Construction of a Novel Shuttle Vector for species based on a Cryptic Plasmid from .

A cryptic plasmid (pTH32) was characterized from 32, an isolate from jeotgal, Korean traditional fermented seafood. pTH32 is 3,198 bp in size with G+C content of 35.84%, and contains 4 open reading frames (ORFs). and are 456 bp and 273 bp in size, respectively, and their translation products showed 65.16% and 69.35% similarities with RepB family plasmid replication initiators, respectively, suggesting the rolling-circle replication (RCR) mode of pTH32. and encodes putative hypothetical protein of 186 and 76 amino acids, respectively. A novel shuttle vector, pMJ32E (7.3 kb, Em), was constructed by ligation of pTH32 with pBluescript II KS(+) and an erythromycin resistance gene (ErmC). pMJ32E successfully replicated in 29212 and 31 but not in other LAB species. A gene, encoding aminopeptidase A (PepA) from CY54, was successfully expressed in 31 using pMJ32E. The transformant (TF) showed higher PepA activity (49.8 U/mg protein) than 31 cell (control). When 31 TF was subculturd in MRS broth without antibiotic at 48 h intervals, 53.8% of cells retained pMJ32E after 96 h, and only 2.4% of cells retained pMJ32E after 14 days, supporting the RCR mode of pTH32. pMJ32E could be useful for the genetic engineering of and species.