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A new, coculture-inducible two-peptide bacteriocin named plantaricin NC8 (PLNC8) was isolated from Lactobacillus plantarum NC8 cultures which had been induced with Lactococcus lactis MG1363 or Pediococcus pentosaceus FBB63. This bacteriocin consists of two distinct peptides, named ␣ and ␤, which were separated by C 2 -C 18 reverse-phase chromatography and whose complementary action is necessary for full plantaricin NC8 activity. N-terminal sequencing of both purified peptides showed 28 and 34 amino acids residues for PLNC8␣ and PLNC8␤, respectively, which showed no sequence similarity to other known bacteriocins. Mass spectrometry analysis showed molecular masses of 3,587 Da (␣) and 4,000 Da (␤). The corresponding genes, designated plNC8A and plNC8B, were sequenced, and their nucleotide sequences revealed that both peptides are produced as bacteriocin precursors of 47 and 55 amino acids, respectively, which include N-terminal leader sequences of the double-glycine type. The mature ␣ and ␤ peptides contain 29 and 34 amino acids, respectively. An open reading frame, orfC, which encodes a putative immunity protein was found downstream of plNC8B and overlapping plNC8A. Upstream of the putative ؊35 region of plNC8B, two direct repeats of 9 bp were identified, which agrees with the consensus sequence and structure of promoters of class II bacteriocin operons whose expression is dependent on an autoinduction mechanism.Many lactic acid bacteria (LAB) produce antimicrobial peptides known as bacteriocins, which are directed mainly to inhibit the growth of related species or species with the same nutritive requirements (14,31,35,52). Hence, bacteriocin production seems to be aimed to compete against other bacteria which are present in the same ecological niche (4,19,20,43). It has been shown that bacteriocin production provides the producer strain with a selective advantage over other nonproducing, isogenic bacteria (46). On the other hand, it might be expected that biosynthesis of bacteriocins is a high-energyconsuming process, providing an advantage to the producer strain only if the cost/benefit ratio is favorable (19,43). It is well known that biosynthesis of proteinaceous substances is a high-energy-consuming process and for this reason is well controlled by molecular regulatory systems such as induction and catabolic repression, for instance. This suggests that bacteriocin production by LAB should also be controlled and regulated by these molecular mechanisms.Bacteria must be able to adapt their metabolism to the changing environmental conditions. This adaptation requires that bacteria sense the multitude of extracellular signals and respond by controlling the expression of an adequate repertoire of genes. Thus, bacteriocin production has been shown to be influenced by pH (1,5,6,32,34), temperature (17, 37, 54), medium composition (3, 13, 26, 27, 55), DNA-damaging agents (24,25,42), and growth conditions (8,15,28,32). However, how the presence of competing microorganisms affects production of bacteriocins is still qu...

Twenty-six strains of LactobaciUlus plantarum isolated from green olive fermentations were tested for cross-antagonistic activities in an agar drop diffusion test. Cell-free supernatants from four of these strains were shown to inhibit the growth of at least one of the L. plantarum indicator strains. L. plantarum LPCO10 provided the broadest spectrum of activity and was selected for further studies. The inhibitory compound from this strain was active against some gram-positive bacteria, including clostridia and propionibacteria as well as natural competitors ofL. plantarum in olive fermentation brines. In contrast, no activity against gram-negative bacteria was detected. Inhibition due to the effect of organic acids, hydrogen peroxide, or bacteriophages was excluded. Since the inhibitory activity of the active supernatant was lost after treatment with various proteolytic enzymes, this substance could be classified as a bacteriocin, designated plantaricin S. Plantaricin S was also sensitive to glycolytic and lipolytic enzymes, suggesting that it was a glycolipoprotein. It exhibited a bactericidal and nonbacteriolytic mode of action against indicator cells. This bacteriocin was heat stable (60 min at 100°C), active in a pH range of 3.0 to 7.0, and also stable in crude culture supernatants during storage. Ultrafiltration studies indicated that plantaricin S occurred as multimolecular aggregates and that the size of the smallest active form is between 3 and 10 kDa. In sodium dodecyl sulfate-polyacrylamide gels, plantaricin S migrated as a peptide of ca. 2.5 kDa. Maximum production of plantaricin S was obtained in a fermentor system in unregulated pH and log-phase cultures of L. plantarum LPCO10 in MRS broth plus 4% NaCl. In these culture conditions, a second bacteriocin (designated plantaricin T) was produced in late-stationary-phase cultures of L. plantarum LPCO10. On the basis of its biological activity, its sensitivity to various enzymes, and its molecular weight (lower than that of plantaricin S) as assessed in sodium dodecyl sulfate-polyacrylamide gel