1520

Journal oj'Food Protection, Vol. 60, No. 12, 1997, Pages 1520-1528 Copyright©, International Association of Milk, Food and Environmental Sanitarians

Isolation and Purification of Colicin ECl 12, a That Inhibits Strains of 0157:H7t

WANDA J. LYONH and DENNIS G. OLSON2 Downloaded from http://meridian.allenpress.com/jfp/article-pdf/60/12/1520/2301394/0362-028x-60_12_1520.pdf by guest on 01 October 2021 IDepartment of Food Science, Louisiana State University Agricultural Center, Louisiana State University, BalOn Rouge, Louisiana 70803; and 2Department oj'Animal Science, 10wa State University, Ames, 10wa 50011, USA

(MS# 96-299: Received 28 October 1996/Accepted 16 March 1997)

ABSTRACT tion of raw milk (6, 8). Vegetables, water, and other meat products have also been linked as vehicles for transmission A swine fecal isolate, identified as Escherichia coli ECLl2, of E. coli 0157:H7 infections (17). was found to produce an antimicrobial substance designated as The work reported in this paper was initiated with the colicin ECLl2. Colicin ECLl2 was inhibitory against 20 strains of purpose of finding bacteriocin-producing strains that could E. coli 0157:H7 previously isolated from both human and bovine effectively inhibit E. coli 0157:H7. Since cattle are reser- feces. Identification of the producer strain was determined pheno- voirs for E. coli 0157:H7, bovine feces were examined for typically by biochemical and morphological tests. Colicin ECLl2 was sensitive to several proteolytic enzymes. Adsorption of colicin bacteria that might have developed a selective advantage in ECLl2 to sensitive cells of E. coli 0157:H7 was bactericidal, animals that have not tested positive for shedding of E. coli resulting in a 2 log reduction in viable cell counts. CoLicinECLl2 0157:H7. In this study a bacteriocin produced by a species was purified from strain ECLl2 by cell extraction and ion- of E. coli was isolated, purified, and found to be a protein exchange chromatography. Sodium dodecyl sulfate-polyacryl- with antimicrobial activity against a narrow spectrum of amide gel electrophoresis of colicin ECLl2 resolved a single microorganisms, including strains of E. coli and Shigella protein with a molecular weight of approximately 65,000. sonnei.

Key words: Colicin, inhibition, E. coli 0157:H7, food preservative MATERIALS AND METHODS

Colicins are antimicrobial proteins synthesized by Esch- Cultures and media erichia coli and active against closely related coliform Escherichia coli ECL 12 was isolated from bovine sewage runoff as described below. The use of colicin ECL 12 in foods and bacteria. Colicin proteins range in molecular weight from the strain that produces it are protected by U.S. patent 5,549,895. 90,000 to 1,500 (12). Colicins are typically encoded on All microorganisms used in this study were grown in media and at with the exception of colicin Land microcin E966 temperatures indicated in Tables 1 and 2. Bacteriocin-producing (12, 18). A large number of colicins have been isolated and strain E. coli ECLl2 and sensitive E. coli 0157:H7 indicator strains characterized by many investigators, For additional perti- were propagated at 37°C in Trypticase soy broth (TSB) (BBL, nent information the reader is directed to previously pub- Becton Dickinson, Cockeysville, MD) supplemented with 0.5% lished reviews (12, 18). yeast extract (TSYE) (Difco Laboratories, Detroit, MI). Frozen Enterohemorrhagic E. coli (EHEC) strains, which in- stocks were maintained at -80°C in the appropriate medium with clude E. coli 0157:H7, are increasingly becoming recog- 50% glycerol. nized as widespread foodbome pathogens. Enterohemor- rhagic E. coli 0157:H7 causes serious illness in humans in Isolation of bacteriocin-producing bacteria the form of bloody diarrhea and hemolytic uremic syndrome Twenty-five fecal samples were obtained from several Iowa (17). EHEC strains are commonly isolated from the feces of farms. The fecal samples (25 g) were weighed into stomacher bags, clinically normal and diarrheic cattle (22). Cattle have been and 250 ml of phosphate buffer (pH 7.0) was added, the bags implicated as a reservoir for E. coli 0157:H7 (8). Human sealed, the material macerated for 5 min in a stomacher. SampLes were removed and serially diluted in 0.2% peptone, spread-plated infections and zoonotic transmission of E. coli 0157:H7 are onto both TSYE agar and violet red bile agar (VRBA) (Difco thought to occur by ingestion of ground beef and consump- Laboratories), and incubated aerobically at 37°C for 18 h. Indi- vidual colonies were transferred from VRBA plates with sterile toothpicks onto duplicate plates of TSYE and reincubated for 18h. * Author for correspondence. Tel: 504-388-5197; Fax: 504-388-5300; One plate was used for replica plating onto fresh TSYE plates and E-mail: [email protected]. the other was retained as a master plate. After growth, each plate t Joumal paper no. J-96-21-0273 of the Louisiana Agricultural Experiment was overlaid with 5 ml of TSYE containing 0.7% agar that had Station, Baton Rouge, LA. Project: S-263. been previously inoculated with different sensitive strains (20 Ill; COLICIN ECL12 1521

