Isolation and characterization of pBM400 : a 53 kb plasmid from Bacillus megaterium QM B1551
Scholle, Michael D.
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Bacillus megaterium QM B1551 has seven indigenous plasmids ranging from 5.5 kb to 165 kb. Our laboratory is studying the role of these plasmids in the cell. We have cloned, sequenced, and characterized five replicons. The fourth largest plasmid, pBM400, has been partially characterized with respect to replication and 18.6 kb sequenced. The sequenced region, designated rrn-clone II, harbors a ribosomal RNA operon (6.3 kb) and a replication clone (12.3 kb). An Xbal fragment of mi-clone II is unique to pBM400. To further characterize pBM400, the plasmid must be isolated into a plasmidless strain. Since the wild-type pBM400 does not contain a phenotypic marker, a kanamycin gene (from pDG792) was introduced into the plasmid by first inserting the Xbal fragment and a temperature-sensitive origin of replication (pE194ts) into the vector pJM103 (CmR). A kanamycin gene was then inserted into the Xbal fragment to generate pSMl, which was transformed into the wild-type strain (seven plasmids) selecting for kanamycin resistance. A transformant was grown at 46°C with kanamycin selection to select for homologous recombination into pBM400. A colony displaying KmR Cms suggested that a double crossover had occurred, thus integrating only the kanamycin gene into pBM400. The integration of the kanamycin gene was further verified by polymerase chain reaction (PCR) and Southern hybridization. The pBM400::kan was then transformed into PY361, a plasmidless strain of B. megaterium. The resultant transformant, PV627 (KmR), was shown to contain only pBM400::kan by agarose gel electrophoresis, PCR, and Southern hybridization analyses. To further analyze the isolated plasmid, restriction analysis of pBM400 was performed with various enzymes. A total size of approximately 53 kb without the kanamycin gene was found. Additionally, partial libraries of EcoRI and Pstl fragments of pBM400 in pJM103 were constructed. Subsequently, all ends of these fragments were sequenced and a restriction map was constructed. Sequence analysis revealed similarities to genes for copper/cadmium resistance, styrene degradation, transposase, integration/recombination, and also to unknown genes from the B. subtilis genome. PV627 was also tested for the presence of a second homologous replicon, as well as for megacin (bacteriocin) production and resistance, neither of which were found on pBM400.