Identification of a replicon from pCC3, a cryptic plasmid from Leuconostoc citreum C4 derived from kimchi, and development of a new host-vector system.

Analysis of the structural properties of pCC3, a cryptic plasmid from Leuconostoc citreum C4 isolated from kimchi, determined its length as 3,338 bp and revealed three open reading frames (ORFs): ORF1-ORF3. ORF3 showed high homology with a replication initiation protein of the theta-type plasmid pTXL1. The fragment encompassing ORF3 and its upstream sequences (nt 1,299-1,634) was found to contain a functional plasmid replicon. A new shuttle vector, pUCC3E1, was constructed based on pCC3. Using Southern hybridization analysis, no single-stranded DNA intermediate was detected from Leu. citreum harboring pUCC3E1, which indicates that pCC3 replicated via the theta mechanism. The pUCC3E1 could be replicated in E. coli TG1 (5.8 x 10(4) CFU/microg DNA) and the developed cloning hosts, Leu. citreum C16 (2.1 x 10(2) CFU/microg DNA) and Leu. citreum GJ7 (8.0 x 10(1) CFU/microg DNA). pUCC3E1 was stably maintained in Leu. citreum C16 (for 100 generations, ca. 94.2%) in the absence of erythromycin (5 microg/ml).