TABLE 1. The inhibitory effect of purified colicin ECLl2 against lated, and all were further characterized and identified as either selected gram-negative bacterial strains using the critical dilution Shigella sonnei, E. coli, or Klebsiella pneumoniae using both the method API 20E analysis as described by the manufacturer (API Analytab Products, Plainview, NY) and phenotypic characteristics listed in Reaction Table 3. Forty-five of the 50 bacteriocin-producing isolates pro- Growth to colicin duced antimicrobial activity that was not effective against E. coli Straina conditions ECL12b 0157:H7. One of the remaining isolates was characterized and Aeromonas hydrophila TSYE,37°C identified as a strain of E. coli and was found to produce an Campylobacter jejuni Thioglycollate, 37°C antimicrobial agent effective against E. coli 0157:H7. This isolate, Escherichia coli JM109 TSYE,37°C ++++ designated E. coli ECLl2, was selected for further characterization, Escherichia coli V517 TSYE,37°C ++++ isolation, and purification of its colicin. Escherichia coli 0157:H7 2903 TSYE,37°C ++++ Escherichia coli 0157:H7 2922 TSYE,37°C ++ Critical dilution assay for colicin activity Escherichia coli 0157:H7 3076 TSYE,37°C +++ Antagonistic activities of colicin ECLl2 preparations were Escherichia coli 0157:H7 2881 TSYE,37°C ++++ confirmed by spotting 5 fll of serially diluted (twofold dilution) Downloaded from http://meridian.allenpress.com/jfp/article-pdf/60/12/1520/2301394/0362-028x-60_12_1520.pdf by guest on 01 October 2021 Escherichia coli 0157:H7 3061 TSYE,37°C +++ colicin solutions onto lawns of indicator as described Escherichia coli 0157:H7 2976 TSYE,37°C +++ previously (13, 14). Approximately 20 fll of an 18-h indicator Escherichia coli 0157:H7 2962 TSYE,37°C +++ culture was added to 5 ml of TSYE soft agar overlays (106 Escherichia coli 0157:H7 2972 TSYE,37°C ++++ CPU/ml), and plates were incubated at 37°C for 18 h. Activity was Escherichia coli 0157:H7 2952 TSYE,37°C +++ defined as the reciprocal of the highest 1:2 dilution causing Escherichia coli 0157:H7 3066 TSYE,37°C +++ complete inhibition of the indicator lawn and was expressed as Escherichia coli 0157: TSYE,37°C ++++ arbitrary units (AU) per m!. All assays were performed in duplicate, H7 NADC 3081 and results presented are the means of duplicate trials. Escherichia coli 0157:H7 2924 TSYE,37°C +++ Escherichia coli 0157:H7 2934 TSYE,37°C ++++ Extraction and purification of colicin ECL12 from producer cells Escherichia coli 0157:H7 2888 TSYE,37°C +++ An 18-h culture of E. coli ECLl2 (3,000 mI) was grown to Escherichia coli 0157:H7 2842 TSYE,37°C ++ early stationary phase (109 cells/ml) in M9 broth (1) at 37°C. The Escherichia coli 0157:H7 FSIS TSYE,37°C +++ cell pellets were washed with 30 ml of 50 mM potassium phosphate 45956-59A buffer (pH 7.2) as described by Herschman and