INFLUENCE OF AMINO-ACID SUBSTITUTIONS IN THE NISIN LEADER PEPTIDE ON BIOSYNTHESIS AND SECRETION OF NISIN BY LACTOCOCCUS-LACTIS

Structural genes for small lanthionine-containing antimicrobial peptid es, known as lantibiotics, encode N-terminal leader sequences which ar e not present in the mature peptide, but are cleaved off at some stage in the maturation process. Leader sequences of the different lantibio tics share a number of identical amino acid residues, but they are cle arly different from sec-dependent protein export signal sequences. We studied the role of the leader sequence of the lantibiotic nisin, whic h is produced and secreted by Lactococcus lactis, by creating site-dir ected mutations at various positions in the leader peptide sequence. M utations at Arg-1 and Ala-4, but not at the conserved Pro-2, strongly affected the processing of the leader sequence and resulted in the ext racellular accumulation of a biologically inactive precursor peptide. Amino acid analysis and H-1 NMR studies indicated that the precursor p eptide with an Ala-4 --> Asp mutation contained a modified nisin struc tural part with the (mutated) unmodified leader sequence still attache d to it. The Ala-4 --> Asp precursor peptide could be activated in vit ro by enzymatic cleavage with trypsin, liberating nisin. These results confirmed that cleavage of the leader peptide is the last step in nis in maturation and is necessary to generate a biologically active pepti de. Several mutations, i.e. Pro-2 --> Gly, Pro-2 --> Val, Asp-7 --> Al a, Lys-9 --> Leu, Ser-10 --> Ala/Ser-12 --> Ala and Val-11 --> Asp/Val -13 --> Glu in the leader peptide did not have any detectable effect o n nisin production and secretion, although some of them affected highl y conserved residues. When mutations were created in the -18 to -15 re gion of the nisin leader peptide (i.e. Phe-18 --> Leu, Leu-16 --> Lys, Asp-15 --> Ala), no secretion or intracellular accumulation could be detected of nisin or its precursors. This suggested that these conserv ed residues are involved in the maturation process and may interact wi th lantibiotic-specific modifying enzymes.