Plasmid Analysis Of Bacillus Thuringiensis Subsp. Chinensis Strain CT-43 By Bioinformatics Method

Bacillus thuringiensis subsp. chinensis strain CT-43 was isolated from China by our laboratory, is the producing strain for the microbial preparation "Double toxins". In addition to expresses a variety of insecticidal crystal protein, it can highly produce nucleotide analogue thuringiensin which has insecticidal activities against a wide range of insects. Bt CT-43 is highly toxic to lepidopterous, Musca domestica of dipterous and Apriona germarii of Coleoptera. Through its whole genome sequencing, a total of 10 plasmids of different sizes was reported which was relatively more plasmid number in reported sequencing Bacillus thuringiensis.10 plasmids were systematically analysed by biological information in terms of plasmid evolution, replication protein, toxin protein, regulatory protein and mobile genetic factor, which provided a good theoretical basis for improving the insecticidal activity of strains, researching insecticidal mechanism and insect resistance, fundamental research of plasmid.The biggest plasmid pBMB281 encoded four insecticidal crystal proteins (Cry1Aa3, Cry Ha14, Cry2Aa9 and Cry2Ab1), one vegetative insecticidal crystal protein (Vip3Aa10) and one hemolytic enterotoxin. Because pBMB281 was highly homologous with the plasmid pBtoxic in replication associated protein, it suggested that it had a specific replication mechanism exist in the presence of insecticidal crystal protein gene of large plasmid. pBMB281 had 57 genes which accounting for 19.86% of the all of genes encoded mobile genetic factors, including 15 complete insertion sequences,2 transposons (Tn554 and Tn1546),7 groupⅡintrons and 5 integrease/recombinase. So many mobile genetic factor showed that the plasmid had a high genetic plasticity. pBMB281 also encoded 19 regulatory proteins, including 3 Rap-Phr regulation systems which could regulate spore formation and expression of extracellular protease. pBMB281 encoded a complete CRISPR/Cas system which containing two CRISPR, suggesting that it might have the function to resist the invasion of foreign genetic material and protect the integrity of cell genome. pBMB281 and pBMB293 of Bacillus thuringiensis YBT-1520 had highly homologous. As strain CT-43 and YBT1520 both had a plurality of mobile genetic factors and conjugal transfer associated genes and most of the differences exist in the sites of mobile genetic factors, suggesting that the two strain whose had different phenotype and serotype occurred plasmid conjugal transfer in the evolution.The second big plasmid pCT127 contained another insecticidal crystal proteins gene crylBal and a gene cluster for thuringiensin biosynthesis. This gene cluster was closely next to a class IV type secretion system.17 genes of pCT127 encoded mobile genetic factors, including 5 complete insertion sequences,2 transposons (Tn554 and Tn1546),2 groupⅡintrons and 1 integrease/recombinase. The S-layer protein encoded by pCT127 might have important implication to phenotype of strain CT-43.11% of 100 ORFs of plasmid pCT83 encoded regulatory proteins, indicating that the presence of this plasmid had an important regulatory role to the strain. Plasmid pCT72 and plasmid pBMB67 of Bacillus thuringiensis YBT-1520 had a extreme high whole genome sequence homology, and 79 of all 86 ORFS of pCT72 which accounted for 91.86% of all coding genes had a very high genetic homology with pBMB67. The main difference of two plasmids was related to mobile genetic factors. The functional similarity ORFs of plasmid pCT51 presented modular distribution, and were divided into three areas: replication control region, mobile genetic factor area, functional area. Plasmid pCT14, pCT8513 and pCT8252 belonged to rolling circle replication Group III plasmid family, and were new members of this plasmid family. Plasmid pCT9547 belonged to rolling circle replication Group VII plasmid family. Though multiple sequence alignment of pCT9547 with plasmid pTXI4-2and pGI2 found conjugal transfer initiation site oriT of pCT9547 which was located in 8674bp to 8697bp of this plasmid. CRISPR is a novel type of microbial defense system, which is unique in that it is invader-specific, adaptive and heritable. It is a recent breakthrough in understanding host-virus interactions. Bioinformatics methods including BLAST, multiple sequence alignment, and RNA structure prediction was used to analyze the CRISPR structures of 24 Bacillus cereus group genomes. CRISPR existed in 42% strains. Two types of RNA secondary structures derived from the repeat sequences were predicted, and demonstrated that stem-loop secondary structure might function in mediating the interaction between foreign genetic elements and CAS-encoded proteins. The sequence homologous among 31% spacer, phage, plasmid and the genomes of Bacillus cereus group further verified that spacer was likely to come from the exogenous mobile genetic factor. cas5 and csdl genes of Bacillus thuringiensis CT-43 in the absence of external environmental stimuli can be transcribed, suggesting that CRISPR/Cas system could also play defense function in the absence of any outside genetic material invasion. As most of the Bacillus cereus group strains contain multiple plasmids and prophages, the CRISPR research in Bacillus cereus group by this study would be help to reveal relationship between host strains with plasmid or host strains with phage.