Partial Purification and Characterization of Bacteriocin ...Partial Purification and...

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Vol. 57, No. 3 APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Mar. 1991, p. 701-706 0099-2240/91/030701-06$02.00/0 Copyright C 1991, American Society for Microbiology Partial Purification and Characterization of a Bacteriocin Produced by Propionibacterium thoeniit WANDA J. LYON1 AND BONITA A. GLATZ2* Department of Microbiology' and Department of Food Science and Human Nutrition,2 Iowa State University, Ames, Iowa 50011 Received 9 July 1990/Accepted 10 December 1990 A partially purified bacteriocin produced by Propionibacterium thoenii designated propionicin PLG-1 was found to be active against closely related species and exhibited a broad spectrum of activity against other microorganisms. Propionicin PLG-1 was found to be heat labile, sensitive to several proteolytic enzymes, and stable at pH 3 to 9. Propionicin PLG-1 was isolated from solid medium, partially purified by ammonium sulfate precipitation, and purified further by gel filtration. Gel filtration experiments revealed that bacteriocin PLG-1 was present as two different protein aggregates with apparent molecular weights of more than 150,000 and approximately 10,000. Resolution of these protein aggregates by sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed the presence of a protein common to both with an apparent molecular weight of 10,000. Propionibacterium species were first described in 1909 by Orla-Jensen, who divided them into two principal groups: the classical, or dairy, propionibacteria and the acnes, or cutaneous, propionibacteria (19). The classical propionibac- teria are used in dairy fermentations and may contribute to natural fermentations of silage and olives (5, 21). As dairy starter cultures, the propionibacteria are used to produce the characteristic eyes and flavor of Swiss-type cheeses (4, 15). Bacteriocins, which are produced by a heterogeneous group of microorganisms, have a wide range of chemical properties, modes of action, and antibacterial spectra (24). Tagg et al. (24) defined bacteriocins as protein-containing molecules that exert a bactericidal mode of action on closely related species. Although numerous bacteriocins from gram- positive organisms have been isolated, characterized, and purified (3, 8, 11), few from the propionibacteria have been characterized and identified. Both classical and cutaneous propionibacteria have been shown to produce bacteriocins (2, 20). Al-Zoreky (2) showed that Microgard (Wesman Foods, Inc., Beaverton, Oreg.), a pasteurized grade A skim milk product fermented by Propionibacterium shermanii, has antagonistic effects against gram-negative bacteria and some yeasts and molds but not against gram-positive bacte- ria. This product has been approved by the Food and Drug Administration and is currently being used as a preservative in cottage cheese (7). Propionibacterium thoenii P127 from the Iowa State Uni- versity culture collection was found to have inhibitory activity against other classical propionibacteria (12). The nature of the inhibitory substance(s) produced by this strain was not defined. In the current study, the inhibitory sub- stance produced by strain P127 was isolated, partially puri- fied, and found to be a protein with bactericidal activity against a wide spectrum of microorganisms. * Corresponding author. t Journal paper no. J-14113 of the Iowa Agriculture and Home Economics Experiment Station, Ames, Project 2827. MATERIALS AND METHODS Bacterial cultures. Strains of microorganisms used in this study (Tables 1 and 2) were obtained from the stock culture collection maintained by the Department of Food Science and Human Nutrition at Iowa State University. Identifica- tion of the various propionibacteria was verified by Gram stain, carbohydrate fermentation patterns (6), and high- performance liquid chromatographic (HPLC) analysis of culture supernatants for the presence of propionic and acetic acids. The propionibacteria were propagated in screw-capped tubes of sodium lactate broth (NLB) (13) at 32°C. Propioni- bacteria and clostridia were grown anaerobically by using the BBL anaerobic Gas-Pak system. All other microorgan- isms used in this study were grown in the media indicated in Tables 1 and 2. Brain heart infusion broth (BHI), Bacto APT broth (APT), Czapek-Dox broth, Lactobacilli MRS broth (MRS), and thioglycolate were obtained from Difco Labora- tories (Detroit, Mich.) and supplemented with 1.5% Bacto- Agar as needed. Frozen stocks were maintained at -80°C in the appropriate medium with 50% glycerol added. Bacteriocin assays. Propionibacterium acidipropionici P5 was routinely used as the indicator strain in bacteriocin assays. The test strain, P. thoenii P127, and the indicator strain were grown for 18 h in NLB before use. Other organisms were grown in the appropriate medium to loga- rithmic phase. Antimicrobial activity of the producer strain was measured by an agar spot assay (10). Activities of partially purified bacteriocin preparations were measured by a well diffusion method (17, 25) or by the critical-dilution method (17). Activity was defined as the reciprocal of the highest dilution causing complete inhibition of the indicator lawn and was expressed as activity units (AU) per milliliter. Approximately 107 cells of indicator organisms were added to soft-agar overlays. Controls for all assays included the examination of dilution buffers for inhibition of the indicator strains. Plates were incubated as appropriate for the indica- tor organisms. Diameters of the clear zones of inhibition were measured in millimeters. 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Vol. 57, No. 3APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Mar. 1991, p. 701-7060099-2240/91/030701-06$02.00/0Copyright C 1991, American Society for Microbiology

