Transcriptional Regulation Of MsmR1 In Paenibacillus Polymyxa SC2

Paenibacillus polymyxa is a kind of plant-growth-promoting rhizobacteria?PGPR?,that has a significant effect on plant growth.It has been widely used in agricultural production.Paenibacillus polymyxa SC2 was isolated from the rhizosphere of pepper and showed unstable state in the laboratory conditions.Two spontaneous mutant strains SC2-M1 and SC2-M2 appeared.SC2-M1 colony morphology is irregular,transparen,no spores,extracellular polysaccharides less,easy to electrotransformation,genomic data is abundant.It is an ideal material for the study of molecular biology of Paenibacillus polymyxa.Preoperative transcript analysis of wild strain SC2 and spontaneous mutant strains SC2-M1 to found,the transcriptional level of the?-galactosidase gene mel A adjacent to msmR1 in the SC2-M1 genome was significantly increased.MsmR1 is a class of transcriptional activators of the AraC/Xyl S family.The AraC/Xyl S family is closely related to the regulation of polysaccharide metabolism,cytotoxicity,stress adaptation and other related gene expression.This study mainly through three aspects analysis of transcriptional regulation of msmR1.First of all,the msmR1 recombinant strain was obtained by knocking out the msmR1 gene of SC2-M1 strain.To detect the effect of msmR1 knockout on carbon source utilization,growth status and antibacterial activity.Secondly,complementation in the msmR1 recombinant strain and to verify the correctness of the knockout result.Thirdly,the changes of antibacterial activity of SC2-M1 and msmR1 recombinant strains were determined,combined with qPCR detection of polymyxin synthesis gene pmxE,select the appropriate fermentation time to carry out transcriptome sequencing analysis.Using the pRN5101 temperature sensitive plasmid,and chloramphenicol as the resistance cassette.The recombinant plasmid pRN-msmR1-cm was constructed.The recombinant plasmid was electrotransformed into the competent cells SC2-M1.The homologous recombination was screened by successive cultivation in 39?.The msmR1recombinant strain SC2-03040 was obtained.The growth of SC2-M1 and SC2-03040 strains was determined,the results showed that compared with SC2-M1,the msmR1 recombinant strain SC2-03040 was slow to grow and the number of viable bacteria was significantly decreased.The utilization ability of SC2-03040 strain to 10 different sugars was determined.The results showed that the knockout of msmR1 resulted in a general decrease in the ability of using carbon source in SC2-03040.The use of fructose,lactose,mannose has a greater impact,compared with SC2-M1,SC2-03040 in lactose as the sole carbon source to cultivate stable period,the OD₆₀₀00 reduced by 57%,fructose OD₆₀₀00 decreased by 54%and mannose OD₆₀₀decreased by 59%.The antibacterial activity of SC2-M1 and msmR1 recombinant strain SC2-03040 on Escherichia coli was determined by plate confrontation and turbidimetric method.The results showed that SC2-03040 had almost lost antagonistic effect compared with SC2-M1.Using the pRN5101 temperature sensitive plasmid,construction of msmR1 complement plasmid pRN-msmR1H,the msmR1 recombinant strain SC2-03040 was completed and homologous recombination removes chloramphenicol resistance cassette.The expression of msmR1 gene was restored and the complementation strain SC2-03040H was obtained.The results showed that the growth traits and antibacterial activities of the complementation strain SC2-03040H recovered to the level of SC2-M1.According to the changes of antibacterial activity and the expression of pmxE transcription of polymyxin synthesis gene of msmR1 recombinant strains and SC2-M1,the bacterial solution was cultivated at 37?for 15h at 180rpm,transcriptome sequencing analysis was performed.The results of transcriptase sequencing showed that compared with SC2-M1 the expression of SC2-03040 was down-regulated by 1454,and the expression of SC2-M1 was up-regulated by 233.Down-regulated genes were mainly associated with carbon source metabolism,amino acid metabolism,transport proteins,and transcriptional regulators.The differential expression genes associated with carbon source metabolism are mainly involved in the metabolic processes such as galactose,fructose,mannose,xylan,dextran and arabinose,pmxA,pmxB,pmxC,pmxD,pmxE,which encoding polymyxin,and fusC,fusD,fusE,fusF,fusG,fusTE,which encoding fusaricidin was significant down-regulated.Encoding histidine synthesis genes hisA,hisB,hisD,hisF,hisG,hisH,hisF,hisZ,his J1 also significant down-regulated.The results showed that the msmR1 gene was involved in the regulation of polysaccharide metabolism,which affected the basis metabolism of the strain and had an indirect effect on the antibiotic metabolism of NRPS.Including the MarR family,AraC family and other transcriptional regulation genes down-regulated,also shows that the mechanism of action of msmR1 transcription regulation is more complex.Found in transcriptome data analysis,more than half of the endogenous plasmid genes in SC2-M1 was not founded transcriptional expression in SC2-03040.Amplification of indigenous plasmid-specific DNA fragments by PCR confirmed the deletion of indigenous plasmid in msmR1 recombinant strain SC2-03040,it is highly probable that the knockout vector pRN5101 is not compatible with the SC2 indigenous plasmid.The results of this study reveal the transcriptional regulation of msmR1 gene,which lays a good foundation for further elucidating its molecular regulation mechanism.