SEQUENCE AND ANALYSIS OF THE 60 KB CONJUGATIVE, BACTERIOCIN-PRODUCINGPLASMID PMRC01 FROM LACTOCOCCUS-LACTIS DPC3147

The complete sequence of pMRC01, a large conjugative plasmid from Lact ococcus lactis ssp, lactis DPC3147, has been determined, Using a shotg un sequencing approach, the 60 232 bp plasmid sequence was obtained by the assembly of 1056 underlying sequences (sevenfold average redundan cy), Sixty-four open reading frames (ORFs) were identified, Analysis o f the gene organization of pMRC01 suggests that the plasmid can be div ided into three functional domains, with each approximately 20 kb regi on separated by insertion sequence (IS) elements. The three regions ar e (i) the conjugative transfer region, including a 16-gene Tra (transf er) operon; (ii) the bacteriocin production region, including an opero n responsible for the synthesis of the novel bacteriocin lacticin 3147 ; and (iii) the phage resistance and plasmid replication region of the plasmid, The complete sequence of pMRC01 provides important informati on about these industrially relevant phenotypes and gives insight into the structure, function and evolution of large Grampositive conjugati ve plasmids in general. The completely sequenced pMRCO1 plasmid should also provide a useful framework for the design of novel plasmids to b e incorporated into starter strain improvement programmes for the dair y industry.