Partial Purification and Characterization of a Bacteriocin Producedby Propionibacterium thoeniitWANDA J. LYON1 AND BONITA A. GLATZ2*

Department of Microbiology' and Department ofFood Science and Human Nutrition,2Iowa State University, Ames, Iowa 50011

Received 9 July 1990/Accepted 10 December 1990

A partially purified bacteriocin produced by Propionibacterium thoenii designated propionicin PLG-1 wasfound to be active against closely related species and exhibited a broad spectrum of activity against othermicroorganisms. Propionicin PLG-1 was found to be heat labile, sensitive to several proteolytic enzymes, andstable at pH 3 to 9. Propionicin PLG-1 was isolated from solid medium, partially purified by ammonium sulfateprecipitation, and purified further by gel filtration. Gel filtration experiments revealed that bacteriocin PLG-1was present as two different protein aggregates with apparent molecular weights of more than 150,000 andapproximately 10,000. Resolution of these protein aggregates by sodium dodecyl sulfate-polyacrylamide gelelectrophoresis revealed the presence of a protein common to both with an apparent molecular weight of10,000.

Propionibacterium species were first described in 1909 byOrla-Jensen, who divided them into two principal groups:the classical, or dairy, propionibacteria and the acnes, orcutaneous, propionibacteria (19). The classical propionibac-teria are used in dairy fermentations and may contribute tonatural fermentations of silage and olives (5, 21). As dairystarter cultures, the propionibacteria are used to produce thecharacteristic eyes and flavor of Swiss-type cheeses (4, 15).

Bacteriocins, which are produced by a heterogeneousgroup of microorganisms, have a wide range of chemicalproperties, modes of action, and antibacterial spectra (24).Tagg et al. (24) defined bacteriocins as protein-containingmolecules that exert a bactericidal mode of action on closelyrelated species. Although numerous bacteriocins from gram-positive organisms have been isolated, characterized, andpurified (3, 8, 11), few from the propionibacteria have beencharacterized and identified. Both classical and cutaneouspropionibacteria have been shown to produce bacteriocins(2, 20). Al-Zoreky (2) showed that Microgard (WesmanFoods, Inc., Beaverton, Oreg.), a pasteurized grade A skimmilk product fermented by Propionibacterium shermanii,has antagonistic effects against gram-negative bacteria andsome yeasts and molds but not against gram-positive bacte-ria. This product has been approved by the Food and DrugAdministration and is currently being used as a preservativein cottage cheese (7).Propionibacterium thoenii P127 from the Iowa State Uni-

versity culture collection was found to have inhibitoryactivity against other classical propionibacteria (12). Thenature of the inhibitory substance(s) produced by this strainwas not defined. In the current study, the inhibitory sub-stance produced by strain P127 was isolated, partially puri-fied, and found to be a protein with bactericidal activityagainst a wide spectrum of microorganisms.

* Corresponding author.t Journal paper no. J-14113 of the Iowa Agriculture and Home

Economics Experiment Station, Ames, Project 2827.

MATERIALS AND METHODS

Bacterial cultures. Strains of microorganisms used in thisstudy (Tables 1 and 2) were obtained from the stock culturecollection maintained by the Department of Food Scienceand Human Nutrition at Iowa State University. Identifica-tion of the various propionibacteria was verified by Gramstain, carbohydrate fermentation patterns (6), and high-performance liquid chromatographic (HPLC) analysis ofculture supernatants for the presence of propionic and aceticacids.The propionibacteria were propagated in screw-capped

tubes of sodium lactate broth (NLB) (13) at 32°C. Propioni-bacteria and clostridia were grown anaerobically by usingthe BBL anaerobic Gas-Pak system. All other microorgan-isms used in this study were grown in the media indicated inTables 1 and 2. Brain heart infusion broth (BHI), Bacto APTbroth (APT), Czapek-Dox broth, Lactobacilli MRS broth(MRS), and thioglycolate were obtained from Difco Labora-tories (Detroit, Mich.) and supplemented with 1.5% Bacto-Agar as needed. Frozen stocks were maintained at -80°C inthe appropriate medium with 50% glycerol added.

