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Transcript of Herranz 2001 Food-Microbiology
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Enterococcus faecium P21: a strainoccurring naturally in dry-fermented
sausages producing the class IIbacteriocins enterocin A and
enterocin B
C. Herranz1, P. Casaus1{, S. Mukhopadhyay1, J. M. Mart|nez1,
J. M. Rodr|guez1, I. F. Nes2, P. E. Herna ndez1and L. M. Cintas1*
Enterococcus faecium P21isolated from a Spanish dry-fermented sausage shows a narrow antimicro-bial activity against closely relatedlactic acid bacteria and strong antimicrobial activity against spoilageand foodborne Gram-positive pathogenic bacteria such as Listeria monocytogenes, Staphylococcusaureus, Clostridium perfringens andClostridium botulinum.The antimicrobialactivity is produced dur-ing growth in MRS broth at 328C; it is heat resistant (20min at 80 ^1008C) and abolished by protease
treatment, and withstands exposure to pH 2^11, freeze-thawing, lyophilization and long term storage at7208C. Purication of the antimicrobial activity by ammonium sulphate precipitation, gel ltration,and cation-exchange, hydrophobic interaction and reverse-phase chromatographies showed that thebroad antimicrobial spectrum exerted byE. faecium P21was indeed due to two peptide bacteriocins.Studies on their N-terminal amino acid sequences and PCR and DNA analyses revealed that thesebacteriocins are identical to the class II bacteriocins enterocin A and enterocin B.The genetic organiza-tion 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 colinearlyarranged. Strikingly, this organization was structurally dierent to that reported for the EntA-producerE. faecium CTC492. # 2001 Academic Press
Introduction
Lactic acid bacteria (LAB) predominate dur-
ing food fermentation because of their ability
to produce bacteriocins and other antimicro-bial 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
ORIGINAL ARTICLE
*Corresponding author. Fax: +34 -913943743.E-mail: [email protected]{Present address: Centros Comerciales Carrefour,
Divisio n de Calidad,c/ Campezon816, 28022-Madrid,Spain.
Received:27 June 2000
1Departamento deNutricion yBromatolog|a III,Facultad deVeterinaria,
UniversidadComplutense deMadrid, 28040-Madrid, Spain2Laboratory ofMicrobial GeneTechnology,Department ofChemistryandBiotechnology,
AgriculturalUniversityof Norway, N-1432
s, Norway
0740-0020/01/020115 + 17 $35.00/0 # 2001 Academic Press
Food Microbiology, 2001, 18, 115^131 doi:10.1006/fmic.2000.0382
Available online at http://www.idealibrary.com on
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such as Listeria monocytogenes and Staphylococ-
cus aureus (Giraa 1995, Jack et al.1995, Casaus
et al.1997, Cintas et al. 1995, 1998a,b).The LAB-
bacteriocins described and characterized to
date show common traits which justify theirclassication into three well-dened classes
(Nes et al. 1996, Moll et al. 1999): class I, the lan-
tibiotics; class II, the small (510 kDa) heat-
stable non-lantibiotics, which are divided
into the subgroups IIa (pediocin-like bacterio-
cins 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) heat-
labile 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 pep-
tide), an immunity gene, and the genes encod-
ing the secretion machinery. Most class II
bacteriocins are synthesized as prebacterio-
cins containing an N-terminal leader sequence
of the so-called double glycine type and are pro-
cessed and translocated through the cytoplas-
mic membrane by ATP-binding cassette (ABC)
transporters and their accessory proteins.
However, recently it has been shown that afew class II bacteriocin precursors contain
the characteristic hydrophobic signal peptide
found in the proteins that are secreted through
the general secretory pathway (or sec-depen-
dent pathway) (Nes et al., 1996). In recent years,
it has been reported that production of some
class II bacteriocins is regulated by a three-
component 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 andEijsink 1999). The IP is a bacteriocin-like pep-
tide 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, Casaus1998); however, most enterocins described to
date have not been characterized at the bio-
chemical 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 entero-
cins 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 bacterio-cins and consist of small (4829^5465 Da), heat-
stable, 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 se-
quence YGNGVxC at the N-terminal region of
the molecule (Nieto-Lozano et al. 1992, Aymer-
ich 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 pedio-
cin-like bacteriocins (class IIa) (Aymerich etal. 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 en-
terocin A (Aymerich et al. 1996) and enterocin
B (Casaus et al. 1997) are of the so called dou-
ble-glycine-type; however, the N-terminal ex-
tension of enterocin P corresponds to a signal
peptide (Cintas et al. 1997, Casaus 1998). In con-
trast to other peptide bacteriocins, enterocinsL50A and L50B are synthesized without an
N-terminal leader sequence or signal peptide
(Cintas et al. 1998a).
