Lactococcin A, a new bacteriocin from Lactococcus lactis subsp. cremoris: isolation and characterization of the protein and its gene

Abstract

A new bacteriocin, termed lactococcin A (LCN-A), from Lactococcus lactis subsp. cremoris LMG 2130 was purified and sequenced. The polypeptide contained no unusual amino acids and showed no significant sequence similarity to other known proteins. Only lactococci were killed by the bacteriocin. Of more than 120 L. lactis strains tested, only 1 was found resistant to LCN-A. The most sensitive strain tested, L. lactis subsp. cremoris NCDO 1198, was inhibited by 7 pM LCN-A. By use of a synthetic DNA probe, kcnA was found to be located on a 55-kb plasmid. The lcnA gene was cloned and sequenced. The sequence data revealed that LCN-A is ribosomally synthesized as a 75-amino-acid precursor including a 21-amino-acid N-terminal extension. An open reading frame encoding a 98-amino-acid polypeptide was found downstream of and in the same operon as kcnA. We propose that this open reading frame encodes an immunity function for LCN-A. In Escherichia coli IcnA did not cause an LCN-A+ phenotype. L. lacfis subsp. lactis IL 1403 produced small amounts of the bacteriocin and became resistant to LCN-A after transformation with a recombinant plasmid carrying kcnA. The other lactococcal strains transformed with the same recombinant plasmid became resistant to LCN-A but did not produce any detectable amount of the bacteriocin.A number of strains of Lactococcus lactis produce bacteriocins. In an extensive survey by Geis et al. (15), it was found that about 5% of the 280 lactococcal strains tested were bacteriocin producers. These workers divided the bacteriocins into eight different classes, but none of them was characterized in detail. Despite numerous reports of lactococcal bacteriocins, little is known about their chemical composition and structure, mode of action, or genetics. Nisin is the only bacteriocinlike compound from L. lactis that has been studied in detail. The molecular structure and genetic determinant of nisin have been identified and, to some extent, its mode of action has been elucidated (5,11,16,21,22,36). Another bacteriocin, termed diplococcin, produced by L. lactis subsp. cremoris, has also been purified and its amino acid composition has been determined (10). Davey (9) showed that the gene coding for diplococcin was located on a 54-MDa conjugative plasmid. Conjugal transfer of bacteriocin plasmids in L. lactis has also been observed by others (30,38). Harmon and McKay (19) identified a Bcll DNA fragment carrying a bacteriocin determinant from a conjugative plasmid. Recently, two bacteriocin genes from another conjugative lactococcal plasmid, previously described by Neve et al. (30), were cloned by van Belkum et al. (46). The clones also carried the immunity factors of the two bacteriocins. The inhibitory spectra of the different lactococcal bacteriocins described vary but are generally more narrow than that of nisin (15). It is therefore plausible that many of the lactococcal bacteriocins described are very different from nisin and thus do not belong to the lanthibiotic family (39) of bacteriocinlike compounds....

Bacteriocins of propionic acid bacteria

“…mL -1 . This is similar to the minimal inhibitory concentrations found for many LAB bacteriocins [3,13,21]. The peptide has a bactericidal mode of action.…”

“…The specific activity of the purified peptide was 2 AU . µg -1 , which is three orders of magnitude less than what is seen for bacteriocins from lactic acid bacteria [3,13,21] and propionicin T1.…”

Pheromone‐induced production of antimicrobial peptides in Lactobacillus

“…Quantitative estimates of bacteriocin activity were obtained using microtitre plate assays as described previously (see Holo et al, 1991). Cell-free culture supernatants were obtained by centrifugation and subsequent filter sterilization.…”

Rapid Two-Step Procedure for Large-Scale Purification of Pediocin-Like Bacteriocins and Other Cationic Antimicrobial Peptides from Complex Culture Medium

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