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Characterization of four plasmids harboured in a Lactobacillus brevis strain encoding a novel bacteriocin, brevicin 925A, and construction of a shuttle vector for lactic acid bacteria and Escherichia coli

¹ Department of Molecular Microbiology and Biotechnology, Graduate School of Biomedical Sciences, Hiroshima University, Kasumi 1-2-3, Minami-ku, Hiroshima 734-8551, Japan
² Frontier Center for Microbiology, Hiroshima University, Kasumi 1-2-3, Minami-ku, Hiroshima 734-8551, Japan
³ Hiroshima Prefectural Food Technology Research Center, Hijiyama-Honmachi 12-70, Minami-ku, Hiroshima 732-0816, Japan

In this study we isolated over 250 lactic acid bacteria (LAB) candidates from fruit, flowers, vegetables and a fermented food to generate an LAB library. One strain, designated 925A, isolated from kimchi (a traditional Korean fermented dish made from Chinese cabbage) produced a novel type of bacteriocin, brevicin 925A, which is effective against certain LAB, including strains of Lactobacillus, Enterococcus, Streptococcus, Bacillus and Listeria. Strain 925A, identified as Lactobacillus brevis, harboured at least four plasmids and we determined the entire nucleotide sequence of each one. The four plasmids were designated pLB925A01–04, and 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. All of these derivatives, which were susceptible to their own antibacterial product, lost the largest plasmid, pLB925A04, suggesting that the genes for bacteriocin biosynthesis (breB and breC) and immunity (breE) are located on pLB925A04. The partial amino acid sequence of purified brevicin 925A and sequence analysis of pLB925A04 showed that breB is the structural gene for brevicin 925A. We constructed a shuttle vector (pLES003, 6134 bp) that can replicate in both Escherichia coli and LAB such as Lactobacillus plantarum, Lb. brevis, Lactobacillus helveticus, Lactobacillus hilgardii and Enterococcus hirae. To determine the function of gene breE, which displays no significant similarity to any other sequences in the BLAST search database, the gene was inserted into pLES003. A pLB925A04-cured derivative transformed with pLES003 carrying breE acquired immunity to brevicin 925A, suggesting that breE encodes an immunity protein.

Correspondence
Masanori Sugiyama
sugi{at}hiroshima-u.ac.jp

The GenBank/EMBL/DDBJ accession numbers for the sequences reported in this paper are AB370334, AB370335, AB370336, AB370337 and AB370338.