During the last years we have isolated sev-
eral bacteriocinogenic LAB from Spanish dry
fermented sausages and biochemically and ge-
netically characterized several bacteriocins
(Sobrino et al. 1992, Cintas 1995, Cintas et al.
1995, 1997, 1998a,b, 2000, Casaus 1998, Casaus
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et al. 1995, 1997, Herranz et al. 1999). In this
work we report on the isolation and identica-
tion ofE. faecium P21 and the characterization
of its bacteriocins by their functional proper-
ties, purication to homogeneity, amino acidsequence and DNA analysis.
Materials and Methods
Bacterial strains and media
Lactic acid bacteriawere 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 pre-
viously described (Cintas et al. 1995, Casaus
1998). Isolate P21 was selected for further stu-dies because of its strong inhibitory activity
against these indicators. The micro-organisms
used for evaluation of the antimicrobial spec-
trum 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 Gram-positive bacteria
Indicator speciesa Strain Sourceb Activityc
Lactobacillus casei 334 ATCC NIZDLactobacillus fermentum 285 CECT 13.9Lactobacillus plantarum 1193 NCDO 16.5Lactobacillus sakei 2714 NCFB NIZDLactobacillus sakei 148 Our strain collection 11.1Pediococcus acidilactici 347 Our strain collection NIZDPediococcus pentosaceus FBB61 TNO 14.1Pediococcus pentosaceus FBB63 TNO NIZD
Pediococcus pentosaceus PC1 TNO NIZDLactococcus cremoris CNRZ117 INRA 14.1Lactococcus lactis BB24 Our strain collection NIZDEnterococcus faecium L50 Our strain collection 15.5Enterococcus faecium T136 Our strain collection NIZDEnterococcus faecium P13 Our strain collection 13.5Enterococcus faecalis EF TNO 16.3
Clostridium perfringens 376 CECT 15.2Clostridium botulinum 551 CECT 15.5Listeria monocytogenes 7973 NCTC 16.4Listeria monocytogenes LI5sv1/2 FVM 17.2Listeria monocytogenes 5105 NCTC 17.7Listeria monocytogenes LI1sv4 FVM 17.5Listeria monocytogenes Scott A FVM 18.4
Staphylococcus aureus 137 FRI 17.7Staphylococcus aureus 196E FRI 17.2Staphylococcus aureus 349 FRI 17.6
aLactococcus lactis subsp. cremoris is abbreviated as Lactococcus cremoris.bAbbreviations: ATCC, American Type Culture Collection (Rockville, USA); CECT, Coleccio n Espaolade Cultivos Tipo (Valencia, Spain); DSM, Deutsche Sammlung von Mikroorganismen und Zell Kulturen,GmbH (Braunschweig, Germany); INRA, Station de Recherches Laitie' res (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, TheNetherlands).cDiameter of inhibition zone in millimeters; NIZD, no inhibition zone detected.
Enterocins A and B from E. faecium P21 117
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Phenotypic identication
Isolate P21 was examined by phase-contrast
microscopy to determine its cell morphology
and tested for Gram-staining reaction and cat-alase activity (Table 2). Preliminary identica-
tion 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 produc-
tion), resistance to 40% (w/v) bile, growth on
media containing 0?04% sodium azide (Bacto
EVA broth, Oxoid; m-Enterococcus, Difco), ur-
ease 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 dehydro-genases as described by the supplier
(Boehringer GmbH, Mannheim, Germany).
