ABSTRACT
The antibiotic peptide nisin produced by Lactococcus lactis is used as a food preservative due to its activity against spores and vegetative cells of Gram-positive bacteria. The post-translational maturation of this secreted peptide includes dehydration of serine and threonine residues, lanthionine formation and a proteolytic processing of 23 amino acids from the N-terminus. Mutations in the nisZ, nisB and nisP genes of the biosynthetic nisZBTCIPRK nisin operon were made by gene replacement or 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. The transcription of the nisin operons and nisin immunity could be partially restored by adding nisin to the growth medium of the cells. Nisin induction of the mutant strains also increased the level of the putative immunity NisI protein. These results showed that the nisZBTCIPRK operon is positively autoregulated and that the nisFEG operon is in the same regulon.
Subject(s)
Gene Expression Regulation, Bacterial , Lactococcus lactis/genetics , Nisin/genetics , Operon , Cloning, Molecular , Mutagenesis , PhenotypeABSTRACT
Lactococcus lactis cells secreting the lantibiotic nisin, commercially used for food preservation, must protect their cell membrane against the pore-forming activity of extracellular nisin. The nisI gene product has been suggested to be a lipoprotein, which due to the location on the extracellular surface would be an ideal candidate for an immunity protein. In vivo labelling of NisI from L. lactis N8 expressed in Escherichia coli proved that NisI is a lipoprotein. Expression of nisI in the nisin-sensitive L. lactis MG1614 strain resulted in immunologically active protein on the cytoplasmic membrane in comparable amounts to the immune strain L. lactis N8, but only to slightly increased nisin immunity, suggesting that additional proteins are needed for full immunity.
Subject(s)
Bacterial Proteins/genetics , Escherichia coli/genetics , Gene Expression/genetics , Lactococcus lactis/genetics , Lipoproteins/genetics , Membrane Proteins , Nisin/immunology , Bacteriocins/immunology , Base Sequence , Drug Resistance, Microbial/immunology , Escherichia coli/drug effects , Immunity , Lactococcus lactis/drug effects , Molecular Sequence DataABSTRACT
In this study, a plasmid was integrated into nisP, creating the first defined mutation in a nisin biosynthetic gene. The mutant strain secreted fully modified nisin with the N-terminal leader still attached. The presence of the leader was confirmed by N-terminal sequencing of the purified precursor. The dehydration and lanthionine formation of the precursor were already completed as active nisin could be formed by cleaving the leader from the inactive precursor by a trypsin treatment or by incubation with wild type cells. Nisin immunity of the NisP mutant strain was lowered to about 10% of the wild type immunity. The results show that NisP is needed fro precursor processing and for development of high immunity of nisin.
