Regulation Of Denitrification By NtrC Protein In Heterotrophic Nitrifying Bacteria NP1

Microbial denitrification is the main technology for the remediation of eutrophication and other water pollution,and is also the main cause of nitrogen fertilizer loss in farmland.Simultaneously,the nitrogen oxides produced from the denitrification metabolism process aggravate the formation of fog and haze.The analysis about key gene expression in microbial ammonia removal process and the study on metabolic regulation will clarify the mechanism of nitrogen oxides formation and impact factors at the molecular level,in order to carry out microbial genetic modification and microbial strains isolation,to remediate water pollution with microbes,It is of great significance to provide theoretical and technical supports for the reduction of nitrogen loss and nitrogen oxides emissions in farmland.In this study,five denitrifying strains from different genus with denitrification function were selected,and the promoters of four key denitrification enzymic genes were analyzed by bioinformatic technology.The results showed that the four key enzymic genes at the denitrification process were existed in different operons.Each of them had a corresponding promoter,and there was some difference between promoters,leading to diversity of four key enzyme expressions.Regardless of aerobic or anaerobic bacteria,the promoters of nitrate reductase and nitrite reductase had strong transcriptional activities.But the promoters of nitric oxide reductase and nitrous oxide reductase had weak transcriptional activities.The intensity difference was 10%-30%,which proved that when the microbes removed nitrogen in the water and caused nitrogen loss in the farmland,the N element could not be completely discharged in the form of N₂ at the molecular level,and the emission was inevitable in the forms of fog and haze factors NO and N₂O,but in different strains,there were differences in the emission proportions of three N elements.NtrC is a nitrogen metabolic regulatory protein,and its research on the regulation of nitrogen element metabolic assimilation was clear basically.However,there was no report on the regulation of nitrogen element metabolisc dissimilation.In this study,the contaminated water body has been isolated from the research group.Using the isolated Sinorhizobium sp.strain NP1 with the ability of remediating water pollution as the initial strain,the ntrC gene of NP1 strain was cloned by homologous clone;the ntrC gene-deficient strain NP1::ntrC was constructed by gene knockout technique;the soe-PCR technique was used to construct the dld-ntrC gene fragments to the ntrC gene over-expressed NP1::dld-ntrC and ntrC gene deletion strain NP1::ntrC:dld-ntrC;and detected the three recombinant strains expressions in nitrogen source cultures with different concentrations by qPCR.The specific results illustrated that the full-length reading frame of the ntrC gene in NP1 was 1455 bp and encoded484 amino acids.The secondary structure mainly consisted of most?-helices and more random coil structures,while the contents of?-sheet and?-turn structure were less.The NP1::ntrC strain grew up slowly when incubated by utilizing potassium nitrate and ammonium sulfate as a sole nitrogen source.Compared to the wild type strain,the denitrification gene expression was down-regulated about 70%and 60%respectively;The growth of the NP1::dld-ntrC strain was not be affected,the denitrifying gene expression was up-regulated by about 1.5-fold compared to the wild-type strain.The NP1::ntrC:dld-ntrC strain recovered when incubated by utilizing potassium nitrate and ammonium sulfate as sole nitrogen source.Compared to the wild-type original strain,the down-regulation of denitrification gene expression reduced from 70%to 20%.The above results fully demonstrated that the ntrC in Sinorhizobium meliloti NP1 regulated the dissimilation on nitrogen element metabolism.