Clpp Proteases Involved In Bacillus Cereus Biofilm Formantion Sporulation And Motility

The ClpP protease is widely present in prokaryotic and eukaryotic cells and plays an important physiological role.The function of ClpP mainly includes two aspects of participating in intracellular protein control to maintain intracellular protein stability and regulate cell development.In the model bacteria Bacillus subtilis,ClpP protein is involved in important physiological processes such as bacterial growth,sporulation and biofilm formation.The protein has not been extensively studied in the function of B.cereus.The use of rhizosphere microorganisms to control plant diseases is an important part of the comprehensive management of plant diseases.The stable presence of biocontrol microorganisms in the natural environment is a prerequisite for their biocontrol effect.Bacillus cereus 0-9 is a biocontrol strain isolated from the roots of healthy wheat and has a control effect on wheat sheath blight.Study the molecular mechanism of biofilm formation and sporulation of Bacillus cereus,reveal its environmental adaptability,and help to improve biological control effects.To further explore the mechanism of clpP gene involved in biofilm formation,sporulation and motility,the B.cereus 0-9 genome sequence was analyzed and two spx related genes were found,named spx1 and spx2.Based on the ?clpP1 and ?clpP2 single knockout strains constructed in the early stage of the laboratory,a double knockout strain ?clpP1?clpP2 of clpP1 and clpP2 was constructed by homologous recombination.By measuring the phenotype of different knockout strains,the results showed that ?clpP1 and ?clpP2 had no significant difference in colony morphology,biofilm formation,growth,protease production and low temperature sensitivity compared with wild type strain 0-9,while ?clpP1?clpP2 the above phenotypes are all defective.It is suggested that clpP1 and clpP2 have functional redundancy in determining the above phenotype,and only when both are missing,they show obvious physiological effects.The phenotypes of motility,low temperature and high temperature sensitivity of different strains were determined.It was found that there was no significant difference between ?clpP1 and wild type strains,and ?clpP2,?clpP1?clpP2 and wild type strains were significantly different.It indicates that the clpP2 gene plays a leading role in bacterial motility and high temperature sensitivity.The sensitivity of different strains to ampicillin was determined.It was found that ?clpP1 and ?clpP1?clpP2 were sensitive to ampicillin,suggesting that clpP1 may be involved in maintaining the normal physiological function of bacterial cell wall.The above results indicate that the two clpP genes present in Bacillus cereus play different roles in colony morphology,growth,sporulation and stress tolerance.To further explore the mechanism of clpP gene involved in biofilm formation,sporulation and motility,the B.cereus 0-9 genome sequence was analyzed and two spx related genes were found,named spx1 and spx2.In view of the accumulation of spx gene in Bacillus subtilis cells affecting sporulation and biofilm formation,the spx1 and spx2 genes were sequentially knocked out based on the double knockout strain ?clpP1?clpP2,construction of a three knockout strain and a four-knockout strain and determination of the phenotype.The results showed that both the ?clpP1?clpP2?spx1 triple knockout strain and the ?clpP1?clpP2?spx1?spx2 four knockout strain were able to restore colony morphology,sporulation and motility to the level of 0-9 wild type strain,while the three knockout strain ?clpP1?clpP2?spx2 could not restore the phenotype.It is suggested that the phenotypic defects that occur after the deletion of the clpP1 and clpP2 double genes may be due to overexpression of the spx1 gene.Polyclonal antibodies that express both Spx1 and Spx2 proteins and prepare two proteins,Western blot analysis of the accumulation of Spx1 and Spx2 in ?clpP1?clpP2.The results showed that the deletion of clpP1 and clpP2 gene did cause accumulation of Spx1 and Spx2 proteins in cells.The above results indicated that the deletion of clpP1 and clpP2 genes in Bacillus cereus 0-9 resulted in the accumulation of Spx1 protein and the phenotypic changes in colony morphology,sporulation and biofilm formation.In order to explore the physiological role of spx in Bacillus cereus,by measuring the biological traits of spx deficient strains,It was found that both ?spx1 and ?spx2 showed resistance to diamide and hydrogen peroxide.?spx1?spx2 is more sensitive to the tolerance of diamides and hydrogen peroxide.It indicates that the spx gene is mainly involved in maintaining the cell redox balance.In this study,?clpX(GFP-ssrA),0-9(GFP-ssrA)labeled strains were constructed,and the green fluorescent electron excitation amount of the labeled strain in the log phase and the stationary phase was determined,and the expression level of GFP-ssrA was determined by Western blotting.The expression level of GFP in ?clpX(GFP-ssrA)was found to be significantly higher than that of wild-type strain 0-9 background strain.The amount of GFP-ssrA transcript levels in ?clpX(GFP-ssrA),0-9(GFP-ssrA)was determined by RT-qPCR and found to be no significant difference.It indicated that the ClpXP proteolytic complex can specifically degrade the abnormal protein GFP-ssrA,and ClpXP directly degrades the GFP-ssrA abnormal protein without affecting the protein synthesis process.