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Characterization of four plasmids harbored in a Lactobacillus brevis strain encoding the new bacteriocin brevicin 925A and construction of a shuttle vector between lactic acid bacteria and E. coli

¹ Hiroshima University;
² Hiroshima Universiry;
³ Hiroshima Prefectural Food Technology Research Center

ABSTRACT

In the present study, we isolated over 250 lactic acid bacteria (LAB) candidates from fruits, flowers, vegetables, and a fermented dish to generate a LAB library. We noticed that a strain, designated 925A, isolated from Kimchi made from Chinese cabbage, a traditional Korean fermented dish, produces a new type of bacteriocin, which was designated as brevicin 925A, and is effective against certain LAB strains, including Lactobacillus, Enterococcus, Streptococcus, Bacillus, and Listeria. The 925A strain, which was identified as Lactobacillus (Lb.) brevis, harbored at least four plasmids. We determined the entire nucleotide sequence of the four plasmids in 925A. These plasmids, designated pLB925A01, pLB925A02, pLB925A03, and pLB925A04, have molecular sizes of 1815, 3524, 8881, and 65 037 bp, respectively. We obtained bacteriocin-non-producing derivatives by treatment of strain 925A with novobiocin. The all derivatives, which were susceptible to their own antibacterial product, lost the largest plasmid, pLB925A04, suggesting that the genes for the bacteriocin-biosynthesis (breB and breC) and immunity (breE) are located on pLB925A04. The partial amino acid sequence of the purified brevicin 925A and sequence analysis of pLB925A04 showed that breB was the structural gene for brevicin 925A. We constructed a shuttle vector (6134 bp), designated pLES003, which can be replicated between E. coli and LAB, such as Lb. plantarum, Lb. brevis, Lb. helveticus, Lb. hilgardii, and Enterococcus hirae. To make up clear the function of breE that displays no significant similarity on the BLAST search database, the gene was inserted into pLES003. The pLB925A04-cured derivative transformed with pLES003 carrying breE acquired immunity to brevicin 925A, suggesting that breE encodes an immunity protein.

⁴ E-mail: sugi{at}hiroshima-u.ac.jp