APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Jan. 1992, p. 143-1490099-2240/92/010143-07$02.00/0Copyright ©3 1992, American Society for Microbiology

Production of an Amylase-Sensitive Bacteriocin by an AtypicalLeuconostoc paramesenteroides Straint

CATHERINE B. LEWUS,' STEPHEN SUN,2 AND THOMAS J. MONTVILLE'*Department of Food Science' and George H. Cook Honors Program,2 Neuw Jersey Agricultural Experiment Station,

Cook College, Ruitgers-The State University, New Brunswick, New Jersey 08903-0231

Received 26 August 1991/Accepted 21 October 1991

An atypical Leuconostoc paramesenteroides strain isolated from retail lamb produced a bacteriocin,leuconocin S, that was inactivated by a-amylase, trypsin, a-chymotrypsin, protease, and proteinase K but notby lipase or heat treatment at 60°C for 30 min. Supernatants from culture broths produced two glycoproteinbands on sodium dodecyl sulfate-polyacrylamide gels; these had molecular weights of 2,000 and 10,000 andactivity against Lactobacilus sake ATCC 15521. The crude bacteriocin preparation was bacteriostatic anddissipated proton motive force. Bacteriocin activity was produced over a wide pH range (5.2 to 7.9) on bufferedagar medium, with an optimum pH of pH 6.15. The optimum pH for production in broth was 6.5 to 7.0.

Gram-positive lactic acid bacteria dominate the microbialflora in vacuum- or modified-atmosphere-packaged refriger-ated meats. Their ability to produce a variety of antimicro-bial agents, including diacetyl, hydrogen peroxide, and lacticacid, makes them excellent starter cultures for food fermen-tations and can give them a competitive edge in a mixedpopulation.

Bacteriocin production by lactococci, lactobacilli, andpediococci has been widely reported (7, 9, 16, 19, 21, 31, 33).While not a highly conserved characteristic, the ability toproduce these protein antimicrobial agents may give a straina competitive edge in a particular ecological niche. Reportsof bacteriocin production by Leluconostoc species are lim-ited to inhibition of Listeria monocytogenes by two strains ofLeuiconostoc geliduim (15, 17). We have also recently re-

ported the production of a bacteriocin by another Leuiconos-toc species (21).

Bacteriocins by definition are proteins (34). The actualmode of action of many of these substances suggests a

dissipation of proton motive force, which leads to stasis ordeath (2, 6, 20).The objectives of this study were to identify to the species

level Leuconostoc strain OX and to further characterize itsbacteriocin(s). The mode of action as well as the ability todissipate proton motive force were examined.

MATERIALS AND METHODS

Bacterial strains and growth media. Leuconostoc strainOX, a bacteriocin-producing strain, was isolated from retaillamb (21). Lactobacillus sake ATCC 15521 was used as theindicator strain in both spot-on-the-lawn and well diffusionassays for bacteriocin activity (22). Lactococcus lactisATCC 11454, which produces nisin, was used as a positivecontrol strain. Stock cultures were maintained at -80°C in20% glycerol. Working cultures were made as stabs on BactoLactobacilli MRS broth (Difco, Detroit, Mich.) preparedwith 0.5% glucose and 1.5% Bacto Agar (Difco). Strainswere maintained as stab cultures and transferred bimonthly

* Corresponding author.t Paper D-10564-3-91 of the New Jersey State Agricultural Exper-

iment Station.

for a maximum of six transfers before new working cultureswere prepared.

Identification to species level of Leuconostoc strain OX. Toidentify Leuconostoc strain OX to the species level, we

performed several biochemical tests (14). These tests in-cluded growth in MRS broth supplemented with 3.0 and6.5% NaCl and 10% ethanol, growth in MRS broth adjustedto pH 4.8 and 6.5, and growth in MRS broth at 37°C. Thecarbohydrate fermentation pattern was determined with APICH tests (Sherwood Medical, Plainview, N.Y.) as directedby the manufacturer.

