Rex Family Repressor Protein Is Involved In Bacillus Cereus Biofilm Formation

The transcriptional repressor protein Rex is conserved in Bacillus subtilis,Staphylococcus aureus,Streptococcus pneumoniae,Streptomyces coelicolor,and the like.It is conserved internally and reflects the redox state by sensing the ratio of NAD⁺/NADH in the cells,maintaining the intracellular redox balance.Under certain conditions,the Rex protein specifically binds to the specific sequence of the target gene promoter?Rex box?-TGTGAAWWWWTTCACA-,regulates the transcription of the target gene,participates in the aerobic metabolism,anaerobic metabolism,morphological differentiation and secondary metabolism of the bacteria.a physiological process.The binding activity of Rex to DNA is affected by the concentration of the cells themselves,the ratio of NADH and NAD⁺.When the concentration of intracellular NADH is increased,the binding of Rex to DNA is inhibited.Conversely,when the concentration of NAD⁺is increased,the binding of Rex to DNA is promoted.Whether Rex has a systemic study in Bacillus cereus.B.cereus 0-9 is a biocontrol fungus that can colonize wheat roots and has good environmental adaptability,which can effectively control wheat sheath blight.Studying the stress response and regulation of strains B.cereus 0-9 helps to reveal the environmental adaptation mechanism of the strain and improve the biocontrol effect.In the present study,the amino acid sequence of the model strain B.subtilis Rex protein was used to compare the coding sequence of the B.cereus 0-9 genome.The amino acid sequence of a putative protein in the B.cereus 0-9 genome was found to be highly homologous,with a consistency of up to 78%.Bioinformatics analysis showed that the two were highly similar in terms of molecular weight,hydrophobicity,hydrophilicity and three-dimensional structure.It is speculated that Rex exists in B.cereus0-9 and named BcRex.To analyze the characteristics of BcRex,BcRex was overexpressed and purified in E.coli,and the binding ability of BcRex to the Rex-specific binding sequence promoter was determined by gel electrophoresis?EMSA?.The results showed that BcRex specifically binds to the Rex box sequence and binds.Activity is affected by Rex protein concentration and NAD⁺/NADH ratio.On the basis of this,the mutant of the BcRex protein-encoding gene was deleted by homologous recombination method,and the transcriptional activity of the Rex box promoter in wild type and mutant was determined by transcriptional fusion.The results showed that the BcRex coding gene was significantly increased after deletion.Activity containing the Rex box sequence promoter.The above results indicate the presence of Rex transcriptional regulators in B.cereus,which regulates gene expression by sensing changes in the intracellular redox environment.To reveal the role of the transcriptional repressor protein Rex in B.cereus,the normal physiological phenotypes of B.cereus 0-9 wild type,rex gene deletion strains and complementary strains and phenotypic changes under different stress conditions were determined.The results showed that after the rex gene was deleted,the spore formation rate of the mutant was reduced by 45%,and the biofilm production was decreased.For erythromycin,chloramphenicol,kanamycin,neomycin sulfate,fosfomycin,and rifampinin antibiotics;for heavy metals such as copper ions,manganese ions,cadmium ions and cobalt ions,the sensitivity to H₂O₂,menaquinone,sodium hypochlorite and other strong oxides and to the anionic surfactant SDS are enhanced.The above results suggest that Rex exerts a multi-effect regulation effect on B.cereus stress adaptation.A biofilm is a structure in which an extracellular matrix composed of proteins,polysaccharides,and DNA is produced by a bacterial population.Biofilm formation plays a key role in the adaptation of bacteria to stress in the natural state.After the rex gene is deleted,the biofilm formed by the mutant is reduced,suggesting that Rex has a regulatory effect on biofilm formation in B.cereus 0-9.To reveal the molecular mechanism of Rex's regulation of biofilm formation,the expression of sipW,calY and tasA encoding the biofilm protein component genes in rex mutants and B.cereus 0-9 was compared by transcriptional fusion.The results showed that the expression of the above gene increased after rex deletion,indicating that Rex does not act by regulating protein components in the biofilm.Homology alignment revealed that there is a homologous gene of lrgB that regulates programmed cell death and cell lysis in S.aureus in the B.cereus 0-9 genome,and the gene of lrgB in rex mutant and wild type B.cereus 0-9 was determined by transcriptional fusion.The change in expression revealed that the expression of lrgB gene was significantly higher than that in B.cereus 0-9 in the rex deletion mutant,suggesting that the biofilm decrease after rex deletion is mainly through up-regulation of lrgB.Biofilm formation of the double mutant was determined by constructing a rex and lrgB double gene mutant,showing biofilm formation recovery of the double mutant.The above results indicate that under the conditions of the assay,Rex in Bacillus cereus adopts a two-bet method to regulate the formation of biofilm.