Functional analysis of promoters in the nisin gene cluster of Lactococcus lactis

The promoters in the nisin gene cluster nisABTCIPRKFEG of Lactococcus lactis were characterized by primer extension and transcriptional fusions to the Escherichia coli promoteless beta-glucoronidase gene (gus4). Three ee promoters preceding the nisA, nisR, and nisF genes, which ail 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 regulates 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 of gus4 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 beta-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 beta-glucuronidase activity directed by the nisF promoter could he detected even after induction with nisin. These results show that, like the nis.4 promoter, the nisF promoter is nisin inducible. The nisF and nis4 promoter sequences have significant similarities and contain a conserved region that could be important for transcriptional control.