Herranz 2001 Food-Microbiology

8/6/2019 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

116 C. Herranz et al.


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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.



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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.



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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.



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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.



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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.



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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



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Communities (Project Contract Bio4-CT96-

5051).

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