Bacteriocin assays. Propionibacterium acidipropionici P5was routinely used as the indicator strain in bacteriocinassays. The test strain, P. thoenii P127, and the indicatorstrain were grown for 18 h in NLB before use. Otherorganisms were grown in the appropriate medium to loga-rithmic phase. Antimicrobial activity of the producer strainwas measured by an agar spot assay (10). Activities ofpartially purified bacteriocin preparations were measured bya well diffusion method (17, 25) or by the critical-dilutionmethod (17). Activity was defined as the reciprocal of thehighest dilution causing complete inhibition of the indicatorlawn and was expressed as activity units (AU) per milliliter.Approximately 107 cells of indicator organisms were addedto soft-agar overlays. Controls for all assays included theexamination of dilution buffers for inhibition of the indicatorstrains. Plates were incubated as appropriate for the indica-tor organisms. Diameters of the clear zones of inhibitionwere measured in millimeters. All assays were performed induplicate, and results presented are the means of duplicatetrials.

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702 LYON AND GLATZ

TABLE 1. Bacterial strains inhibited by partially purifiedbacteriocin from strain P127 in the well diffusion assay

Organism Growth conditions of inhibitiona

Gram positiveLactobacillus bulgaricus AR2 MRS, 37°C +2Lactobacillus bulgaricus IT6 MRS, 37°C +1Lactobacillus casei MRS, 370C +2Pediococcus cerevisiae MRS, 320C +2Lactococcus lactis subsp. APT, 37°C +2

cremorisLactococcus lactis subsp. APT, 370C +2

lactis DRC-1Lactococcus lactis subsp. APT, 370C +3

lactis C2

Gram negativeCampylobacter jejuni Thioglycolate, 370C +2Escherichia coli JM109 TSB,b 370C +l1Escherichia coli V517 TSB, 37-C +1Pseudomonas fluorescens BHI, 370C +4Pseudomonas aeruginosa BHI, 370C +4Vibrio parahaemolyticus BHI, 320C +3

a -, No inhibition; +1, s10 mm; +2, 11 to 14 mm; +3, 15 to 17 mm; +4,.18 mm.

b TSB, Tryptic soy broth.c Zone of inhibition was hazy, not clear.

Mitomycin C and UV light induction of bacteriocin. StrainP127 was inoculated into screw-capped test tubes of NLBand incubated at 32°C for 18 h. Mitomycin C (Sigma Chem-ical Co., St. Louis, Mo.) was added at a final concentrationof 1.0 p.g/ml, and incubation was continued at 320C. Samples(1 ml each) were removed at 60, 120, 240, and 360 min andcentrifuged to remove the cells, and the supernatants (200 ,uleach) were analyzed by the well diffusion method.A 10-ml sample of P127 culture grown for 18 h in NLB was

placed in a sterile petri dish and exposed to short-wave UVlight from a 15-W General Electric germicidal bulb at adistance of 20 cm. Times of exposure ranged from 0 to 30 s.After exposure, the cell suspension was reincubated at 320Cfor 12 h and centrifuged, and the supernatant (100 ,ul) wasanalyzed for bacteriocin activity by the well diffusionmethod.

TABLE 2. Yeast and mold strains inhibited by partially purifiedbacteriocin from strain P127 in the well diffusion assay

Organism Growth Diam of zonemediuma of inhibition'

Aspergillus wentii ATCC 1778 Czapek Dox +2Apiotrichum curvatum Czapek Dox +1Candida utilis Czapek Dox +3Candida lipolytica Czapek Dox + 1Fusarium tricinctum Czapek Dox +1Phialophora gregata Czapek Dox +3Saccharomyces cerevisiae ATCC 24702 TSBd +lCSaccharomyces cerevisiae TSB +l1Saccharomycopsis fibuligera Czapek Dox +3Scopulariopsis sp. Czapek Dox +3Trichoderma reesi Czapek Dox +3

a All media were held at 32°C.b -, No inhibition; +1, '10 mm; +2, 11 to 14 mm; +3, 15 to 17 mm; +4,

.18 mm.c Zone of inhibition was hazy, not clear.d TSB, Tryptic soy broth.

