Abstract
Lacticin 3147 is a two-component bacteriocin produced by Lactococcus lactis subspecies lactis DPC3147. In order to further characterize the biochemical nature of the bacteriocin, both peptides were isolated which together are responsible for the antimicrobial activity. The first, LtnA1, is a 3,322 Da 30-amino acid peptide and the second component, LtnA2, is a 29-amino acid peptide with a mass of 2,847 Da. Conventional amino acid analysis revealed that both peptides contain the thioether amino acid, lanthionine, as well as an excess of alanine to that predicted from the genetic sequence of the peptides. Chiral phase gas chromatography coupled with mass spectrometry of amino acid composition indicated that both LtnA1 and LtnA2 contain D-alanine residues and amino acid sequence analysis of LtnA1 confirmed that the D-alanine results from post-translational modification of a serine residue in the primary translation product. Taken together, these results demonstrate that lacticin 3147 is a novel, two-component, D-alanine containing lantibiotic that undergoes extensive post-translational modification which may account for its potent antimicrobial activity against a wide range of Gram-positive bacteria.
Highlights
Lacticin 3147 is a two-component bacteriocin produced by Lactococcus lactis subspecies lactis DPC3147
Purification of Lacticin 3147—We previously reported the partial purification of lacticin 3147, a broad spectrum bacteriocin produced by L. lactis DPC3147 [15]
We reveal that the two-component antimicrobial peptide lacticin 3147 undergoes a series of complex posttranslational modifications including conversion of serine residues to D-alanine and the formation of lanthionine bridges
Summary
The producing strain L. lactis DPC3147, as well as the sensitive indicator strains L. lactis HP and L. lactis AM2 were maintained by weekly subculture in M17 (Difco, Detroit, MI) supplemented with 0.5% lactose (LM17) and all strains were stocked at Ϫ70 °C in 40% glycerol. Bacteriocin activity was assayed after each step using L. lactis HP as the indicator strain and the method previously described by McAuliffe et al [20]. In order to locate complementary activity, 10-l aliquots of putative A1 fractions were cross-tested with isolated peptide A2 and vice versa. 10-l aliquots of isolated fractions, A1, A1Ј, and A2 were each dispensed into separate, triangularly arranged agar wells formed in an agar plate which had previously been seeded with the sensitive indicator strain L. lactis AM2. Synergistic activity between the peptides was indicated by a zone of inhibition between the wells, while activity from individual peptides was observed as a zone around a well on the side facing away from all other fractions
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