Two mutant strains of Desulfovibrio vulgaris Hildenborough lacking either the sod gene for periplasmic superoxide dismutase or the rbr gene for rubrerythrin, a cytoplasmic hydrogen peroxide (H 2 O 2 ) reductase, were constructed. Their resistance to oxidative stress was compared to that of the wild-type and of a sor mutant lacking the gene for the cytoplasmic superoxide reductase. The sor mutant was more sensitive to exposure to air or to internally or externally generated superoxide than was the sod mutant, which was in turn more sensitive than the wild-type strain. No obvious oxidative stress phenotype was found for the rbr mutant, indicating that H 2 O 2 resistance may also be conferred by two other rbr genes in the D. vulgaris genome.

Genes for superoxide reductase (Sor), rubredoxin (Rub), and rubredoxin:oxygen oxidoreductase (Roo) are located in close proximity in the chromosome of Desulfovibrio vulgaris Hildenborough. Protein blots confirmed the absence of Roo from roo mutant and sor-rub-roo (srr) mutant cells and its presence in sor mutant and wild-type cells grown under anaerobic conditions. Oxygen reduction rates of the roo and srr mutants were 20 to 40% lower than those of the wild type and the sor mutant, indicating that Roo functions as an O 2 reductase in vivo. Survival of single cells incubated for 5 days on agar plates under microaerophilic conditions (1% air) was 85% for the sor, 4% for the roo, and 0.7% for the srr mutant relative to that of the wild type (100%). The similar survival rates of sor mutant and wild-type cells suggest that O 2 reduction by Roo prevents the formation of reactive oxygen species (ROS) under these conditions; i.e., the ROS-reducing enzyme Sor is only needed for survival when Roo is missing. In contrast, the sor mutant was inactivated much more rapidly than the roo mutant when liquid cultures were incubated in 100% air, indicating that O 2 reduction by Roo and other terminal oxidases did not prevent ROS formation under these conditions. Competition of Sor and Roo for limited reduced Rub was suggested by the observation that the roo mutant survived better than the wild type under fully aerobic conditions. The roo mutant was more strongly inhibited than the wild type by the nitric oxide (NO)-generating compound S-nitrosoglutathione, indicating that Roo may also serve as an NO reductase in vivo.Desulfovibrio spp. are anaerobically living, sulfate-reducing bacteria (SRB) that generate energy via dissimilatory sulfate reduction in the absence of air. Nevertheless, because Desulfovibrio spp. may be periodically exposed to air in their natural environment, they have evolved oxygen survival strategies. Completion of the genome sequence (18) has indicated that D. vulgaris Hildenborough has genes for oxygen reductases, including those for membrane-bound cytochrome c oxidase (Cox, DVU1811-1815) and cytochrome bd oxidase (Cbd, DVU3270-3271) and cytoplasmic rubredoxin:oxygen oxidoreductase (Roo, DVU3185); genes for inactivation of reactive oxygen species (ROS), including those for superoxide dismutase (Sod, DVU2140), catalase (DVUA0091), superoxide reductase (Sor, DVU3183), and rubrerythrins Rbr1 (DVU3094), Rbr2 (DVU2310), and Ngr (DVU0019); and genes for oxygen chemotaxis (13, 33), including those for DcrA (DVU3182) and DcrH (DVU3155). The importance of D. vulgaris Sor has been demonstrated by comparing the survival of a sor mutant with that of the wild type upon incubation in air-saturated medium (11, 21, 31). The genome sequence has indicated that roo is immediately downstream from the sor-rub operon, encoding Sor (formerly referred to as rubredoxin oxidoreductase [Rbo]; 3) and rubredoxin (Rub, DVU3184). Roo has been proposed to be the terminal oxidase of a cytoplasmic, non-energy-conserving chain (8,12), whereas t...

A 47 kb genomic island (GEI) bracketed by 50 bp direct repeats, containing 52 annotated genes, was found to delete spontaneously from the genome of Desulfovibrio vulgaris Hildenborough. The island contains genes for site-specific recombinases and transposases, rubredoxin:oxygen oxidoreductase-1 (Roo1) and hybrid cluster protein-1 (Hcp1), which promote survival in air and nitrite stress. The numbering distinguishes these from the Roo2 and Hcp2 homologues for which the genes are located elsewhere in the genome. Cells with and without the island (GEI(+) and GEI(-) cells respectively) were obtained by colony purification. GEI(-) cells arise in anaerobic cultures of colony-purified GEI(+) cells, indicating that the site-specific recombinases encoded by the island actively delete this region. GEI(+) cells survive better in microaerophilic conditions due to the presence of Roo1, whereas the Hcps appear to prevent inhibition by sulfur and polysulfide, which are formed by chemical reaction of sulfide and nitrite. Hence, the island confers resistance to oxygen and nitrite stress. However, GEI(-) cells have a higher growth rate in anaerobic media. Microarrays and enzyme activity stains indicated that the GEI(-) cells have increased expression of genes, which promote anaerobic energy conservation, explaining the higher growth rate. Hence, while lowering the efficiency of anaerobic metabolism, the GEI increases the fitness of D. vulgaris under stress conditions, a feature reminiscent of pathogenicity islands which allow more effective colonization of environments provided by the targeted hosts.

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Inactivation of PerR by oxidative stress and a corresponding increase in expression of the perR regulon genes is part of the oxidative stress defense in a variety of anaerobic bacteria. Diluted anaerobic, nearly sulfide-free cultures of mutant and wild-type Desulfovibrio vulgaris (10(5)-10(6) colony-forming units/ml) were treated with 0 to 2,500 μM H(2)O(2) for only 5 min to prevent readjustment of gene expression. Survivors were then scored by plating. The wild type and perR mutant had 50% survival at 58 and 269 μM H(2)O(2), respectively, indicating the latter to be 4.6-fold more resistant to killing by H(2)O(2) under these conditions. Significantly increased resistance of the wild type (38-fold; 50% killing at 2188 μM H(2)O(2)) was observed if cells were pretreated with full air for 30 min, conditions that did not affect cell viability. The resistance of the perR mutant increased less (4.6-fold; 50% killing at 1230 μM H(2)O(2)), when similarly pretreated. Interestingly, no increased resistance of either was achieved by exposure with 10.6 μM H(2)O(2) for 30 min, the highest concentration that could be used without killing the cells. Hence, in environments with low D. vulgaris biomass only the presence of external O(2) effectively activates the perR regulon. As a result, mutant strains lacking one of the perR regulon genes ahpC, dvu0772, rbr1 or rbr2 displayed decreased resistance to H(2)O(2) stress only following pretreatment with air.