CHARACTERIZATION OF THE NISIN GENE-CLUSTER NISABTCIPR OF LACTOCOCCUS-LACTIS - REQUIREMENT OF EXPRESSION OF THE NISA AND NISI GENES FOR DEVELOPMENT OF IMMUNITY

The nisin gene cluster nisABTCIPR of Lactococcus lactis, located on a 10-kbp DNA fragment of the nisin-sucrose transposon Tn5276, was charac terized. This fragment was previously shown to direct nisin-A biosynth esis and to contain the nisP and nisR genes, encoding a nisin leader p eptidase and a positive regulator, respectively [van der Meer, J. R., Polman, J., Beerthuyzen, M. M., Siezen, R. J., Kuipers, 0. P. & de Vos , W. M. (1993) J. Bacteriol. 175, 2578-25881. Further sequence analysi s revealed the presence of four open-reading frames, nisB, nisT, nisC and nisI, downstream of the structural gene nisA. The nisT, nisC and n isI genes were subcloned and expressed individually in Escherichia col i, using the T7-RNA-polymerase system. This resulted in the production of radiolabelled proteins with sizes of 45 kDa (NisC) and 32 kDa (Nis I). The nisT gene product was not detected, possibly because of protei n instability. The deduced amino acid sequence of NisI contained a con sensus lipoprotein signal sequence, suggesting that this protein is a lipid-modified extracellular membrane-anchored protein. Expression of nisI in L. lactis provided the cells with a significant level of prote ction against exogeneously added nisin, indicating that NisI plays a r ole in the immunity mechanism. In EDTA-treated E. coli cells, expressi on of nisI conferred up to a 170-fold increase in immunity against nis in A compared to controls. Moreover, a lactococcal strain deficient in nisin-A production, designated NZ9800, was created by gene replacemen t of nisA by a truncated nisA gene and was 10-fold less resistant to n isin A than the wild-type strain. A wild-type immunity level to nisin and production of nisin was obtained in strain NZ9800 harboring comple menting nisA and nisZ plasmids. Transcription analyses of several L la ctis strains indicated that an expression product of the nisA gene, to gether with NisR, is required for the activation of nisA transcription .