Preparation of partially purified bacteriocin. Eighteen-hourcultures of strain P127 were swabbed across the entiresurfaces of petri plates that each contained 15 ml of sodiumlactate agar (NLA) made with 0.4% agar. The plates wereincubated anaerobically for 5 days at 32°C, stored at -20°Cfor 24 h, and thawed at room temperature for 3 h. Thecollapsed agar mixture was poured into sterile Whirl-Pakbags (Nasco, Fort Atkinson, Wis.), massaged to break upthe agar fragments, and allowed to equilibrate for 18 h at 4°C.The agar suspension was filtered through Whatman No. 1filter paper (Fisher Scientific Co., Pittsburgh, Pa.), and thefiltrate was centrifuged at 12,000 x g for 20 min to removeagar fragments and bacterial cells. Ammonium sulfate wasadded slowly to the supernatant until the final concentrationwas 60% (wt/vol). The mixture was slowly stirred at 4°C for1 h and filtered through cheesecloth. Precipitated proteinwas removed by centrifugation at 25,000 x g for 30 min,suspended in 0.05 M potassium phosphate buffer (pH 7.0),and dialyzed against 2 liters of the same buffer for 18 h inSpectra/Por no. 4 dialysis tubing (Spectrum Medical Indus-tries, Los Angeles, Calif.; molecular weight (MW) cutoff,12,000 to 14,000). The preparation was concentrated to S mlby dialysis against polyethylene glycol (Carbowax 20,000;Sigma) and filtered through a 0.45-p.m-pore-size Duraporefilter (Millipore Product Division, Bedford, Mass.). Thispreparation was designated partially purified bacteriocin.

Sensitivity to enzymes and heat. A sample of partiallypurified bacteriocin was assessed for its sensitivity to vari-ous enzymes and heat treatments. Enzymes (all obtainedfrom Sigma) and their respective buffers were pronase E(type XXV, 4.1 U/mg), 0.01 M sodium borate-0.05 M HCl-5mM CaCl2-1 mM CoCl2 (pH 7.2); a-chymotrypsin (type II,47 U/mg), 0.05 M Tris hydrochloride (pH 8.0)-0.01 M CaCl2;protease (type V, 1 U/mg), 0.05 M Tris hydrochloride (pH8.0); catalase (2,000 U/mg), 10 mM potassium phosphate (pH7.0); phospholipase C (type I, 10 U/mg), 0.05 M Tris hydro-chloride (pH 7.0)-0.01 M CaCl2; pepsin (3,200 IU/ml), 0.2 Mcitrate (pH 6.0); trypsin (type 1X, 15,000 U/mg), 0.05 M Trishydrochloride (pH 8.0); lipase (type 1, 8.6 U/mg), 0.05 MTris hydrochloride (pH 8.0)-0.01 M CaCl2; papain (EC3.4.22.2, 16 U/mg), 0.05 M acetate (pH 4.5)-0.2 M NaCI.The bacteriocin preparation (500 ,ul, 64 AU/ml) was incu-

bated with 500 p.g of each enzyme per ml for 60 min at 37°Cexcept for samples containing trypsin, a-chymotrypsin, andcatalase, which were incubated at 25°C. Prior to beingassayed for bacteriocin activity, preparations containingpapain were adjusted to pH 6.0 and those containing trypsinand chymotrypsin were treated with trypsin-chymotrypsininhibitor (Sigma) according to the manufacturer's instruc-tions.Temperature stability was determined by heating a 1-ml

sample of partially purified bacteriocin (protein concentra-tion, 500 pig/ml; 64 AU/ml) in glass test tubes (10 by 75 mm)at the times and temperatures listed in Table 3. After heattreatment, 500-pI samples were assayed for remaining activ-ity by the well diffusion method.

