Food Microbiology, 2001, 18, 115^131 Available online at http://www.idealibrary.

com on

doi:10.1006/fmic.2000.0382

ORIGINAL ARTICLE

Enterococcus faecium P21: a strain occurring naturally in dry-fermented sausages producing the class II bacteriocins enterocin A and enterocin B
C. Herranz1, P. Casaus1{, S. Mukhopadhyay1, J. M. Mart| nez1, J. M. Rodr| guez1, I. F. Nes2, P. E. Hernandez1 and L. M. Cintas1*

Enterococcus faecium P21isolated from a Spanish dry-fermented sausage shows a narrow antimicrobial activity against closely related lactic acid bacteria and strong antimicrobial activity against spoilage and foodborne Gram-positive pathogenic bacteria such as Listeria monocytogenes, Staphylococcus aureus, Clostridium perfringens and Clostridium botulinum.The antimicrobial activity is produced during growth in MRS broth at 328C; it is heat resistant (20 min at 80 ^1008C) and abolished by protease treatment, and withstands exposure to pH 2^11, freeze-thawing, lyophilization and long term storage at 7208C. Purication of the antimicrobial activity by ammonium sulphate precipitation, gel ltration, and cation-exchange, hydrophobic interaction and reverse-phase chromatographies showed that the broad antimicrobial spectrum exerted by E. faecium P21 was indeed due to two peptide bacteriocins. Studies on their N-terminal amino acid sequences and PCR and DNA analyses revealed that these bacteriocins are identical to the class II bacteriocins enterocin A and enterocin B.The genetic organization of the enterocin A (EntA) operon in E. faecium P21 shows that the bacteriocin structural gene (entA), the immunity gene (entiA) and the induction peptide gene (entF) are clustered and colinearly arranged. Strikingly, this organization was structurally dierent to that reported for the EntA-producer # 2001 Academic Press E. faecium CTC492.

Received: 27 June 2000 Departamento de Nutricion y Bromatolog| a III, Facultad de Veterinaria, Universidad Complutense de Madrid, 28040Madrid, Spain 2 Laboratory of Microbial Gene Technology, Department of Chemistry and Biotechnology, Agricultural University of Norway, N-1432 s, Norway
1

Introduction
Lactic acid bacteria (LAB) predominate during food fermentation because of their ability
*Corresponding author. Fax: +34 -913943743. E-mail: lcintas@eucmax.sim.ucm.es { Present address: Centros Comerciales Carrefour, Division de Calidad, c/ Campezo n816, 28022 -Madrid, Spain.
0740 -0020/01/020115 + 17 $35.00/0

to produce bacteriocins and other antimicrobial compounds (Daeschel 1989, Stiles and Hastings 1991). Bacteriocins constitute a large and heterogeneous group of ribosomally synthesized proteins or peptides displaying antimicrobial activity against other bacteria (Klaenhammer 1993, Nes and Eijsink 1999). Many LAB-bacteriocins inhibit a broad range of Gram-positive bacteria, including spoilage and foodborne pathogenic micro-organisms
# 2001 Academic Press

116 C. Herranz et al.

such as Listeria monocytogenes and Staphylococcus aureus (Giraa 1995, Jack et al. 1995, Casaus et al. 1997, Cintas et al. 1995, 1998a,b).The LABbacteriocins described and characterized to date show common traits which justify their classication into three well-dened classes (Nes et al. 1996, Moll et al. 1999): class I, the lantibiotics; class II, the small (510 kDa) heatstable non-lantibiotics, which are divided into the subgroups IIa (pediocin-like bacteriocins with strong anti-Listeria activity), IIb (bacteriocins whose activity depends on the complementary action of two peptides), IIc (sec-dependent bacteriocins) and IId (class II bacteriocins not included in the previous groups); and class III, large (430 kDa) heatlabile bacteriocins. Genetic studies of bacteriocin synthesis have revealed that production and secretion of most class II bacteriocins require a structural gene of the prebacteriocin (inactive precursor peptide), an immunity gene, and the genes encoding the secretion machinery. Most class II bacteriocins are synthesized as prebacteriocins containing an N-terminal leader sequence of the so-called double glycine type and are processed and translocated through the cytoplasmic membrane by ATP-binding cassette (ABC) transporters and their accessory proteins. However, recently it has been shown that a few class II bacteriocin precursors contain the characteristic hydrophobic signal peptide found in the proteins that are secreted through the general secretory pathway (or sec-dependent pathway) (Nes et al., 1996). In recent years, it has been reported that production of some class II bacteriocins is regulated by a threecomponent signal transduction pathway (Hoch and Silhavy 1995), consisting of an induction peptide (IP) (peptide pheromone), a histidine protein kinase (HPK) and a response regulator (RR) (Diep et al. 1996, Nes et al. 1996, Nes and Eijsink 1999). The IP is a bacteriocin-like peptide synthesized as a prepeptide including a double-glycine leader sequence and shares many of the physico-chemical properties of the bacteriocins (Diep et al. 1995, Nes et al. 1996). The secreted IP acts as a signal for triggering and maintaining transcription of the genes required for bacteriocin production (Nes et al. 1996, Nes and Eijsink 1999).

Bacteriocin production by Enterococcus spp. of food origin has been known for many years (McKay 1990, Villani et al. 1993, Ben Embarek et al. 1994, Olasupo et al. 1994, Torri Tarelli et al. 1994, Cintas 1995, Giraa 1995, Casaus 1998); however, most enterocins described to date have not been characterized at the biochemical and molecular levels. The exceptions are enterocin A (Ent A) from E. faecium CTC492 (Aymerich et al. 1996), enterocin B (Ent B) from E. faecium T136 (Casaus et al. 1997, Casaus 1998), enterocin P (Ent P) from E. faecium P13 (Cintas et al. 1997) and enterocins L50A and L50B (Ent L50) from E. faecium L50 (Cintas 1995, Cintas et al. 1998a,b). All these enterocins show a broad antimicrobial activity spectrum like other class II bacteriocins and consist of small (4829^5465 Da), heatstable, cationic, amphiphilic and hydrophobic peptides without modied amino acid residues. However, biochemical and genetic data reveal that they dier in several features, such as the presence of the pediocin-like consensus sequence YGNGVxC at the N-terminal region of the molecule (Nieto-Lozano et al. 1992, Aymerich et al. 1996, Ennahar et al. 2000) and the secretion pathway used to externalize the bacteriocins to the extracellular medium. Enterocin A and enterocin P are both pediocin-like bacteriocins (class IIa) (Aymerich et al. 1996, Cintas et al. 1997), while enterocin B and enterocins L50A and L50B are class IId bacteriocins (Nes et al., 1996; Moll et al., 1999). As shown for other bacteriocins (Nes et al. 1996), enterocins A, B and P are synthesized as prebacteriocins. The leader sequences of enterocin A (Aymerich et al. 1996) and enterocin B (Casaus et al. 1997) are of the so called double-glycine-type; however, the N-terminal extension of enterocin P corresponds to a signal peptide (Cintas et al. 1997, Casaus 1998). In contrast to other peptide bacteriocins, enterocins L50A and L50B are synthesized without an N-terminal leader sequence or signal peptide (Cintas et al. 1998a). During the last years we have isolated several bacteriocinogenic LAB from Spanish dry fermented sausages and biochemically and genetically characterized several bacteriocins (Sobrino et al. 1992, Cintas 1995, Cintas et al. 1995, 1997, 1998a,b, 2000, Casaus 1998, Casaus

Enterocins A and B from E. faecium P21 117

et al. 1995, 1997, Herranz et al. 1999). In this work we report on the isolation and identication of E. faecium P21 and the characterization of its bacteriocins by their functional properties, purication to homogeneity, amino acid sequence and DNA analysis.