Preparation of crude bacteriocin. Leuconostoc strain OXwas propagated overnight at 30°C in APT broth (Difco).Cells were removed by centrifugation at 13,000 x g for 10min, and the supernatant fluid was decanted. One-milliliterportions of the supernatant fluid were placed in Eppendorftubes, which were heated at 60°C for 0, 10, 15, 30, and 60 minor at 100°C for 5, 10, 15, and 20 min and placed immediatelyon ice. Bacteriocin activity was assayed by both the welldiffusion (29) and the critical dilution (23) assays. Superna-tant heated at 60°C for 30 min was used as the crudebacteriocin preparation for further assays. Arbitrary unitswere defined as the reciprocal of the highest dilution showinginhibition of the indicator lawn.

Sensitivity of the bacteriocin to a-amylase and lipase. Thesensitivity of the bacteriocin to a-amylase (type II-A; Sigma)and to lipase (Sigma) was determined by a modification (21)of the method developed by van Belkum et al. (38).

Determination of the glycoprotein nature of the bacteriocin.A Leuconostoc strain OX crude bacteriocin preparation (1ml) was mixed with 100 ,ul of a-amylase (10 mg/ml) preparedin 0.1 M phosphate buffer (pH 6.9), and the mixture was

incubated at 30°C for 0.5 h. Control tubes contained 100 ,ul of0.1 M phosphate buffer in place of the a-amylase. Thesolutions were heated to 100°C for 20 min to inactivate thea-amylase. Bacteriocin activity was determined by the welldiffusion assay.As a further control to ensure that a-amylase was not

acting on L. sake bacteriocin receptors, we treated L. sakecells with oa-amylase as described below. An overnight MRSbroth culture of L. sake ATCC 15521 propagated at 30°C was

adjusted to an A6. of 0.3. Three-milliliter aliquots were

treated with 500 p.l of a-amylase (10 mg/ml) in 0.1 Mphosphate buffer (pH 6.9) or with 500 p.l of phosphate buffer

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alone and allowed to react for 1 h at 30°C. Cells wereremoved by centrifugation and washed four times withphosphate buffer to remove a-amylase. To each assay tube,5 ml of crude bacteriocin preparation or 5 ml of APT brothwas added. The A660 was observed at 0, 1, 2, 4, 12, 24, and48 h. At the same time, samples were removed and the welldiffusion assay was performed. To remove L. sake cells, wecentrifuged the preparations at 13,000 x g for 10 min andthen heat treated them at 100°C for 10 min prior to carryingout the well diffusion assay.

Cidal versus static effect of the bacteriocin. A plate diffusionassay was used to determine whether leuconocin S caused acidal or static effect by two separate methods. Spot-on-the-lawn agar plates (22) were inoculated with Leiconostocstrain OX and L. lactis ATCC 11454. The plates wereoverlaid with brain heart infusion agar (1% agar) which hadbeen seeded with L. sake ATCC 15521 and were placed at4°C. An uninoculated spot-on-the-lawn agar plate was alsooverlaid as described above. With a sterile straw, an agarplug was removed from the zone of inhibition produced bythe bacteriocin on the indicator lawn plate and from therefrigerated seeded plates. The agar plugs were dissolved in1 ml of 50 mM phosphate buffer (pH 6.5) and used toinoculate duplicate tubes which contained 10 ml of MRSbroth and which were incubated at 30°C for up to 48 h todetermine whether any viable cells remained. Protease res-cue was used on another set of spot-on-the-lawn agar plates.o-Chymotrypsin (2 p.1; 10 mg/ml) in 10 mM phosphate buffer(pH 7.8) was spotted adjacent to the Leuconostoc strain OXcolony in the zone of inhibition. The plates were incubatedovernight at 4°C to allow protease diffusion and then incu-bated anaerobically overnight at 30°C. Growth of the L. sakeindicator cells which had been in the zone of inhibition andprotease rescue of these cells would indicate a static, ratherthan a cidal, effect of the bacteriocin.Measurement of ApH and A+. The membrane potential