Tests for effects of organic acids. The concentrations ofpropionic and acetic acid produced by strain P127 weremeasured by HPLC. The strain was grown in NLB for 5days, the culture was centrifuged, and the supernatant wasfiltered through a 0.22-p.m-pore-size Durapore filter. A 20-p.lsample was injected into a Waters HPLC model 501 (WatersInc., Milford, Mass.) equipped with an Aminex HPX-87Hcolumn (Bio-Rad Chemical Division, Richman, Calif.) thatwas run at 65°C. The mobile phase was 0.012 N HCl at a flowrate of 0.8 ml/min. Organic acids were detected by a refrac-

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PROPIONICIN PLG-1 FROM P. THOENII 703

TABLE 3. Sensitivity of propionicin PLG-1 to various enzymaticand heat treatments

Treatment ~~~~~Diam (mm) of zoneTreatment of inhibitiona

Control (phosphate buffer) ............................... 14a-Chymotrypsin ..................................... 0Protease ..................................... 0Papain ..................................... 0Pepsin ..................................... 0Trypsin ..................................... 0Pronase..................................... 9Catalase ..................................... 14Lipase ..................................... 14Lysozyme ..................................... 14Phospholipase C ..................................... 14

Control (no heat) ..................................... 1775°C for 60 min ............................. ........ 1780°C for 30 min ............................. ........ 1685°C for 15 min ............................. ........ 1085°C for 30 min ............................. ........ 090°C for 3 min ..................................... 0100°C for 1 min ............................. ........ 0

a All diameters are the means of duplicate tests.

tive index detector (model R401, Waters). Peaks were iden-tified by comparing retention times with those of standards.pH stability of bacteriocin activity. Partially purified bacte-

riocin samples (1 ml, 64 AU/ml) were individually dialyzedfor 24 h against 2-liter volumes of the following buffers: 0.05M citrate buffer (pHs 3, 4, 5, and 6), 0.05 M potassiumphosphate buffer (pH 7), 0.05 M Tris hydrochloride (pHs 8and 9). Three changes of each buffer were made duringdialysis. After dialysis, the bacteriocin solutions (200 ,ul)were assayed for activity by the well diffusion method.

Adsorption studies. Adsorption of partially purified bacte-riocin to bacterial cells was studied by modifying the methodof Barefoot and Klaenhammer (3). Strains P5 and P127 weregrown in NLB for 18 h, harvested by centrifugation, washedin 0.05 M potassium phosphate buffer (pH 7.0), and sus-

pended at a final concentration of approximately 108 cells perml in 500 ,ul of a preparation of partially purified bacteriocin(32 AU/ml). The suspensions were incubated for 0, 30, and60 min at 32°C, the A6. was measured, the cells wereremoved by centrifugation, and the supernatants were fil-tered through a 0.45-,um-pore-size Durapore filter. The bac-teriocin activity of the filtrate was assayed by the welldiffusion method. The cell pellet was washed with 0.05 Mpotassium phosphate buffer (pH 7.0) and suspended in 1 mlof phosphate buffer, and viable counts were obtained onNLA plates by incubating the plates anaerobically at 32°Cfor 5 days.MW determination. The MW of the bacteriocin was deter-

mined by gel filtration. A 5-ml sample of partially purifiedbacteriocin in 0.05 M potassium phosphate buffer (pH 7.0)was applied to a descending Sephadex G-200 column (2.5 by42.8 cm; Pharmacia Fine Chemicals, Piscataway, N.J.) at4°C and eluted with 0.05 M potassium phosphate buffer (pH7.0) at a flow rate of 0.40 ml/min. Protein in the eluentfractions was measured by determining the A280. Fractionswith protein were pooled, concentrated 10-fold by dialysisagainst polyethylene glycol, exhaustively dialyzed in no. 4Spectra/Por tubing against 0.05 M potassium phosphatebuffer (pH 7.0) at 4°C, filter sterilized through a 0.45-,um-pore-size Durapore membrane, and assayed for bacteriocin

activity by the well diffusion method. MWs were determinedby comparison of elution volumes with those of knownstandards (14). Protein standards (Sigma) used were apofer-ritin, 6,500; carbonic anhydrase, 29,000; cytochrome c,12,400; and alcohol dehydrogenase, 150,000.

Protein determinations. Protein in 50-,ul samples of par-tially purified bacteriocin was determined by the modifiedmethod of Lowry et al. (16) according to the specifications ofthe manufacturer (Sigma). Bovine serum albumin was usedto construct a standard curve.SDS-PAGE. Polyacrylamide gel electrophoresis (PAGE)

in the presence of 0.1% sodium dodecyl sulfate (SDS) wasperformed on a vertical slab gel (1 mm) as described byLaemmli (14). Polyacrylamide and N,N'-methylenebisacryl-amide (Sigma) concentrations were 5 and 0.15%, respec-tively, in the stacking gel (10 ml) and 20 and 0.5% in theseparating gel (30 ml). Electrophoresis was conducted at aconstant current of 30 mA for 4 h at 4°C. The gel was stainedwith Coomassie brilliant blue-R (Sigma) and enhanced withsilver stain (Sigma) as described by Merril et al. (18). Proteinstandards and their MWs were ovalbumin, 43,000; carbonicanhydrase, 29,000; ,-lactoglobulin, 18,400; lysozyme,14,300; bovine trypsin inhibitor, 6,200; a and P insulin, 3,000(Bethesda Research Laboratories, Gaithersburg, Md.).