Materials and Methods

Bacterial strains and media
Lactic acid bacteria were isolated from chorizo, a typical Spanish dry-fermented sausage, manufactured with no added starter cultures, and screened for antimicrobial activity by the

stab-on-agar test using Listeria monocytogenes Scott A, Lactobacillus sakei 148 and Pediococcus acidilactici 347 as indicator strains, as previously described (Cintas et al. 1995, Casaus 1998). Isolate P21 was selected for further studies because of its strong inhibitory activity against these indicators. The micro-organisms used for evaluation of the antimicrobial spectrum of strain P21 are listed in Table 1. The LAB were propagated in MRS broth (Oxoid Ltd., Basingstoke, UK) at 328C. Clostridium spp. were propagated anaerobically (Oxoid Anaerobic System) in RCM broth (Oxoid) at 378C. All other strains were grown in APT broth (Difco Laboratories, Detroit, USA) at 32 or 378C.

Table 1. Antimicrobial activity of cell-free culture supernatant from E. faecium P21 against Grampositive bacteria Indicator speciesa Lactobacillus casei Lactobacillus fermentum Lactobacillus plantarum Lactobacillus sakei Lactobacillus sakei Pediococcus acidilactici Pediococcus pentosaceus Pediococcus pentosaceus Pediococcus pentosaceus Lactococcus cremoris Lactococcus lactis Enterococcus faecium Enterococcus faecium Enterococcus faecium Enterococcus faecalis Clostridium perfringens Clostridium botulinum Listeria monocytogenes Listeria monocytogenes Listeria monocytogenes Listeria monocytogenes Listeria monocytogenes Staphylococcus aureus Staphylococcus aureus Staphylococcus aureus
a b

Strain 334 285 1193 2714 148 347 FBB61 FBB63 PC1 CNRZ117 BB24 L50 T136 P13 EF 376 551 7973 LI5sv1/2 5105 LI1sv4 Scott A 137 196E 349

Sourceb ATCC CECT NCDO NCFB Our strain collection Our strain collection TNO TNO TNO INRA Our strain collection Our strain collection Our strain collection Our strain collection TNO CECT CECT NCTC FVM NCTC FVM FVM FRI FRI FRI

Activityc NIZD 13.9 16.5 NIZD 11.1 NIZD 14.1 NIZD NIZD 14.1 NIZD 15.5 NIZD 13.5 16.3 15.2 15.5 16.4 17.2 17.7 17.5 18.4 17.7 17.2 17.6

Lactococcus lactis subsp. cremoris is abbreviated as Lactococcus cremoris. Abbreviations: ATCC, American Type Culture Collection (Rockville, USA); CECT, Coleccion Espaola de Cultivos Tipo (Valencia, Spain); DSM, Deutsche Sammlung von Mikroorganismen und Zell Kulturen, ' GmbH (Braunschweig, Germany); INRA, Station de Recherches Laitieres (Jouy-en-Josas Cedex, France); FRI, Food Research Institute (Madison, USA); FVM, Facultad de Veterinaria (Madrid, Spain); NCTC, National Collection of Type Cultures (London, UK); TNO, Nutrition and Food Research (Zeist, The Netherlands). c Diameter of inhibition zone in millimeters; NIZD, no inhibition zone detected.

118 C. Herranz et al.

Phenotypic identication
Isolate P21 was examined by phase-contrast microscopy to determine its cell morphology and tested for Gram-staining reaction and catalase activity (Table 2). Preliminary identication was determined according to the tests proposed by Schleifer and Kilpper-Blz (1984) and Devriese et al. (1993), which included the ability to grow at 10, 45 and 508C, growth on media containing 6?5 and 10% NaCl, growth at dierent pHs, arginine dihydrolase activity, Voges-Proskauer reaction (acetoin production), resistance to 40% (w/v) bile, growth on media containing 0?04% sodium azide (Bacto

EVA broth, Oxoid; m-Enterococcus, Difco), urease activity, and hemolysis on 5% calf blood agar plates (Oxoid). Conguration of lactic acid produced from glucose was determined enzymatically with D- and L -lactate dehydrogenases as described by the supplier (Boehringer GmbH, Mannheim, Germany). Fermentation patterns were determined with API Rapid CH fermentation strips (API, Bio' merieux, Montallieu Vercieu, France) in CHL medium. Total proteins were analysed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) (Laemmli 1970), and the pattern obtained was compared with those of reference strains as described by

Table 2. Phenotypic characteristics of isolate P21

Test Morphology Gram Calf blood hemolysis Catalase pHa Growth at 108C at 458C at 508C at pH 4?5^9?6 in 40% bile in 6?5% NaCl in 10% NaCl in 0?04% sodium azide Urease Arginine hydrolysis CO2 production H2S production Voges^Proskauer Lactic acid b Glycerol Erythritol D-Arabinose L -Arabinose Ribose D-Xylose L -Xylose Adonitol b Methyl-Xyloside Galactose D-Glucose D-Fructose D-Mannose L -Sorbose
a b

Reaction or characteristic Cocci 7 7 4?1 7 7 7 7 7 L 7 7 7 7 7 7 7

Test Rhamnose Dulcitol Inositol Mannitol Sorbitol a-Methyl-D-Mannoside a-Methyl-D-Glucoside N Acetil glucosamine Amygdalin Arbutin Esculin Salicin Cellobiose Maltose Lactose Melibiose Saccharose Trehalose Inulin Melezitose D-Ranose Starch Glycogen Xylitol b-Gentibiose D-Turanose D-Lyxose D-Tagatose D-Fucose L -Fucose D-Arabitol L -Arabitol Gluconate 2 -Keto-Gluconate

Reaction or characteristic 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7

Final pH of stationary growth phase cultures grown in MRS broth at 328C. Conguration of lactic acid produced from glucose.

Enterocins A and B from E. faecium P21 119

Kersters and de Ley (1975) and Pot et al. (1994) by B. Pot, University of Ghent, Ghent, Belgium.