(iA4) and proton gradient (ApH) were determined on thebasis of the distributions of [3H]tetraphenylphosphonium ionand ["4C]salicylic acid, respectively, as described by Tsenget al. (36). The cell suspensions were energized to a finalconcentration of 0.01 M glucose for 15 min. Crude bacteri-ocin preparation (100 ,ul/ml) was added to the energizedcells, and the mixture was incubated for 30 min prior to theaddition of the radiolabeled probes. An equal volume ofdiluent was added to the untreated controls. To rule outnonspecific binding of the probes, we added valinomycin (25pLI; 2 mM) and nigericin (25 RI; 1 mM) to the controls (1). Torule out dissipation of proton motive force due to themetabolism and transport of lactate, we added 25 RI of 0.5 MD-lactate to the controls. The intracellular volume of L. sakeATCC 15521 cells was determined by using 1.5 p.Ci of H20for the total aqueous space and subtracting the space occu-

pied by 0.5 ,uCi of [14C]inulin.SDS-PAGE. The crude bacteriocin preparation was sub-

jected to sodium dodecyl sulfate-polyacrylamide gel electro-phoresis (SDS-PAGE) on a 20% uniform-pore gel (HoefferScientific). Samples were applied to four lanes: one for a

molecular weight determination, one with S pul of 1% Alcianblue (10) for a molecular weight determination, and two forbacteriocin activity assays. Seventy-five microliters of sam-

ple and 10 ,u1 of molecular weight standard 442472L (BDHLtd., Poole, England) were applied to the gel. The samplewas prepared by mixing equal parts of sample and samplebuffer and incubating the mixture at 4°C for 3 h. Themolecular weight standard was prepared in sample buffer as

directed by the manufacturer. The gel was run at 13 mA until

the sample entered the separating gel, at which point thecurrent was increased to 16 mA for a total running time of 8to 10 h.The side of the gel with samples and molecular weight

standards was silver stained as directed by the manufacturer(Bio-Rad, Richmond, Calif.). One of the lanes for activitywas fixed for 2 h in 20% isopropanol-10% acetic acid andthen washed in distilled water for 6 h (4). The lane was cut inhalf horizontally, and the pieces were placed in two squaresterile petri dishes and each overlaid with 10 ml of brainheart infusion agar (1% agar) seeded with 104 to 105 L. sakeATCC 15521 cells per ml. The plates were incubated over-night at 30°C and examined for inhibition zones. Alterna-tively, to determine activity, we sliced the remaining laneinto 5-mm portions, placed the pieces in 300 p.l of stackingbuffer, and allowed them to elute overnight at 4°C (3). A welldiffusion assay was performed on these samples. Using themolecular weight standards and linear regression analysis,we determined the approximate molecular weight of thebacteriocin in conjunction with the position of the zone ofinhibition.Optimum pH for bacteriocin production in liquid and solid

media. APT broth was adjusted to pH 4.5, 5.0, 5.5, and 6.0with 0.2 M citrate buffer and to pH 6.5, 7.0, and 7.5 with 0.2M phosphate buffer. Samples were removed at 0, 1, 2, 4, 8,12, and 17.5 h. Cells were removed by centrifugation andtreated as for the preparation of crude bacteriocin andassayed for activity by the well diffusion assay. Growth ofLeuconostoc strain OX at the various pHs was monitoredover the same time period by observing the A660. The sampleshowing the greatest activity in the well diffusion assay wasfurther assayed for arbitrary units.A basal medium consisting (in grams per 100 ml) of

tryptone (Difco; 1.7), soytone (Difco; 0.3), NaCl (0.5), yeastextract (0.6), glucose (0.5), and Noble agar (Difco; 1.5) wasprepared with 0.2 M MES [2-(N-morpholino)-ethanesulfonicacid; Calbiochem, LaJolla, Calif.] and 0.2 M HEPES (N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid; Calbio-chem). The medium prepared with MES buffer was adjustedto pH 5.2, 5.8, 6.15, 6.5, and 6.8. The medium prepared withHEPES buffer was adjusted to pH 6.9, 7.55, and 7.9. Tenmilliliters of each medium was poured into petri plates, driedovernight, and used for spot-on-the-lawn bacteriocin assays.After 24 h of Leuconostoc strain OX growth, the media wereeach overlaid with 10 ml of the basal medium that had beenbuffered with 0.2 M MES buffer (pH 6.15) and had beenseeded with L. sake ATCC 15521 as described previously.The plates were incubated anaerobically overnight at 30°C.The initial pH of the media and the final pH of the mediaafter growth of the L. sake cells were measured.