RESULTS

Conditions for production of inhibitory activity. Inhibitoryactivity of strain P127 against strain P5 was observed onlywhen cultures were grown on solidified agar. Activity wasnever seen in cell-free supernatants of cultures grown inNLB, even after fivefold concentration by dialysis againstpolyethylene glycol. Attempts to isolate propionicin PLG-1from NLA yielded low concentrations of the bacteriocin thatseemed to be associated with protein components of themedium (data not shown). Treatment of P127 broth cultureswith mitomycin C or UV light did not induce production ofinhibitory activity. Inhibitory activity could be measuredonly after strain P127 had been grown for at least 24 h onNLA; maximum inhibitory activity was seen after approxi-mately 140 h.

Inhibitory activity could be separated from cells of pro-ducer strain P127 by growing the strain in NLA that con-tained 0.4% agar, freezing the agar to collapse the gelstructure, and removing cells and agar debris by centrifuga-tion. This method was used to produce a crude preparationof bacteriocin that was further purified by ammonium sulfateprecipitation and dialysis against 0.05 M potassium phos-phate buffer (pH 7.0). The resulting partially purified bacte-riocin was then used in further tests.

Inhibitory activity against other microorganisms. Partiallypurified bacteriocin was tested in well diffusion assays toevaluate its inhibitory activity against various microorgan-isms. Thirty-one strains of classical propionibacteria fromthe culture collection were tested. All strains of Propioni-bacterium freudenreichii subsp. freudenreichii and P.freudenreichii subsp. shermanii were insensitive to thissubstance, but all strains of P. thoenii, P. acidipropionici,and Propionibacterium jensenii were sensitive. Of the 15tested strains of P. freudenreichii, none produced antimicro-bial substances against other selected indicator strains ofpropionibacteria (data not shown). Producer strain P127 wasnot inhibited by its own inhibitory factor.

Table 1 presents results of well diffusion assays conductedwith other bacteria. Lactic acid bacteria were inhibited, buttested strains of Staphylococcus aureus, Streptococcus

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704 LYON AND GLATZ

faecalis (group D), six species ofBacillus, and two species ofClostridium were not inhibited. Of the gram-negative organ-isms tested, strains of Pseudomonas were most sensitive.Representative strains of Aeromonas hydrophila, Yersiniaenterocolitica, and two species of Salmonella were notinhibited.

Inhibitory activity was also observed against some moldsand yeasts (Table 2). Hazy zones of inhibition observed withstrains of Saccharomyces suggest that partial inhibition, orperhaps fungistatic rather than fungicidal activity, occurred.Of three species of Aspergillus tested, only Aspergilluswentii was inhibited. One strain each of Fusarium roseumand Penicillium chrysogenum were not inhibited.

Effects of enzymes and heat on the inhibitory substance.Samples of partially purified inhibitory substance were foundto be sensitive to protease, pronase E, pepsin, trypsin, andot-chymotrypsin but were not affected by phospholipase C,lipase, or catalase (Table 3). Inactivation of bacteriocin in itsnative structural conformation by pronase E was incompleteand may be attributed to the proteolytic requirement ofpronase E for a free N-terminal amino acid in its substrate(23). Inhibitory activity was unaffected by heating at moder-ate temperatures but was rapidly lost by heating at temper-atures greater than 85°C (Table 3). Buffers and enzymepreparations alone had no effect on the indicator lawns.These data suggest that the inhibitory substance is a proteinwhich is active against other strains of bacteria. We proposethat it be designated a bacteriocin and given the name

propionicin PLG-1.Other potential inhibitory substances. Propionibacteria can

produce other inhibitory substances such as organic acids(propionate and acetate), diacetyl (2,3-butanedione), andhydrogen peroxide (7). Therefore, it was important to elim-inate these compounds as the source of inhibitory activityproduced by strain P127. These inhibitory substances are oflow MW. In the preparation of partially purified bacteriocin,samples were exhaustively dialyzed against buffer in dialysistubing with an MW cutoff of 12,000 to 14,000. The inhibitoryactivity was retained in the dialysis bag, which suggests thatit is not caused by low-MW molecules.

