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

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Abstract
A new, coculture-inducible two-peptide bacteriocin named plantaricin NC8 (PLNC8) was isolated fromLactobacillus plantarumNC8 cultures which had been induced withLactococcus lactisMG1363 orPediococcus pentosaceusFBB63. This bacteriocin consists of two distinct peptides, named α and β, which were separated by C2-C18reverse-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 PLNC8α and PLNC8β, respectively, which showed no sequence similarity to other known bacteriocins. Mass spectrometry analysis showed molecular masses of 3,587 Da (α) and 4,000 Da (β). The corresponding genes, designatedplNC8AandplNC8B, 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 α and β peptides contain 29 and 34 amino acids, respectively. An open reading frame, orfC, which encodes a putative immunity protein was found downstream ofplNC8Band overlappingplNC8A. Upstream of the putative −35 region ofplNC8B, 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.