RESULTS

Identification to the species level of Leuconostoc strain OX.Using the protocol developed by Schillinger and Lucke (30)and Garvie (14), we identified Leuconostoc strain OX asLeuconostoc paramesenteroides. The organism was a gram-positive coccus which was catalase negative, produced gasfrom glucose, and -did not grow in MRS broth supplementedwith 10% ethanol or in MRS broth at 37°C. Growth of theorganism was observed in MRS broth adjusted to pH 4.8 and6.5 and in MRS broth supplemented with 3.0 and 6.5% NaCl.The carbohydrate fermentation pattern determined with APICH tests was negative for adonitol, amygdalin, arabinose,arabitol, arbutin, dulcitol, erythritol, galactose, glycerol,glycogen, inositol, inulin, lactose, D-xylose, melibiose,

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L. PARAMESENTEROIDES AMYLASE-SENSITIVE BACTERIOCIN 145

00

s0 0'0I 0.4

0.2

'< 0.6

0.0 *0 10 20 30 40

Time (hours)FIG. 1. Growth of L. sake ATCC 15521 treated with the L. paramnesenteroides OX bacteriocin. Symbols:

bacteriocin; *, L. sake, bacteriocin; O, L. sake, ox-amylase; A. L. sake.

,B-methylxyloside, ao-methylmannoside, raffinose, rhamnose,ribose, sorbose, xylose, and L-xylose and positive for cello-biose, esculin, fructose, glucose, maltose, mannitol, man-

nose, ot-methylglucoside, saccharose, salicin, trehalose, andturanose. While this fermentation pattern most closely re-

sembles that of L. paramnesenteeroides, the reactions forarabinose, cellobiose, galactose, mannitol, melibiose, sali-cin, and xylose were atypical (14).Heat sensitivity. Leuconocin S was stable against heating

at 60°C for 30 min. After it was heated at 60°C for 60 min,50% of the activity was lost. When it was heated at 100°C,the activity was reduced by 75%.

Glycoprotein nature of the bacteriocin. ot-Amylase negatedthe inhibition zone in the spot-on-the-lawn assay. Lipase didnot. Pretreatment of L. sake ATCC 15521 with a-amylasedid not influence the amount of the bacteriocin bound to thecells, as indicated by the residual amount of the bacteriocinin the medium in the well diffusion assay. Comparableamounts of the bacteriocin bound to receptors of cellstreated with ot-amylase or not treated with a-amylase. Inaddition, the growth of L. sake cells pretreated with ox-amy-lase was similar to that of untreated cells in the presence orabsence of the bacteriocin (Fig. 1). In the presence of thebacteriocin, the growth of the culture was inhibited, while inthe absence of the bacteriocin, the cells continued to multi-ply.Mode of action. L. sake ATCC 15521 cells were recovered

from the zone of inhibition produced by L. paramesenter-oi-des OX, while no cells were recovered from the zone ofinhibition produced by L. lactis ATCC 11454. Additionally,protease rescue of the L. sake cells was observed on thezones produced by L. paramesenteroides but not on thezones produced by L. lactis. These results indicate thatleuconocin S had a static effect while nisin had a cidal effect.The cell volume of L. sake ATCC 15521 was 1.54 ul/

u I

50

0. L. sake, ox-amylase,

mg. The effect of the crude leuconocin S preparation on theproton motive force of L. sake ATCC 15521 is shown inTable 1. The metabolism and transport of lactate did notaffect the proton motive force (data not shown). At both pH7.0 and 5.5, total proton motive force was dissipated in thepresence of leuconocin S. However, at pH 7.0, the effectwas a dissipation of Ak4, while at pH 5.5, the effect appearedto be dissipation of ApH.SDS-PAGE. When the crude bacteriocin preparation was

electrophoresed on 20% polyacrylamide gels in the presenceof 0.1% SDS, activity migrated in two bands. Figure 2 showsthe results of the well diffusion assay. The portion of the geloverlaid with brain heart infusion agar (1% agar) seeded withL. sake ATCC 15521 demonstrated one distinct, diffuse zoneof inhibition corresponding to an approximate molecularweight of 10,000. Molecular weight standards were easilydetected with silver staining (Fig. 3, lane C); however, thelane containing only the bacteriocin showed diffuse silverstaining (Fig. 3, lane B). In the presence of Alcian blue, twobands (Fig. 3, lane A), corresponding to the two areas ofactivity, showed heavy silver staining. The approximatemolecular weights of these two bands were 10,000 and 2,000.Optimum pH for bacteriocin production in liquid and solid