Fermentation patterns were determined with
API Rapid CH fermentation strips (API, Bio-
me' rieux, Montallieu Vercieu, France) in CHL
medium. Total proteins were analysed by so-
dium dodecyl sulfate-polyacrylamide gel elec-
trophoresis (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 Reaction or characteristic Test Reaction or characteristic
Morphology Cocci Rhamnose 7Gram Dulcitol 7Calf blood hemolysis 7 Inositol 7Catalase 7 Mannitol pHa 4?1 Sorbitol 7Growth a-Methyl-D-Mannoside 7
at 108C a-Methyl-D-Glucoside 7at 458C NAcetil glucosamine at 508C 7 Amygdalin at pH 4?5^9?6 Arbutin
in 40% bile Esculin in 6?5% NaCl Salicin in 10% NaCl Cellobiose in 0?04% sodium azide Maltose
Urease 7 Lactose Arginine hydrolysis Melibiose 7CO2 production 7 Saccharose H2S production 7 Trehalose Voges^Proskauer 7 Inulin 7Lactic acid b L Melezitose 7Glycerol D-Ranose 7Erythritol 7 Starch 7D-Arabinose 7 Glycogen 7L-Arabinose Xylitol 7Ribose b-Gentibiose 7
D-Xylose 7 D-Turanose 7L-Xylose 7 D-Lyxose 7Adonitol 7 D-Tagatose 7b Methyl-Xyloside 7 D-Fucose 7Galactose L-Fucose 7D-Glucose D-Arabitol 7D-Fructose L-Arabitol 7D-Mannose Gluconate 7L-Sorbose 7 2-Keto-Gluconate 7
a Final pH of stationary growth phase cultures grown in MRS broth at 328C.b Conguration of lactic acid produced from glucose.
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Kersters and de Ley (1975) and Pot et al.
(1994) by B. Pot, University of Ghent, Ghent,
Belgium.
Bacteriocin assays
Cell-free culture supernatants of the bacterio-
cinogenic 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 subse-
quently centrifuged at 12 000g 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 (MilliporeCorp., Bedford, Massachussets, USA).The anti-
microbial 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 ali-
quots 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 un-
der 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 purica-
tion processes was quantied by a microtiter
plate assay (Holo et al. 1991). Briey, two-
fold serial dilutions (50 ml) of bacteriocin
extracts in MRS broth were prepared in
microtiter plates. The wells were then lled
up to 200ml by the addition of 150ml o f a
diluted (in MRS) fresh overnight culture of
E. faecium P13 (Cintas et al. 1997, Casaus 1998).
Growth inhibition was measured spectropho-tometrically 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).
Eect of enzymes, heat treatments, pH,freeze-thaw, lyophilization and storage onbacteriocin activity
Proteolytic, lipolytic and amylolytic enzymesused in our studies and their sources are listed
in Table 3. All enzymes were added to cell-free
culture supernatants ofE. faecium P21 at a nal
concentration of 1 mg ml71. Controls consisted
of samples of enzymes in sterile medium and
untreated bacteriocin solution. All prepara-
tions were incubated at 378C for 6h and en-
zymes were heat-inactivated at 1008C for
10 min. Stability of the antagonistic activity to
heat was determined by heating aliquots of cell-
free culture supernatants at 80 and 1008C for
20 min. Eect of pH on bacteriocin activity
was tested adjusting the pH of cell-free culturesupernatants 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
Table 3. Physico-chemical stability of the bac-teriocin activity of cell-free culture supernatantsfrom E. faecium P21
Treatment Bacteriocinactivitya
Enzymes:Pepsin (Serva, No. 318200) 7Trypsin (Merck, No. 82130 7Papain (Merck, No.7144) 7Protease Type II (Sigma,No. P- 4755)
7
Lipase Type VII (Sigma,No. L-1754)
a-Amylase (BoehringerMannhein, No. 161764)
Heat:20 min at 808C 20 min at 1008C
pH:2?0^11?0
Freeze-thaw Lyophilization Storage at 7208Cb
aBacteriocin activity was determined against E. fae-cium P13 by the microtiter plate assay (see Materialsand Methods). Symbols:, inhibition zone; ^, no inhi-bition zone.b Supernatants were stored at7208C for 12 months.
Enterocins A and B from E. faecium P21 119
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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 other-
wise stated. The bacteriocins were puried
from a 1-l E. faecium P21 culture which was
grown in MRS broth at 328C until the late loga-rithmic phase (A620 = 0?9). The cells were re-
moved by centrifugation at 12 000g 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 000gfor 30 min at 48C, the
pellet and oating material were combined and
solubilized in 100ml of 20 mM sodium phos-
phate 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 wasfurther subjected to cation-exchange (SP
Sepharose Fast Flow) and hydrophobic-
interaction (Octyl-Sepharose CL-4B) chroma-
tographies, followed by reverse-phase chroma-
tography in a C2 to C18 column (PepRPC HR5/5)
integrated in a fast-performance liquid chro-
matography 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?5mlmin71. Fractions with
high bacteriocin activity were mixed and re-
chromatographed on the same reverse phasecolumn to obtain chromatographically pure
bacteriocins. Puried bacteriocins were stored
in 60% 2-propanol containing 0?1% TFA
at7208C.
