Characterization and Heterologous Expression of the Genes Encoding Enterocin A Production, Immunity, and Regulation in Enterococcus faecium DPC1146

Abstract

Enterocin A is a small, heat-stable, antilisterial bacteriocin produced by Enterococcus faecium DPC1146. The sequence of a 10,879-bp chromosomal region containing at least 12 open reading frames (ORFs), 7 of which are predicted to play a role in enterocin biosynthesis, is presented. The genes entA,entI, and entF encode the enterocin A prepeptide, the putative immunity protein, and the induction factor prepeptide, respectively. The deduced proteins EntK and EntR resemble the histidine kinase and response regulator proteins of two-component signal transducing systems of the AgrC-AgrA type. The predicted proteins EntT and EntD are homologous to ABC (ATP-binding cassette) transporters and accessory factors, respectively, of several other bacteriocin systems and to proteins implicated in the signal-sequence-independent export of Escherichia colihemolysin A. Immediately downstream of the entT andentD genes are two ORFs, the product of one of which, ORF4, is very similar to the product of the yteI gene ofBacillus subtilis and to E. coli protease IV, a signal peptide peptidase known to be involved in outer membrane lipoprotein export. Another potential bacteriocin is encoded in the opposite direction to the other genes in the enterocin cluster. This putative bacteriocin-like peptide is similar to LafX, one of the components of the lactacin F complex. A deletion which included one of two direct repeats upstream of the entA gene abolished enterocin A activity, immunity, and ability to induce bacteriocin production. Transposon insertion upstream of the entF gene also had the same effect, but this mutant could be complemented by exogenously supplied induction factor. The putative EntI peptide was shown to be involved in the immunity to enterocin A. Cloning of a 10.5-kb amplicon comprising all predicted ORFs and regulatory regions resulted in heterologous production of enterocin A and induction factor in Enterococcus faecalis, while a four-gene construct (entAITD) under the control of a constitutive promoter resulted in heterologous enterocin A production in both E. faecalis and Lactococcus lactis.

Production of enterocin A byEnterococcus faeciumMMRA isolated from ‘Rayeb’, a traditional Tunisian dairy beverage

“…faecium species (Nes et al 2007). Enterocin A producing strains have also been isolated in many other locations and food products such as dairy products (O'Keeffe et al 1999;Ennahar et al 2001), black olives (Franz et al 1999a), from nuka (Losteinkit et al 2001). The isolation of enterocin A producers from traditional made 'Rayeb' in Tunisia reinforced the idea that enterocin A is widespread among enterococcal strains all over the world and may play a significant role in controlling Listeria growth in fermented foods.…”

Diversity of enterococcal bacteriocins and their grouping in a new classification scheme

“…These characteristics make it equivalent to pediocin PA-1/AcH (Marugg et al, 1992). EntA is produced by several E. faecium strains: CTC492, T136 and P21, which were isolated from Spanish fermented sausages (Aymerich et al, 1996;Casaus et al, 1997;Herranz et al, 2001); by strain BFE 900 from black olives; by strains DPC 1146, WHE 81 and EFM01 from dairy sources (Franz et al, 1999b;O'Keeffe et al, 1999;Ennahar & Deschamps, 2000;Ennahar et al, 2001); and by N5 from nuka (a Japanese rice-bran paste) (Losteinkit et al, 2001). EntA is active against Enterococcus, Lactobacillus and Pediococccus spp., as well as Listeria spp., including L. monocytogenes.…”

Preliminary characterization of bacteriocins produced by Enterococcus faecium and Enterococcus faecalis isolated from pig faeces

“…The best characterized bacteriocins from enterococci (enterocins) were described from strains originating from food sources. These include the class II enterocins A, B, P, CRL35 and bacteriocin 31 (Aymerich et al 1996;Farías et al 1996;Tomita et al 1996;Casaus et al 1997;Cintas et al 1997;Franz et al 1999a;O'Keeffe et al 1999). Enterocins L50A and L50B, also produced by an enterococcal food isolate, do not belong to any of the three classes of bacteriocins as defined by Nes et al (1996), but show homology to the staphylococcal peptide toxins (Cintas et al 1998).…”