Impacts Of Nitrate And Nitrite On Physiology Of Shewanella Oneidensis

It is well known that nitrite and nitrate are widespread in nature, and have a significant impact on biological systems. Shewanella oneidensis, a facultative anaerobe of the y-proteobacterium family, possesses remarkably diverse respiratory capacities for reducing various organic and inorganic substrates and becomes a model for investigating redox transformations. Recent studies of the microorganism have elucidated that nitrate and nitrite, as electron acceptors, are reduced to ammonia under anaerobic conditions whereas function as growth inhibitors at high concentrations. Given that the oxygen is ubiquitous in environments, in this study we focus on the effect of nitrate and nitrite on S. oneidensis under aerobic conditions. The results are as follows:1. NaNO3affects growth of S. oneidensis under aerobic conditions by two distinct mechanisms, which are dose-dependent. At high concentrations (≥100mM), NaNO3impeded growth through sodium as a salt/osmotic stress agent; at low concentrations (≤50mM), NaNO3induced aberrant growth because nitrate is reduced to nitrite and accumulated nitrite inhibits growth. Nitrate per se is not the cause for the aberrant growth of S. oneidensis. Nitrite is much more toxic than nitrate to cells.2. The lack of the global regulator Crp (Δcrp) sensitizes cells to nitrite. To screen mutants that restore the growth defect of the Acrp strain in the presence of nitrite, we performed the transposon mutagenesis in the Δcrp strain. Terminal cytochrome bd oxidase (Cyd) is found to have a critical role in the resistance to the nitrite and cytochrome cbb3, oxidase (Cco) appears to be direct inhibition target of nitrite. Subsequent analysis revealed that the inhibition is likely imposed via NO. Under aerobic conditions, S. oneidensis have a relatively strong resistant to NO because of NO-reduction capacity of NrfA. Neither Cco nor Cyd is sensitive to NO, suggesting a difference in inhibitory mechanisms resulting from nitrite and NO.3. Under aerobic conditions, nitrate reduction occurs immediately and may not have a role in dissipation of excess redox energy. In contrast, respiration of nitrite does not commence until cells enter the stationary phase, suggesting that nitrite reduction is dependent on the physiological status of cell.4. A promoter-embedded transposon was used to mutagenize the Acyd strain in order to screen for nitrite resistance suppressors. Over-expressed ScyA caused by the transposon insertion in Acyd restored the growth defect resulting from nitrite. We then confirmed the result by overexpressing the scyA gene using multiple-copy plasmid, indicating that ScyA is involved in nitrite resistance. As a component of the aerobic respiration pathway, ScyA transfers electrons from CymA and/or PetABC to Ceo. A large amount of ScyA in Δcyd enhances the electron flux to Cco, leading to the restoration of the growth defect of Acyd in the presence of nitrite.