Amino acid sequence analysis
The N-terminal amino acid sequence of puri-
ed bacteriocins was determined by Edman de-
gradation with an Applied Biosystems (Foster
City, California, USA) model 477A automatic
sequence analyser by Dr J. Va zquez, ProteinChemistry Facility, Centro de Biolog|a Molecu-
lar Severo Ochoa, Madrid, Spain.
PCR analysis and DNA sequencing
Total DNA ofE. faecium P21 and E. faeciumT136
(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 reac-
tions. Oligonucleotide primers used for PCR
and DNA sequencing (Table 4) were obtained
from KEBOLab (Spanga, Sweden). Specicprimers 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 immu-
nity (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 Sequence
TH10 5 GAT TAT GAA ACATTT AAA AAT TTT GTC 3LA1 5 AAA ACC ACC TAT AGA CAT TCC TGC 3ENTB3 5 AGA CCT AAC AAC TTA TCT AAA G 3ENTB5 5 GTT GCA TTT AGA GTA TAC ATT TGC 3SK2 5 CCG CTC TAG AAC TAG TGG ATC 3
Primer TH10 was designed by Aymerich et al. (1996); primers LA1, ENTB3 and ENTB5 were designed byCasaus (1998).
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positive Voges^Proskauer reaction and pro-
duced ammonia from thehydrolisis 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, sorbitolor gluconate. The strain did not show urease
activity and it was non-hemolytic on calf blood.
By comparison of its SDS-PAGE protein pat-
tern 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 culturesupernatants ofE. faecium P21
Table 1 shows the antimicrobial activity of neu-tralized and lter-sterilized cell-free culture
supernatants ofE. faecium P21 against selected
Gram-positive bacteria. Of the 15 LAB tested,
only three strains of Lactobacillus spp., one
strain ofPediococcus pentosaceus, one strain of
Lactococcus lactis subsp. cremoris, two strains
ofE. faecium and one strain of E. faecalis were
inhibited by culture supernatants, with inhibi-
tion zone diameters ranging from 11?1 to
16?5 mm. However, all the spoilage and food-
borne Gram-positive bacteria tested, which in-
cluded ve strains of L. monocytogenes, one
strain of Clostridium perfringens, one strain ofC. botulinum, and three strains of S. aureus
were sensitive to cell-free culture superna-
tants, with inhibition zone diameters ranging
from 15?2 to 18?4 mm. None of the Gram-nega-
tive bacteria tested (Salmonella typhimurium,
Escherichia coli, Pseudomonas uorescens, Yersi-
nia enterocolitica, Enterobacter aerogenes and
Aeromonas hydrophila) were inhibited by cell-
free culture supernatants ofE. faecium P21 un-
der the stated experimental conditions (results
not shown).
Physico-chemical stability of the bacteriocinactivity
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-
sin, papain and protease II), but was not
aected bylipolytic or amylolytic enzymes such
as lipase VII or a-amylase, respectively. Inhibi-
tory activity of cell-free culture supernatants
was maintained completely after heat treat-ment at 80 and 1008C for 20 min. These results
suggested that the inhibitory compound(s) pro-
duced by E. faecium P21 is a heat-stable protei-
naceous compound(s).The inhibitory protein(s)
withstood exposure to pH values between 2
and 11 for 24 h at 4 and 328C, and its antimicro-
bial activity was not lost by freezing and thaw-
ing, 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. faeciumP13 by the agar well diusion assay. Bacterio-
cin 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,
Figure 1. Growth kinetics (*), pH (&) and anti-microbial activity (~) of E. faecium P21 grown inMRS broth at 328C. Antimicrobial activity of cell-free culture supernatants was assayed by an agardiusion assay using E. faecium P13 as indicatormicro-organism.
122 C. Herranz et al.
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although it decreased rapidly during the
stationary phase.
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 sul-
phate precipitation allowed a seven-fold in-
crease 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 interac-
tion column represented a 5% recovery of bac-
teriocin activity.The rst run of the last step inthe purication procedure, reverse-phase chro-
matography, revealed two well separated frac-
tions 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 rechromato-
graphed three times on the same column,
which resulted in each case in a single protein
absorbance peak, coinciding with the antimi-
crobial activity peak (fractions A and B) (Figs
3 and 4, respectively). Fractions A and B eluted
when the 2-propanol gradient had reached 28?0and 29?0%, respectively, and represented a
recovery of 1?25 and 1?81%, respectively, of the
initial bacteriocin activity in the cell-free cul-
ture supernatant.
