Enterocin 96, a Novel Class II Bacteriocin Produced by Enterococcus faecalis WHE 96, Isolated from Munster Cheese

Abstract

Enterococcus faecalis WHE 96, a strain isolated from soft cheese based on its anti-Listeria activity, produced a 5,494-Da bacteriocin that was purified to homogeneity by ultrafiltration and cation-exchange and reversedphase chromatographies. The amino acid sequence of this bacteriocin, named enterocin 96, was determined by Edman degradation, and its structural gene was sequenced, revealing a double-glycine leader peptide. After a comparison with other bacteriocins, it was shown that enterocin 96 was a new class II bacteriocin that showed very little similarity with known structures. Enterocin 96 was indeed a new bacteriocin belonging to class II bacteriocins. The activity spectrum of enterocin 96 covered a wide range of bacteria, with strong activity against most gram-positive strains but very little or no activity against gram-negative strains.Bacteriocins are a heterogeneous group of ribosomally synthesized antibacterial peptides that inhibit strains and species that are usually, but not always, closely related to the producing bacteria (16). Enterococcal bacteriocins, often termed enterocins, have been widely investigated, mainly because they are active against gram-positive food-borne pathogens, such as Listeria monocytogenes, Staphylococcus aureus, and Bacillus cereus. The vast majority of enterocins are active only against gram-positive bacteria (10, 17); however, some exceptions with broad activity spectra described in recent years showed the ability to inhibit the growth of gram-negative microorganisms (5,11,13).The increasing number of enterocins reported in the literature and the emergence of novel structures that could not be included in classical bacteriocin classifications (12,14,17) prompted the grouping of enterocins into a new four-class scheme by Franz et al. (8). Most enterocins known so far were included in class II (small, nonlantibiotic peptides), which was divided into three subgroups: class II.1, enterocins of the pediocin family; class II.2, enterocins synthesized without a leader peptide; and class II.3, other linear, non-pediocin-like enterocins.The fact that numerous Enterococcus strains found in a variety of fermented and nonfermented foods produce bacteriocins, often more than one per strain, has sparked interest in their use in food preservation (4). Despite the concerns over enterococci as opportunistic pathogens and indicators of fecal contamination, they are indigenous species in the gastrointestinal tract and have long been used as human and/or animal probiotics (1, 2, 7).In this work, we describe and characterize a new class II enterocin produced by Enterococcus faecalis WHE 96, previously isolated from Munster cheese, for its anti-Listeria properties. The amino acid sequence, the structural gene, and the spectrum of activity of this bacteriocin are reported. MATERIALS AND METHODSBacterial strains and cultures. The bacteriocin producer E. faecalis WHE 96 was isolated from Munster cheese. E. faecalis CECT 481 was chosen as the indicator strain to monitor bacteriocin activity dur...

Enterocin F4-9, a Novel O -Linked Glycosylated Bacteriocin

“…4). The N-terminal leader sequence of class II bacteriocins has a role in the recognition of the precursor bacteriocin by transport and maturation components of bacteriocin-dependent secretion systems (26). The calculated mass of enterocin F4-9 was smaller than the observed mass by 403.5 Da.…”

Cysteine S -glycosylation, a new post-translational modification found in glycopeptide bacteriocins

“…ORFs encoding proteins with cd02511 domains are present in a few characterised bacteriocin gene clusters, notably those associated with the production of sublancin [12], PlnN [19] and enterocin 96 [24]. Although a role for peptide glycosyltransferases in the maturation of bacteriocins from Gram-positive bacteria has not previously been proposed, the molecular weights of sublancin and enterocin 96 are greater than predicted, suggesting that they could be glycopeptides.…”

“…DNA sequence for the enterocin 96 gene cluster of E. faecalis WHE 96 was not available; the E. faecalis TX0104 gene cluster encoding an identical prebacteriocin (gi:227517439) is shown here. (C) AA sequences of preglycocin F and putative preglycocins, including ASM1 [28], sublancin [12] and enterocin 96 [24]. Leader peptides (lowercase italics), and known (for GccF and SunA) and hypothetical disulfide bonds and glycosylation sites (bold, underlined) are indicated.…”

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