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

Abstract

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 ofLactobacillus,Enterococcus,Streptococcus,BacillusandListeria. Strain 925A, identified asLactobacillus 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 (breBandbreC) and immunity (breE) are located on pLB925A04. The partial amino acid sequence of purified brevicin 925A and sequence analysis of pLB925A04 showed thatbreBis the structural gene for brevicin 925A. We constructed a shuttle vector (pLES003, 6134 bp) that can replicate in bothEscherichia coliand LAB such asLactobacillus plantarum,Lb. brevis,Lactobacillus helveticus,Lactobacillus hilgardiiandEnterococcus hirae. To determine the function of genebreE, which displays no significant similarity to any other sequences in theblastsearch database, the gene was inserted into pLES003. A pLB925A04-cured derivative transformed with pLES003 carryingbreEacquired immunity to brevicin 925A, suggesting thatbreEencodes an immunity protein.