Helinski (9) with Escherichia coli 0157:H7 FSIS TSYE,37°C +++ the following modifications. After the cells had been washed and 45753-57B-31 pelleted by centrifugation at 12,000X g at 4°C, the colicin was Escherichia hermanii TSYE,37°C extracted from the cell pellet by resuspending the pellets in a total ATCC 33650 volume of 100 ml of 50 mM potassium phosphate buffer (pH 7.2) Escherichia fergusonii TSYE,37°C containing 1.0 M NaCl, 1 mM EDTA, 1 mM amidinophenylmeth- ATCC 35469 anesulfonyl fluoride (APMSF), and 1 mM pepstatin (all purchased Pseudomonas fluorescens BRr,32°C from Boehringer Mannheim, Indianapolis, IN). The suspension Pseudomonas aeruginosa BRr,32°C was homogenized at low speed with a Waring blender for 30 min at Salmonella derby TSYE,37°C Salmonella dublin TSYE,37°C Salmonella newington TSYE,37°C TABLE 2. The inhibitory effect of purified colicin ECLl2 against Salmonella typhimurium ATCC TSYE,37°C selected gram-positive bacterial strains using the critical dilution 14028 method Shigella dysenteriae TSYE,37°C Reaction Shigella sonnei TSYE,37°C ++++ Growth to colicin b Shigella sonnei ATCC 9290 TSYE,37°C +++ Selected strainsa conditions ECL12 Vibrio parahaemolyticus BRr,32°C Yersinia enterocolitica ATCC TSYE,37°C Bacillus cereus BRr,37°C 23715 Clostridium perfringens Meat, 37°C Enterococcus faecium TSYE,37°C a Strains with no designation are from the culture collection of the Listeria monocytogenes Scott A TSYE,32°C Department of Microbiology at Iowa State University. E. coli Lactobacillus bulgaricus MRS, 37°C . 0157:H7 strains were obtained from the National Animal Disease Lactococcus casei MRS, 37°C Center, Ames, IA. Pediococcus cerevisiae MRS, 37°C b The reaction was assayed by the critical dilution method de- Propionibacterium shermanii TSYE,32°C scribed in the text. Testing against each indicator strain was done Staphylococcus au reus BRr,37°C by spotting 256 AU of activity onto cell lawns (105 CPU/ml). -, Streptococcus faecalis BRr,37°C no inhibition; +, zone of inhibition :55 mm; + +, zone of inhibition 6-10 mm; +++, zone of inhibition 11-15 mm; a Strains are from the culture collection of the Department of ++++, zone of inhibition >15 mm. Microbiology at Iowa State University. b The reaction was assayed by the critical dilution method de- scribed in the text. Testing a each indicator strain was done by 106 CFU/mI), and plates were reincubated at 37°C for 18 h. After spotting 1:2 dilutions of purified colicin onto cell lawns (105 development of the overlay lawn, individual colonies were evalu- CFU/ml). -, no inhibition; +,zone of inhibition :55 mm; + +, ated for zones of inhibition against selected pathogens and spoilage zone of inhibition 6-10 mm; +++, zone of inhibition 11-15 organisms. A total of 50 bacteriocin-producing strains were iso- mm; + + + +,zone of inhibition> 15 mm. 1522 LYON AND OLSON