Amino acid sequence analyses
Puried bacteriocins from the last reverse-
phase chromatography step (fractions A and B
(Figs 3 and 4, respectively)) were subjected toEdman degradation analyses in order to deter-
mine their partial amino acid sequences
(Fig. 5). The rst 30 amino acid residues of the
Table 5. Purication of activity A (enterocin A) and activity B (enterocin B) from E. faecium P21
Volume(ml)
TotalA254
a
Totalactivity
(103 BU)b
Specicactivity
(sp. activity) c
Increase insp. activity
(fold)
Yield(%)
Culture supernatant 1 000 13 300 521 39 1 100
FractionAmmonium sulfate precipitation 100 860 228 265 7 44Gel ltration chromatography 200 1340 377 282 7 72Cation-exchange chromatography 50 6?75 29 4316 110 6Hydrophobic-interaction chromatography 10 9?7 24 2492 64 5Reverse-phase chromatographyFraction A 1?2 0?024 6?5 272 250 6950 1?25Fraction B 2?0 0?030 9?5 314 667 8033 1?81
a Optical density at 254 nm multiplied by the volume in ml.b Bacteriocin units (BU).c BU per ml divided by optical density at 254nm.
Figure 2. Reverse-phase chromatography of theantimicrobial activity from E. faecium P21. Theamount applied to the column was obtained from 1 lof culture. BU, bacteriocin units.
Enterocins A and B from E. faecium P21 123
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N-terminus of fraction A included nine uniden-
tied positions and the pediocin-like bacterio-
cin consensus amino acid sequence YGNGV
(Nieto-Lozano et al. 1992, Aymerich et al. 1996,
Ennahar et al. 2000) in positions 8^12. Homol-
ogy searches for the partial amino acid se-
quence of fraction A revealed a high homology
with enterocin A from E. faecium CTC492
(Aymerich et al. 1996). The partial amino acidsequence 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 entero-
cin B from E. faecium T136 (Casaus et al. 1997,
Casaus 1998).
Genetic evidence for enterocin A andenterocin B production in E. faecium P21
The amino acid sequences of the puried
peptides with antimicrobial activity from
E. faecium P21 strongly suggested that entero-
cins A and B, or closely related bacteriocins,
are produced by this enterococcal strain. Con-
sequently, based on the amino acid sequence si-
milarity 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 oligo-
nucleotides for both enterocins and total DNA
from E. faecium P21 were used as primersand 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, respec-
tively, 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 P21contains entA and entB (Fig. 6). Automated
DNA sequencing of both strands of these
PCR fragments revealed that these two part-
ially 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 chromatographyof fraction A from E. faecium P21.
Figure 4. Last reverse-phase chromatographyof fraction B from E. faecium P21.
124 C. Herranz et al.
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with the EcoRV ligation reaction as template
and the polylinker primer SK2 in combination
with the entA-specic primer TH10, and a280 bp fragment obtained with the DraI liga-
tion reaction as template and the polylinker
primer SK2 in combination with the entB-
specic primer ENTB5. Automated DNA se-
quencing 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 bac-
teriocins enterocin A and enterocin B.
Further DNA analysis of the sequence down-
stream of entA revealed the presence of twoconsecutive ORFs with the same polarity
(Fig. 7). The rst ORF, located two nucleotides
downstream of the stop codon ofentA, resulted
identical to entiA from E. faecium CTC492 (Ay-
merich et al. 1996). EntiA encodes a translation
product of 103 amino acid residues correspond-
ing to the immunity protein of enterocin A
(Aymerich et al. 1996, OKeee et al. 1999).
The 41-bp DNA region ofE. faecium P21 located
Figure 5. N-terminal amino acid sequence of puried peptide fractions from E. faecium P21. The x indi-cates that the residue could not be identied. Letters above and below the amino acid sequences indicatethat the residue could not be determined with certainty. For comparison, the rst 30 and 49 amino acid re-sidues of enterocin A (Aymerich et al.1996) and enterocin B (Casaus et al.1997), respectively, are also shown.
Figure 6. Agarose gel electrophoresis of PCRfragments generated from total DNA of E. faeciumP21 (1) and E. faecium T136 (2) with specic oligonu-cleotide primers for enterocin A (A) and enterocin B(B) structural genes. M refers to the molecularweight marker.
