Molecular analysis of multiple bacteriocin production by Enterococcus faecium CRL1879

.N. SUÁREZ, ; J. BONACINA, ; F. SESMA; L. SAAVEDRA.

Tucumán

Simposio; Simposio Internacional de Bacterias Lácticas (SIBAL); 2013

Centro Referencia para Lactobacilos

Enterococci are associated with fermented foods, especially with artisanal dairy products such as cheese, where they are involved in flavor and aroma development. Furthermore, species of Enterococcus produce a wide array of structurally diverse antimicrobial peptides named enterocin and often it is easy to find production of more than one per strain. Such attractive traits have increased the interest for their biotechnological use in food preservation, especially since these compounds are particularly active against food-borne pathogens, such as Listeria. In a previous work, 151 isolates from homemade traditional cheeses manufactured in the province of Tucumán were evaluated for their anti-listerial potential. Ten strains exhibited antimicrobial activities and they were identified as Enterococcus faecium by sequencing their 16S rDNA. E. faecium CRL 1879 was selected for further analysis because of its inhibitory spectrum. The aim of the present work was to characterize genetically and biochemically the bacteriocin production by E. faecium CRL1879. This strain evidenced the presence of genes encoding for enterocin A, enterocin B, enterocin P and enterocin SE-K4 by using PCR strategies using specific bacteriocin primes. The analysis of bacteriocin expression was evaluated by quantitative real time PCR (qRT-PCR). RNA was extracted from a 12 h bacterial culture and total RNA (free of DNA) was used for cDNA synthesis. qRT-PCR reactions with SYBR green detection were run on an IQTM 5 Multicolor Real- Time PCR Detection System (BioRad). Absolute quantification was performed using a standard calibration curve. Results of qRT-PCR analysis confirmed the presence of each enterocin gene transcript (ent A, ent B, ent P, and ent SE-K4), being enterocin P the most abundant transcript (2,4x107 copies μl -1). Since this strain showed a unique pattern of bacteriocin genes and it represents a good candidate for future food application, we submitted it to a whole-genome sequencing procedure. The genome sequence (AN AOUK00000000) was obtained using a whole-genome shotgun (WGS) strategy with an Ion Torrent Personal Genome Machine. Genomic analysis was performed using the RAST Server, Bactibase database and Blast algorithms. Genome sequence analysis of E. faecium CRL 1879 confirmed the presence of enterocin biosynthetic gene clusters previously described. Additionally, it also revealed the presence of four open reading frames (ORFs) encoding for the two-component bacteriocin enterocin X and a novel two-component bacteriocin not described yet. We report here that E. faecium CRL 1879 possess the genetic machinery necessary to produce several antimicrobial peptides, including the discovery of a novel two-peptide bacteriocin.