TABLE 3. Phenotypic characteristics of isolate ECLl2

Parameter or test Reaction or characteristic Parameter or test Reaction or characteristic

Morphology Small rods Acid produced from: Gram reaction Adonitol Catalase + Arabitol Pigmentation White Cellobiose Motility -1+ Dextrin Indole production + Esculin Methyl red + Fructose + Voges- Proskauer Galactose + Citrate, Simmons Glucose + Hydrogen sulfide on TSI Glycerol + Downloaded from http://meridian.allenpress.com/jfp/article-pdf/60/12/1520/2301394/0362-028x-60_12_1520.pdf by guest on 01 October 2021 Urease Lactose Phenylalanine deaminase Maltose Lysine decarboxylase + Maltotriose Arginine dihydrolase + Mannitol + Ornithine decarboxylase Mannose + Glucose, gas production (X-Methy 19lucoside NO;- toNO;- + Raffinose + Oxidase, Kovac's Rhamnose 0- Nitropheny l-I3-D-galactosidase + Salicin Deoxyribonuclease + Sorbitol + Gelatin liquefaction Sucose Lambda receptor Trehalose +

4°C. The cell suspension was centrifuged at 27,000X g for 30 min Protein in the eluent fractions was measured by determining the at 4°C, and the supernatants were decanted, dialyzed against absorbance at 280 nm. One-milliliter fractions containing protein dissociation buffer (6 M guanidine HCl, 0.2 M NaCl, and 1 mM were assayed for bacteriocin activity by spotting 20 fIl of the dithiothreitol), and concentrated in a stirred cell against a PM-lO suspension onto lawns of indicator organisms as described previ- membrane (Amicon, Beverly, MA; MW cutoff, 10,000). The buffer ously (15). Molecular weight (MW) values were determined by was exchanged with either 20 mM 2-(N-morpholino)ethanesul- comparison of elution volumes with those of known standards. fonic acid (MES; pH 6.5) or 20 mM Tris·Cl (pH 8.0), and MW standards (Bio-Rad, Hercules, CA) used were thyroglobulin, concentrated in a stirred cell against a PM-I0 membrane. Each of 670,000; bovine gamma globulin, 158,000; chicken ovalbumin, these preparations were assayed by the critical dilution method (13) 44,000; equine myoglobin, 17,000; and vitamin B-12, 1,350. using various E. coli indicator strains. These preparations were designated as partially purified colicin ECL12 and were applied to Bactericidal action of colicin ECLl2 descending ion-exchange columns as indicated below or used in Bactericidal activity of purified colicin against sensitive mode of action studies. bacterial cells was studied by modifying the method of Lyon et al. a. lon-exchange chromatography. Partially purified colicin (15). Cells from 18-h TSYE cultures of strains E. coli ECL12 and ECL12 (51,200 AU/ml; 50 mg protein) was applied to both a selected strains of E. coli 0157:H7 were harvested by centrifuga- descending Econo-Pac High Q ion-exchange column (1 by 5 cm) tion (9,000 X g), washed in 50 mM potassium phosphate buffer (pH equilibrated with 20 mM MES buffer (pH 6.5) at 5°C and a CM 7.2), and added at final concentrations of 2.8 X 105 CFU/ml and sepharose ion-exchange column equilibrated with 20 mM Tris·Cl 3.2 X 105 CFU/ml, respectively, to a purified colicin ELC12 buffer (pH 8.0) at 5°C. Proteins were eluted from the columns with preparation (500 f1l; 1,024 AU/mI). The suspensions were incu- a linear salt gradient (0 to 0.5 M NaCl; 75 ml) in their respective bated for 0, 30, and 60 min at 37°C, the absorption was measured at buffers at a flow rate of 0.5 ml/min, and refractive indices were 600 nm, the cells were removed by centrifugation, and the determined. Fractions (1 ml) containing protein were assayed for supernatants were evaluated for residual colicin ECL12 activity by bacteriocin activity by spotting 20 J.llof the suspension onto lawns serially diluting 1:2 in 50 mM potassium phosphate buffer (pH 7.2) of indicator organisms as described previously (14). Fractions (1 before assaying by the critical dilution method (13). The cell pellet mI) containing colicin were pooled (total of six fractions), dialyzed was washed with 50 mM potassium phosphate buffer (pH 7.2) and against distilled water containing 1 mM EDTA, and concentrated resuspended in 1.0 ml of 50 mM potassium phosphate buffer (pH tenfold in a stirred cell against a PM -10 membrane. Concentrated 7.2), and viable counts were obtained on both TSYE and MacCon- active protein fractions were analyzed by sodium dodecyl sulfate- key sorbitol (Difco Laboratories) plates by incubating the plates at polyacrylamide gel electrophoresis (SDS-PAGE) as described 37°C for 18 h. Controls included buffer with and without cells. below. b. Gelfiltration and MW determination. The molecular weight of purified colicin ECL12 was determined by gel filtration. A Sensitivity to proteolytic enzymes and heat 0.5-ml sample of purified bacteriocin (51,200 AU/ml; 0.75 mg A sample of purified colicin ECL12 was assessed for its protein) in 20 mM MES buffer (pH 6.5) was applied to a sensitivity to various proteolytic enzymes as described by Lyon et descending Bio-Gel P-30 column (1 by 5 cm) at 5°C and eluted al. (15). Enzymes (all obtained from Sigma, St. Louis, MO) and with 20 mM MES buffer (pH 6.5) at a flow rate of 1.0 ml/min. buffers were prepared as described by Lyon and Glatz (13). Purified COLICIN ECLl2 1523