Enterocins A and B from E. faecium P21 125
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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 entero-
cin A operon in E. faecium P21 encodes a pri-
mary translation product of 48 amino acid
residues. Orf3 is preceded eight nucleotides
Figure 7. Nucleotide sequence of a 811-bp fragment from E. faecium P21 containing the structural geneof enterocin A (entA), the immunity gene (entiA ) and the induction peptide (IP) gene (entF). The deducedamino 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 pro-moter sequences and ribosome binding sites (RBS) areunderlined; direct repeats right (DRR) and left (DRL)within the conserved regulatory-like boxes are overlined.
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upstream by a potential ribosome binding site,
5 GAGGGG 3. A likely 710 consensus promo-
ter region (Pribnow box) (TATAGT) is located
four nucleotides upstream of the ribosome
binding site, an a sequence (TTGAAT) showingresemblance to s70 promoter 735 region is lo-
cated at the optimal distance of 18 nucleotides
upstream the 710 region. One set of direct re-
peats consisting of 10 nucleotides with the se-
quence 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 pre-viously 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 bacteriocino-
genic LAB and/or their bacteriocins as natural
biopreservatives to suppress growth of spoi-
lage and pathogenic bacteria in foods, we have
focused our studies on the characterization ofbacteriocins produced by LAB isolated from
Spanish dry-fermented sausages. In this re-
spect, 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 ofE. faecium from
dry-fermented sausages has been frequently
reported. The enterocin A and B producers
E. faecium CTC492 and E. faecium T136 were iso-lated 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 pro-
ducts 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 iso-
lated 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 suitabil-
ity 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 Desma-
zeaud 1992). These observations suggest that
this antimicrobial activity is truly due to pep-
tide bacteriocin(s). To further characterize the
bacteriocin activity, a purication procedure
including ammonium sulphate precipitation,
gel ltration, and cation-exchange, hydropho-
bic interaction and reverse-phase chromato-graphies 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 sequen-
cing by Edman degradation showed that frac-
tion 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 iden-
tied in E. faecium CTC492 and T136 (Aymerich
1996, Casaus et al. 1997, Casaus 1998) (Fig. 5). A
number of PCR products containing the bacter-iocin 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 bacterio-
cin 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 characteristicamongst the class II bacteriocin regulated sys-
tems; 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 ofentFa set of
direct repeats of 10 bp (8 bp identical) spaced by
an AT-rich region. Similar features have also
Enterocins A and B from E. faecium P21 127
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Communities (Project Contract Bio4-CT96-
5051).
References
Anderson, D. G. and McKay, L. L. (1983) Simple andrapid method for isolation of large plasmid DNAfrom lactic streptococci. Appl. Environ. Microbiol.46, 549^552.
Anderssen,E. L., Diep, D. B., Nes, I. F., Eijsink,V. andNissen-Meyer, J. (1998) Antagonistic activity ofLactobacillus plantarum C11: two new two-peptidebacteriocins, plantaricin EF and JK, and theinduction 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 andgenetic characterization of enterocin A fromEnterococcus faecium, a new antilisterial bacter-iocin 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 ofEnterococcus
faecium strains isolated from sous-vide cookedsh llets. Food Microbiol.11, 525^536.
Bennik, M. H. J., Smid, E. J. and Gorris, L. G. M.(1997) Vegetable-associated Pediococcus parvu-lus produces pediocin PA-1. Appl. Environ. Micro-biol. 63, 2074^2076.
Casaus, M. P. (1998) Aislamiento e identicacio n debacterias la cticas de origen ca rnico productoras
de bacteriocinas. Caracterizacion bioqu|mica ygene tica de la enterocina P de Enterococcusfaecium P13 y de la enterocina B de Enterococcusfaecium T136. Ph.D. Thesis. Universidad Complu-tense de Madrid, Madrid, Spain.
Casaus, M. P., Cintas, L. M., Sua rez, A., Rodr|guez,J. M., Holo, H., Herna ndez, P. E. and Nes, I. F.(1995) Enterococcus faeciumT136: a two anti-Lister-ia active bacteriocins producer, isolated fromSpanish dry fermented sausages, abstract p-156.In Abstracts of the 2nd Contractors Meeting ofParticipants in the EC Biotechnology ProgrammeG-Project, Cork, Ireland.
Casaus, P., Nilsen, T., Cintas, L. M., Nes, I. F.,Herna ndez, P. E. and Holo, H. (1997) Enterocin
B, a new bacteriocin from Enterococcus faeciumT136 which can act sinergistically with enterocinA. Microbiology 143, 2287^2294.
