Mechanisms Of Genes Rema And Mrbf Governing Biofilm Formation In Bacillus Cereus

Bacteria are often viewed as simple prokaryotic creatures.There are two survival forms of bacteria in natural environment,one is planktonic form,and the other form is multicellular aggregates.It is now widely recognized that bacterial cells are most often found in close association with surfaces and interfaces,in the form of multicellular aggregates commonly referred to as biofilms.Biofilm formation contributes microorganisms to improve their resistance to various stresses in the environment.The components of the biofilm,the metabolic regulatory network,and signaling pathways have been well characterized in the model bacteria Bacillus subtilis.B.cereus is an another spore-producing gram-positive bacterium widely distributed in nature,and its biofilm composition and formation regulation mechanism need further study.B.cereus 0-9 is a biocontrol bacteria isolated from the roots of wheat,which can effectively control eyespot disease of wheat and other soil-borne diseases.Uncovering the molecular mechanisms of biofilm formation by the biocontrol bacteria 0-9 contributes to understand the colonization and biocontrol mechanism of the strain in wheat rootsIn this study,a transposon TnYLB-1 insertion mutant library of B.cereus 0-9 strain constructed previously were screened to find biofilm formation mutants,two mutants with changes in biofilm yield were obtained from 600 transposon insertion mutant strains.The biofilm formation ability of mutant No.239 was higher than that of wild type,and meanwhile,the biofilm formation ability of mutant No.55 1 was lower than that of wild type.The flanking sequences of the transposon insertion site of the two mutants were isolated by reverse PCR and the homology comparison showed that mutant 239 and 551 might result from mutation in DUF370 domain-containing protein encoding gene and a biofilm formation regulator respectively.As the putative amino acid sequence of the former gene products has highly similarity to RemA in B.subtilis,which is a transcriptional gene regulating biofilm formation,we name this gene as remA.Meanwhile,we named the second gene as mrbF based on its product annotated as a master regulator of biofilm formation in chromosome of B.cereus 0-9To disclose whether the mutants isolated previously were indeed from remA and mrbF mutation,non-scar gene deletion strategy was conducted to delete remA and mrbF from B.cereus 0-9 chromosome respectively,and the gene knockout strain of ?remA and ?mrbF were constructed.Biofilm formation ability of the AremA and ?mrbF were assayed,the results showed same phenotype change as the transposon insertion mutant.Following gene complementation,the biofilm formation phenotype can be restored to the wild-type 0-9 level,which indicate that the phenotypes of biofilm formation in the screened transposon insertion mutants are the result of the inactivation of remA and mrbF genesIn order to reveal the role of remA in the formation of B.cereus biofilm,transcriptome sequencing(RNAseq)of ?remA and the wild-type B.cereus 0-9 were performed under stationary growth phase.The results showed that 857 genes up-regulated and 543 genes down-regulated in ?remA compared to wild-type 0-9.The transcriptome data also showed that the expression of sipW-tasA opern and the gene calY,which involves in biofilm protein components production and regulated by the biofilm formation transcription regulation repressor SinR and AbrB,were down regulated.These data indicates that the roles of remA in bio film formation is impossible to work by SinR and AbrB-mediated biofilm formation signaling pathways.To test this hypothesis,double-gene deletion mutants of AremAAsinR and ?remA?abrB were constructed respectively and biofilm formation was assayed.The results showed that biofilm formation or colony characteristics of two double-gene deletion mutants were different from any of single gene deletion mutant or wild-type,which indicated the hypothesis raised above is correct.Moreover,gene expression patten of operon sipW-tasA and calY in ?remA and its wild type B.cereue 0-9 were determined by qRT-PCR and transcriptional fusion,which showed the same expression dynamics as in transcriptome data,that is,after remA deletion,expression of sipW-tasA and calY down regulated.These data further demonstrates the role of RemA in biofilm formation in B.cereus is independent on SinR or AbrB mediated biofilm formation signaling pathwaysIt has been demonstrated that extracellular DNA(eDNA)is one of the components of biofilm and the operon of lrgAB regulates cell lysis and programmed death in a variety of bacteria.Based on the transcriptome data,it was found that the expression of the B.cereus lrgAB operon decreased significantly in ?remA compaired with wild type.In order to analyze whether RemA affects biofilm formation by regulating the IrgAB operon,IrgAB knockout strains and overexpression strains were constructed respectively.It was found that the biofilm formation of knockout strains increased,and the biofilm decreased significantly after overexpression.The proportion of dead cells and extracellular DNA content in the biofilm formed by remA deletion strains,lrgAB deletion strains and overexpression strains were further determined.It was found that after the deletion of remA and lrgAB,cell death in the biofilm increased,and the eDNA content increased,while IrgAB over expression strains showed the opposite results,indicating that RemA positively regulates lrgAB to control cell death and eDNA release,thereby affecting biofilm formation.Based on the above results,a model for RemA to regulate biological formation is proposed:RemA positively regulates the expression of IrgAB,and the encoding product of IrgAB controls cell death and eDNA release.When remA is deleted,the expression of lrgAB decreases,and through an unknown mechanism,it promotes cell lysis and release of eDNA,thereby increasing the production of biofilmsIn order to reveal the role of gene mrbF in biofilm formation,abrB and sinR were knocked out on the basis of mrbF gene deletion background to construct double gene deletion strains ?mrbFAsinR and?mrbF?abrB.The results of biofilm formation measurement showed that the biofilm formation of?mrbF?sinR was similar ?inrbF,biofilm formation of ?mrbFAabrB is similar to the ?mrbF,while SinR and AbrB both regulate biofilm formation through negative regulation of sipW-tasA and calY gene expression Therefore,it is speculated that mrbF plays a role through the biofilm formation signal pathway regulated by SinR and AbrB.To test this hypothesis,a fusion vector with calY promoter and green fluorescent protein encoding gene gfp was constructed and inserted into the genomes of B.cereue 0-9 and ?mrbF respectively.Transcription fusion assays reveals the expression time and maximum expression of calY in the AmrbF strain were lower than its expression in B.cereue 0-9,which proved that mrbF affects biofilm formation by acting on the signaling pathways regulated by SinR and AbrB.The major regulator of spore formation SpoOA plays a central role in spore development and biofilm formation.It affects biofilm formation by regulating the expression of sinR and abrB genes.In order to analyze whether mrbF affects biofilms via SpoOA,transcriptional fusion was used to determine the expression dynamics of the abrB promoter.The results showed that the abrB promoter is more active in the AmrbF strain in the early growth stage than in B.cereue 0-9 active.In the later stage,it is lower than its activity in B.cereue 0-9.The expression of the gene encoding the stage II regulatory protein SpoIIG in spore development regulated by the phosphorylated form of SpoOA(SpoOA?P)was determined,showing that the expression level of spoIIG in AmrbF was also significantly lower than its expression in B.cereue 0-9.Combined with the delayed sporulation phenotype of the AmrbF mutant,it indicates that mrbF affects biofilm formation and spore production by regulating spoOA gene expression or inhibiting the phosphorylation of its product SpoOA.Based on the above results,a model for mrbF regulating biofilm formation is proposed:mrbF product promotes the expression of spoOA or the phosphorylation of its products,thereby inhibiting and promoting the expression of abrB and sinR,respectively,which regulate the transcription of sipW-tasA and calY To regulate the formation of biofilms.