Both methods yielded similar results with estimated copy number of 154–170 copies/cell and of 56–60 copies/cell for pMyBK1 and pMG2B-1, respectively (Figure 5B). Such a difference strongly suggests that the two plasmids have distinct replication and /or regulation systems. Together the 2 M. yeatsii plasmids represent a total extrachromosomal DNA amount of 636 kbp per cell, which is approximately 37% of the total cell DNA. Next, the genetic structure of pMyBK1 was analyzed. The 2 CDSs found in the pMyBK1 sequence (CDSA and B, encoding polypeptides of respectively 519 and 272 aa) showed no homolog

with other mycoplasma plasmids (Figure 2A). The presence of a 192-bp intergenic region buy YM155 between the CDSs as well as the predicted rho-independent

transcription terminator immediately downstream of each CDS strongly suggests that the 2 CDSs are transcribed independently rather than as a single operon. The deduced amino acid sequence of pMyBK1 CDSA exhibits low but significant similarity with mobilization proteins of various bacteria. The N-terminal part of the CDSA protein contains a Mob/Pre domain (pfam01076) typical for relaxases of the MobV superfamily that includes proteins involved in conjugative mobilization and plasmid intramolecular recombination [49]. Sequence alignments with representatives of the MobV family clearly showed that the CDSA protein did possess the three conserved motifs of the family [50] (data not shown). Subsequent phylogenetic analyses

of the CDSA polypeptide with the complete set of MobV proteins described see more by Garcillan-Barcia [51] classified the pMyBK1 protein Florfenicol mTOR inhibitor within the MobV4 relaxase family (data not shown). In contrast to CDSA, no functional domain or characteristic secondary structure was identified in the CDSB-encoded protein. Blast searches revealed that the CDSB protein of pMyBK1 shared significant homology with five chromosome-encoded proteins of Mcc, strain California Kid, or M. leachii, strain PG50 and 99/014/6 but with no known associated function. Identification of the replication protein and the mode of replication of pMyBK1 Since none of the pMyBK1-encoded proteins share homology to known replication proteins, CDSA and CDSB were both regarded as putative candidates. To identify the replication protein and delineate the replication region of pMyBK1, a series of deletion and frameshift mutations were introduced in a shuttle plasmid (E. coli/M. yeatsii), named pCM-H, that was constructed by combining pMyBK1 to a colE1 replicon carrying the tetM tetracycline resistance gene as the selection marker (Figure 2A). The mutated plasmids were then introduced into a plasmid-free M. yeatsii strain (#13156 from the Anses collection) by PEG-transformation, and their replication capacity was measured by the number of resulting tetracycline resistant colonies.