Characterization of the nisFEG operon of the nisin Z producing Lactococcus lactis subsp. lactis N8 strain.

Abstract

Biosynthesis of the food additive nisin, a posttranslationally modified peptide antibiotic existing as two natural variants (A and Z), requires eleven genes (nisA/ZBTCIPRKFEG) involved in modification, secretion, regulation and self-immunity. The suggested self-immunity genes (nisFEG) of the nisin Z producer Lactococcus lactis subsp. lactis N8 were cloned and sequenced. Putative binding sites of the NisR transcription factor were recognized upstream of the nisF promoter. The hydrophilic NisF protein was expressed in Escherichia coli and shown to be associated with the membrane. Expression of the nisF gene from a plasmid in L. lactis MG1614, a strain lacking the nisin operons, did not increase the nisin resistance of the cells. This showed that NisF alone does not protect against nisin. Overexpression of the nisF gene in the N8 nisin producer did not affect the level of nisin immunity, indicating that the wild-type amount of NisF is not limiting the level of nisin immunity. Production of antisense-nisEG or antisense-nisG RNA in L. lactis N8 resulted in severe reduction in the level of nisFEG mRNA and a clearly reduced immunity showing that the nisFEG transcript is important for development of nisin self-immunity.