Nisin produced by Lactococcus lactis 6F3 is used as a food preservative and is the most important member of a group of
peptide-
antibiotics containing
lanthionine bridges (
lantibiotics) (N. Schnell, K.-D. Entian, U. Schneider, F. Götz, H. Zähner, R. Kellner, and G. Jung, Nature [London] 333:276-278, 1988).
Nisin is ribosomally synthesized, and its structural gene, nisA, encodes a prepeptide that is posttranslationally modified, revealing the active
lantibiotic (C. Kaletta and K.-D. Entian, J. Bacteriol. 171:1597-1601, 1989). Adjacent to nisA, the additional genes nisB, nisT, and nisC were identified. Over their entire sequences, these genes were homologous to genes recently identified as important for the biosynthesis of
lantibiotics, that is,
subtilin from Bacillus subtilis ATCC 6633 and
epidermin from Staphylococcus epidermidis Tü 3298. Genes nisB, nisT, and nisC corresponded to open reading frames of 993, 600, and 418
amino acid residues, respectively. The nisT open reading frame is homologous to
proteins of the HlyB (
hemolysin B protein of Escherichia coli) subfamily.
Proteins of this subfamily are responsible for the secretion of a variety of compounds, including large
polypeptides,
polysaccharides, and anti-
drug tumors, indicating that NisT may be involved in
nisin transport. Northern (
RNA) blot analysis revealed a 0.3-kb transcript for the nisA structural gene, and the transcriptional start point of the nisA gene was determined by primer extension. Additionally, a
mRNA of at least 3 kb was identified by using a hybridization probe specific to nisB.
Antibodies were raised against the NisB
protein, and Western blot (immunoblot) analysis revealed a molecular weight of about 115 kDa, which is in accordance with the theoretical
protein size of 117.5 kDa as calculated from the nisB open reading frame. Several amphipathic transmembrane alpha-helices indicated that NisB is associated with the membrane. This was confirmed by preparing L. lactis vesicles. The NisB
protein was tightly associated with the vesicle fraction and was released by
sodium dodecyl sulfate treatment only. These results suggest that NisB is membrane associated and that
nisin biosynthesis occurs at the cell membrane.