colicin ECLl2 (500 J.ll, 51,200 AU/ml) was incubated with 500 ECLl2 (0.25 J.lg and 0.5 J.lg) with two different concentrations of J.lg/ml of each enzyme for 60 min at 37°C except for samples lambda DNA (1.0 and 2.0 J.lg) at 37°C for 1 h in 100 J.llof reaction containing trypsin and a-chymotrypsin, which were incubated at mixture as described by Schaller and Nomura (19). Twenty-five 25°e. Prior to assaying for bacteriocin activity, preparations microliters from each reaction mixture was analyzed by electropho- containing pepsin were adjusted to pH 6.0 and those containing resis in 0.9% agarose gel; gels were stained with ethidium bromide trypsin and chymotrypsin were treated with trypsin-chymotrypsin and examined for degradation of DNA. The ability of some colicins inhibitor (Sigma) according to the manufacturer's instructions and to inactivate ribosomes of susceptible bacteria was determined by as described by Lyon and Glatz (13). Temperature stability was utilizing a cell-free poly-(U)-directed protein synthesis as de- determined by heating I-ml samples of purified colicin ECLl2 in scribed by Pugsley and Oudega (18). Appropriate controls were sealed glass test tubes at the times and temperatures listed in Table included in all experiments by utilizing purified colicin E3 (Sigma), 4. After heat treatment, samples were serially diluted in potassium colicin B, and colicin E2. phosphate buffer (pH 6.5) and assayed for remaining activity by the critical dilution method (14). N- Terminal amino acid sequencing and amino acid composition Purified colicin ECLl2 was transferred from polyacrylamide Downloaded from http://meridian.allenpress.com/jfp/article-pdf/60/12/1520/2301394/0362-028x-60_12_1520.pdf by guest on 01 October 2021 Sodium dodecyl sulfate-polyacrylamide gel electrophoresis gels onto modified PVDF membranes (Sigma) for N-terminal SDS-PAGE was carried out with 12% homogenous gels as sequencing analyses as described by the method of Ausubel (1). described by Laemmli (10). Partially purified colicin ECLl2 The colicin ECLl2 protein band was stained with SYPRO Orange preparations (20,500 AU/ml) were resuspended in 50 J.llof 50 mM fluorescent protein stain using the method described by the sodium phosphate buffer (pH 7.2), and 30-J.llsamples (0.2 J.lg) were manufacturer (Bio-Rad). The protein band was cut out of the analyzed in a 12% sodium dodecyl sulfate-polyacrylamide gel. membrane and sequenced using a automated sequencer (Perkin- Electrophoresis was conducted at a constant voltage (50 V) for 4.0 Elmer Corp., Applied Biosystem, Foster City, CA) at the Iowa State h at 15°e. The gel was stained with Coomassie brilliant blue R as University Protein and Nucleic Acid Facility. Derivatization and described by Merril et al. (16). Protein standards and their detection of phenylthiocarbamyl amino acids by reversed-phase individual MWs are as follows: phosphorylase b, 94,000; albumin, high-performance liquid chromatography was done at the Iowa 67,000; ovalbumin, 43,000; carbonic anhydrase, 30,000; trypsin State University Protein and Nucleic Acid Facility. inhibitor, 20,000; and a-lactalbumin, 14,400 (Pharmacia Biotech- nology, Piscataway, NJ). Colicin ECLl2 activity in sterile ground beef Beef patties (95% lean; 25 g; 1 by 5 cm) were sterilized by Protein determinations irradiation (40 kGy) at the Iowa State University Linear Accelator Protein was determined by the modified method of Lowry et Facility (LAF). The use of irradiated meat allowed us to directly al. (11), according to the specifications of the manufacturer select for the indicator strain and provide means to monitor E. coli (Sigma). Bovine serum albumin was used to construct a standard 0157:H7 populations in a model meat system. Beef slurries were curve. prepared as described by Degnan and Luchansky (5). One part sterile ground beef was combined with 1 part sterile distilled water Mode of action ofpurifed colicin ECLl2 and blended to homogeneity at high speed in a Waring blender for 3 The mode of action of purified colicin ECLl2 was determined min. For each assay 1 ml of whole, homogenized slurry was added by measuring the effect the bacteriocin has on susceptible cells. to a sterile tube. Five hundred microliters of partially purified De-energization of the cytoplasmic membrane of sensitive cells colicin ECLl2 (51,200 AU) and 104 cells of sensitive strain 3066 or was measured by the ability of the cells to accumulate radioactive the producer strain E. coli ECLl2 were aseptically added to each amino acids as described by Pugsley and Oudega (18). Deoxyribro- tube of slurry and mixed throughly. After addition of colicin, the activity was measured by incubating purified colicin meat slurries were incubated at both 4 and 100e. Periodically, samples were removed, serially diluted, and plated onto both MacConkey agar supplemented with sorbitol and Trypticase soy TABLE 4. Sensitivity of colicin ECLl2 to various enzymatic and agar and incubated at 37°C for 18 h. Random individual E. coli heat treatments colonies were selected and verified as coliform cells by API 20E Inhibitory activity" analysis (API Analytab Products) and by E. coli OI57:H7-specific Treatment (AU/ml) ELISA EHEC- TEK tests (Organon Teknika, Durham, NC).

