Biochemical and genetic evidence for production of enterocins A and B by Enterococcus faecium WHE 81

Enterococcus faecium WHE 81, isolated from cheese, has been reported to pro duce a bacteriocin called "enterocin 81" [J. Appl. Microbiol. 85 (1998) 521 .]. Purification of "enterocin 81" was carried out using ammonium sulfate p recipitation, desalting on ODP-90 reverse-phase column, and purification th rough SP Sepharose HP cation exchange and C-2/C-18 reverse-phase chromatogr aphies. The antimicrobial was eluted from the C-2/C-18 column as four indiv idually active fractions, designated ASI, B81, C81 and D81. The purificatio n procedure used proved particularly efficient for the bacteriocin in fract ion D81, with a yield of 46%, while only 3.8% the bacteriocin in fraction B 81 could be collected. MALDI-TOF mass spectrometry of the bacteriocins in f ractions B81 and D81 showed respective masses of 4833.0 and 5462.2 Da. Amin o acid sequencing of the two peptides revealed two class-II bacteriocins wh ose sequences were similar to those of enterocin A and enterocin B, respect ively. Using proper primers, chromosomal fragments of 212 and 216 bp enclos ing bacteriocin structural genes were PCR-amplified. Cloning of the amplico ns and their sequencing revealed two genes with sequences identical to the structural genes of enterocins A and B, respectively. It was therefore clea rly established that E. faecium WHE 81 produces bacteriocins respectively i dentical to enterocins A and B. Our results, combined with data from previo us reports, suggest that the two bacteriocins may be widespread among enter ococcal strains and may play an important role in controlling the growth of pathogens and other undesirable bacteria in certain fermented food product s. (C) 2001 Elsevier Science BN. All rights reserved.