Characterization of the indigenous plasmid pvy103 of Bacillus megaterium

Includes bibliographical references (pages [89]-97)The indigenous plasmid pVY103 of Bacillus megaterium QM B1551 was partially characterized in this study. It was 5.5 kb in size, and was mapped by restriction endonuclease digestion. Two previously cloned fragments from this plasmid (the origin fragment and the fusiform fragment, both can autonomously replicate) were shown to overlap. A shuttle plasmid was constructed that contained pVY103 combined with Escherichia coli plasmid pUC18 and a neomycin resistance gene, and was functional in both E. coli and B. megaterium. Plasmid stability assays revealed the instability of pVY103 when pUC18 was inserted into it. Moreover, sporulation efficiency tests using strains containing different combinations of plasmid origins suggested that the origin from plasmid pVY103 might interfere with host sporulation. This effect could be reduced when this origin coexisted with a theta replication origin. DNA sequencing of the 2.3-kb origin fragment of pVY103 revealed two putative ORFs with little homology to known sequences. ORF 1 could encode a protein of 298 amino acids with a molecular weight of 34,505 and a pi of 9.51. ORF 2 could encode a protein of 110 amino acids with a molecular weight of 12,912 and a pi of 10. A 25-bp region was found within the 5' end of ORF 1 to have 76% identity to the consensus sequence of the plus strand origin of the pUB110/pC194 family, which replicates by the rolling circle mechanism. A aA promoter region and a ribosomal binding site were found preceding ORF 1. No such sequences could be identified preceding ORF 2. No rho-independent transcription terminator could be found following ORF 1 or ORF 2. Subcloning showed that ORF1 was necessary for replication. In addition, a surprising number of direct and inverted repeats was found spanning the entire origin fragment, with a very low probability (< 0.0001) according to statistical analysis that these would be found by chance. These results suggest that pVY103 replicates by the rolling circle mechanism, and that the cloned origin of this plasmid interferes with host sporulation.M.S. (Master of Science