Genomics,Transcriptomics,and Regulatory Mechanisms Of Iturin A Biosynthesis Of A Biocontrol Strain Bacillus Velezensis CC09

Bacillus velezensis CC09 is a highly effective endophytic and biocontrol strain with a broad antifungal spectrum,plant growth promotion capability,and high efficiency in prevention and control of soil-borne disease such as continuous cropping problem of Rehmannia glutinosa Libosch as well as powdery mildew of wheat,grapes,strawberries.Cyclic lipopeptide?CLPs?iturin A,which is synthesized by non-ribosomal peptide synthase,is one of the key active compound in responsible for the induced systemic resistance and antimicrobial activities.Although the biochemical properties and operon structure of iturin A synthetases have been clearly characterized,the regulatory mechanisms at transcriptional and post-transcriptional level have not been reported yet.In this study,we mainly focus on the biological characteristics,biocontrol basis,and regulatory mechanism of iturin A biosynthesis by non-ribosomal peptide synthase in Bacillus velezensis CC09 using genomics,comparative transcriptomics,RNA polymerase mutation,molecular biology and bioinformatics techniques.The results would provide us new idea and new way in studying the biocontrol mechanisms of beneficial Bacillus strains.Data of Hiseq2000 sequencing showed that the genome size of B.velezensis CC09 is 4 167 153 bp,with GC content of 46.1%and 4 231 genes,of which 4 141 are protein coding sequences,taking a proportion of 97.9%to the total genes.About 677 genes directly or indirectly associated with the biocontrol function taking a proportion of 16.3%to the total CDSs,of which 567 genes?taking a proportion of 83.8%to the total biocontrol involved genes?related to the biosynthesis of 13 secondary metabolites,which included 4 novel secondary metabolites and 2 unique metabolites in B.velezensis;besides,53 genes involved in the function of motility and chemotaxis,14 genes involved in the function of polysaccharide degradation,36 genes involved in the function of biofilm formation and 7 genes involved in the function of synthesizing plant growth promotion hormone.In addition,a large genome island?139.512 kb?was found in the genome of strain CC09,which encodes drug transport and toxin synthesis related proteins.Phylogenomic analysis based on core genome sequences suggested that strain CC09 was closest to strains B.velezensis FZB42 and Bacillus sp.Pc3,with a sequences identity of 99%,while far from Bacillus amyloliquefaciens.In addition,many strains in B.amyloliquefaciens might belong to B.velezensis,which deserve to be determined in the future.Iturin A is one of the major antimicrobial compounds synthesized through non-ribosomal synthesis pathway in B.velezensis CC09.According to genome sequence,the gene cluster encoding the non-ribosomal peptide synthase of iturin A is 37 249 bp consisting of four structural genes designated as ituD,ituA,ituB and ituC,and a ?A recongnized promotor.Moreover,two binding sites of transcriptional regulator DegU were found at the 64 bp and 137 bp upstream sequence of the promotor.Using Red/ET recombination system,gene cluster encoding iturin A synthetase was cloned from strain CC09 and expressed in E.coli with the yield of iturin A 47?g/mL.Eight RNA polymerase ? subunit mutants with the same genetic backgroud of B.velezensis CC09 were constructed using homologous recombination method.The mutations in ? subunit were H485Y,H485C,H485R,H485D,S490L,Q472R,S490L/S617F and S617F;correspondin to strains of CC09-RIF1,CC09-RIF2,CC09-RIF3,CC09-RIF4,CC09-RIF5,CC09-RIF6,CC09-RIF7 and CC09-617F,respectively.Except CC09-617F?S617F?,all the other seven mutants are rifampin resistant.Based on iturin A production that was detected by HPLC in the metabolites,CC09-RIF3?H485R?and CC09-RIF4?H485D?were the positive mutants,the production of iturin A was 1.67 and 2.03 times higher than that of the wild type strain CC09,respectively;while CC09-RIF1?H485Y?,CC09-RIF5?S490L?,CC09-RIF6?Q472R?,and CC09-RIF7?S490L/S617F?were the negative mutants,which was 0.81,0.44,0.56 and 0.80 times as high as that of the wild type strain CC09 respectively;and H485C was an neutral mutant,the production of iturin A was not different from that of the wild type strain CC09.In addition,different rifampin-resistant mutants had different biological phenotypes,of which mutations of H485 at ? subunit showed significantly impacts on rifampin tolerance,swarming motility,and antimicrobial activity of B.velezensis CC09,suggesting H485 plays an important role on maintaining the normal growth and metabolism in B.velezensis.Using RNA-seq technique,we conducted a comparative transtcriptomics analysis of a iturin A-producing positive mutant CC09-RIF3?H485R?and a negative mutant CC09-REF6?Q472R?as well as a wild-type strain CC09 in the exponential growth phase.Result showed that approximately 25%genes were differentially expressed?DEGs?in both the positive and negative mutants relative to the genes in the wild type strain.The DEGs mainly involved in cell motility and chemotaxis,ribosomal structural proteins,carbonhydrate metabolism?pyrimidine metabolism,fructose and mannose metabolism?,and cell membrane/wall components were up-regulated in the positive mutant;while those associated with cell motility and chemotaxis,inorganic ion transport,ribosomal structural proteins,cells division and differentiation,and purine and fatty acids were down-regulated in the negative mutant.Moreover,eight DEGs related to non-ribosome synthesis pathway were down-regulated in both mutants except 2 of thme were up-regulated in the positive mutant.Three DEGs associated with type I polyketide synthesis pathway were down-regulated in both the positive and negative mutants.In addition,effects of H485R and Q472R on the physiology,biochemical activity and morphology of B.velezensis were studied using Biolog identification plate and laser confocal microscope observation,and the relationship between DEGs and phenotypes were analyzed.All these results suggest that the mutation of H485R and Q472R globally regulate the biological,physiological and metabolic profiles of B.velezensis.Based on the above transtcriptomics data of CC09-RIF3?H485R?and CC09-RIF6?Q472R?,3593 genes were classified into eight functional modules using weighted gene co-expression network?WGCNA?package from R language.The correlations between the "modules" and the "phenotyes" were fully studied.Genes that were co-expressed with gene cluster encoding iturin A synthetase included SB24₀0605,SB24₁0655,SB24₁3140,SB24₁3145,SB24₁3150,SB24₁3155,and SB24₁3160,which encode the proteins related to carbonhydrate metabolism,and the genes of phrF,phrA,rapA,rapB,and degU,which encode the transcriptional regulators.Besides,20 genes were identified probably to be involved in the post-transcriptional regulation of iturin A synthesis.Based on the results we obtained in this study,we proposed a hypothesis about iturin A synthesis regulation in B.velezensis CC09.