Functional Identification Of General Nitrogen Regulatory Protein NtrC In Pseudomortas Stutzeri A1501

Pseudomonas stutzeri A1501is a nitrogen-fixing bacterium which was isolatedfrom rice field in South China.This strain can convert N2to ammonia undermicroaerophilic conditions. Environmental ammonium and the supply of energy are thetwo critical factors that affect the nitrogenase activity. NtrC is the global nitrogenregulator and it was suggested to maintain the carbon-nitrogen balance. In order toanalyze the biological function of NtrC in Pseudomonas stutzeri A1501, ntrC mutant wasconstructed and its phenotype traits were analysed. Then different expression level ofnitrogen and carbon metabolism-associated genes between A1501and its ntrC mutantunder different conditions were determined by real-time quantitative PCR. Finally, themechanism of regulation of nitrogen fixation by NtrC was preliminary studied. The mainresults are described as follows:1. Disruption of the ntrC gene was achieved using a double cross-overrecombination with the pK18mobsacB by inserting chloramphenicol-resistance gene andthen ntrC deletion mutant of A1501was constructed successfully, named ΔntrC. Growthof the wild-type and ntrC mutant in A15medium containing different nitrogen sourceswere monitored. The growth of ΔntrC was shown to be similar to the wild-type strainunder normal conditions. The growth of ΔntrC was impaired, with the amendment ofKNO3or urea as a nitrogen source in the A15medium as compared to the wild typestrain. The results proved that NtrC was involved in regulating the genes and proteinsparticipated in nitrate and urea assimilation.2. The nitrogenase activity of ΔntrC was reduced to10%as compared to thewild-type under microaerobic conditions in the free-living state. RT-qPCR revealed thatthe expression of nifA and nifH gene was reduced more than5fold in ntrC. For findingout the biological function of ntrC further, we measured nitrogenase ability in ntrC thatoverexpresses cbrB gene ΔntrC（cbrB）. The result indicated that ΔntrC（cbrB） mutanthave more nitrogenase ability. The result also showed that NtrC might have aninteraction with catabolic regulation protein CbrB.3. Deletion of the ntrC gene significantly reduced the capacity of chemotaxis anddenitrification, which suggest that NtrC is directly or indirectly involved in regulating thegenes and proteins participated in chemotaxis and denitrification. However, ΔntrC hadmore vitality under oxidative stress condition. The increased expression of katE and sodCby RT-qPCR might confer resistance against the ntrC mutant. These results indicated that NtrC not only affects nitrogen source metabolism, but also regulate other metabolicpathways.4. The His-NtrC protein was over-expressed in E. coli BL21(DE3) carrying plasmidpET28a and was used to be made antibody; Determination of the consensus NtrC bindingsite in P.stutzeri by the method of bioinformatics. The purified protein will be used tofurther verify molecular interacting analysis between NtrC protein and its target genes.Our study demonstrates that the function of NtrC is diversity in P. stutzeri, whichprovide a theoretical foundation for the genome-wide analysis of NtrC-dependent geneexpression and global regulation mechanism.