Enterocin X, a Novel Two-Peptide Bacteriocin from Enterococcus faecium KU-B5, Has an Antibacterial Spectrum Entirely Different from Those of Its Component Peptides

Abstract

Enterocin X, composed of two antibacterial peptides (X␣ and X␤), is a novel class IIb bacteriocin fromEnterococcus faecium KU-B5. When combined, X␣ and X␤ display variably enhanced or reduced antibacterial activity toward a panel of indicators compared to each peptide individually. In E. faecium strains that produce enterocins A and B, such as KU-B5, only one additional bacteriocin had previously been known.Bacteriocins are gene-encoded antibacterial peptides and proteins. Because of their natural ability to preserve food, they are of particular interest to researchers in the food industry. Bacteriocins are grouped into three main classes according to their physical properties and compositions (11,12). Of these, class IIb bacteriocins are thermostable non-lanthionine-containing two-peptide bacteriocins whose full antibacterial activity requires the interaction of two complementary peptides (8,19). Therefore, two-peptide bacteriocins are considered to function together as one antibacterial entity (14).Enterocins A and B, first discovered and identified about 12 years ago (2, 3), are frequently present in Enterococcus faecium strains from various sources (3,5,6,9,13,16). So far, no other bacteriocins have been identified in these strains, except the enterocin P-like bacteriocin from E. faecium JCM 5804 T (18). Here, we describe the characterization and genetic identification of enterocin X in E. faecium KU-B5. Enterocin X (identified after the enterocin P-like bacteriocin was discovered) is a newly found class IIb bacteriocin in E. faecium strains that produce enterocins A and B.Enterocins A and B in Enterococcus faecium KU-B5. E. faecium KU-B5, a thermotolerant lactic acid bacterium screened from sugar apples in Thailand, has the ability to multiply and express antibacterial activity across a wide range of temperatures (20 to 43°C). Bacteriocin activity was tested by the critical dilution spot-on-lawn method (21) and expressed in activity units (AU) per milliliter. The strongest antibacterial activity of E. faecium was observed when it was cultured at 37°C in de Man-Rogosa-Sharpe (MRS) broth, with a wide spectrum of targets, including Enterococcus, Lactobacillus, Lactococcus, Bacillus, and Listeria (Table 1). Since enterocins A and B are frequently found in many antibacterial E. faecium strains, we initially examined the enterocin A-and B-encoding genes in E. faecium KU-B5 by using PCR with specific primers (5Ј-TGTACGAAGTGCATTCTCAA-3Ј and 5Ј-TA TTAAAGGACCGGGATCTA-3Ј for enterocin A; 5Ј-ACT CTAAAAGGAGCGAGTTT-3Ј and 5Ј-AGAGCTGGGGA TGAAATATT-3Ј for enterocin B). As expected, these genes were detected in genomic DNA of E. faecium KU-B5. However, the pooled activities from enterocins A and B could not explain the total activities in the culture supernatant of E. faecium KU-B5, such as activity toward Bacillus circulans JCM 2504 T (Table 1). On the other hand, the gene encoding the enterocin P-like bacteriocin (18) was not detected in E. faecium KU-B5. Thus, we assumed that other bacteriocins exist in E. faecium KU-B5,...

Purification and Characterization of Multiple Bacteriocins and an Inducing Peptide Produced byEnterococcus faeciumNKR-5-3 from Thai Fermented Fish

“…E. faecium NKR-5-3 was stored at À80 C in M17 medium (Merck, Darmstadt, Germany) with 15% glycerol, and propagated in M17 medium at 30 C for 22 h before use. Indicator strains for the determination of antimicrobial spectra were propagated at an appropriate temperature (30 C or 37 C), recommended by the culture collections, for 18 h before use.…”

“…Bacteriocins were purified by a 4 step procedure from a 1-liter culture grown to stationary phase in M17 medium at 30 C for 22 h. The cells were removed by centrifugation at 9;000 Â g for 10 min, and 20 g of activated Amberlite XAD-16 resin (Sigma-Aldrich, St. Louis, MO) was added to the supernatant. The resin matrix was slowly shaken for 3 h, and the resin matrix was washed with Milli Q water and then with 50% (v/v) ethanol.…”

“…E. faecium NKR-5-3 was propagated in TSBYE at 30 C for 22 h before use. In the analysis of the bacteriocin induction assay, the TSBYE culture inoculated with 1% of pre-culture of strain NKR-5-3 was incubated at 30 C, and 0.23 mM purified enterocin NKR-5-3D was added to the culture after 4 h of inoculation.…”

Produção de bacteriocina por Bifidobacterium lactis a partir de soro de leite

“…Tais estratégias de triagem são fundamentais para a detecção e a identificação de bacteriocinas potentes de várias subclasses. Exemplos recentes incluem: uma bacteriocina de classe IIa, chamada avicina A, que foi identificada a partir de Enterococcus avium, estirpes isoladas de amostras de fezes de bebês humanos amamentados e saudáveis da Etiópia e da Noruega (BIRRI el al., 2010); uma bacteriocina de estrutura cíclica chamada garvicina ML, produzida por uma cepa de Lactococcus garvieae, isolado do pato-real (BORRERO et al, 2011); uma bacteriocina de classe IIb, a enterocina X, de Enterococcus faecalis, isolada a partir de maçãs (HU et al, 2010), e uma bacteriocina glicolisada (glicocina F) de Lb.…”

“…Such a strategy is still fundamental for detection and identification of powerful bacteriocins of various subclasses, and recent examples of this include a) a class IIa bacteriocin named avicin A that was identified from Enterococcus avium strains isolated from faecal samples of healthy human infants from both Ethiopia and Norway (Birri, Brede, Forberg, Holo, & Nes, 2010), b) a circular bacteriocin named garvicin ML produced by a Lactococcus garvieae strain isolated from mallard duck (Borrero et al, 2011), c) a class IIb bacteriocin named enterocin X isolated from an Enterococcus faecium strain from sugar apples (Hu, Malaphan, Zendo, Nakayama, & Sonomoto, 2010) and d) a glycosylated bacteriocin (glycocin F) from Lactobacillus plantarum isolated from fermented corn (Kelly, Asmundson, & Huang, 1996).…”