The Resident Plasmid May Contribute To The Phenomenon Of Spore-crystal Connection In Bacillus Thuringiensis Subsp. Finitimus

Bacillus thuringiensis subsp finitimus YBT020 was isolated, which harbored three resident plasmids. YBT020 produced at least two parasporal inclusions. One inclusion formed somewhat later exterior to the exosporium. Another inclusion was formed within the exosporium and remained with the spore after mother cell lysis.In this work, the relationship between the phenomenon of spore-crystal connection and the plasmids was investigated. 75 colonies curing of the gene cry26Aα, and 19 colonies curing of both cry26Aα and cry28Aα were obtained from 2162 colonies by PCR amplification. Mutants BMB1151 curing of the plasmid harboring cry26Aα, and BMB1152 curing of all plasmids were obtained. Then the morphological, physiological, biochemical and SDS-PAGE diversities between wild-type strain YBT020 and its mutants BMB1151, BMB1152. The spore-crystal connection didn't appear when cry26Aα and cry28Aα were transformed back into the strain BMB1152 by shuffle vector alone or in combination together. It was suggested that the plasmids of YBT020 contribute to the phenomenon. When gene cry26Aα was transformed back into in the strain BMB1151, spore-crystal connection didn't form either. So the plasmid harboring cry26Aα may contribute to the phenomenon of spore-crystal connection in B. thuringiensis subsp finitimus YBT020.The results was indicated that the direction for our further investigation as well as providing a good material on study of the molecular mechanism of spore-crystal connection in Bacillus thuringiensis subsp finitimus。Transformation by electroporation was often used in the gene engineering of Bacillus thuringiensis. But this method had some disadvantages; this method easily arose to some mutant, such as plasmid cured. And it was very difficult to transformate large plasmid to the wild strains. The mobilizable shuttle plasmid pBMB0951, carrying the OriT site of (IncP) RK2 was constructed. Conjugal transfer pBMB0951 was performed from E. coli to BMB171 by the strain E.coli S17-1 or the helper plasmid pRK2073. Results show that the plasmid carrying the OriT site of (IncP) RK2 was able to be transfered from E. coli to Bacillus thuringiensi. So it was possible to transferring the plasmid to wild strains of B. thuringiensis by conjugation.