moura et al. (18) indicated that these physical properties aresimilar to those of rubrerythrin (Rbr), which contains a rubre-doxin-like FeS4 site and one nonsulfur, oxobridged di-iron site.

FIG. 2. Maps of the dcrA and rbo-rub region in D. vulgaris wild type, R1, and L2. (WT) Numbering is as for accession no. M81168. The 1.1-kb SalI (S) fragmentis divided in the upstream (up), downstream (down), and deleted (D) regions. The hybridization positions and directions of polymerization of several primers are shown.(R1) Plasmid pNotDRboCmMOB is located at nt 2800 to 12600. The deleted region (D) of the rbo gene is replaced by a 1.4-kb BamHI (B) insert (I) containing thecat gene. P128 and P129 are cat-specific primers. The oriT and sacB genes (from pMOB2) and the bla gene (from pUC) are located on a 7.4-kb fragment. (L2)Replacement mutant lacking a functional rbo gene.

FIG. 3. Southern blot of PstI-digested chromosomal DNAs of Cmr and Sucr

derivatives of D. vulgaris R1. The blot was hybridized with a radiolabeled sacBprobe. M, bacteriophage l DNA digested with HindIII. The sizes of the bandsare indicated in kilobases.

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Rbr, of which the three-dimensional structure is known, formsan operon with genes for a Fur-like and a rubredoxin-likeprotein. Despite this extensive knowledge, Lumppio et al. re-cently described Rbr as a non-heme iron protein of unknownfunction (16).

The dcrA gene, present immediately upstream from the rbo-rub operon (4), was shown to encode a chemoreceptor proteinthat functions as a sensor of the oxygen concentration or redoxpotential of the environment (7). D. vulgaris F100, a dcrA

deletion mutant, appeared to be more resistant to oxygen in-activation than the wild type (5). The findings by Pianzzola etal. (19) that rbo complements sodAB deficiency in E. coli pro-vided a possible explanation for this puzzling phenotype.Northern blotting studies indicated that deletion of dcrA in-creased expression of the rbo-rub operon (5). At the proteinlevel, this effect can be seen in Fig. 5 (compare lanes 1 and 3 or2 and 4); D. vulgaris F100 appears to have a ca. twofold-increased content of Rbo over the wild type. Although thisimplicated Rbo in repair or prevention of oxygen damage in D.vulgaris, the question of whether this is its only function inDesulfovibrio spp. and other anaerobic bacteria remained. Ourpresent results indicate that deletion of the rbo gene does notaffect growth under anaerobic conditions (Fig. 6A) but makesD. vulgaris clearly more sensitive to oxygen inactivation (Fig.6B; Fig. 7). It appears, therefore, that the main physiologicalfunction of Rbo is that of an oxygen defense protein in Desul-fovibrio spp. and possible also in other anaerobic bacteria and

FIG. 4. PCR analysis of chromosomal DNAs from D. vulgaris wild type, R1,and L2. DNA was amplified with primers P122-rbo-f and P123-rbo-r. The PCRproducts were run on an 0.7% (wt/vol) agarose gel which was stained withethidium bromide. M, 100-bp marker ladder.

FIG. 5. Protein blotting of cells of D. vulgaris F100 (lanes 1 and 2), wild type(lanes 3 and 4), and L2 (lanes 5 and 6). Identical amounts of cells, correspondingto ca. 150 mg of protein (lanes 1, 3, and 5) or 75 mg of protein (lanes 2, 4, and6) were loaded together with ca. 50, 100, 200, and 400 ng of purified Rbo (lanes7 to 10).

FIG. 6. Effect of air on the growth of D. vulgaris wild type (h) and L2 ({) inliquid culture. Medium C (5 ml) was inoculated with 50 ml of an overnight culture(ca. 109 CFU/ml). Growth was then monitored under strictly anaerobic condi-tions (A) or following 2 h of exposure to air (B). The zero time point for thegrowth curves in panel B is the time at which the cultures were returned toanaerobic conditions.

FIG. 7. Survival of D. vulgaris wild type (h) and L2 ({) exposed to air.Anaerobic cultures of the wild type and L2 in medium C (5 ml; 1.2 3 109

CFU/ml) were diluted 100-fold in aerobic medium C at zero time. Samples werereturned to anaerobic conditions after 4 to 24 h (aeration time) and platedimmediately at various dilutions. Surviving cells were counted after 1 week ofanaerobic incubation of the plates.

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that the physiological function of rubredoxin is to assist in theelectron transport required for this defense function. Assum-ing that the redox potential of the D. vulgaris cytoplasm rises tohigher values under the aerobic conditions in which this de-fense system operates, the enigma of the high redox potentialof rubredoxin is finally explained.

The mechanism by which Rbo protects an E. coli sodABmutant from superoxide was recently studied in some detail(15). Rbo does not have significant SOD activity but functionsin E. coli by serving as a preferred target for superoxide orderived radicals and possibly also by contributing iron-sulfurcluster-repair activity. Interestingly, the rbr gene encoding Rbrof Clostridium perfringens was similarly found to be capable ofcomplementing sodAB deficiency in E. coli. Rbr was thereforealso proposed to function as a scavenger of oxygen radicals(14). The recent completion of several genomic sequencingprojects has indicated that Rbo and Rbr may be widespread inanaerobic bacteria. For instance, the sulfate-reducing ar-chaeon Archaeoglobus fulgidus has a single Rbo homolog andfour Rbr homologs (12). D. vulgaris Hildenborough has at leastone other Rbr homolog, nigerythrin, for which the gene wasrecently cloned (16). Whether all of these novel redox proteinsfunction primarily in oxygen defense, as does Rbo in D. vul-garis, or in anaerobic metabolism can be determined by furthergene deletion studies, as presented here.

ACKNOWLEDGMENTS

We thank Karie-Lynn Lutz for assistance in the preparation ofRbo-specific antibody, Ian Chisholm for preparation of factor Xa-cleaved MalE-Rbo fusion protein, and Anita Telang for sequencingplasmid pJK34.

This work was supported by a grant from the Natural Science andEngineering Research Council of Canada (NSERC) to G.V.

REFERENCES

1. Brumlik, M. J., G. Leroy, M. Bruschi, and G. Voordouw. 1990. The nucle-otide sequence of the Desulfovibrio gigas desulforedoxin gene indicates thatthe Desulfovibrio vulgaris rbo gene originated from a gene fusion event. J.Bacteriol. 172:7289–7292.

2. Brumlik, M. J., and G. Voordouw. 1989. Analysis of the transcriptional unitencoding the genes for rubredoxin (rub) and a putative rubredoxin oxi-doreductase (rbo) in Desulfovibrio vulgaris Hildenborough. J. Bacteriol. 171:4996–5004.

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