Cintas, L. M.1995. Caracterizacio n bioqu|micay gen-e tica parcial de la pediocina L50, una nueva bac-teriocina producida por Pediococcus acidilacticiL50 aislado de embutidos crudos curados. Ph.D.Thesis. Universidad Auto noma de Madrid,Madrid, Spain.
Cintas, L. M., Rodr|guez, J. M., Ferna ndez, M. F.,Sletten, K., Nes, I. F., Herna ndez, P. E. and Holo,H. (1995) Isolation and characterization of pedio-
cin L50, a new bacteriocin from Pediococcus acid-ilactici with a broad inhibitory spectrum. Appl.Environ. Microbiol. 61, 2643^2648.
Cintas, L. M., Casaus, P., Hvarstein, L. S., Herna n-dez, P. E. and Nes, I. F. (1997) Biochemical andgenetic characterization of enterocin P, a novelsec-dependent bacteriocin from Enterococcus fae-cium P13 with a broad antimicrobial spectrum.Appl. Environ. Microbiol. 63, 4321^4330.
Cintas, L. M., Casaus, P., Holo, H., Herna ndez, P. E.,Nes, I. F. and Hvarstein, L. S. (1998a) EnterocinsL50A and L50B, two novel bacteriocins fromEnterococcus faecium L50, are related to staphylo-coccal hemolysins. J. Bacteriol. 180, 1988^1994.
Cintas, L. M., Casaus, P., Ferna ndez, M. F.and Herna ndez, P. E. (1998b) Comparative antimi-crobial activity of enterocin L50, pediocin PA-1,nisin A and lactocin S against spoilage and food-borne pathogenic bacteria. Food Microbiol. 15,289^298.
Cintas, L. M., Casaus, P., Herranz, C., Hvarstein, L.S., Holo, H., Herna ndez, P. E., and Nes, I. F. (2000)Biochemical and genetic evidence that Enterococ-cus faecium L50 produces enterocins L50A andL50B, the sec-dependent enterocin P, and a novelbacteriocin secreted without an N-terminal ex-tension termed enterocin Q. J. Bacteriol. 182,6806^6814.
Daeschel, M. A. (1989) Antimicrobial substancesfrom lactic acid bacteria for use as food preserva-tives. Food Technol.43, 164^167.
Devereux, J., Haeberli, P. and Smithies, O. (1984) Acomprehensive set of sequence analysis programsfor the VAX. Nucleic Acids Res. 12, 387^395.
Devriese, L. A., Pot, B. and Collins, M. D. (1993) Phe-notypic identication of the genus Enterococcusand dierentiation of phylogenetically distinctenterococcal species and species groups. J. Appl.Bacteriol. 75, 399^400.
Diep, B. D., Hvarstein, L. S. and Nes, I. F. (1995) Abacteriocin-like peptide induces bacteriocinsynthesis 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 thebacteriocin production in Lactobacillus plantar-um C11. J. Bacteriol. 178, 4472^4483.
Ennahar, S., Sashihara, T., Sonomoto, K. and Ishiza-ki, 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 technol-ogy. Food Microbiol. 12, 291^299.
Hanlin, M. B., Kalchayanand, N., Ray, P. and Ray, B.(1993) Bacteriocins of lactic acid bacteria in com-bination 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 Herna ndez, P. E. (1999) Biochemical and
Enterocins A and B from E. faecium P21 129
-
8/6/2019 Herranz 2001 Food-Microbiology
16/17
geneticevidence of enterocin P production by twoEnterococcus faecium-like strains isolated fromfermented sausages. Curr. Microbiol. 39, 282^290.
Hoch, J. A. and Silhavy, T. J. (1995) Two-componentSignalTransduction. Washington, ASM Press.
Holo, H., Nilssen, O. and Nes, I. F. (1991) LactococcinA, a new bacteriocin from Lactococcus lactissubsp. cremoris: isolation and characterization ofthe protein and its gene. J. Bacteriol. 173,3879^3887.
Jack, R.W.,Tagg, J. R. and Ray, B. (1995) Bacteriocinsof Gram-positive bacteria. Microbiol. Rev. 59,171^200.
Jime nez-D|az, R., R|os-Sa nchez, R. M., Desmazeaud,M., Ruiz-Barba, J. L. and Piard, J. C. (1993) Plan-taricin S andT, two newbacteriocins produced byLactobacillus plantarum LPCO10 isolated from agreen olive fermentation. Appl.Environ.Microbiol.59, 1416^1424.
