CHARACTERIZATION OF THE LACTOCOCCUS-LACTIS NISIN-A OPERON GENES NISP,ENCODING A SUBTILISIN-LIKE SERINE PROTEASE INVOLVED IN PRECURSOR PROCESSING, AND NISR, ENCODING A REGULATORY PROTEIN INVOLVED IN NISIN BIOSYNTHESIS

Biosynthesis of the lantibiotic peptide nisin by Lactococcus lactis NI ZO R5 relies on the presence of the conjugative transposon Tn5276 in t he chromosome. A 12-kb DNA fragment of Tn5276 including the nisA gene and about 10 kb of downstream DNA was cloned in L. lactis, resulting i n the production of an extracellular nisin precursor peptide. This pep tide reacted with antibodies against either nisin A or the synthetic l eader peptide, suggesting that it consisted of a fully modified nisin with the nisin leader sequence still attached to it. This structure wa s confirmed by N-terminal sequencing and H-1-nuclear magnetic resonanc e analysis of the purified peptide. Deletion studies showed that the n isR gene is essential for the production of this intermediate. The ded uced amino acid sequence of the nisR gene product indicated that the p rotein belongs to the family of two-component regulators. The deduced amino acid sequence of NisP, the putative product of the gene upstream of nisR, showed an N-terminal signal sequence, a catalytic domain wit h a high degree of similarity to those of subtilisin-like serine prote ases, and a putative C-terminal membrane anchor. Cell extracts of Esch erichia coli overexpressing nisP were able to cleave the nisin precurs or peptide, producing active, mature nisin. A similar activation was o btained with whole cells but not with membrane-free extracts of L. lac tis strains carrying Tn5276 in which the nisA gene had been inactivate d. The results indicate that the penultimate step in nisin biosynthesi s is secretion of precursor nisin without cleavage of the leader pepti de, whereas the last step is the cleavage of the leader peptide sequen ce from the fully maturated nisin peptide.