Eect of enzymes, heat treatments, pH, freeze-thaw, lyophilization and storage on bacteriocin activity
Proteolytic, lipolytic and amylolytic enzymes used in our studies and their sources are listed in Table 3. All enzymes were added to cell-free culture supernatants of E. faecium P21 at a nal concentration of 1 mg ml71. Controls consisted of samples of enzymes in sterile medium and untreated bacteriocin solution. All preparations were incubated at 378C for 6 h and enzymes were heat-inactivated at 1008C for 10 min. Stability of the antagonistic activity to heat was determined by heating aliquots of cellfree culture supernatants at 80 and 1008C for 20 min. Eect of pH on bacteriocin activity was tested adjusting the pH of cell-free culture supernatants to pH-values ranging from 2 to 11. Samples were incubated at 4 and 328C for 24 h. Aliquots of sterile MRS broth with the pH adjusted to these pH-values were used as

Bacteriocin assays
Cell-free culture supernatants of the bacteriocinogenic strain E. faecium P21 were obtained essentially as previously described (Cintas et al. 1995). Briey, E. faecium P21 was grown in MRS broth at 328C until early stationary phase (A620 = 1?0). The culture was subsequently centrifuged at 12 000 g for 10 min at 48C, and the supernatant was neutralized to 6?2 with 1 M NaOH and lter-sterilized through 0?22 -mm-pore-size lters (Millipore Corp., Bedford, Massachussets, USA).The antimicrobial activity of the cell-free culture supernatant was determined by the agar well diusion assay, performed essentially as described by Cintas et al. (1995); 100 ml aliquots of supernatants were placed in wells (7-mm diameter) cut in cooled soft MRS, RCM or APT agar plates (20 ml) previously seeded (105 cfu ml71) with the appropriate indicator strains (Table 1).The plates were kept at 48C for 2 h and subsequently incubated under optimal conditions for growth of the target micro-organisms. After 24 h, the diameters (mm) of the growth inhibition zones were measured. The bacteriocin activity during the purication processes was quantied by a microtiter plate assay (Holo et al. 1991). Briey, twofold serial dilutions (50 ml) of bacteriocin extracts in MRS broth were prepared in microtiter plates. The wells were then lled up to 200 ml by the addition of 150 ml of a diluted (in MRS) fresh overnight culture of E. faecium P13 (Cintas et al. 1997, Casaus 1998). Growth inhibition was measured spectrophotometrically at 620 nm with a microtiter plate reader (Labsystems iEMS Reader MF, Labsystems, Helsinki, Finland) after 12 h of incubation at 328C. One bacteriocin unit was dened as the reciprocal of the highest dilution of bacteriocin which inhibited the growth of the indicator micro-organism by 50% (50% of the turbidity of the control culture without bacteriocin).

Table 3. Physico-chemical stability of the bacteriocin activity of cell-free culture supernatants from E. faecium P21 Treatment Enzymes: Pepsin (Serva, No. 318200) Trypsin (Merck, No. 82130 Papain (Merck, No. 7144) Protease Type II (Sigma, No. P- 4755) Lipase Type VII (Sigma, No. L-1754) a-Amylase (Boehringer Mannhein, No. 161764) Heat: 20 min at 808C 20 min at 1008C pH: 2?0^11?0 Freeze-thaw Lyophilization Storage at 7208Cb
a

Bacteriocin activitya 7 7 7 7

Bacteriocin activity was determined against E. faecium P13 by the microtiter plate assay (see Materials and Methods). Symbols:, inhibition zone; ^, no inhibition zone. b Supernatants were stored at7208C for 12 months.

120 C. Herranz et al.

controls. After each treatment, samples and controls were tested for antimicrobial activity against E. faecium P13 by the microtiter plate assay described above.

Bacteriocin purication
Purication of bacteriocins was achieved using a modication of the procedure described by Nissen-Meyer et al. (1992) and Cintas et al. (1995). All the chromatographic equipment was obtained from Pharmacia-LKB (Uppsala, Sweden) and all the purication steps were performed at room temperature if not otherwise stated. The bacteriocins were puried from a 1-l E. faecium P21 culture which was grown in MRS broth at 328C until the late logarithmic phase (A620 = 0?9). The cells were removed by centrifugation at 12 000 g for 10 min at 48C, and ammonium sulphate was gradually added to achieve 45% saturation. The sample was kept at 48C with stirring for 30 min. After centrifugation at 12 000 g for 30 min at 48C, the pellet and oating material were combined and solubilized in 100 ml of 20 mM sodium phosphate buer, pH 5?8 (buer A). To remove any trace of ammonium sulphate, the bacteriocin preparation was passed through gel ltration G-25 PD-10 columns previously equilibrated with buer A. The gel-ltered fraction was further subjected to cation-exchange (SP Sepharose Fast Flow) and hydrophobicinteraction (Octyl-Sepharose CL- 4B) chromatographies, followed by reverse-phase chromatography in a C2 to C18 column (PepRPC HR5/5) integrated in a fast-performance liquid chromatography system (FPLC system) (Cintas et al. 1995, Casaus 1998). The bacteriocins were eluted from the reverse phase column with a

linear gradient of 2 -propanol (Merck) in aqueous 0?1% (v/v) triuoroacetic acid (TFA) at a ow rate of 0?5 ml min71. Fractions with high bacteriocin activity were mixed and rechromatographed on the same reverse phase column to obtain chromatographically pure bacteriocins. Puried bacteriocins were stored in 60% 2 -propanol containing 0?1% TFA at 7208C.

Amino acid sequence analysis

The N-terminal amino acid sequence of puried bacteriocins was determined by Edman degradation with an Applied Biosystems (Foster City, California, USA) model 477A automatic sequence analyser by Dr J. Vazquez, Protein Chemistry Facility, Centro de Biolog| a Molecular Severo Ochoa, Madrid, Spain.

PCR analysis and DNA sequencing

Total DNA of E. faecium P21 and E. faecium T136 (enterocins A and B producer) (Casaus et al. 1997, Casaus 1998) was obtained by the alkaline lysis method of Anderson and McKay (1983), and was used as DNA template for PCR reactions. Oligonucleotide primers used for PCR and DNA sequencing (Table 4) were obtained from KEBOLab (Spanga, Sweden). Specic primers TH10 and LA1 were designed from the single-strand DNA sequence of the region of E. faecium CTC492 and E. faecium T136 containing the enterocin A structural (entA) and immunity (enti) genes (Aymerich et al. 1996, Casaus 1998). The 5 end of the coding strand primer (TH10) is located four nucleotides upstream of the start codon of entA, and the 5 end of the complementary strand primer (LA1) is located

Table 4. Primers used for PCR and DNA sequencing

Primers TH10 LA1 ENTB3 ENTB5 SK2 5 5 5 5 5 Sequence GAT TAT GAA ACA TTT AAA AAT TTT GTC 3 AAA ACC ACC TAT AGA CAT TCC TGC 3 AGA CCT AAC AAC TTA TCT AAA G 3 GTT GCA TTT AGA GTA TAC ATT TGC 3 CCG CTC TAG AAC TAG TGG ATC 3

Primer TH10 was designed by Aymerich et al. (1996); primers LA1, ENTB3 and ENTB5 were designed by Casaus (1998).

