Characterization of pMRI 5.2, a rolling-circle-type plasmid from Lactobacillus plantarum BFE 5092 which harbours two different replication initiation genes.

Plasmid pMRI 5.2 from Lactobacillus plantarum BFE 5092 was sequenced and analysed. The sequence consists of 5206bp with a mol% G+C content of 35.8%. Nine putative open reading frames were identified. A typical pC194 family double strand origin (dso) and a putative single strand origin (sso) were predicted upstream of a rep gene. This rep gene encoded a replication protein of 314 amino acids exhibiting 98% amino acid sequence identity to the Rep protein of plasmid pLAB1000 from Lactobacillus hilgardii. A mob gene encoding a mobilization protein was also identified and this protein showed high amino acid similarity to Mob proteins from various L. plantarum plasmids. Downstream of the mob gene, a second putative replication region was identified that is similar to the pMV158 family of plasmids. It contains a dso as well as a putative sso, and encodes the 52 amino acid repressor-like protein RepA, the replication initiation protein RepB of 215 amino acids, and the 48 amino acid RepC that is similar to ORFD of the lactococcal plasmid pWVO1. RT-PCR and qRT-PCR expression analyses of the rep and repB genes showed that the repB gene was expressed at a higher level. To confirm that the plasmid replicated by the rolling-circle-type mechanism, the presence of a characteristic single strand intermediate DNA was shown to be produced during replication. Plasmid copy number was ca. 30 per equivalent chromosome copy number based on qRT-PCR analyses. The plasmid also encodes four additional putative proteins of unknown function. The unusual feature of a rolling-circle plasmid having two different plasmid-encoded replication initiation proteins from different replicon families suggests that the genes for these may have originated from different plasmids.