Differentiation And Evolution Of Bacillus Cere U S Group Based On CSAB Gene And Plasmidome

Bacillus cereus group includes B. anthracis which can cause anthrax, B. cereus which is an opportunistic pathogen, B. thuringiensis which can produce crystal proteins that can be used as microbial insecticides and other more5species of Bacillus. As some members of this group could cause severe illnesses to human and some others can be used as biological pesticide, identification and differentiation for strains from this group were frequently studied by many researchers. But many of these typing systems could discriminate strains effectively. For B. thuringiensis, more and more novel insecticidal crystal protein genes need to be cloned to overcome insects’resistance, so novel strains need to be isolated. But the most effective method for this, H-serotype identification was not used any more. New method is needed to be explored to do this. On the other hand, many genome sequences of B. cereus group were published, which enables us to explore new methods on the basis of comparative genomics.In this study, we focused on new typing marker with higher resolution for identification, differentiation of members of B. cereus group.Horizontal genetic transfer (HGT) plays an important role in bacterial evolution, and one of the most important contributors to HGT is plasmid, which can transfer between cells as a vector of genes and provide a basis for genomic rearrangements via homologous recombination. So the origin, evolution, dynamics and their relationships with chromosomes are needed to be studied. Different strains of B. cereus group contain different numbers, different types and different sizes of plasmids. Moreover, more than100genome sequences of plasmids from B. cereus group were published. So they can be used as a model to study the topics about bacterial plasmids. In this study, we focused on the origin and dynamics of megaplasmids from B. cereus group and taking all the plasmids as a whole to study the relationship between plasmid and chromosome.We predicted many putative gene clusters involving in synthesis of antibiotics, among which the most abundant members belong to bacteriocin. Bacteriocins are important in the ecology of bacterial communities. Moreover, strains of Bacillus are important members of human microbiome. In this study, we focused on the diversity of bacteriocins from human microbiome of different sites and studied the ecological roles of them in different bacterial commuties.1) Differentiation of Bacillus anthracis, Bacillus cereus, Bacillus thuringiensis based on csaB gene reflect their hosts’originVarious gene markers have been studied for differentiation of the Bacillus cereusgroup, but their discrimination power has remained insufficient for their low level of diversity. This study described the use of the S-layer protein anchoring related gene csaB for differentiation among the B. anthracis, B. cereus and B. thuringiensis strains. When compared with other marker genes (>85%), csaB showed higher diversity (78-100%). The topology of its phylogenetic tree was similar with but not identical to those previously reported. Based on this typing system, all the B. cereus s.l. tested strains (n=198) were found to be clustered into two major groups. All the B. anthracis strains have identitical csaB sequence, were clustered in one branch of Group Ⅰ, and strains of B. cereus and B. thuringiensis distributed discretely around all the groups. Quite interestingly, these two groups reflected two types of animal hosts:more than80%strains isolated from higher animals in group Ⅰ,21out of22strains isolated from insects in group Ⅱ. When the cell wall outmost proteins (S-layer homology or SLH proteins) were analyzed, we found that group I strains have more SLH protein genes than those from group Ⅱ and many SLH proteins involving in interacting with animal environment were mainly contained by strains of group I.2) Gene csaB can be used for differentiation of B. thuringiensis strains In addition, regarding the B. thuringiensis strains tested (n=115) representing70serotypes, we found that the strains belonging to the same B. thuringiensis serovar were grouped together and that the different serotypes and the different serovars in the same serotype could be discriminated. Moreover, analysis of csaB gene along with flagellin amino acid sequences could discriminate all serotypes and serovars of B. thuringiensis strains. Taken together, these results indicate that these csaB-based (sub-)groupings reflect the differential host environmental pressures on their bacterial hosts.3) Megaplasmids larger than100kb of Bacillus cereus group may originate from integrated envents of samller plasmidsAs important vessels of horizontal gene transfer, plasmids play significant roles in the evolution of bacterial genomes. Many bacterial strains contain multiple plasmids, but the dynamics and evolution of these plasmids, as well as the relationships between plasmids and chromosomes, are unclear. This work studied the origin and evolution of31B. cereus group megaplasmids (>100kb) focusing on the most conserved regions on plasmids, minireplicons. Sixty-five putative minireplicons were identified and classified to six types on the basis of proteins that are essential for replication. Twenty-nine of the31megaplasmids contained two or more minireplicons. Phylogenetic analysis of the protein sequences showed that different minireplicons on the same megaplasmid have different evolutionary histories. Therefore, we speculated that these megaplasmids are the results of fusion of smaller plasmids. All plasmids of a bacterial strain must be compatible. In megaplasmids of the B. cereus group, individual minireplicons of different megaplasmids in the same strain belong to different types or subtypes. Thus, the subtypes of each minireplicon they contain may determine the incompatibilities of megaplasmids. A broader analysis of all1285bacterial plasmids with putative known minireplicons whose complete genome sequences were available from GenBank revealed that34%(443plasmids) of the plasmids have two or more minireplicons. This indicates that plasmid fusion events are general among bacterial plasmids.4) Plamids of B. cereus group are vectors of chromosome redundant genesWe focused on plasmids from the Bacillus cereus group because most strains contain several plasmids. We collected genome sequences of104plasmids from B. cereus group, and studied relationships between plasmids and chromosomes by focusing on the shared genes. Plasmids’genes show more similar basic features (base composition and codon usage) to the chromosomal variable genes (distributed genes and unique genes) than to the chromosomal core genes. Although all the functional categories of chromosomal genes are presented by plasmids genes, the proportions of each category for these two gene sets are different. When focusing on the596shared gene families between chromosomes and plasmids, they display uniform distribution among chromosomes and plasmids, respectively. Moreover, these shared genes between plasmids and chromosomes usually have different promoters and terminators, which indicate they are regulated by different elements in the transcriptional level. We speculate for the whole group, as the environments they survived are changing frequently, the adaptive genes they contained must be preserved on plasmids or chromosomes.5) Diversity and dynamics of bacteriocins from human microbiomeAs gene-encoded peptides that usually show toxic to bacteria closely related to the producing strain, bacteriocins play very important roles in the ecology of bacterial communities. Human body sites are different natural habitats for huge bacteria to form different microbiota, and bacterial communities in this microbiota usually keep the dynamic balance. So the distributions and dynamics of bacteriocins among these microbiota are interesting to be studied. We used the public sequences of class Ⅰ and Ⅱ bacteriocins to search against the amino acid sequences predicted from the WGS data of the human microbiome project. We predicted predicted3850putative bacteriocins with802belonging to small modified bacteriocins Class Ⅰ and the rest3048belonging to small unmodified bacteriocins Class Ⅱ. Samples from different body sites contain different abundances of bacteriocins with the most abundant bacteriocin-containing samples from oral cavity sites, and actually most of these putative bacteriocins were from samples of oral cavity sites. Based on the sequence similarity networks, bacteriocins from samples of the same body sites are clustered together, and for each site several types are dominant. When we focused on the reference genomes for the predicted bacteriocins, we found that for those from sites of oral cavity, most of them are from Streptococcus which is the most dominant genus in oral cavity. The situations for bacteriocins from other body sites are similar; the reference genomes are usually from the dominant genera of those sites, such as Lactobacillus for vagina and Bacteroides for gut. We suggest that bacteriocins play an important role for different bacterial genera or species to occupy dominant positions in different human body sites under different living environments.