Enterocins A and B from E. faecium P21 121

27 nucleotides upstream of the stop codon of entA. Specic primers ENTB3 and ENTB5 were designed from the single-strand DNA sequence of the region of E. faecium T136 containing the enterocin B structural gene (entB) (Casaus et al. 1997, Casaus 1998). The 5 end of the coding strand primer (ENTB3) is located 84 nucleotides downstream of the start codon of entB, and the 5 end of the complementary strand primer (ENTB5) is located 1 nucleotide upstream of the stop codon of entB. These primer pairs were used in PCR reactions to amplify a 172 -bp and a 126 -bp DNA fragment of the enterocin A and enterocin B structural genes, respectively, from total DNA of E. faecium P21 and E. faecium T136 (positive control). The PCR conditions included a hot start at 978C (2 min), annealing at 558C (30 s), elongation at 728C (45 s), and denaturation at 948C (1 min). Amplication reactions (35 cycles) were carried out with the Gene Amp PCR kit in a DNA-Thermal Cycler according to the suppliers instructions (Perkin-Elmer Cetus, Norwalk, Connecticut, USA). PCR products were analyzed by electrophoresis on 2% (w/v) agarose gels [16Tris-acetate-EDTA buer (pH 8.0)].The gels were run at 80 V for 90 min, using the pBR322 DNA-MspI digest ladder (New England Biolabs Inc., Beverly, Massachussets, USA) as a molecular weight standard. The 172 and 126 -bp PCR fragments were further puried by using a QIAquick PCR Purication Kit (Qiagen GmbH, Hilden, Germany). DNA sequencing was performed on both strands by using the ABI Prism Dye Terminator Cycle Sequencing Ready Reaction Kit with AmpliTaq DNA polymerase and dye-labeled terminators (Perkin-Elmer, Applied Biosystems Division, Foster City, California, USA) and an ABI Prism 377 DNA Sequencer (Perkin-Elmer). Analysis of the sequence was performed with the ABI Prism Sequencing Analysis (version 3 Ofc1) and the Autoassembler (version 1.4) software package (Perkin-Elmer). For further DNA sequencing of the enterocin A and enterocin B operons, dierent samples of total DNA of E. faecium P21 were cleaved with the blunt-end cutters DraI, EcoRV, or HincII, and ligated (T4 DNA ligase, Promega Corp., Madison, Wisconsin, USA) to dephosphorylated pBluescript II SK (Stratagene,

La Jolla, California, USA) digested with HincII. Restriction enzymes were obtained from New England Biolabs and Promega and were used in accordance with the suppliers instructions. Samples from these ligation reactions were subsequently used as templates to amplify DNA segments of EntA and Ent B operons using entA- or entB-specic primers in combination with the polylinker primer SK2 (Table 4). PCR reactions were carried out as described above, except that elongation time at 728C was increased to 2min. A specic PCR product consisting of approximately 800 bp was obtained with the EcoRV ligation reaction when the polylinker primer SK2 was used in combination with the entA-specic primer TH10. A specic PCR product consisting of approximately 280 bp was obtained with the DraI ligation reaction when the polylinker primer SK2 was used in combination with the entBspecic primer ENTB5. Automated DNA sequencing of both PCR products was performed as described above and allowed us to obtain the sequence of entA and entB reported in this paper.

Computer analysis of DNA and protein sequences

Analyses of DNA and protein sequences were performed by using the PC/Gene program package (version 6.8; Intelligenetics, Inc., Mountain View, California, USA) and the Sequence Analysis Software Package (version 7.2) licensed from the Genetics Computer Group, University of Wisconsin, Madison, USA (Devereux et al. 1984).

Results
Identication of bacteriocinogenic isolate P21
As shown in Table 2, isolate P21 is a catalasenegative, Gram-positive, facultatively anaerobic coccus with the ability to grow at 10 and 458C, at pH 9?6, in 6?5 and 10% NaCl broth, and in the presence of both 40% (v/v) bile and 0?04% sodium azide. It fermented glucose to L -lactic acid but did not produce gas, gave a

122 C. Herranz et al.

positive Voges^Proskauer reaction and produced ammonia from the hydrolisis of arginine. The nal pH in MRS broth was 4?1 and acid was produced from ribose and L -arabinose but not from glycogen, D-arabitol, D-tagatose, sorbitol or gluconate. The strain did not show urease activity and it was non-hemolytic on calf blood. By comparison of its SDS-PAGE protein pattern with a database of protein patterns from other LAB and considering all of the features cited above, isolate P21 was identied as E. faecium.

Antimicrobial spectrum of cell-free culture supernatants of E. faecium P21

Table 1 shows the antimicrobial activity of neutralized and lter-sterilized cell-free culture supernatants of E. faecium P21 against selected Gram-positive bacteria. Of the 15 LAB tested, only three strains of Lactobacillus spp., one strain of Pediococcus pentosaceus, one strain of Lactococcus lactis subsp. cremoris, two strains of E. faecium and one strain of E. faecalis were inhibited by culture supernatants, with inhibition zone diameters ranging from 11?1 to 16?5 mm. However, all the spoilage and foodborne Gram-positive bacteria tested, which included ve strains of L. monocytogenes, one strain of Clostridium perfringens, one strain of C. botulinum, and three strains of S. aureus were sensitive to cell-free culture supernatants, with inhibition zone diameters ranging from 15?2 to 18?4 mm. None of the Gram-negative bacteria tested (Salmonella typhimurium, Escherichia coli, Pseudomonas uorescens, Yersinia enterocolitica, Enterobacter aerogenes and Aeromonas hydrophila) were inhibited by cellfree culture supernatants of E. faecium P21 under the stated experimental conditions (results not shown).

sin, papain and protease II), but was not aected by lipolytic or amylolytic enzymes such as lipase VII or a-amylase, respectively. Inhibitory activity of cell-free culture supernatants was maintained completely after heat treatment at 80 and 1008C for 20 min. These results suggested that the inhibitory compound(s) produced by E. faecium P21 is a heat-stable proteinaceous compound(s).The inhibitory protein(s) withstood exposure to pH values between 2 and 11 for 24 h at 4 and 328C, and its antimicrobial activity was not lost by freezing and thawing, lyophilization and long-term storage (12 months) at 7208C. The antagonistic activity of E. faecium P21 was measured after 16 h of incubation at 328C in MRS, BHI and APT broth against E. faecium P13 by the agar well diusion assay. Bacteriocin production and secretion was observed in all media tested, but the highest activities were obtained in MRS broth (results not shown). Figure 1 shows the growth kinetics (log cfu ml71), pH evolution and antimicrobial activity of cell-free culture supernatants of E. faecium P21 grown in MRS broth at 328C. Maximum antimicrobial activity in the growth medium was obtained in the late logarithmic phase of growth (12 h after inoculation), when the pH of the medium had dropped to 4?4,

Physico-chemical stability of the bacteriocin activity

The eect of enzymes, heat treatment, pH, freeze-thaw, lyophilization and storage on the antimicrobial activity of cell-free culture supernatants of E. faecium P21 is summarized in Table 3. Bacteriocin activity was completely abolished by protease treatment (pepsin, tryp-

Figure 1. Growth kinetics (*), pH (&) and antimicrobial activity (~) of E. faecium P21 grown in MRS broth at 328C. Antimicrobial activity of cellfree culture supernatants was assayed by an agar diusion assay using E. faecium P13 as indicator micro-organism.

Enterocins A and B from E. faecium P21 123

although it decreased rapidly during the stationary phase.