Control (phosphate buffer) 51,200 RESULTS a-Chymotrypsin o Pronase E o Papain o Isolate identification Trypsin o Various phenotypic traits of the producer strain were Lipase 51,200 evaluated. On the basis of biochemical and morphological Lysozyme 51,200 characteristics, the bacteriocin-producing isolate was identi- Phospholipase C 51,200 fied as Escherichia coli as described by the procedures of Control (no heat) 51,200 Brenner (3) and further vertified by API 20E analysis (API 75°C for 60 min 51,200 Ana1ytab Products). 80°C for 30 min 16,384 85°C for 15 min 4,096 Inhibitory activity against other microorganisms 90°C for 15 min o Purified colicin ECL12 was tested by the critical a Colicin activity was measured by the critical dilution method and dilution method to evaluate its inhibitory activity against all measurements are the mean of triplicate tests. various microorganisms (Table 1 and 2). Of the gram- 1524 LYON AND OLSON

negative strains tested, 2 strains of Shigella sonnei, 17 ,-... strains of E. coli 0157:H7, and 2 strains of other E. coli -.-= serotypes were sensitive to this purified colicin, but tested 0 ,-... r.. 6 1250 strains of Aeromonas hydrophila, Campylobacter jejuni, U S '-'...• -.0 Escherichia hermanii, Escherichiafergusonii, Shigella dys- < enteriae, Vibrio parahaemolyticus, Yersinia enterocolitica, = 5 1000 '-' =0 (.,l ...••... two species of Pseudomonas, and four species of Salmonella .;: were not inhibited. None the tested gram-positive strains ~- 4 750 ...... • were inhibited (Table 2). (.,l (.,l

FIGURE 2. Adsorption of purified colicin ECLl2 (1,064 AU/ml) by A sensitive strain E. coli 0157:H7 (2.8 X 1()5 CFU/ml) and producer 0.25 strain ECLl2 (3.2 X 105 CFU/ml) cells in 50 mM potassium ---- phosphate buffer (pH 6.5) for 120 min at 37°C. Points are the ---- 'f? 20000 8 0.2 ~ average of triplicate plates not differing by more than ±10 >= ~ CFU/ml. Symbols: _, surviving CFU/ml for producer strain E. 0 '-' 'i" 15000 ;>-. coli ECLl2; D, surviving CFU/ml for indicator strain E. coli ('.l 0.15 .•.... '-' 0157:H7; e, residual colicin ECLl2 activity after incubation with a) :9 :~.•.... u u the producer strain ECLl2; 0, residual colicin ECLl2 activity ~>= 0.1 0\ 10000 ~ 13 ('.l after incubation with the indicator strain E. coli 0157:H7. 0 ell -....l 0.05 9 5000 U ~ i ~ i:.Ll 0 >= 0 0 :9 demonstrated that the immunity protein could be separated 0 10 20 30 40 50 60 "0 U from the complexed E3 with 6 M guanidine-HCl (12, 18). Volume (ml) We used this observation in our purification scheme by dialyzing against a dissociation buffer containing with 6 M B guanidine-HCI. The preparation was assayed for activity by ,-... the critical dilution method (13) and was found to have a S 0.075 20000 ~ twofold increase in activity when compared to partially >= 0 S purified colicin preparations that were not treated with 'i" ;>-. ('.l 15000 .•.... '-' 0.05 guanidine-HCl (data not shown). Guanidine-HCl-treated <1.l :~.•.... u u partially purified colicin (51,200 AU/ml; 50 mg protein per ~>= 10000 ~ ..0 ('.l ml) obtained by cell extraction was applied to a descending l-< 0 0.025 CM sepharose ion-exchange column and eluted with a salt ell ....l- ..0 5000 U ~ i:.Ll gradient (Fig. 1). Proteins were eluted in two peaks using an >= NaCl gradient (0 to 0.5 M) in 20 mM MES buffer (pH 6.5). 0 0 :9 0 10 20 30 40 "0 The first protein peak was eluted with buffer containing no u NaCl, the second peak was eluted with buffer approximately Volume (ml) 0.2 M in NaCl (data not shown). All five fractions within the FIGURE 1. (A) Elution of partially purified colicin ECLl2from a second peak contained colicin activity (ranging from 2,560 CM sepharose ion-exchange column with 20 mM MES (pH 6.5). AU/ml to 20,500 AU/ml; 80 to 120 J.lgprotein per ml). All Colicin ECLl2 was eluted with a linear gradient of 0 to 0.5 M NaCl fractions containing activity were combined, dialyzed, and in 20 mM MES (pH 6.5). Fractions (1 ml) were collected, and concentrated to 2.0 ml in an Amicon stirred cell as described colicin activity for each fraction was determined by the critical above and applied to a descending Bio-Gel P30 column. dilution method. (B) Pooled colicin fractions were dialyzed, Bacteriocin activity was eluted from the column in one peak. concentrated, and reapplied to a CM sepharose ion-exchange column and eluted with 20 mM MES (pH 6.5) containing 0.1 M The mean elution volume of the bacteriocin was compared NaCl. Fractions (1 ml) were collected, and colicin activity for each to those of standards and the MW of the bacteriocin was fraction was determined by the critical dilution method. Symbols: determined to be approximately 66,000. Colicin samples D absorbance at 240 nm; °colicin ECLl2 activity (A U/ml). were analyzed for purity by SDS·PAGE. COLICIN ECL12 1525

