Characterization of pFMBL1, a small cryptic plasmid isolated from Leuconostoc mesenteroides SY2

Abstract

A 4661 bp cryptic plasmid, pFMBL1, was isolated from Leuconostoc mesenteroides SY2, an isolate from Kimchi, and characterized. Nucleotide sequence analysis revealed two open reading frames, orf1 and orf2. orf2 was 453 bp in size and its translation product had 58% identity with a putative protein possibly involved in the replication of pTXL1, a cryptic plasmid from L. mesenteroides ssp. mesenteroides Y110. RNA transcript from orf2 was detected but not from orf1 or intergenic region. Minimum 3.5 kb fragment encompassing orf1 and orf2 was required for the replication of pFMBL1 and employed for the construction of Escherichia coli-Leuconostoc shuttle vector, pSJ33E. L. mesenteroides SY1 (another Kimchi isolate), Leuconostoc ssp., and Lactobacillus brevis were successfully transformed with pSJ33E, and the transformation efficiencies were ranged between 1.1 × 10 1 and 4 × 10 5 transformants/μg DNA. No single-stranded DNA intermediate was detected from L. mesenteroides SY1 cells harboring pSJ33E, indicating that pFMBL1 probably replicated via θ-type mechanism. pSJ33E was stably maintained in L. mesenteroides SY1 in the absence of erythromycin (Em, 5 μg/ml) and after 1 month of daily subculturing in MRS broth without selective pressure, three percent of cells still retained pSJ33E.