Amino acid sequence analyses

Puried bacteriocins from the last reversephase chromatography step (fractions A and B (Figs 3 and 4, respectively)) were subjected to Edman degradation analyses in order to determine their partial amino acid sequences (Fig. 5). The rst 30 amino acid residues of the

Purication of the bacteriocins

Results of bacteriocin purication obtained from a late-logarithmic-growth phase culture of E. faecium P21 grown in MRS broth at 328C are summarized in Table 5. Ammonium sulphate precipitation allowed a seven-fold increase in specic antimicrobial activity and 44% recovery of the initial bacteriocin activity in the cell-free culture supernatant. The 10 -ml fraction eluted from the hydrophobic interaction column represented a 5% recovery of bacteriocin activity.The rst run of the last step in the purication procedure, reverse-phase chromatography, revealed two well separated fractions with bacteriocin activity (fractions A and B), eluting at 36?5 and 40?0% 2 -propanol, respectively (Fig. 2). In order to obtain puried fractions for amino acid sequence analysis, both fractions were separately rechromatographed three times on the same column, which resulted in each case in a single protein absorbance peak, coinciding with the antimicrobial activity peak (fractions A and B) (Figs 3 and 4, respectively). Fractions A and B eluted when the 2 -propanol gradient had reached 28?0 and 29?0%, respectively, and represented a recovery of 1?25 and 1?81%, respectively, of the initial bacteriocin activity in the cell-free culture supernatant.

Figure 2. Reverse-phase chromatography of the

antimicrobial activity from E. faecium P21. The amount applied to the column was obtained from 1 l of culture. BU, bacteriocin units.

Table 5. Purication of activity A (enterocin A) and activity B (enterocin B) from E. faecium P21
Volume (ml) Culture supernatant Fraction Ammonium sulfate precipitation Gel ltration chromatography Cation-exchange chromatography Hydrophobic-interaction chromatography Reverse-phase chromatography Fraction A Fraction B
a b

Total A254a 13 300 860 1340 6?75 9?7 0?024 0?030

Total Specic activity activity (103 BU)b (sp. activity) c 521 228 377 29 24 6?5 9?5 39 265 282 4316 2492 272 250 314 667

Increase in Yield sp. activity (%) (fold) 1 7 7 110 64 6950 8033 100 44 72 6 5 1?25 1?81

1 000 100 200 50 10 1?2 2?0

Optical density at 254 nm multiplied by the volume in ml. Bacteriocin units (BU). c BU per ml divided by optical density at 254 nm.

124 C. Herranz et al.

Ennahar et al. 2000) in positions 8^12. Homology searches for the partial amino acid sequence of fraction A revealed a high homology with enterocin A from E. faecium CTC492 (Aymerich et al. 1996). The partial amino acid sequence of the rst 49 residues of fraction B was determined, revealing the presence of 13 unidentied residues and the absence of the pediocin-like consensus sequence. Homology searches for this sequence in protein data banks revealed a high homology with enterocin B from E. faecium T136 (Casaus et al. 1997, Casaus 1998).

Genetic evidence for enterocin A and enterocin B production in E. faecium P21

The amino acid sequences of the puried peptides with antimicrobial activity from E. faecium P21 strongly suggested that enterocins A and B, or closely related bacteriocins, are produced by this enterococcal strain. Consequently, based on the amino acid sequence similarity of fractions A and B with enterocin A and enterocin B, respectively (Fig. 5), two PCRs were conducted to elucidate the presence of the structural genes of these enterocins in E. faecium P21. For this purpose, specic oligonucleotides for both enterocins and total DNA from E. faecium P21 were used as primers and template, respectively. Total DNA from E. faecium T136 (enterocin A and B producer) (Casaus et al. 1997) was used as a positive control. Reactions carried out using the primer combinations LA1 TH10 and ENTB3 ENTB5 would be expected to amplify, respectively, a 172 and a 126 bp fragment in case that E. faecium P21 contains the enterocin A and enterocin B structural genes. Agarose gel electrophoresis of PCR products allowed the visualization of two amplication bands of these sizes, suggesting that E. faecium P21 contains entA and entB (Fig. 6). Automated DNA sequencing of both strands of these PCR fragments revealed that these two partially sequenced genes were identical to the corresponding regions of entA and entB in E. faecium T136. In order to determine the complete sequence of these two genes from E. faecium P21, two new specic PCR fragments were constructed: an 800 bp fragment obtained

Figure 3. Last reverse-phase chromatography

of fraction A from E. faecium P21.

Figure 4. Last reverse-phase chromatography

of fraction B from E. faecium P21.

N-terminus of fraction A included nine unidentied positions and the pediocin-like bacteriocin consensus amino acid sequence YGNGV (Nieto-Lozano et al. 1992, Aymerich et al. 1996,

Enterocins A and B from E. faecium P21 125

Figure 5. N-terminal amino acid sequence of puried peptide fractions from E. faecium P21. The x indicates that the residue could not be identied. Letters above and below the amino acid sequences indicate that the residue could not be determined with certainty. For comparison, the rst 30 and 49 amino acid residues of enterocin A (Aymerich et al. 1996) and enterocin B (Casaus et al. 1997), respectively, are also shown.

Figure 6. Agarose gel electrophoresis of PCR

fragments generated from total DNA of E. faecium P21 (1) and E. faecium T136 (2) with specic oligonucleotide primers for enterocin A (A) and enterocin B (B) structural genes. M refers to the molecular weight marker.

with the EcoRV ligation reaction as template and the polylinker primer SK2 in combination with the entA-specic primer TH10, and a 280 bp fragment obtained with the DraI ligation reaction as template and the polylinker primer SK2 in combination with the entBspecic primer ENTB5. Automated DNA sequencing of these two PCR fragments revealed that entA and entB from E. faecium P21 were identical to the corresponding genes from E. faecium T136 (results not shown), indicating that E. faecium P21 produces, at least, the bacteriocins enterocin A and enterocin B. Further DNA analysis of the sequence downstream of entA revealed the presence of two consecutive ORFs with the same polarity (Fig. 7). The rst ORF, located two nucleotides downstream of the stop codon of entA, resulted identical to entiA from E. faecium CTC492 (Aymerich et al. 1996). EntiA encodes a translation product of 103 amino acid residues corresponding to the immunity protein of enterocin A (Aymerich et al. 1996, OKeee et al. 1999). The 41-bp DNA region of E. faecium P21 located

126 C. Herranz et al.

Figure 7. Nucleotide sequence of a 811-bp fragment from E. faecium P21 containing the structural gene
of enterocin A (entA), the immunity gene (entiA ) and the induction peptide (IP) gene (entF). The deduced amino acid sequences of EntA, EntiA and EntF are shown below the DNA sequence. The cleavage sites of the prebacteriocin and the precursor of IP are indicated by vertical arrows. The putative 710 and 735 promoter sequences and ribosome binding sites (RBS) are underlined; direct repeats right (DRR) and left (DRL) within the conserved regulatory-like boxes are overlined.

immediately downstream of the stop codon of entiA was identical to that found in E. faecium CTC492 (Aymerich et al. 1996); however, from the nucleotide at position 553 (adenine), the DNA sequence identity between these two

enterococcal strains was no longer observed. The second ORF, orf3, identied in the enterocin A operon in E. faecium P21 encodes a primary translation product of 48 amino acid residues. Orf3 is preceded eight nucleotides

Enterocins A and B from E. faecium P21 127

upstream by a potential ribosome binding site, 5 GAGGGG 3. A likely 710 consensus promoter region (Pribnow box) (TATAGT) is located four nucleotides upstream of the ribosome binding site, an a sequence (TTGAAT) showing resemblance to s70 promoter 735 region is located at the optimal distance of 18 nucleotides upstream the 710 region. One set of direct repeats consisting of 10 nucleotides with the sequence TCTCAA(T/A)(T/A)TT was seen in the promoter region. The two repeats were spaced by an AT-rich region of 25 bp. The repeat close (23 nucleotides) to the 735 site was termed right (DRR), whereas the second repeat located 25 nucleotides upstream was termed left (DRL). The deduced amino acid sequence of the gene product of orf3 resulted identical to that previously reported for the induction peptide (EntF) of enterocin A from E. faecium CTC492 (Nilsen et al. 1998) and E. faecium DPC1146 (OKeee et al. 1999).

