Purification and Genetic Characterization of Plantaricin NC8, a Novel Coculture-Inducible Two-Peptide Bacteriocin fromLactobacillus plantarumNC8

Abstract

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...

Production of plantaricin NC8 by Lactobacillus plantarum NC8 is induced in the presence of different types of gram-positive bacteria

“…On the other hand, the presence of an autoinducing activity suggests the 22 existence of a quorum sensing mechanism which regulates bacteriocin production the 23 population of L. plantarum NC8. Actually, the presence of two direct repeats upstream of 24 the putative -35 region of plNC8B, the gene encoding PLNC8β (Maldonado et al 2003)18 which agrees with the consensus sequence and structure of promoters of class II 1 bacteriocins regulated by autoinduction suggests a similar mechanism. 2 3 Acknowledgments 1 2 We thank L. Axelsson (MATFORSK,Norwegian Food Research Institute,Osloveien,3 Norway) for providing the L. plantarum NC8 strain.…”

“…Finally, in order to investigate whether or not PLNC8 itself was involved in the 11 autoinducing activity, the purified peptides PLNC8α and PLNC8β generated from PLNC8 12 by C 2 -C 18 reverse-phase chromatography (Maldonado et al 2003) The influence of pH (Ahn and Stiles 1990;Barefoot et al 1994b;Jiménez-Díaz et al 1993;5 Kaiser and Montville 1993;Kleerebezem et al 1997), temperature (Diep et al 2000;Leal-6 Sánchez et al 2002;Mørtvedt-Abildgaard et al 1995;Tichaczeck et al 1992), and growth 7 conditions (Biswas et al 1991;de Vuyst et al 1996;Gutowski-Ecked et al 1994;Jiménez-8 Díaz et al 1993;Leal-Sánchez et al 2002), previously described as enhancers of 9 bacteriocin production by LAB, was studied. None of these factors appeared to promote 10 production of PLNC8 by L. plantarum NC8 growing alone.…”

Induction of Plantaricin Production inLactobacillus plantarumNC8 after Coculture with Specific Gram-Positive Bacteria Is Mediated by an Autoinduction Mechanism

“…Interestingly, this bacteriocin was produced by L. plantarum NC8 only after cocultivation with specific grampositive strains or the addition of heat-killed cells from some of the inducing strains (33,34). However, no cell-free supernatants (CFS) from any of the inducing strains had any effect (34).…”

“…Recently, a novel class IIb bacteriocin, plantaricin NC8 (PLNC8), has been biochemically and genetically characterized (33). Interestingly, this bacteriocin was produced by L. plantarum NC8 only after cocultivation with specific grampositive strains or the addition of heat-killed cells from some of the inducing strains (33,34).…”

Antibacterial activity Lactobacillus plantarum isolated from fermented vegetables and investigation of the plantaricin genes

“…Bacteriocins produced by L. plantarum are known as plantaricins (Omar et al, 2008). The genetic determinants for plantaricins were reported as shown for strains L. plantarum strain C11 (Diep et al, 2003), WCFS1 (Kleerebezem et al, 2003), NC8 (Maldonado et al, 2003) and J23 (Rojo-Bezares et al, 2008). The L. plantarum C11 plantaricin cluster contains five operons Chang et al 797 (Diep et al, 2003).…”