TABLE 1. Effect of the crude leuconocin S preparation onproton motive force

Sample ApH AtliTotal proton

(pH) (mV) (mV) motive force

Control (7.0) -29.0 -90.0 -119.0Treated (7.0) -23.3 -72.1 -95.4Control (5.5) -82.6 -69.6 -152.2Treated (5.5) -23.3 -59.1 -82.4

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6

51-

4

3

21-

I

01 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 2

Slice Number

FIG. 2. Activity bands for the L. pacr-atesenteroides OX bacteriocin in SDS-PAGE.

media. Growth (as measured by A660) and bacteriocin pro-duction in broth of L. paramesenteroides OX over time atvarious pHs are shown in Fig. 4 and 5, respectively. At aninitial pH of <6.0, the growth of strain OX was greatlyinhibited. Bacteriocin production was detectable at pH 6.0and appeared to be optimal at pH 7.0 and 6.5 after 17.5 h.

A B C

FIG. 3. Molecular weight standards and the L. paaranesenteroi-des OX bacteriocin silver stained after SDS-PAGE. (A) Bacteriocin,Alcian blue; B, bacteriocin; C, molecular weight standards: a, 2,512;b, 6,214; c, 8,159; d, 14,401; e, 16,949.

The activity in these samples was 400 arbitrary units per ml.Growth and bacteriocin production were slightly depressedat pH 7.5.The results of the buffered-agar assay are presented in

Table 2. While zones were slightly larger on medium buff-ered with HEPES, bacteriocin production reached a maxi-mum at pH 6.15 and declined slightly in either direction.Bacteriocin production occurred over the entire pH range(5.2 to 6.9) measured.

DISCUSSION

Leiiconostoc species ferment dulcitol, erythritol, glycerol,inositol, inulin, rhamnose, and sorbose (14). Variationsbetween the fermentation patterns of L. paramesenteroidesOX and other strains of the same species only indicatestrain-to-strain differences. Unlike L. gelidum, L. parame-senteroides OX is unable to produce acid from amygdalinand ribose (15, 17).

Proton motive force is not essential for the growth offermenting cells, but the maintenance of the cytoplasmic pHabove a given critical value is essential for the functioning ofthe internal enzymes of the cells (18). The dissipation of ApHin L. sake cells at pH 5.5 is most likely the reason that thecells were unable to grow in the presence of leuconocin S.Other bacteriocins produced by lactic acid bacteria may alsodissipate proton motive force. Bhunia et al. (6) demonstratedthat the treatment of sensitive cells with pediocin AcHresulted in the leakage of potassium ions. Colicins, A, El,Ia, lb, and K form ion-permeable channels in the bacterialcytoplasmic membrane which lead to the collapse of mem-brane proton motive force (20).The cx-amylase sensitivity of the bacteriocin is unusual.

Pretreatment of the indicator organism with a-amylase hadno effect, indicating that the bacteriocin receptors on the L.

E-,

0

NCA

c0

23 24

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L. PARAMESENTEROIDES AMYLASE-SENSITIVE BACTERIOCIN

1.2

e 0.80

C

o, 0.6

.0

.0

It~ 0.4

20

Time (hours)FIG. 4. Growth of L. paramesenteroides OX at pH 4.5 (x), 5.0 (C), 5.5 (0) (superimposed datum points); 6.0(0), 6.5 (A), 7.0 (A), and

7.5 (U).