Discussion
Sensitized by the possible use of bacteriocinogenic LAB and/or their bacteriocins as natural biopreservatives to suppress growth of spoilage and pathogenic bacteria in foods, we have focused our studies on the characterization of bacteriocins produced by LAB isolated from Spanish dry-fermented sausages. In this respect, a new LAB isolate was identied as E. faecium P21 and shown to display a narrow antimicrobial activity against closely related LAB and strong antimicrobial activity against many foodborne Gram-positive bacteria (Table 1). During the past several years, the isolation of bacteriocinogenic strains of E. faecium from dry-fermented sausages has been frequently reported. The enterocin A and B producers E. faecium CTC492 and E. faeciumT136 were isolated byAymerich et al. (1996) and Casaus et al. (1997); the enterocin P producers E. faecium P13, E. faecium AA13 and E. faecium G16 were isolated from the same type of fermented products by Cintas et al. (1997), Casaus (1998) and Herranz et al. (1999); and very recently, it has been reported that E. faecium L50, a strain isolated from Spanish dry fermented sausages, produces enterocins L50A and L50B, enterocin

P and enterocin Q (Cintas et al. 1998a, Cintas et al. 2000). These results indicate the suitability of dry-fermented sausages as a source for the isolation of bacteriocinogenic enterococcal strains. The antimicrobial activity in the growth media of E. faecium P21 was lost by protease treatment but withstood heat treatments and exposition to a wide range of pH values like most LAB-bacteriocins (Piard and Desmazeaud 1992). These observations suggest that this antimicrobial activity is truly due to peptide bacteriocin(s). To further characterize the bacteriocin activity, a purication procedure including ammonium sulphate precipitation, gel ltration, and cation-exchange, hydrophobic interaction and reverse-phase chromatographies was carried out (Table 5). After the last run on the reverse-phase column, two fractions containing bacteriocin activity were obtained (fractions A and B, Figs 3 and 4, respectively). N-terminal amino acid sequencing by Edman degradation showed that fraction A contained a bacteriocin peptide similar to the class IIa enterocin A, while peptide in fraction B was closely related to the class IId enterocin B, both bacteriocins previously identied in E. faecium CTC492 and T136 (Aymerich 1996, Casaus et al. 1997, Casaus 1998) (Fig. 5). A number of PCR products containing the bacteriocin structural genes were also sequenced, revealing that E. faecium P21 indeed produces enterocins A and B. The genetic organization of the enterocin A locus in E. faecium P21 shows that the bacteriocin structural gene (entA), the immunity gene (entiA) and the induction peptide gene (entF ) are clustered and colinearly arranged (Fig. 7). This organization was identical to that reported for the EntA-producer E. faecium DPC1146 (OKeee et al. 1999). The clustering of these three genes is a common characteristic amongst the class II bacteriocin regulated systems; however, the location of the IP gene with respect to the bacteriocin structural genes is dierent depending on the bacteriocin system described (Nes et al. 1996, Nes and Eijsink 1999). The EntA operon in E. faecium P21 includes in the promoter region of entF a set of direct repeats of 10 bp (8 bp identical) spaced by an AT-rich region. Similar features have also

128 C. Herranz et al.

been identied in the conserved regulatorylike boxes in the promoters of other genes within bacteriocin operons and may serve as a binding site for a transcription regulator (Diep et al. 1996, Nes et al. 1996) Strikingly, the EntA operon in E. faecium P21 and E. faecium CTC492 is structurally dierent. In E. faecium P21, the IP gene (entF ) is located immediately downstream of the immunity gene (entiA) (Fig. 7); however, in E. faecium CTC492, both entiA and entFare spaced by an intergenic region of aproximately 1 100 bp (Nilsen et al. 1998). Computer analysis of the DNA sequence located upstream of entF in E. faecium CTC492 (Nilsen et al. 1998) allowed us to identify an incompletely sequenced ORF encoding a truncated peptide of 134 amino acid residues. Homology search of this gene product in protein data banks revealed signicant similarities to several enterococcal transposases (results not shown). Recent studies have shown that production of multiple bacteriocins by a single strain is a phenotype more widespread in nature than initially believed. Examples of LAB producing several bacteriocins abound within the genera Carnobacterium (Quadri et al. 1994, Pilet et al. 1995, Saucier et al. 1997), Lactobacillus (Jimenez-D| az et al. 1993, Diep et al. 1996, Anderssen et al. 1998), Lactococcus (van Belkum et al. 1989, 1991, 1992), Leuconostoc (RevolJunelles et al. 1996) and Enterococcus (Casaus et al. 1997, Cintas et al. 1998a, 2000). In recent years, with the advent of many applications of bacteriocins as biopreservatives in food, a gradually emerging concern is the potential development of bacteriocin resistance among bacteria (Muriana 1996). From an applied point of view, the combined use of several bacteriocins with dierent mechanisms of action would enhance their antimicrobial eect and, therefore, reduce the appearance of bacteriocin resistant populations (Hanlin et al. 1993). In this context, it is interesting to note that enterocin A and B are two broad spectrum bacteriocins lacking in primary structure similarities, showing dierent target specicity and acting synergistically (Casaus et al. 1997), which suggests that both bacteriocins may possess dierent mechanisms of action on sensitive cells.

Several studies have described the identication of independently isolated LAB genera and strains producing identical bacteriocins (Remiger et al. 1996, Bennik et al. 1997, Cintas et al. 1998b, Herranz et al. 1999). Although E. faecium P21 and E. faecium T136 produce enterocin A and enterocin B, both strains are dierent according to SDS-PAGE analyses, carbohydrate fermentation patterns and plasmid proles (results not shown). The fact that several independently isolated enterococcal strains produce enterocins A and B (Aymerich et al. 1996, Casaus et al. 1997, Casaus 1998, Nilsen et al. 1998, present work) led us to speculate that production of these bacteriocins may confer an adaptive advantage to the producer strains by increasing their ability to control the competing microora. The broad spectrum and the synergistic action of enterocins A and B make them a promising agent to control, as a part of a multihurdle preservation system, the growth of undesirable bacteria, such as L. monocytogenes, in foods. However, elucidation of the molecular mode of action of enterocins A and B is required before application of these bacteriocins to control the presence of spoilage and foodborne pathogens in foods.