TABLE 5. Comparison of the N-terminal sequence, molecular weight, and mode of action of colicin ECL12 with other colicins of similar molecular weight

N-Tenninal amino PurifiedMW Mode of Colicin acid sequence of colicina actionb

ECLl2 MVAYYKGAP 65,000 DNA nuclease El MVLIVALYY 57,279 Membrane depolar- ization E2 MSGGDGRGH 61,561 DNA nuclease E3 MVAYYKLAP 60,000 Ribosomal inacti- vation DF13 MSGGDGRGPG 59,283 Ribosomal inacti- vation Downloaded from http://meridian.allenpress.com/jfp/article-pdf/60/12/1520/2301394/0362-028x-60_12_1520.pdf by guest on 01 October 2021 A VPMTWGGMKG 62,989 Membrane depolarc ization K MIGGFVGILV 69,000 Membrane depolar- ization

a Molecular weights were determined by either SDS-PAGE or from the sequences (4, 7,9, 11, 21). FIGURE 3. Coomassie blue-stained SDS-PAGE gel of purified b Mode of action of various colicins are described elsewhere (9,11, colicin ECL12 obtained from a CM sepharose ion-exchange 18). column. Lanes: lane A, purified colicin ECL12 (0.4 /-Lg);lane B, molecular weight standards (1.0 /-LI;1.0 /-Lg/d]ml of each protein; Temperature sensitivity was assessed by heating par- top to bottom: ovalbumin, MW 43,000; carbonic anhydrase, MW tially purified colicin ECL12 preparations at various tempera- 29,000; 13-lactoglobulin, MW 18,400; lysozyme, MW 14,300; tures and evaluating residual activity. Inhibitory activity was bovine trypsin inhibitor, MW 6,200; a-insulin, MW 3,400; and unaffected by heating at 75°C for 60 min and some loss of (3-insulin, MW 2,300). activity was noted after 30 min at 80°C (Table 4).

Effect of colicin ECL12 on sensitive and nonsensitive cells SDS-PAGE Samples (500 ~) of purified colicin ECL12 (1,024 Samples (0.4 f.l.g)of colicin ECL12 preparations isolated AU/ml) were mixed with 500 III of cell suspensions of by the cell extraction method and purified by ion exchange indicator strain E. coli 0157:H7 isolate 3066 and producer were analyzed by SDS-PAGE in denaturing 12% polyacryl- strain ECL12 (2.8 X 105 CFU/ml, and 3.2 X 105 CFU/ml, amide gels (Fig. 3). The Coomassie blue-stained SDS-PAGE respectively). After incubation for 30 and 60 min, the cells were pelleted by centrifugation and enumerated for viable TABLE 6. Amino acid compositiona of colicin ECL12 counts. Residual inhibitory activity of the supernatant was Amino acids measured by the critical dilution method. Viable counts of Amino acids determined indicator strain E. coli 0157:H7 isolate 3066 were reduced by 99.9% after exposure to colicin ECL12, but viable counts Asparagine 84 of producer strain ECL12 were unchanged (Fig. 2). Incuba- Threonine 22 tion times longer than 30 min significantly (P < 0.05) Serine 41 reduced the number of viable microorganisms detected from Glutamine 52 Proline 31 1.5 X 103 CFU/ml at 30 min to 1.3 X 101 at 60 min. Residual Glycine 64 inhibitory activity was detected in the supernatant of prepa- Alanine 60 rations mixed with E. coli 0157:H7 isolate 3066 and E. coli Cysteine o ECL12 cells (Fig. 2). Similar results were obtained with Valine 40 several E. coli 0157:H7 isolates (data not shown). These results Methionine 7 suggest that colicin ECL12 adsorbs to the indicator strain 3066 Isoleucine 19 but not to the producer strain ECL12. The absorption of colicin Leucine 24 CL12 is specific, and the kinetics of death of indicator cells Tryosine 7 treated with colicin ECL12 is similar to that observed for Phenylalanine 17 colicins (18). Application of buffer without colicin ECL12 Tryptophan 10 did not affect the growth of the indicator strain. Hisidine 11 Lysine 45 Arginine 31 Effects of enzymes and heat on colicin ECL12 Samples of partially purified colicin ECL12 were found Total 544 to be sensitive to protease, pronase E, pepsin, trypsin, and a The data are given as the average of two separate colicin ECL12 a-chymotrypsin but were not affected by phospholipase C preparations with a sample difference no greater than ::!::0.5for all and lipase (Table 4). amino acids detected. 1526 LYON AND OLSON gel revealed a single protein band with a molecular weight of approximately 65,000. 5 Mode of action Purified colicin ECL12 was incubated with lambda ·······0·······0·······0·······0·..····0·······0· ..····0·······0 DNA. Figure 4 shows the results of an ethidium bromide- .•..•tI.l 4 stained 0.9% agarose gel. We found that 0.25 flg of purified ::: ECL12 when incubated with either 1.0 or 2.0 flg of lambda ='o C.l DNA degrades the DNA, and it is clear that this conversion a.l is almost complete after 1 h of incubation at 37°C. No rRNA 3 ,.Q degradation or depolarization was noted in .•..•-~ separate experiments (data not shown). •• :::