sake cells were not a-amylase sensitive. The results indicatethat the bacteriocin produced by L. paramesenteroides OXwas a glycoprotein which required both the glyco portionand the protein portion of the molecule for activity. Alcianblue staining of the SDS-PAGE bands associated with inhib-itor activity further confirmed the glycoprotein nature of thebacteriocin. The results indicate that two inhibitors, bothglycoprotein in nature, may exist, one with a molecular

12

10

8U)

6 -

34

weight of approximately 10,000 and the other with a molec-ular weight of approximately 2,000. Alternatively, and moreplausibly, leuconocin S may have formed an aggregate thatwas dispersed by 0.1% SDS in the gel or may have beencleaved to form the lower-molecular-weight band. The bac-teriocins or bacteriocinlike inhibitors produced by otherLeiconostoc spp. have not yet been characterized as to theirmolecular weights (15, 17). Lactocin, produced by Lactoba-

0 10 20Time (hours)

FIG. 5. Production of the L. paramesenteroides OX bacteriocin at pH 4.5 (x), 5.0 (C1), 5.5 (0) (superimposed datum points); 6.0 (0), 6.5(A), 7.0 (A), and 7.5 (U).

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TABLE 2. Bacteriocin production on solid media bufferedto various pHs

pH (buffer) Zone size (mm) Final pH"

5.2 (MES) 9.2 5.35.8 (MES) 10.2 5.36.15 (MES) 12.7 5.56.5 (MES) 11.9 5.76.8 (MES) 11.3 5.8

6.9 (HEPES) 12.77.55 (HEPES) 12.67.9 (HEPES) 11.1

" Measured after growth of the indicator cells.

cillus helveticus LP27 (37), is the only other cited glycopro-tein bacteriocin produced by a lactic acid bacterium. Sta-phylococcin (13) and thermocin (12) are also glycoproteinbacteriocins. Patek et al. (26) suggested that the bacteriocin-like substance produced by Corynebaeteriutm gliutarniculn isa glycoprotein, on the basis of nuclear magnetic resonancedata.The protein nature of leuconocin S was previously re-

ported (21). Leuconocin S is sensitive to a-chymotrypsin,pronase E, trypsin, and proteinase K. Originally, Tagg et al.(34) defined six characteristics for bacteriocins. Over time, ithas become clear that these criteria are met only by theprototype bacteriocins, the colicins. The term bacteriocinhas since been generalized to include all microbially pro-duced inhibitors that are proteinaceous and do not actagainst the producing cells (20). However, quite often thedesignation of cidal versus static effect is dependent uponaspects of the assay system, including the number of arbi-trary units, the buffer or broth, the purity of the inhibitor,and the indicator species and cell concentration used. Staticagents may cause growth to cease even though viability ismaintained. Pediocin AcH reduces Lactobacillits planta-itinpopulations by 1 to 4 logs, depending on the growth phase ofthe cells (5). Similar results against L. monocyvogenes at-tached to meat surfaces were seen with the bacteriocinproduced by Pediococcus acidilaatici which had been iso-lated from Lactacel 110, a commercial starter culture (25).Only under certain circumstances do these bacteriocins actin a cidal fashion. There is precedence for proteinaceousinhibitors that cause a static effect to be labelled bacteriocins(27, 28, 35). Even nisin in certain cases has a static effect, nota cidal one (8, 11, 24, 32). Thus, we believe that the onlyclear-cut definition of bacteriocins is that they are inhibitorsthat are proteinaceous in nature. On the basis of thiscurrently proposed definition, the bacteriocin produced byL. paramesenteroides OX, although bacteriostatic, is truly abacteriocin.Leuconocin S has a broad spectrum of activity (21, 25a). It

is active against L. monocytogenes, Staphylococcus au-reu.s,Yersinia enterocolitica, and some strains of Clostridiirnbotulinum and is heat stable. On the basis of these charac-teristics, the use of L. paramnesenteroides OX and its bacte-riocin as a natural means of preservation in a variety of foodsparticularly susceptible to psychrotrophic pathogens maymeet consumer demands for foods free of chemicals andpreservatives and deserves further research.

ACKNOWLEDGMENTS

This work was supported by state appropriations, U.S. Hatch Actfunds, and a grant from the Cattlemen's Beef Promotion and

Research Board and was conducted in cooperation with the BeefIndustry Council.

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