The post-translationally modified, antimicrobial peptide nisin is secreted by strains of Lactococcus lactis that contain the chromosomally located nisin biosynthetic gene cluster nisABTCIPRKFEG. When a 4-base pair deletion is introduced into the structural nisA gene (⌬nisA), transcription of ⌬nisA is abolished. Transcription of the ⌬nisA gene is restored by adding subinhibitory amounts of nisin, nisin mutants, or nisin analogs to the culture medium, but not by the unmodified precursor peptide or by several other antimicrobial peptides. Upon disruption of the nisK gene, which encodes a putative sensor protein that belongs to the class of twocomponent regulators, transcription of ⌬nisA was no longer inducible by nisin. Fusion of a nisA promoter fragment to the promoterless reporter gene gusA resulted in expression of gusA in L. lactis NZ9800 (⌬nisA) only upon induction with nisin species. The expression level of gusA was directly related to the amount of inducer that was added extracellularly. These results provide insight into a new mechanism of autoregulation through signal transduction in prokaryotes and demonstrate that antimicrobial peptides can exert a second function as signaling molecules.

Quorum sensing in lactic acid bacteria (LAB) involves peptides that are directly sensed by membrane-located histidine kinases, after which the signal is transmitted to an intracellular response regulator. This regulator in turn activates transcription of target genes, that commonly include the structural gene for the inducer molecule. The two-component signal-transduction machinery has proven to be indispensable for transcription activation and production of several autoinducers found in LAB, which are predominantly bacteriocins or bacteriocin-like peptides. In the nisin autoregulation process in Lactococcus lactis the NisK protein acts as the sensor for nisin and the NisR protein as the response regulator, activating transcription of target genes. The cis-acting elements for NisR were identified as the nisA and nisF promoter fragments and these were further analysed for inducibility. Based on this knowledge efficient nisin-controlled expression (NICE) systems were developed for several different lactic acid bacteria. A promising application of the NICE system is the development of autolytic starter lactococci, which will lyse in an early stage during cheese ripening thereby facilitating the release of intracellular enzymes which can contribute to flavour formation.

The kinetics, control, and efficiency of nisin-induced expression directed by the nisA promoter region were studied in Lactococcus lactis with transcriptional and translational fusions to the gusA reporter gene. In the nisin-producing L. lactis strain NZ9700, the specific ␤-glucuronidase activity increased very rapidly after mid-exponential growth until the maximum level at the start of the stationary phase was reached. Expression of the gusA gene was also studied in L. lactis NZ9800, an NZ9700 derivative carrying a deletion in the structural nisA gene that abolishes nisin production, and in L. lactis NZ3900, an MG1363 derivative containing the regulatory nisRK genes integrated in the chromosome. In both strains, ␤-glucuronidase activity was linearly dependent on the amount of nisin added to the medium. Without nisin, no ␤-glucuronidase production was observed. To optimize translation initiation, an expression vector was constructed by fusing the gusA gene translationally to the start codon of the nisA gene. Use of the translational fusion vector yielded up to six times more ␤-glucuronidase activity than the transcriptional fusion vector in these strains after induction by nisin. In this way, gene expression can be achieved in a dynamic range of more than 1,000-fold. The ␤-glucuronidase activity was found to be up to 25-fold higher in extracts of strain NZ3900 than in extracts of strain NZ9800. This translational fusion vector was used for high-level production of aminopeptidase N, up to 47% of the total intracellular protein. These results clearly illustrate the potential of the nisin-inducible expression system for overproduction of desired proteins.

The promoters in the nisin gene cluster nisABTCIPRKFEG of Lactococcus lactis were characterized by primer extension and transcriptional fusions to the Escherichia coli promoterless ␤-glucuronidase gene (gusA). Three promoters preceding the nisA, nisR, and nisF genes, which all give rise to gusA expression in the nisin-producing strain L. lactis NZ9700, were identified. The transcriptional autoregulation of nisA by signal transduction involving the sensor histidine kinase NisK and the response regulator NisR has been demonstrated previously (O. P. Kuipers, M. M. Beerthuyzen, P. G. G. A. de Ruyter, E. J. Luesink, and W. M. de Vos, J. Biol. Chem. 270: 27299-27304, 1995), and therefore the possible nisin-dependent expression of gusA under control of the nisR and nisF promoters was also investigated. The nisR promoter was shown to direct nisin-independent gusA expression in L. lactis MG1363, which is a nisin-transposon-and plasmid-free strain. L. lactis NZ9800, which does not produce nisin because of a deletion in the nisA gene, containing the nisF-gusA fusion plasmid, gave rise to ␤-glucuronidase production only after induction by nisin. A similar regulation was found in L. lactis NZ3900, which contains a single copy of the nisR and nisK genes but no other genes of the nisin gene cluster. In contrast, when the nisK gene was disrupted, no ␤-glucuronidase activity directed by the nisF promoter could be detected even after induction with nisin. These results show that, like the nisA promoter, the nisF promoter is nisin inducible. The nisF and nisA promoter sequences have significant similarities and contain a conserved region that could be important for transcriptional control.

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