Studies of producer self-protection and nisin biosynthesis of Lactococcus lactis

Just before the discovery of penicillin by Fleming, reports in the literature appeared that described potent antimicrobial substances produced by lactic acid bacteria. It was found that these substances were specifically active against a wide range of other gram-positive bacteria. These characteristics meant that these compounds were attractive candidates for application in either food preservation e.g. by preventing food spoilage or by inhibiting growth of food pathogens, or for pharmaceutical use, e.g. to prevent or fight infections in humans or animals. In the late sixties and early seventies, it was shown that part of the inhibitory activity of some lactic acid bacteria was due to the production of a special class of antimicrobial peptides today called lantibiotics. After the finding that lantibiotics are ribosomally synthesized peptides, a wealth of novel information was generated in the late eighties and early nineties by sequencing several of the gene clusters involved in the biosynthesis of various lantibiotics including the gene clusters for nisin. The biological system behind production, secretion and self-defence of nisin has proven to be both extensive and delicately regulated, requiring the interplay between at least eleven genes, namely nisA/Z/QBTCIPRKFEG. In this work regulation and biosynthesis of nisin was studied and the role of NisI in nisin immunity evaluated. In order to analyse the biosynthesis of nisin, mutations in the nisZ, nisB and nisP genes of the nisZBTCIPRK operon were made by gene replacement or with the integration of a plasmid. The mutations caused a drastic decrease of the transcription from the promoters upstream of the nisZBTCIPRK and nisFEG operons resulting in loss of nisin production and nisin immunity. Adding nisin externally to these mutant strains partly restored the transcriptional activity and the tolerance to nisin, but not the ability to produce active nisin. Our results show that the nisZBTCIPRK operon and the nisFEF operon are positively autoregulated via nisin and are in the same regulon. The role of NisI protein in the nisin immunity was studied by protein expression, labelling, immuno blotting and in vitro interaction methods. The results showed that NisI could exist in two forms i.e. a membrane-bound and a soluble form. NisI can interact in vitro with purified nisin and might have two biological functions-as an immunity protein and as an enhancer of the activity of nisin. The function of NisP for the maturation of nisin was studied by analysing the nisP mutant strain. It was shown …