| Bacillus thuringiensis strain YBT-1520,which is highly toxic to lepidopteran pests, was isolated by our lab.In this thesis,we reported the sequence determination and initial analysis of the genome of this strain.The results revealed that this genome consisted of one chromosome and nine plasmids.TA systems have been extensively studied on low-copy-number plasmids in Gram-negative bacteria as addiction systems because of their ability to maintain the plasmids during cell division and a few TA systems have been described on small plasmids in Gram-positive bacteria.We identified 562 TA loci belonging to the seven known TA gene families and present the results from an exhaustive search for TA loci in the completely sequenced Bacillus genomes.The putative TA loci from YBT-1520 genomes were studied and the putative TA loci from other 6 completely sequenced Bacillus genomes were found by bioinformatics analyse.1.The study on the putative kyAB TA system from plasmid pBMB8240Based on the shotgun sequence analysis of the strain YBT-1520 plasmid pBMB8240 was found to be a novel cryptic plasmid.In pBMB8240,two contiguous orfs(this gene pair was tentatively named kyAB for strain kurstaki YBT-1520) were noticed.The putative protein KyA and KyB displays sequence similarities with other antitoxin and toxin protein,respectively.Attempts to clone the kyA in E.coli DH5αwere easy and successful.In kyB cloning case,all recombinants harbored mutations in the kyB gene,suggesting the toxic activity of KyB protein in E.coli.Based on sequence homology with other toxin-antitoxin system genes and the lethal activity of KyB in Escherichia coli,the segregational stability kyAB cassette was identified to be the first functional putative TA segregational stability system from B. thuringiensis.2.The study on the putative kyCD TA system from plasmid pBMB7635A database sequence search revealed that the B.thuringiensis H1.1 plasmid pGI1 (8254 bp) had DNA sequences and organizations similar to plasmid pBMB9741 except for a fragment of 1.6 kb located upstream from the rep-gene.We designed a pair of contiguous primers to verify the DNA sequence upstream from the rep-gene.Here we named the 'new' plasmid as pBMB7635(7635 bp).Two orfs(orf94 and orf133) present in pBMB7635 but absent in pBMB9741. ORF94 shares 100%amino acid identity with the TasA putative antitoxin protein of pGI1, while its neighbor ORF133 shares 98%amino acid identity with the TasB putative toxin protein of pGI1.The orf94/orf133 system is denoted kyCD system.Attempts to clone the kyC in E.coli DH5αwere easy and successful,while continuing efforts to clone kyD gene and kyCD system in E.coli DH5αhave not yet been successful,suggesting the toxic activity of KyD protein,suggesting the KyD protein failed to inhibit the lethal activity of the cognate toxin and kyCD system appeared to be functional but unregulated in E.coli.3.Plasmid pBMB2062 from YBT-1520 strain was cloned and characterizedA multicopy plasmid pBMB2062 from Bacillus thuringiensis subsp,kurstaki YBT-1520 strain was cloned and characterized by dot-blot analysis with the ORF1 fragment as a probe,and it was found to be present in 41%B.thuringiensis strains.The sequences of 7 pBMB2062-like plasmids from randomly selected B.thuringiensis strains (positive signal in the dot-blot analysis) were highly conserved.Two orfs,orf1 and orf2, were present in this plasmid.Orf1 was found to be necessary for plasmid replication, whereas Orf2 did not play a role in replication or stability.Based on its sequence homology,ORF2 was putative solitary antitoxin belonged to MetJ/Arc/CopG family.4.The mazEF system from YBT-1520 chromosome was cloned and characterizedBased on the sequences homology with other mazEF toxin-antitoxin systems,a novel mazEF system was identified on the YBT-1520 chromosome.To investigate the role of mazEF system in Bacillus thuringiensis,the mazEF system on B.thuringiensis strain BMB171 was knocked out.The conclusion was that the mazEF system was involved in the PCD of the B.thuringiensis strain BMB171.
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