Characterization of Bacteriocin N15 Produced by Enterococcus faecium N15 and Cloning of the Related Genes.

Abstract

Enterococcus faecium N15 was isolated from nuka (Japanese rice-bran paste), which is utilized as starter in the fermenting of vegetables, and was found to produce a bacteriocin that exhibited a broad spectrum of activity, including activity against Listeria monocytogenes and Bacillus circulans JCM2504. The bacteriocin was sensitive to proteases (alpha-chymotrypsin, proteinase K, trypsin, and pepsin) and alpha-amylase, but it was resistant to lipase. The bacteriocin was resistant to heat treatment at 100 degrees C for 2 h, but its activity was completely lost after autoclaving at 121 degrees C for 15 min. It was active over a wide pH range from 2.0 to 10.0. The bacteriocin showed bactericidal activity against Lactobacillus sake JCM1157 at a concentration of 40 AU/ml. Its molecular weight was estimated by SDS-PAGE to be about 3-5 kDa. PCR primers were designed based on the conserved amino acid sequences of class IIa bacteriocins. A 3-kb DNA fragment was amplified and three open reading frames (ORFs) were found. The first encodes a probable immunity protein of 103 amino acid residues and shows complete homology with the putative immunity protein of E. faecium DPC1146. The second and third ORFs respectively encode a probable transposase gene and an inducing factor. The upstream region of the immunity gene, in which the bacteriocin structural gene is located, was amplified. A homology search revealed that the bacteriocin produced by E. faecium N15 exhibits complete identity to enterocin A, a bacteriocin produced by E. faecium DPC1146. PCR using the primers designed in this study is a rapid and sufficient method of screening for bacteriocin-producing strains.