Comparative Genome And Proteome Analysis Of Three Bacillus Cereus Strains

Bacillus cereus is wide spread in nature.On one side,it is an opportunistic pathogen causing food poisoning.On the other side,as a microecological additive,Bacillus cereus exerts important functions on clinic.However,lack of a definite distinguishing criteria on Bacillus cereus brings about an argument of food safety for its application.Thus constructing a dual-model and undertaking basic research on Bacillus cereus,is of important theoretical exploration value.Therefore,we makes an in-depth study on Bacillus cereus from aspects of biological characteristics,genomics and proteomics,aiming at finding out an accurate method for intraspecific classification and differences between probiotic and pathogenic of Bacillus cereus,and provide basic data and technical basis for establishment of a scientific evaluation system.Firstly,two strains of Bacillus cereus P0021 and P0011 for probiotic application and one strain HN001 isolated from food poisoning patients,were applied for our study.The virulence determinant distinguish of three strains from Bacillus thuringiensis and Bacillus anthracis was performd.Various parameters,including mycelial morphology,growth curve,carbohydrate metabolism,drug resistance,and haemolysis and lecithinase activity were used to analyze the biological characteristics of the three strains.Results confirmed that the three strains were Bacillus cereus,its biological characteristics are similar,especially for P0021 and P0011.The three strains own haemolysis and lecithinase activity,and the drug resistance ability against 13 kinds of commonly used antibiotic is poor except for penicillin,cefazolin and cefalotin,however,three strains have obvious differences in sucrose metabolism,the strain HN001 can utilize sucrose,but P0021 and P0011 can not.Secondly,based on the above results,to deeply explore the biological difference among Bacillus cereus strain P0021,P0011 and HN001,complete genomes of the three strains was sequenced by Pacbio.Protein profile of three strains was analyzed by two-dimensional polyacrylamide gel electrophoresis(2-DE)and tandem mass spectrometry(MS/MS).With the aid of bioinformatics analysis,there were signifcant difference on plasmid and protein expression profile between HN001 and P0021&P0011.HN001 contains two plasmids showing low homology with the large plasmid of P0021 and P0011.There were great difference on functions,spot distribution and expression level of whole cell and supernatant protein.Moreover,the genomic characteristics of strains P0021 and P0011 is similar.The genomic and plasmid homology is as high as 99.9%and its evolution distance on the phylogenetic tree is zero,without any insertion,deletion,reverse and inversion.The protein expression profiles of P0021 and P0011 are almost the same,and there is no difference in the 117 identified protein spots,indicating that Bacillus cereus P0021 and P0011 come from the same clone.Considering the difference of sucrose metabolism between HN001 and P0021&P0011,to disclose the relationship between sucrose utilization and pathogenicity of Bacillus cereus,sucrose metabolic pathways of the three strains and other 13 completed sequencing strains from KEGG database were analyzed by bioinformatics analysis,and further verified with 18 reported strains of Bacillus cereus from different sources.Results showed that partial of non-pathogenic(probiotic)strains of Bacillus cereus can not utilize sucrose due to absence of whole operon,whereas most of those pathogenic strains made use of sugar,indicating that sucrose metabolism was likely to be related to pathogenicity.Moreover,by prediction of gene of virulence factors and analyse of toxic proteins it was found enterotoxin genes of hbl and nhe,and other virulence gene of hly,cyt and plc,but not that of vomiting toxins gene ces existing in the three strains.Besides,the virulence proteins of PLC,Hbl,InhA and Nhe were also detected according to the protein expression profiles.Hence,it was concluded that probiotic and pathogenic properties of Bacillus cereus might be influenced by environmental and other factors.In conclusion,analysis on biological properties,genomics and proteomics by comparison of probiotic strain P0021&P0011 with pathogenic strains of HN001,was performed to ascertain the difference of phenotype and nature molecular biology of Bacillus cereus,and set up a protocol for differentiate the probiotic and pathogenic strains of Bacillus cereus,founding up a preliminary basis for application of probiotic Bacillus cereus and monitoring the potential pathogenic effect of Bacillus cereus,differences between probiotic and pathogenic bacteria were found out,the method to distinguish different strains of Bacillus cereus was also set up,and the differential analysis would be helpful for microecological preparation and potential danger control of Bacillus cereus.