Nitrate is an inhibitor of sulfate-reducing bacteria (SRB). In petroleum production sites, amendments of nitrate and nitrite are used to prevent SRB production of sulfide that causes souring of oil wells. A better understanding of nitrate stress responses in the model SRB, Desulfovibrio vulgaris Hildenborough and Desulfovibrio alaskensis G20, will strengthen predictions of environmental outcomes of nitrate application. Nitrate inhibition of SRB has historically been considered to result from the generation of small amounts of nitrite, to which SRB are quite sensitive. Here we explored the possibility that nitrate might inhibit SRB by a mechanism other than through nitrite inhibition. We found that nitrate-stressed D. vulgaris cultures grown in lactate-sulfate conditions eventually grew in the presence of high concentrations of nitrate, and their resistance continued through several subcultures. Nitrate consumption was not detected over the course of the experiment, suggesting adaptation to nitrate. With high-throughput genetic approaches employing TnLE-seq for D. vulgaris and a pooled mutant library of D. alaskensis, we determined the fitness of many transposon mutants of both organisms in nitrate stress conditions. We found that several mutants, including homologs present in both strains, had a greatly increased ability to grow in the presence of nitrate but not nitrite. The mutated genes conferring nitrate resistance included the gene encoding the putative Rex transcriptional regulator (DVU0916/Dde_2702), as well as a cluster of genes (DVU0251-DVU0245/Dde_0597-Dde_0605) that is poorly annotated. Follow-up studies with individual D. vulgaris transposon and deletion mutants confirmed high-throughput results. We conclude that, in D. vulgaris and D. alaskensis, nitrate resistance in wild-type cultures is likely conferred by spontaneous mutations. Furthermore, the mechanisms that confer nitrate resistance may be different from those that confer nitrite resistance.

Sulfate-reducing microbes, such as Desulfovibrio vulgaris Hildenborough, cause “souring” of petroleum reservoirs through produced sulfide and precipitate heavy metals, either as sulfides or by alteration of the metal reduction state. Thus, inhibitors of these microbes, including nitrate and nitrite ions, are studied in order to limit their impact. Nitrite is a potent inhibitor of sulfate reducers, and it has been suggested that nitrate does not inhibit these microbes directly but by reduction to nitrite, which serves as the ultimate inhibitor. Here we provide evidence that nitrate inhibition of D. vulgaris can be independent of nitrite production. We also show that D. vulgaris can use nitrite as a nitrogen source or terminal electron acceptor for growth. Moreover, we report that use of nitrite as a terminal electron acceptor requires nitrite reductase (nrfA) as a D. vulgaris nrfA mutant cannot respire nitrite but remains capable of utilizing nitrite as a nitrogen source. These results illuminate previously uncharacterized metabolic abilities of D. vulgaris that may allow niche expansion in low-sulfate environments. Understanding these abilities may lead to better control of sulfate-reducing bacteria in industrial settings and more accurate prediction of their interactions in the environment.