Establishment of a new replicon generated from an integrational plasmid and a cryptic pUB110 origin-like region in Bacillus subtilis.

A Bacillus subtilis integrational plasmid pVG4 (7.3 kb) was constructed. It was composed of a part of pBR322 (Ap, OriC), a part of pUB110 (Nm), and a part of the spoOA gene. The origin region of pUB110 had been deleted over 1.4 kb. Surprisingly, the replicative plasmids (8 kb) were generated by transformation of B. subtilis BG83 with pVG4 on selection with Nm. Analysis of the total DNA indicated the presence of repeated DNA and that pVG4 did not appear to integrate at the spoOA locus. The new replicon termed pYV exhibited a quite different restriction pattern from pVG4 due to dramatic DNA rearrangements. Although some components initially present in pVG4 were either present in pYV (Ap, oriC, and Nm) or lost (spoOA), other ones were newly gained such as a chromosomal fragment and a pUB110-type origin-like region. The latter was identified by restriction sites mapping and limited sequence analyses. By PCR amplification, the origin region of pYV was shown to be present as a cryptic sequence in the chromosome of strain BG83. The chromosomal fragment integrated into pYV was at least 0.25 kb long and located in a 19-kb SfiI fragment mapping at 106 degrees. We propose that the establishment of the new replicon pYV is the result of a genetic recombination between the pUB110 part present in the integrational plasmid pVG4 and the cryptic origin region of pUB110 harbored in the chromosome of the recipient strain BG83 in relation with a particular role of neomycin selection.