Regulatory Mechanisms Of Nitrogen Fixation Gene By Transcriptional Regulator GlnR In Paenibacillus Polymyxa

Nitrogen-fixing Paenibacillus is a genus of endospore-forming microorganism,which has shown great advantage in agriculture due to strong stress resistance and production of plant growth promoting and antimicrobial substances.Being similar to other nitrogen-fixing microorganisms,Paenibacillus fix nitrogen under nitorgen-limited condition,and grow well with no nitrogenase activity under nitrogen-excess condition.The affect of ammonium to nitrogen fixation in Gram-negative bacteria was intensively studied,the transcription of nif genes in which was strictly controlled by the concentration of ammonium in medium.Due to the difficulty of genetic transformation in Gram-positive bacteria,the regulatory mechanism of nitrogen fixation in Paenibacillus remains unknown.Clarifying this mechanism will help to reveal the similarities and differences between the regulatory mechanisms of nitrogen fixation in Gram-negative and positive bacteria,and provide theoretical basis for construction and application of engineering strain that fixes nitrogen despite of nitrogen availability.GlnR is the global regulator of nitrogen metabolism in Bacillus subtilis,which represses the transcription of glnRA(encoding GlnR and glutamine synthetase),tnrA(encoding the other nitrogen metabolism regulator TnrA)and ureABC(encoding urease)under nitrogen-excess condition.There is one glnR gene in the genome of Paenibacillus polymyxa WLY78 which is co-transcripted with glnA,while there is another glnA gene(glnAl)scattered in the genome.Deletion analysis demonstrates that GlnR is essentially required for nif gene transcription under nitrogen limitation,and GlnR together with glutamine synthetase(GS)encoded by glnA within glnRA operon is involved in repressing nif expression under excess nitrogen.Electrophoretic mobility shift assays(EMSA),Surface plasnmon resonance(SPR)spectroscopy and Chromatin immunoprecipitation-qPCR(Chip-qPCR)reveal two GlnR-biding sites flanking the-35/-10 regions of nif promoter.The affinity of GlnR to site ? located downstream of the nifB transcription start site is stronger than to site ? seated upstream of the-35 region.GlnR alone binds to the two GlnR-binding sites,but the binding ability of GlnR,especially to site ? is greatly enhanced and stabilized when GlnR is in complex with feedback-inhibited glutamine synthetase(FBI-GS).Mutation and deletion analyses demonstrate that the GlnR-binding site ? is specially required for activating nif transcription,while GlnR-binding site ? is for repressing nif expression.These results reveal for the first time that nif transcription of P.polymyxa WLY78 is positively and negatively regulated by GlnR by binding to two different loci in response to nitrogen availability.This study not only reveals the novel regulation mechanism of nif expression in Paenibacillus,but also provides insight into dual active and repressive functions of GInR.Prediction and verification of GlnR binding sites in the genome of P.polymyxa WLY78 re'veal several genes directly regulated by GlnR,including glnAl,appABCDF and ldh.Transcriptome analysis of WT and ?glnR strains under nitrogen-limited and nitrogen-excess conditions indicates that GlnR functions as both activator and repressor under both conditions.Differential expression of genes involved in nitrogen and cabon metabolisms indicates the role of GlnR in the crosstalk between nitrogen and carbon metabolism-associated pathways.Differential expression of related genes indicates that GlnR controls the process of nitrogen fixation not only by regulating nif cluster at transcription level,but also by affecting transportation of elements essential for nitrogenase synthesis.This work expands the acknowledge of GlnR's regulatory function.In this work,the regulatory mechanism of nitrogen fixation in P.polymyxa was revealed,and the multiple regulatory role of GlnR was indicated.These investigations will help to gain insights into the complex regulatory network of nitrogen metabolism in nitrogen-fixing Paenibacillus,and provided theoretical basis for construction of engineering nitrogen-fixing strain suitable for extensive environment.