Therefore, replication of all mycoplasma plasmids is likely to be driven through a rolling-circle mechanism by a Rep protein of the pMV158 family type. Mosaic structure of the mycoplasma plasmids is indicative of recombination events In spite of a conserved structure, multiple pair-wise DNA sequence comparisons indicated that mycoplasma plasmids are in fact a mosaic of rep, dso, copG, and sso blocks. This was evidenced by the occurrence of several local regions of homology detected by using the BLAST program (Figure 5). Pairs of plasmids that show a high level of identity for the Rep sequence (e.g. pKMK1 and check details pMG1B-1; pMG2D-1 and pMG2B-1) do not necessarily share a high degree of identity
for the region upstream of copG. Interestingly, high sequence identity for the region spanning sso was found to be indicative of plasmids being hosted by the same mycoplasma species. For instance, the following plasmid-pairs, pADB201 and pKMK1, pMG1B-1 and pMG2D-1, and pMG2B-1 and pMG2F-2 were isolated from Mmc, Mcc, and M. yeatsii, respectively (Figure 5). This result is consistent
with the fact that during replication this region interacts with chromosome-encoded components . Further degrees of mosaicism were found in particular cases such as for pMG2D-1, in which two putative dso showing sequence heterogeneity are found. Other BMS202 examples of genetic variability are the small size of pBG7AU and the unusual location of the dso in pMG2F-2. Such a mosaic structure is clearly indicative of successive recombination
events between replicons. Figure 5 Analysis of plasmid Rabusertib supplier content of Mycoplasma yeatsii type strain GIH (TS). A. Agarose gel electrophoresis of total DNA. Lanes were loaded after twofold dilution series of the DNA preparation obtained as described in Methods. Bands corresponding to the chromosome and the 2 plasmids are identified. Lane M, DNA ladder. B. Estimated plasmid copy number of pMyBK1 and pMG2B-1 as estimated by gel assay (Panel A) and relative real-time PCR as described in Methods. pMyBK1 is a unique representative of a new replicon family As indicated above, M. yeatsii strain GIH TS was the only strain that yielded a banding pattern of extrachromosomal DNA that suggested the presence of two distinct Lck plasmids (Figure 5A). The small plasmid, pMG2B-1, was shown to belong to the pMV158 family like all other mycoplasma plasmids (Figure 3). In contrast, the larger plasmid (3,432 bp) named pMyBK1 (GenBank Accession number EU429323; ) has a genetic organization that sets it apart from the other mycoplasma plasmids. Initial database searches using pMyBK1 sequence as a query indicated low identity with other plasmids and prompted us to further analyze this plasmid that might represent a new family of replicons. First, the relative copy number of each plasmid of M.