Strain P127 was grown to stationary phase (144 h) in NLB,and the culture supernatant was analyzed for propionic andacetic acids by HPLC. Only 0.4% propionate and 0.2%acetate were present. In contrast, indicator strain P5 toler-ated up to 1.5% propionic acid when grown on gradientplates containing 0 to 2.0% propionic acid. Partially purifiedbacteriocin preparations contained only 0.008% propionicacid and no acetic acid.The fact that catalase had no effect on inhibitory activity

indicates that hydrogen peroxide is not the inhibitory factor(Table 3). These collective data indicate that the observedinhibitory activity is not due to low-MW substances such asorganic acids or hydrogen peroxide or to intolerance of theindicator strain P5 to propionic acid.pH stability of propionicin PLG-1. Partially purified bacte-

riocin was equilibrated in buffers with various pH values andassayed by the well diffusion method. Activity was greatestat pH 7.0 and was detected throughout the range ofpH 3.0 to9.0. At pH 7.0, the diameter of the zone of inhibition againstindicator strain P5 was 22 mm, while at acidic pHs (3 to 4),there was partial lost of activity (diameter of the zone ofinhibition was 11 mm). At pHs 5 to 6 and 8 to 9, the zones ofinhibition were 14 and 18 mm, respectively.

Effect of propionicin PLG-1 on sensitive and nonsensitivecells. Aliquots (500 jil each) of partially purified bacteriocinwere mixed with 500 ,ul of cell suspensions of strains P127

TABLE 4. Culture viability and bacteriocin activity afterincubation of bacteriocin preparations with indicator

or producer cells'

Diam (mm) of zone Viable countsTest mixture ofihbtobOf inhibition CFU/ml % Reductionb

Bacteriocin + bufferc 14Cells + buffer

Strain P127 3.3 x 108 0Strain P5 1.3 x 108 0

Bacteriocin + cellsStrain P127 14 3.2 x 108 3.0Strain P5 0 5.2 x 105 99.6

a Initial cell concentrations were 3.3 x 101 CFU/ml for strain P127 and 1.3x 108 CFU/ml for P5. Cell suspensions (500 .l) and/or partially purifiedbacteriocin (500 p.d; 32 AU/ml) were used in each test. After incubation for 30min at 32°C, residual bacteriocin activity was assayed by the well diffusionmethod. Zones were measured as diameters from the edge of the clearingzone.

b Calculated as [(CFU prior to incubation - CFU after incubation)/CFUprior to incubation] x 100.

c 0.05 M potassium phosphate buffer (pH 7.0).

and P5. After incubation for 30 min, the cells were pelletedby centrifugation and enumerated for viable counts. Resid-ual inhibitory activity of the supernatant was measured bythe well diffusion method. Results are presented in Table 4.

Viable counts of indicator strain P5 were reduced by99.6% after exposure to propionicin PLG-1, but viablecounts of strain P127 were unchanged. Incubation longerthan 30 min did not significantly change the number of viablemicroorganisms detected. Residual inhibitory activity wasnot detected in the supernatant of preparations mixed withstrain P5 cells but was unchanged in preparations mixed withP127 cells. These results suggest that propionicin PLG-1 didnot adsorb to producer strain P127 but did adsorb to indica-tor strain P5. The effect on sensitive cells was bactericidal,but no obvious cell lysis was seen.MW determination. Partially purified bacteriocin was ap-

plied to a descending Sephadex G-200 column. Protein waseluted in two peaks, and pooled fractions from these twopeaks had antimicrobial activity. On the basis of compari-sons of mean elution volumes with those of protein stan-dards (data not shown), MWs of active fractions weredetermined to be greater than 150,000 and approximately10,000.The bacteriocin appeared to be associated with contami-

nating proteins from the medium. Applying the bacteriocinto a descending Sephadex G-50 column prior to applicationto a Sephadex G-200 column allowed some, but not com-plete, separation from these contaminants (data not shown).SDS-PAGE. When active fractions (60 jig of protein) from

the second peak eluted from a Sephadex G-200 column wereelectrophoresed in a denaturing 20% polyacrylamide gel, asingle diffused protein band with an MW of approximately10,000 was resolved (Fig. 1). Similarly, a fraction from thefirst peak resolved a protein band with an MW of approxi-mately 10,000, but this fraction also contained approxi-mately 12 contaminating proteins.