Acknowledgements
We are indebted to Dr. Bruno Pot for his assistance in the identication of strain P21 and to Dr Jesus Vazquez for amino acid sequencing analyses. This work was partially supported by grants ALI97- 0559 and AGL2000 - 0707 from the Comi sion Interministerial de Ciencia y Tecnolog| a (CICYT). Carmen Herranz and Pilar Casaus hold fellowships within the National Plan for Training Researchers (FPI) from the Minister io de Educacion y Ciencia, Spain. Sanghamitra Mukhopadhyay holds a fellowship from the Agencia Espaola de Cooperacion Internacional (AECI). Jose Mar| a Mart| nez holds a fellowship from the Comunidad de Madrid, Spain. Luis M. Cintas was recipient of a Postdoctoral Biotechnology Research Training Grant from the Commission of the European

Enterocins A and B from E. faecium P21 129

Communities (Project Contract Bio4 -CT96 5051).

References
Anderson, D. G. and McKay, L. L. (1983) Simple and rapid method for isolation of large plasmid DNA from lactic streptococci. Appl. Environ. Microbiol. 46, 549^552. Anderssen, E. L., Diep, D. B., Nes, I. F., Eijsink,V. and Nissen-Meyer, J. (1998) Antagonistic activity of Lactobacillus plantarum C11: two new two-peptide bacteriocins, plantaricin EF and JK, and the induction factor plantaricin A. Appl. Environ. Microbiol. 64, 2269^2272. Aymerich, T., Holo, H., Hvarstein, L. S., Hugas, M., Garriga, M. and Nes, I. F. (1996) Biochemical and genetic characterization of enterocin A from Enterococcus faecium, a new antilisterial bacteriocin in the pediocin family of bacteriocins. Appl. Environ. Microbiol. 62, 1676^1682. Ben Embarek, P. K., From Jeppesen, V. and Hus, H. H. (1994) Antibacterial potential of Enterococcus faecium strains isolated from sous-vide cooked sh llets. Food Microbiol. 11, 525^536. Bennik, M. H. J., Smid, E. J. and Gorris, L. G. M. (1997) Vegetable-associated Pediococcus parvulus produces pediocin PA-1. Appl. Environ. Microbiol. 63, 2074^2076. Casaus, M. P. (1998) Aislamiento e identicacion de bacterias lacticas de origen carnico productoras de bacteriocinas. Caracterizacion bioqu| mica y genetica de la enterocina P de Enterococcus faecium P13 y de la enterocina B de Enterococcus faecium T136. Ph.D. Thesis. Universidad Complutense de Madrid, Madrid, Spain. Casaus, M. P., Cintas, L. M., Suarez, A., Rodr| guez, J. M., Holo, H., Hernandez, P. E. and Nes, I. F. (1995) Enterococcus faecium T136: a two anti-Listeria active bacteriocins producer, isolated from Spanish dry fermented sausages, abstract p-156. In Abstracts of the 2nd Contractors Meeting of Participants in the EC Biotechnology Programme G-Project, Cork, Ireland. Casaus, P., Nilsen, T., Cintas, L. M., Nes, I. F., Hernandez, P. E. and Holo, H. (1997) Enterocin B, a new bacteriocin from Enterococcus faecium T136 which can act sinergistically with enterocin A. Microbiology 143, 2287^2294. Cintas, L. M. 1995. Caracterizacion bioqu| mica y gen etica parcial de la pediocina L50, una nueva bacteriocina producida por Pediococcus acidilactici L50 aislado de embutidos crudos curados. Ph.D. Thesis. Universidad Autonoma de Madrid, Madrid, Spain. Cintas, L. M., Rodr| guez, J. M., Fernandez, M. F., Sletten, K., Nes, I. F., Hernandez, P. E. and Holo, H. (1995) Isolation and characterization of pedio-

cin L50, a new bacteriocin from Pediococcus acidilactici with a broad inhibitory spectrum. Appl. Environ. Microbiol. 61, 2643^2648. Cintas, L. M., Casaus, P., Hvarstein, L. S., Hernandez, P. E. and Nes, I. F. (1997) Biochemical and genetic characterization of enterocin P, a novel sec-dependent bacteriocin from Enterococcus faecium P13 with a broad antimicrobial spectrum. Appl. Environ. Microbiol. 63, 4321^4330. Cintas, L. M., Casaus, P., Holo, H., Hernandez, P. E., Nes, I. F. and Hvarstein, L. S. (1998a) Enterocins L50A and L50B, two novel bacteriocins from Enterococcus faecium L50, are related to staphylococcal hemolysins. J. Bacteriol. 180, 1988^1994. Cintas, L. M., Casaus, P., Fernandez, M. F. and Hernandez, P. E. (1998b) Comparative antimicrobial activity of enterocin L50, pediocin PA-1, nisin A and lactocin S against spoilage and foodborne pathogenic bacteria. Food Microbiol. 15, 289^298. Cintas, L. M., Casaus, P., Herranz, C., Hvarstein, L. S., Holo, H., Hernandez, P. E., and Nes, I. F. (2000) Biochemical and genetic evidence that Enterococcus faecium L50 produces enterocins L50A and L50B, the sec-dependent enterocin P, and a novel bacteriocin secreted without an N-terminal extension termed enterocin Q. J. Bacteriol. 182, 6806^6814. Daeschel, M. A. (1989) Antimicrobial substances from lactic acid bacteria for use as food preservatives. Food Technol. 43, 164^167. Devereux, J., Haeberli, P. and Smithies, O. (1984) A comprehensive set of sequence analysis programs for the VAX. Nucleic Acids Res. 12, 387^395. Devriese, L. A., Pot, B. and Collins, M. D. (1993) Phenotypic identication of the genus Enterococcus and dierentiation of phylogenetically distinct enterococcal species and species groups. J. Appl. Bacteriol. 75, 399^400. Diep, B. D., Hvarstein, L. S. and Nes, I. F. (1995) A bacteriocin-like peptide induces bacteriocin synthesis in L. plantarum C11. Mol. Microbiol. 18, 631^639. Diep, B. D., Hvarstein, L. S. and Nes, I. F. (1996) Characterization of the locus responsible for the bacteriocin production in Lactobacillus plantarum C11. J. Bacteriol. 178, 4472^4483. Ennahar, S., Sashihara, T., Sonomoto, K. and Ishizaki, A. (2000) Clas IIa bacteriocins: biosynthesis, structure and activity. FEMS Microbiol. Rev. 24, 85^106. Giraa, G. (1995) Enterococcal bacteriocins: their potential as anti-Listeria factors in dairy technology. Food Microbiol. 12, 291^299. Hanlin, M. B., Kalchayanand, N., Ray, P. and Ray, B. (1993) Bacteriocins of lactic acid bacteria in combination have greater antibacterial activity. J. Food Prot. 56, 252^255. Herranz, C., Mukhopadhyay, S., Casaus, P., Mart| nez, J. M., Rodr| guez, J. M., Nes, I. F., Cintas, L. M. and Hernandez, P. E. (1999) Biochemical and

