MOLECULAR CHARACTERIZATION AND MODE OF ACTION OF A BACTERIOCIN PRODUCED BY Lactococcus lactis subsp. lactis CRL 1584

PASTERIS, S.E.; ALE, C. E.; VERA PINGITORE, E.; NADER-MACÍAS, M. E.

Simposio; IV SIMPOSIO INTERNACIONAL DE BACTERIAS LÁCTICAS (SIBAL) ALIMENTOS, SALUD Y APLICACIONES.; 2013

Lactococcus lactis CRL 1584 isolated from a Lithobates catesbeianus hatchery inhibits the growth of Citrobacter freundii (bullfrog´s pathogen) and Listeria monocytogenes (food-borne bacteria) by a synergic effect among lactic acid, hydrogen peroxide and a bacteriocin. On the basis of their technological characteristics, the bacteriocin could be used to control pathogenic microorganisms in raniculture and food-spoilage bacteria. The aim of this work was to advance in the knowledge of the bacteriocin through the determination of the molecular localization of the gene encoding the molecule and its mode of action including L. monocytogenes viability and ultrastructure cell damages. To identify the bacteriocin genes in L. lactis CRL 1584, degenerate primers were designed. The DNA was isolated and amplified by PCR and agarose electrophoresis gels were performed to identify the amplicons. The amplified fragments were purified, sequenced and analyzed. The presence of extrachromosomal elements was also studied. Plasmid curing experiments using novobiocin, were performed to obtain a plasmid-free strains. The PCR amplification using the nisin family oligonucleotides amplified a DNA fragment of 450 bp. Its sequence revealed a bacteriocin gen with a leader peptide of 23 amino acids and a propeptide of 34 amino acids identical to nisin Z. Moreover, L. lactis possess one plasmid and the plasmid-free strain maintained its antimicrobial activity against L. monocytogenes that was similar to the wild-type strain activity. Thus, the bacteriocin genes would be encoded in the chromosome. On the other hand, supernatants of L. lactis cultures were collected and concentrated to achieve 4,200 AU/mL of bacteriocin. Then, fractions of supernatants were treated as follows: a-neutralized supernatant+catalase (N+C), b- Neutralized+catalase+chymotrypsin (N+C+Chym). Chymotrypsin+heating (Chym+h) was used as control. Fractions were supplemented with BHI broth and inoculated with 5 x 105 cfu/ml L. monocytogenes Scott A. Samples were taken for viability and ultrastructural evaluations. Bacteriocin has a bactericidal effect on L. monocytogenes cells. After 60 min of incubation, L. monocytogenes viability decreased in 0.7 log units and no viable cells were detected after 150 min in N+C. In both, N+C+Chym and Chym+h did not exhibit differences in the bacterial growth reaching 3 log units over the initial cell number at the end of the assay (300 min). The ultrastructural studies on pathogenic cells revealed a clumping of the cytoplasmic material with an increased periplasmic space and an altered pattern of electron density in the cell wall at 60 min. These changes were more evident at 120 min of incubation. These results, described for the first time in L. lactis from a L. catesbeianus hatchery, support the potential application of the bacteriocin as biopreservative of bullfrog carcasses and its inclusion in beneficial products for raniculture, as well.