DISCUSSION

The inhibitory substance produced by strain P127 seemsto be a large, heat-labile protein that we have designatedpropionicin PLG-1. Inhibitory activity produced by strainP127 was not detected in cell-free supernatants of broth

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PROPIONICIN PLG-1 FROM P. THOENII 705

FIG. 1. SDS-PAGE of fractions from protein peaks eluted from a

Sephadex G-200 column to which partially purified bacteriocinpreparation had been applied. Lanes: 1, fraction from first peak(estimated MW, 150,000); 2, MW standards (top to bottom, ovalbu-min [MW 43,000], carbonic anhydrase [MW 29,000], ,-lactoglobulin[MW 18,400], lysozyme [MW 14,300], bovine trypsin inhibitor [MW6,200], and a and ,B insulin (MW 3,000]; 3 and 5, fractions fromsecond peak (estimated MW, 10,000); 4, empty.

cultures but was isolated from semisolid medium. Similarly,Barefoot and Klaenhammer (3) were unable to isolate thebacteriocin lactacin B from broth cultures of Lactobacillusacidophilus and could isolate only small quantities from agar

plate cultures.One characteristic of classical bacteriocins is a narrow

spectrum of activity (1, 24). More recent reports (8, 24) haveshown that some bacteriocins produced by gram-positivebacteria have a broad spectrum of activity. The bacteriocinproduced by strain P127 is active against some, but not all, ofthe classical Propionibacterium species, some other gram-

positive organisms (especially lactic acid bacteria), some

gram-negative organisms, and some yeasts and molds. Pro-pionicin PLG-1 is different from Microgard, which containsan inhibitory substance of low MW produced by a strain ofP. freudenreichii subsp. shermanii. Microgard inhibits manygram-negative organisms and some molds and yeasts but no

gram-positive organisms. More-extensive testing of thespectrum of activity will be performed with purified propi-onicin PLG-1.

Propionicin PLG-1 was active over a wide pH range (pH 3to 9), with greatest activity at pH 7. Similarly, Gonzalez andKunka (11) found that pediocin PA-1, a bacteriocin producedby Pediococcus acidilactici, was active over a wide pHrange (4 to 7), with some loss of activity at pHs 2, 3, 9, and10. This stability over a wide range of pH values may beuseful if propionicin PLG-1 is used as an antimicrobial agentin fermented foods or other products.Tagg et al. (24) described bacteriocin action as a single-hit

mechanism in which the bacteriocin adsorbs to, penetrates,and kills sensitive cells in an irreversible process. Propioni-cin PLG-1 was found to adsorb to sensitive cells of strain P5and to be bactericidal. Tagg et al. (24) suggested thatbacteriocin resistance results from a lack of bacteriocin-specific receptors in the cell membrane, whereas bacteriocinimmunity results from production of a substance, perhaps a

protein, that binds to the bacteriocin and prevents lethalaction on the producer cells. Propionicin PLG-1 did notadsorb to its producer strain and did not inhibit other closely

related strains, but the mechanism of resistance is yet to bedetermined. Propionicin PLG-1 seems to be different fromsome bacteriocins isolated from other gram-positive organ-isms, which bind nonspecifically to both sensitive and non-sensitive cells (3, 11).Crude preparations of bacteriocin applied to a Sephadex

G-200 column were eluted in two peaks that containedbacteriocin activity. Proteins in these peaks were estimatedto have MWs of approximately 10,000 and greater than150,000. After resolution by SDS-PAGE, a common diffusedprotein of approximate MW 10,000 was found in activefractions. This protein may be the actual propionicin mole-cule. Its identity and activity await further determination.Results in the current study parallel those reported forlactacin B (MW 100,000) isolation (3). Both propionicinPLG-1 and lactacin B were first isolated as large proteinaggregates, and both bacteriocins were dissociated fromother protein components by SDS-PAGE. The apparentassociation of this bacteriocin with other proteins, some ofwhich probably originated in the culture medium, mayrequire the development of a defined medium to optimizebacteriocin production and purification. Further purificationand characterization of this bacteriocin are currently inprogress.

ACKNOWLEDGMENTS

We acknowledge Dale Grinstead, who first observed the inhibi-tory activity of strain P127, and Patricia Murphy, for her helpfuladvice and assistance in protein purification.

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