Cloning and expression of the genes involved in the production of and immunity against the bacteriocin lacticin RM

The production of lacticin RM, a novel bacteriocin produced by Lactococcus lactis subsp. lactis EZ26, is associated with the presence of a 6-kb plasmi d, pHU1. The information necessary for lacticin RM production and immunity was localized to a 2.5-kb SalI-Eco47III fragment. Sequencing analysis of th is fragment revealed the presence of six open reading frames (ORFs), Deleti on and mutation analyses showed that orfX and orfY are not required for lac ticin RM production or immunity, whereas the other ORFs (lacA, lacF, lacG a nd lacI) are necessary for the bacteriocin's production. Transcription anal ysis indicated that lacA, lacF and lacG are organized in an operon. lacA is probably the lacticin RM structural gene. It putatively encodes a 134-amin o acid peptide, and it does not share homology with known bacteriocins. The deduced LacG protein is hydrophobic and consists of six potential trans-me mbrane helices. lacF encodes a conserved ATP-binding domain homologous to A BC transporters known in bacteriocin immunity systems. LacF and LacG may fo rm an active ABC transporter. Gene-disruption mutations indicated that both are required for immunity against lacticin RM. lacI encodes a small cation ic protein, which is required for the production of and immunity to lactici n RM. Protection was obtained only when lacF, lacG and lacI were present to gether. (C) 2000 Elsevier Science B.V. All rights reserved.