The Comparative Transcriptomic Analysis And The Functional Analysis Of Anaerobic Regulatory Protein FNR In Paenibacillus

Paenibacillus polymyxa WLY78 has a known smallest nitrogen-fixing gene cluster,containing nine nitrogen-fixing genes(nifBnifHnifDnifKnifEnifNnifXhesAnifV).This whole nitrogen fixation gene cluster can make Escherichia coli has the ability to fix nitrogen,but the level of nitrogenase activity in recombinant E.coli is only about 10%of that of Paenibacillus.At the same time,the efficiency of biological nitrogen fixation is mainly regulated by oxygen and ammonium in environmental,which means oxygen and ammonium in the high concentration can inhibit the transcription of nitrogen fixation gene.But at present regulatory mechanism of nitrogen fixation in Paenibacillus is still unclear.In P.polymyxa WLY78 and the recombinant E.coli 78-7,with the method of transcriptome analysis,this study aims to explore their differences in gene expression of nitrogen fixation.After that,the anaerobic regulatory protein FNR in Paenibacillus,which is highly expressed in the nitrogen fixation condition,and its role in regulation of Paenibacillus under anaerobic conditions was mainly discussed.In order to explore the reasons of low nitrogenase activity in recombinant E.coli,the transcriptome of P.polymyxa WLY78 and E.coli 78-7 cultivated in N2-fixing(no ammonium and no oxygen)and non-N2-fixing(100 mM NH4C1 and air)conditions was analyzed.Our results demonstrate that the expression of the nif gene operon was highly induced in Paenibacillus in N2-fixing condition.The non-nif genes specially required for nitrogen fixation,such as transporters of Fe,S,Mo and electron and of Fe-S cluster assembly systems were coordinately transcribed with nif genes in Paenibacillus.Although the Paenibacillus nif genes were significantly transcribed in E.coli 78-7 in both N2-fixing and non-N2-fixing condition,transcript levels of Fe2+/Fe3+ transporters,electron transporters and Fe-S cluster assembly systems were transcribed at low level in N2-fixing condition in E.coli.This study will provide novel information for engineering nitrogenase biosynthetic pathway intonon-N2-organisms and valuable guidance for improving nitrogenase activity in the heterologous host.Based on analysis of the transcriptome,4 fur of Paenibacillus encoding the anaerobic regulatory proteins were highly up-regulated in N2-fixing condition.To explore the relationships between 4 fnr,and their regulation function in anaerobic conditions,we successfully construct the fnr single and multiple deletion mutant strains and complementary strains using the homologous recombination technology.Through sequence alignment and phenotypic analysis,we found that fnrl and fnr3 are similar to B.subtilis fnr in structure and function,and they impact the nitrogenase activity of Paenibacillus.After FNR binding sites prediction and transcriptomic analysis of ?fnrl3 double deletion mutant strains,we found that fnr1 and fnr3 can activate or inhibit lots of genes in nif gene clusters,iron transport,energy metabolism,electron transport and metabolism of carbon.And the EMSA results show that fur1 and fnr3 can bind the promoter region of qoxABCD,narGHJI,ndh,hemN3,hydEG,nrdDG,pflBA and resDE.This study revealed that FNR 1 and FNR3 were global regulatory proteins under anaerobic conditions,which laid a foundation for further study on the oxygen regulation mechanism of Paenibacillus.