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

Abstract

Biosynthesis of the lantibiotic peptide nisin by Lactococcus lactis NIZO R5 relies on the presence of the conjugative transposon TnS276 in the chromosome. A 12-kb DNA fragment of T M276 including the nisA gene and about 10 kb of downstream DNA was cloned in L. lactis, resulting in the production of an extracellular nisin precursor peptide. This peptide reacted with antibodies against either nisin A or the synthetic leader peptide, suggesting that it consisted of a fully modified nisin with the nisin leader sequence still attached to it. This structure was confirmed by N-terminal sequencing and 'H-nuclear magnetic resoqnnce analysis of the purified peptide. Deletion studies showed that the nisR gene is essential for the production of this intermediate. The deduced amino acid sequence of the nisR gene product indicated that the protein 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 with a high degree of similarity to those of subtilisin-like serine proteases, and a putative C-terminal membrane anchor. Cell extracts of Escherichia coli overexpressing nisP were able to cleave the nisin precursor peptide, producing active, mature nisin. A similar activation was obtained with whole cells but not with membrane-free extracts ofL. lactis strains carrying TnS276 in which the nisA gene had been inactivated. The results indicate that the penultimate step in nisin biosynthesis is secretion of precursor nisin without cleavage of the leader peptide, whereas the last step is the cleavage of the leader peptide sequence from the fully maturated nisin peptide.Nisin is a 34-residue antibacterial peptide that is produced by several strains of Lactococcus lactis and strongly inhibits the growth of a broad range of gram-positive bacteria (24). Insertion of the peptide into the cytoplasmic membrane of the target cell renders the membrane permeable to monovalent cations (18) and dissipates the proton motive force of the cell (18, 37). The mature peptide displays several unusual features, such as the dehydrated residues dehydroalanine and dehydrobutyrine, which are derived from Ser and Thr residues, respectively (22, 24), and lanthionine and P-methyllanthionine residues, which form five intramolecular thioether bridges. Molecular structures similar to those in mature nisin are found in several other antibacterial peptides secreted, e.g., by Bacillus subtilis (subtilin) (21) or Staphylococcus epidermidis (epidermin and Pep5) (2, 42), together forming the group of the lantibiotics (47; for a review, see reference 25).Recently, it was reported that two widely distributed natural variants of nisin exist, named nisin A and nisin Z, which differ in a single amino acid residue (11,36). The structural genes for both nisin A, nisA, and nisin Z, nisZ, have been cloned and sequenced elsewhere (5,14,26,36).These studies showed that both nisin variants are ribosomally synthesized as 57-amino-acid pre...

A two-component signal-transduction cascade in Carnobacterium piscicola LV17B: two signaling peptides and one sensor-transmitter

“…Nevertheless, it is tempting to speculate that CbnK acts as a peptide pheromone sensor involved in regulation of CbnR activity, probably by modulation of its phosphorylation state. The results described here would then suggest that CbnK maintains CbnR in a non-activated form under non inducing conditions, which has been reported for other response regulators [2,23,28].…”

Characterization of the nisin gene cluster nisABTCIPR of Lactococcus lactis

“…The gene product of nisP is involved in the extracellular processing of a fully matured precursor nisin (van der Meer et al, 1993). Finally, the nisR gene (van der Meer et al, 1993) and its homologues in the spa and epi operons, encode regulatory proteins involved in gene expression. In the epidermin biosynthetic operon the epiD gene encodes a flavoprotein, which is probably involved in the formation of S-[(Z)-2-aminovinyl]-~-cysteine in epidermin (Kupke et al, 1992).…”

“…The protein encoded by the nisT gene and its counterparts in the epi and spa gene clusters, have been implicated in the translocation process of lantibiotics, since their deduced sequences show a high degree of similarity to those of transport ATPases, exemplified by HlyB of Escherichia coli (Mackman et al, 1986). The gene product of nisP is involved in the extracellular processing of a fully matured precursor nisin (van der Meer et al, 1993). Finally, the nisR gene (van der Meer et al, 1993) and its homologues in the spa and epi operons, encode regulatory proteins involved in gene expression.…”

“…Several genes have been characterized that encode proteins involved in biosynthetic processes of different lantibiotics, e.g. epidermin (epi; Schnell et al, 1992), subtilin (spa; Banerjee and Hansen, 1988;Chung and Hansen, 1992;Klein et al, 1992;Klein et al, 1993), pep5 (pep; Reis and Sahl, 1991) and nisin (nis; Buchman et al, 1988;Steen et a)., 1991 ;Mulders et al, 1991 ;van der Meer et al, 1993). The proteins encoded by the nisB and nisC genes , and their counterparts in other lantibiotic-encoding gene clusters, might be involved in modification reactions of the prelantibiotic.…”

“…The biosynthesis of nisin and other lantibiotics is thought to take place by successive enzyme-catalyzed modifications of the ribosomally synthesized precursor peptide (Schnell et al, 1988). These specific modifications of precursor nisin include the following: dehydration of serine and threonine residues, resulting in dehydroalanine and dehydrobutyrine residues, respectively ; the addition of free thiol groups of cysteine residues to the double bonds of dehydrated residues, resulting in meso-lanthionine and P-methyllanthionine residues; cleavage of the leader peptide from the mature precursor nisin after secretion into the medium (van der Meer et al, 1993). Several genes have been characterized that encode proteins involved in biosynthetic processes of different lantibiotics, e.g.…”