Nitrate reduction pathway in Shewanella oneidensis MR-1

Shewanella oneidensis MR-1 is a gram-negative bacterium with an extraordinary metabolic versatility in anion reduction, including the reduction of NO3-, Fe(III), U(VI), Mn(IV), Se(VI), Cr(VI). While reduction of nitrate and nitrite has been described for this microorganism, it is not known whether the reduction is by denitrifcation or dissimilatory nitrate reduction into ammonium (DNRA). By both physiological and genetic evidence, I proved that DNRA is the nitrate reduction pathway in this organism. Using the complete genome sequence of S. oneidensis MR-1, I identified a gene encoding a periplasmic nitrate reductase based on its 72% sequence identity with the napA gene in E. coli. Anaerobic growth of MR-1 on nitrate was abolished in a site directed napA mutant I constructed, indicating that NapA is the only nitrate reductase present. The anaerobic expression of the napA and nrfA, a homolog of the cytochrome b552 nitrite reductase in E. coli, increased with increasing nitrate concentration until a plateau was reached at 3 mM KNO3. This indicates that these genes are not repressed by increasing concentrations of nitrate.; The reduction of nitrate generates intermediates that can be toxic to the microorganism. To determine the genetic response of MR-1 to high concentrations of nitrate, DNA microarrays were used to obtain a complete gene expression profile of MR-1 at low (1 mM) versus high (40 mM) nitrate concentrations. Genes encoding transporters and efflux pumps were up-regulated, perhaps as a mechanism to export toxic compounds. In addition, the gene expression profile of MR-1, grown anaerobically with nitrate as the only electron acceptor, suggested that this dissimilatory pathway contributes to N assimilation. Hence the nitrate reduction pathway could serve a dual purpose.; The role of EtrA, a homolog of Fnr (global anaerobic regulator in E. coli) was examined using an etrA deletion mutant I constructed, S. oneidensis Etra7-1. The global transcriptome suggested a starvation response for anaerobic cultures of EtrA7-1 when nitrate was the electron acceptor. Genes involved in the activation and synthesis of the LambdaSo, MuSol and MuSo2 prophages of MR-1 were up-regulated, suggesting a phage infection. This could be responsible for the low growth yields observed for EtrA7-1 when compared to the wild type. Starvation is a stress condition that is known to induce viral infections. Even though starvation was not directly targeted for examination, the results in this study suggest that EtrA might play an important role in the survival of MR-1 under starvation. Moreover, the low biomass suggests a greater sensitivity of MR-1 to starvation than the toxicity associated with high nitrate concentrations. Down-regulation of genes involved in the nitrate reduction pathway was also observed for EtrA7-1 relative to the wild type, which suggests a positive regulatory role for this protein in the nitrate reduction pathway of S. oneidensis MR-1.