The Regulation Mechanism Of Nitrate/nitrite Respiration In Shewanella Oneidensis

Shewanella oneidensis is a facultative y-bacterium which has received enormous attention for its potential applications in bioremediation of heavy metals and energy generation via fuel cells. It could reduce many kinds of organic and inorganic compounds with its versatile respiration abilities. Presently, many of its respiration pathways and their regulatory mechanisms have been elucidated. However, we don’t know much about the regulation of nitrate/nitrite respiration in S. oneidensis. In this study, we first searched for cultivation conditions that allow occurrence of nitrate/nitrite respiration and a biomass enough for examination of mutants defective in nitrate/nitrite respiration. Then with this cultivation approach, we tried to illustrate the roles of two-component regulatory system and global regulator in regulating nitrate/nitrite respiration in S. oneidensis through physiological characterization and mutational analyses. The results are summarized as below.Cultivation conditions suitable for characterizing S. oneidensis mutants defective in nitrate/nitrite respiration. We made attempts to controlled chemostat cultures and uncontrolled batch cultrues to determine the growth and nitrate/nitrite respiration of S. oneidensis in the presence of nitrate/nitrite under aerobic and anaerobic conditions. Under anaerobic condition, the cell densities of controlled chemostat cultures were quite low, especially in the case of nitrite with which the biomass was hardly visible when cells reached the steady state. Although aerobic chemostat cultures could grow well in the presence of nitrate/nitrite, cells only respired on nitrate not nitrite. The uncontrolled batch cultures, unexpectedly, opened a window for investigation of nitrate/nitrite respiration in S. oneidensis. The growth of cells in the presence of nitrate was good; unlike nitrate, nitrite reduced the growth rate (approximately25%by generation times), in agreement with its highly toxic feature, but the cells could reach enough biomass at last. With regard to nitrate/nitrite reduction, the batch cultures were able to carry out reduction of both nitrate and nitrite. However, nitrite reduction was unlike nitrate reduction in that it could not commence until cells entered the stationary phase, indicating that the physiological status of cells was crucial to the process. In addition, the culture condition conferred a sensitivity that enabled differentiation of mutants with impaired nitrate/nitrite respiration. These data, collectively, indicated that the uncontrolled batch cultures, which not only allowed reaching the biomass required for biochemical and genetic analyses but also conferred sensitivity high enough for differentiating the degree of defectiveness of mutants, were suitable for the investigation in this study.The regulation mechanism of nitrate/nitrite respiration by two-component regulatory system(TCS) NarQ-NarP in S. oneidensis. In model organism Escherichia coli, homologous TCS NarQEc-NarPEc and NarXEc-NarLEc, distinguished from each other by a cysteine cluster found in NarXEc only, cross-function asymmetrically in regulation of nitrate and nitrite respiration. A BLAST search using NarQEc, NarPEc, NarXEc and NarLEc sequences against the S. oneidensis genome returned three significant hits that have at least a90%coverage and over50%sequence similarity. These proteins were SO3981, SO3982and SO1860. According to genome annotation, SO3981was annotated to be NarQ and since SO3982was predicted to be in the same operon with SO3981, it was designated as NarP. The trans-phosphorylation assay found that i) NarQ was able to undergo auto-phosphorylation in the presence of ATP and autophosphorylated NarQ in turn was able to phosphorylate NarP but not SO1860;ⅱ) neither NarP nor SO1860could phosphorylate itself when ATP was present. These data suggested that S. oneidensis possesses a NarP-NarQ TCS with NarQ being the sensor kinase and NarP being the response regulator. Point mutational assay demonstrated that Asp57within NarP was its phosphorylation site and hence essential for its activity. Results of physiological characterization and mutational analyses showed that this TCS was involved in regulation of nitrate/nitrite respiration through directly controlling the transcription of nap and nrfA (genes encoding nitrate and nitrite reductases, respectively), but regulated nrfA more tightly that nap.Global regulator Crp activated expression of NarP-NarQ as well as NRF and NAP directly. Global regulator is a kind of transcriptional factor which can regulate the expressions of many genes and take part in respirational regulation in bacteria. Three global regulators ArcA, Fnr and Crp in S. oneidensis had been shown to be involved in the regulation of respiration. We constructed in-frame deletion mutants for genes encoding these three global regulators and found that AarcA and Afnr were indistinguishable from the wild type in their ability to reduce nitrate/nitrite, by contrast, Acrp displayed complete loss of nitrate reduction and only retained a small share of nitrite-reducing capacity. The qRT-PCR results showed that expression pattern of nap and nrfA was not significantly altered by the removal of Fnr compared with the wild type. By contrast, the loss of Crp resulted in a complete shutdown of transcription of nap and reduced expression of nrfA to roughly5%. These data illustrated that Crp played a predominant role in regulation of nitrate/nitrite reduction in S. oneidensis. In contrast to NarP-NarQ, Crp was more essential for nap rather than nrfA. Besides, the defect in nitrate/nitrite reduction introduced by the removal of Crp was more severe than AnarP, indicating that Crp not only regulated expresion of narQ-narP, but also controlled expression of nap and nrfA directly.