Kersters, K. and de Ley, J. (1975) Identication andgrouping of bacteria by numerical analysis oftheir electrophoretic protein patterns. J. Gen.Microbiol. 87, 333^342.
Klaenhammer, T. R. (1993) Genetics of bacteriocinsproduced by lactic acid bacteria. FEMS Microbiol.Rev. 12, 39^86.
Laemmli, U. K. (1970) Cleavage of structural pro-teins during the assembly of the head of bacter-iophage T4. Nature 227, 680^685.
McKay, A. M. (1990) Antimicrobial activity ofEnterococcus 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 in-
sertion and pore formation. AntonieVan Leeuwn-hoek 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 quor-um-sensing mechanisms. In Cell-Cell Signalingin 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) Biosynthesisof bacteriocins in lactic acid bacteria. Antonievan Leeuwenhoek 70,113^128.
Nieto-Lozano, J. C., Nissen-Meyer, J., Sletten, K.,Pela ez, C. and Nes, I. F. (1992) Purication and
amino acid sequence of a bacteriocin producedby Pediococcus acidilactici. J. Gen. Microbiol. 138,1^6.
Nilsen,T., Nes, I. F. and Holo, H. (1998) An exportedinducer peptide regulates bacteriocin productionin 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 bacter-iocin whose activity depends on the complemen-tary action of two peptides. J. Bacteriol. 174,5686^5692.
OKeee,T., Hill, C. and Ross, P. (1999) Characteriza-tion and heterologous expression of the genes en-coding enterocin A production, immunity, andregulation in Enterococcus faecium DPC1146. Appl.Environ. Microbiol.65, 1506^1515.
Olasupo, N. A., Schillinger, U., Franz, C. M. A. P. andHolzapfel, W. H. (1994) Bacteriocin production byEnterococcus faecium NA01from waraa fermen-ted skimmed cow milk product fromWest Africa.Lett. Appl. Microbiol.19,438^441.
Piard, J. C. and Desmazeaud, M. (1992) Inhibitingfactorsproduced bylactic acid bacteria. 2. Bacter-iocins and other antibacterial substances. Lait72, 113^142.
Pilet, M. F., Dousset, X., Barre, R., Novel, G., Desma-zeaud, M. and Piard, J. C. (1995) Evidence of twobacteriocins produced by Carnobacterium piscico-la and Carnobacterium divergens isolated fromsh and active against Listeria monocytogenes.J. Food Prot. 58, 256^262.
Pot, B., Vandamme, P. and Kersters, K. (1994)Analysis of electrophoretic whole organism pro-tein ngerprints. In Chemical Methodin Prokaryo-tic 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 geneticcharacterization 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 inlactobacilli 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)Leuconostocmesenteroides subsp. mesenteroides synthesizestwo 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 analysisand regulation of carnobacteriocin productionin Carnobacterium piscicola LV17. Gene 188,271^277.
Schleifer, K. H. and Kilpper-Blz, R. (1984) Transferof Streptococcus faecalis and S. faecium to thegenus 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., Ferna ndez, M. F., Sanz, B. and Her-na ndez, P. E. (1992) Sakacin M, a bacteriocin-likesubstance from Lactobacillus sake 148. Int. J. FoodMicrobiol.16, 215^225.
Stiles, M. E., and Hastings, J. W. (1991) Bacteriocinproduction by lactic acid bacteria: potential foruse in meat preservation. Trends Food Sci. Tech-nol. 2, 247^251.
Torri Tarelli, G., Carminati, D. and Giraa, G. (1994)Production of bacteriocins active against Listeria
130 C. Herranz et al.
-
8/6/2019 Herranz 2001 Food-Microbiology
17/17
monocytogenes and Listeria innocua from dairyenterococci. Food Microbiol. 11, 243^252.
Van Belkum, M. J., Hayema, B. J., Geis, A., Kok, J.and Venema, G. (1989) Cloning of two bacteriocingenes 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 andnucleotide sequence of two lactococcal bacterio-cin operons. Appl. Environ. Microbiol.57, 492^498.
Van Belkum, M. J., Kok, J. and Venema, G. (1992)Cloning, sequencing, and expression in Escheri-chia coli oflcnB, a third bacteriocin determinantfrom 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) Enterocin226NWC, a bacteriocin produced by Enterococcus
faecalis 226, active against Listeria monocytogenes.J. Appl. Bacteriol.74, 380^387.
Enterocins A and B from E. faecium P21 131