Research Of The Plasmids From Bacillus Thuringiensis Subsp. Tenebrionis Strain YBT-1765

B. thuringiensis subsp, tenebrionis YBT-1765(H_8ab) was isolated from a warehouse.This strain harbored three indigenous plasmids. In this study, the replicon of the largestdetected plasmid pBMB 165 and the smallest plasmid pBMB 175 of strain YBT-1765 werecharacterized. The research results were summarized as following:1. Characterization of pBMB165 repliconA 20kb DNA fragment containing plasmid pBMB165 replicon was isolated fromBacillus thuringiensis subsp, tenebrionis YBT-1765 and characterized. Deletion analysisrevealed that a replication initiation protein (Rep165), an origin of replication (ori165)and an iteron region were required for replication. In addition, two overlapping ORFs(ORF6 and ORF10) were found to be key elements for the stability control of plasmidreplication. As far as we know, the replicon of the plasmid pBMB 165 is the first instanceof a plasmid replicon being isolated from subsp. tenebrionis and characterized.Sequence comparison showed that the replicon of pBMB165 was homologous toreplicons of the pAMβ1 family, indicating that pBMB165 replicon belongs to this family.Furthermore, the pAMβ1 family replicons that have so far been identified from the B.cereus group (pBMB165, pBT9727, pXO2 and pAW63) have been shown to share thesame organization; however, this organization scheme is distinctly different from the onefound in the pAMβ1 family replicons that are of other origin. It was suggested thatgenetic variability is present among the pAMβ1 family replicons.There were five transposable elements or remnants thereof in close proximity to andwithin the replicon control region. An IS231-like element was identified and namedIS231U; ISBth165, ISBth166 and ISBth167 were tentatively classified as belonging to theIS5, IS110 and IS3 family of transposable elements, respectively, on the basis of theirsequence similarities; TnBMB165 was a remnant classⅡtransposon. The presence ofthese transposable elements or remnants provided the useful genetic information and ledus to speculate that genetic exchange and recombination are potentially responsible forthe divergence among the replicons of the pAMβ1 family.2. Screening of plasmid-cured mutant of YBT-1765By gradually elevating growth temperature and adding sodium dodecyl sulfate (SDS)as the plasmid-curing agent, we obtained two partially plasmid-cured mutant ofYBT-1765. In which, BMB193 harbored two large plasmids (plasmid pBMB165 wascured) and BMB175 harbored only the smallest plasmid pBMB175. 3. Cloning and characterization analysis of pBMB175A cryptic plasmid pBMB175 was the smallest from Bacillus thuringiensis subsp.tenebrionis YBT-1765 was isolated and characterized. Sequence analysis showed thatpBMB175(1, 4841 bp) contained at least eighteen putative open reading frames (ORFs),among which nine ORFs displayed the homology with the hypothetical proteins inrolling-circle replication plasmid pGI3.Deletion analysis revealed that the pBMB175 minireplicon located in a novel 1,151bpfragment. This fragment contains ORF7 coding sequence, which encodes a protein(Rep175, 149 amino acids [aa]) indispensable for plasmid replication. Rep175 has not anysignificant similarity with known proteins. Furthermore, a putative double strand origin(dso), having no DNA similarity to characterized dso of other replicon so far, wasidentified in this mini-replicon fragment. These features showed that pBMB175 could beplaced into a new plasmid family.