Genetic basis for nitrate resistance in Desulfovibrio strains

Abstract

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.

Regulation of Nitrite Stress Response in Desulfovibrio vulgaris Hildenborough, a Model Sulfate-Reducing Bacterium

“…Because SRB can occur in environments where they may be exposed to nitrite as a result of the activity of nitrate-reducing bacteria and also because nrfHA-carrying bacteria often have the ability to reduce nitrate, we tested the effect of sodium nitrate on nrfH gene expression. D. vulgaris cannot reduce nitrate, and growth is moderately inhibited by an unknown mechanism at 100 mM sodium nitrate (10,48). The WT, nrfR mutant, and JW9382 strains were exposed to 100 mM sodium nitrite at the mid-log phase for 1 h. We observed that the expression levels of the reference genes of choice, rplS and rpoD, were strongly decreased (ϳ5-to 10-fold) upon the addition of nitrate (Fig.…”

“…Some SRB, such as Desulfovibrio alaskensis G20, lack any means for reducing the nitrite and are highly sensitive to small amounts of nitrite (10,11). However, other SRB can reduce nitrite via a cytochrome c-type nitrite reductase, NrfA, and are able to tolerate low millimolar amounts of nitrite (7,12).…”

Rex (Encoded by DVU_0916) in Desulfovibrio vulgaris Hildenborough Is a Repressor of Sulfate Adenylyl Transferase and Is Regulated by NADH

“…In fact, we recently reported that the Rex DvH mutant was inhibited for growth with thiosulfate as the terminal electron (33), an observation that deserves examination. In addition, Rex DvH has been predicted to regulate more than 50 genes (21).…”

“…All plasmid constructs were made by the SLIC procedure (32). The protocol used to generate the strain lacking the sat promoter (⌬P sat ), JW9293, conferring kanamycin resistance and 5-FU sensitivity, has previously been described (33). Briefly, 150 bp upstream of sat (Ϫ150 to Ϫ1) was replaced with a cassette conferring resistance to kanamycin and a counterselectable marker (upp, the gene encoding uracil phosphoribosyltransferase that confers 5-FU sensitivity to the 5-FU r parental strain).…”

“…To examine the role of Rex DvH , a marker-exchange deletion of rex and a complemented deletion strain were constructed (33). These two strains, in addition to the parental strain, were grown by sulfate respiration in MOLS4 or by pyruvate fermentation in MOYPyr.…”