130 C. Herranz et al.

genetic evidence of enterocin P production by two Enterococcus faecium-like strains isolated from fermented sausages. Curr. Microbiol. 39, 282^290. Hoch, J. A. and Silhavy, T. J. (1995) Two-component Signal Transduction.Washington, ASM Press. Holo, H., Nilssen, O. and Nes, I. F. (1991) Lactococcin A, a new bacteriocin from Lactococcus lactis subsp. cremoris: isolation and characterization of the protein and its gene. J. Bacteriol. 173, 3879^3887. Jack, R.W.,Tagg, J. R. and Ray, B. (1995) Bacteriocins of Gram-positive bacteria. Microbiol. Rev. 59, 171^200. Jimenez-D| az, R., R| os-Sanchez, R. M., Desmazeaud, M., Ruiz-Barba, J. L. and Piard, J. C. (1993) Plantaricin S and T, two new bacteriocins produced by Lactobacillus plantarum LPCO10 isolated from a green olive fermentation. Appl. Environ. Microbiol. 59, 1416^1424. Kersters, K. and de Ley, J. (1975) Identication and grouping of bacteria by numerical analysis of their electrophoretic protein patterns. J. Gen. Microbiol. 87, 333^342. Klaenhammer, T. R. (1993) Genetics of bacteriocins produced by lactic acid bacteria. FEMS Microbiol. Rev. 12, 39^86. Laemmli, U. K. (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227, 680^685. McKay, A. M. (1990) Antimicrobial activity of Enterococcus faecium against Listeria spp. Lett. Appl. Microbiol. 11, 15^17. Moll, G. N., Konings, W. N. and Driessen, A. J. M. (1999) Bacteriocins: mechanism of membrane insertion and pore formation. AntonieVan Leeuwnhoek 76, 185^198. Muriana, P. M. (1996) Bacteriocins for control of Listeria spp. in food. J. Food Prot. 59, suppl. 54^63. Nes, I. F., and Eijsink, V. G. H. (1999) Regulation of group II peptide bacteriocin synthesis by quorum-sensing mechanisms. In Cell-Cell Signaling in Bacteria (Eds G. M. Dunny, and S. C. Winans) pp. 175^192.Washington, Am. Soc. Microbiol. Nes, I. F., Bao Diep, D., Hvarstein, L. S., Brurberg, M. B., Eijsink,V. and Holo, H. (1996) Biosynthesis of bacteriocins in lactic acid bacteria. Antonie van Leeuwenhoek 70, 113^128. Nieto-Lozano, J. C., Nissen-Meyer, J., Sletten, K., Pelaez, C. and Nes, I. F. (1992) Purication and amino acid sequence of a bacteriocin produced by Pediococcus acidilactici. J. Gen. Microbiol. 138, 1^6. Nilsen, T., Nes, I. F. and Holo, H. (1998) An exported inducer peptide regulates bacteriocin production in Enterococcus faecium CTC492. J. Bacteriol. 180, 1848^1854. Nissen-Meyer, J., Holo, H., Hvarstein, L. S., Sletten, K. and Nes, I. F. (1992) A novel lactococcal bacteriocin whose activity depends on the complementary action of two peptides. J. Bacteriol. 174, 5686^5692.

OKeee,T., Hill, C. and Ross, P. (1999) Characterization and heterologous expression of the genes encoding enterocin A production, immunity, and regulation in Enterococcus faecium DPC1146. Appl. Environ. Microbiol. 65, 1506^1515. Olasupo, N. A., Schillinger, U., Franz, C. M. A. P. and Holzapfel, W. H. (1994) Bacteriocin production by Enterococcus faecium NA01 fromwaraa fermented skimmed cow milk product from West Africa. Lett. Appl. Microbiol. 19, 438^441. Piard, J. C. and Desmazeaud, M. (1992) Inhibiting factors produced by lactic acid bacteria. 2. Bacteriocins and other antibacterial substances. Lait 72, 113^142. Pilet, M. F., Dousset, X., Barre, R., Novel, G., Desmazeaud, M. and Piard, J. C. (1995) Evidence of two bacteriocins produced by Carnobacterium piscicola and Carnobacterium divergens isolated from sh and active against Listeria monocytogenes. J. Food Prot. 58, 256^262. Pot, B., Vandamme, P. and Kersters, K. (1994) Analysis of electrophoretic whole organism protein ngerprints. In Chemical Method in Prokaryotic Systematics (Eds M. Goodfellow, and A. G. ODonnell) pp. 493^521. Chichester, J. Wiley & Sons, Inc. Quadri, L. E. N., Sailer, M., Roy, K. L.,Vederas, J. C. and Stiles, M. E. (1994) Chemical and genetic characterization of bacteriocins produced by Carnobacterium piscicola LV17B. J. Biol. Chem. 269, 12204^12211. Remiger, A., Ehrmann, M. A. and Vogel, R. (1996) Identication of bacteriocin-encoding genes in lactobacilli by polymerase chain reaction (PCR). Syst. Appl. Microbiol. 19, 28^34. Revol-Junelles, A. M., Mathis, R., Krier, F., Fleury, Y., Delfour, A. and Lefebvre, G. (1996) Leuconostoc mesenteroides subsp. mesenteroides synthesizes two distinct bacteriocins. Lett. Appl. Microbiol. 23, 120^124. Saucier, L., Paradkar, A. S., Frost, L. S., Jensen, S. E. and Stiles, M. E. (1997) Transcriptional analysis and regulation of carnobacteriocin production in Carnobacterium piscicola LV17. Gene 188, 271^277. Schleifer, K. H. and Kilpper-Blz, R. (1984) Transfer of Streptococcus faecalis and S. faecium to the genus Enterococcus nom. rev. as Enterococcus faecalis comb. nov. and Enterococcus faecium comb.nov. Int. J. Syst. Bacteriol. 34, 31^34. Sobrino, O. J., Rodr| guez, J. M., Moreira, W. L., Cintas, L. M., Fernandez, M. F., Sanz, B. and Her nandez, P. E. (1992) Sakacin M, a bacteriocin-like substance from Lactobacillus sake 148. Int. J. Food Microbiol. 16, 215^225. Stiles, M. E., and Hastings, J. W. (1991) Bacteriocin production by lactic acid bacteria: potential for use in meat preservation. Trends Food Sci. Technol. 2, 247^251. Torri Tarelli, G., Carminati, D. and Giraa, G. (1994) Production of bacteriocins active against Listeria

Enterocins A and B from E. faecium P21 131

monocytogenes and Listeria innocua from dairy enterococci. Food Microbiol. 11, 243^252. Van Belkum, M. J., Hayema, B. J., Geis, A., Kok, J. and Venema, G. (1989) Cloning of two bacteriocin genes from a lactococcal bacteriocin plasmid. Appl. Environ. Microbiol. 55, 1187^1191. Van Belkum, M. J., Hayema, B. J., Jeeninga, R. E., Kok, J. and Venema, G. (1991) Organization and nucleotide sequence of two lactococcal bacteriocin operons. Appl. Environ. Microbiol. 57, 492^498.

Van Belkum, M. J., Kok, J. and Venema, G. (1992) Cloning, sequencing, and expression in Escherichia coli of lcnB, a third bacteriocin determinant from the lactococcal bacteriocin plasmid p9B4 - 6. Appl. Environ. Microbiol. 53, 572^577. Villani, F., Salazano, G., Sorrentino, E., Pepe, O., Marino, P. and Coppola, S. (1993) Enterocin 226NWC, a bacteriocin produced by Enterococcus faecalis 226, active against Listeria monocytogenes. J. Appl. Bacteriol. 74, 380^387.