Atypical genetic locus associated with constitutive production of enterocin B by Enterococcus faecium BFE 900

A purified bacteriocin produced by Enterococcus faecium BFE 900 isolated fr om black olives was shown by Edman degradation and mass spectrometric analy ses to be identical to enterocin B produced by E. faecium T136 from meat (P . Casaus, T. Nilsen, L. M. Cintas, I. F. Nes, P. E. Hernandez, and H. Holo, Microbiology 143:2287-2294, 1997). The structural gene was located on a 2. 2-kb HindIII fragment and a 12.0-kb EcoRI chromosomal fragment. The genetic characteristics and production of EntB by E. faecium BPE 900 differed from that described so far by the presence of a conserved sequence like a regul atory box upstream of the EntB gene, and its production was constitutive an d not regulated. The 2.2-kb chromosomal fragment contained the hitherto und etected immunity gene for EntB in an atypical orientation that is the rever se of that of the structural gene. Typical transport and other genes associ ated with bacteriocin production were not detected on the 12.0-kb chromosom al fragment containing the EntB structural gene. This makes the EntB geneti c system different from most other bacteriocin systems, where transport and possible regulatory genes are clustered. EntB was subcloned and expressed by the dedicated secretion machinery of Carnobacterium piscicola LV17A. The structural gene was amplified by PCR, fused to the divergicin A signal pep tide, and expressed by the general secretory pathway in Enterococcus faecal is ATCC 19433.