Characterization of the Replicon Region of the Enterococcus faecium Plasmid pEV105 Potentially Used in the Construction of Cloning Vector

摘要

A replication probe vector (pUB380) was constructed for detecting the replicon of LAB plasmid. A cryptic plasmid, pEVlO5, was isolated from Enterococcus faecium KLDS 6.0718. Multiple restriction endonucleasc fragments of pEV105 were separately ligased to pUB380. After a series of subcloning and elcctrotransformation, the minimal replicon of pEV 105 was isolated on a 2.5 kb Pst I- Xbal I fragment. Replicon based on this region followed a theta-type mechanism of replication in Enterococcus faecium KLDS 6.0718. The minimal replicon DNA fragment was sequenced and the result shows that the gene sequence homology has 99.8 % compared with partial sequence of pCIZ2 from Enterococcus faecium L50. Five putative ORFs were concluded by software. The result of alignments indicated two ORFs, repA105 and repB105, encoding two putative proteins RepA105 and RepB105 of 245 and 178 amino acids respectively. The 2.5 kb fragment of minimal replicon as a stable replicon is feasible for constructing food-grade cloning and expressing vector of lactic acid bacteria.