....• Downloaded from http://meridian.allenpress.com/jfp/article-pdf/60/12/1520/2301394/0362-028x-60_12_1520.pdf by guest on 01 October 2021 N-Terminal sequencing and amino acid composition 2 The colicin ECL12 band was transferred onto ProBlot, cut out, and subjected to N-terminal sequence analysis. The 1 1 2 3 o 25 50 75 100 125

Time (h)

FIGURE 5. Inhibition ofE. coli OI57:H7 in beef slurries. Points are the average of triplicate plates not differing by more than ± 10 CFU/ml. Symbols: 0, surviving CFUlml for producer strain E. coli ECLl2; e, surviving CFU/ml for indicator strain E. coli OI57:H7 at 4°C; D, surviving CFU/ml for indicator strain E. coli OI57:H7 at IO°e.

sequence of the first nine amino acids of colicin ECL12 was determined, and the results of these analyses are shown in Table 5. The amino acid composition was determined from two different preparations of purified colicin ECLI2, and the average amino acid composition is shown in Table 6. The protein consists of 544 residues and contains a large number of charged residues (arginine, lysine, asparagine, glutamine) (Table 6), which accounts for its high polarity. The molecu- lar weight of colicin ECLl2 (60,415) is in reasonable agreement with the molecular weight determined by SDS- PAGE (65,000; Table 5).

Activity of colicin ECL12 in beef slurries The inhibitory activity of partially purified colicin ECL12 (ca. 51,200 AU per slurry sample) against a strain of E. coli 0157:H7 was evaluated in sterile beef slurries (Fig. 5). Colicin ECL12 activity decreased in all tested slurries with significant loss (ca. 40 to 50% decrease) occurring within 30 min after addition of the colicin (data not shown). After the addition of colicin ECL12 to the beef slurries, E. coli 0157:H7 viable cell counts were reduced by 2.3 log FIGURE 4. Degradation of lambda DNA by purified colicin ECLl2 within 1 h when incubated at both 4 and lo°C. E. coli in vitro. Lambda DNA was incubated with purified colicin ECLl2 0157:H7 viable counts stayed at that level for 124 h at both in a total volume of 100 ILlfor 1 hat 37"C as described by Pugsley 4 and lo°C (Fig. 5). and Oudega (18). Twenty-five microliters from each reaction mixture was analyzed by 0.9% agarose gel electrophoresis. Lanes: DISCUSSION lane 1, 0.25 ILg of lambda DNA without purified colicin ECLl2; lane 2, 2.0 ILg of lambda DNA incubated with 0.25 ILg of colicin ECL12 (protein concentration determined by the Lowry method); Escherichia species are known to produce colicins that lane 3, 1.0 ILg of lambda DNA incubated with 0.25 ILg of colicin are inhibitory against closely related species (12, 18). A ECLl2. bovine sewage isolate, identified as Escherichia coli ECLI2, COLICIN ECLl2 1527 was classified on the basis of both phentotypic and morpho- other isolated from other gram-negative organ- logical data. The colicin produced by strain ECL12 was isms, which have been shown to bind specifically to active against other closely related gram-negative organisms sensitive cells (18). (especially E. coli 0157:H7), but none of the tested gram- Preliminary data presented in this paper indicate that positive organisms. bacteriocins in combination with other food additives could Colicin ECL12 (MW 65,000) was purified from contami- provide an effective method for controlling E. coli 0157:H7 nating protein by applying partially purified colicin to a in both fresh and processed meats, thus assuring a safer descending CM sepharose ion-exchange column. Evidence product for consumption that has an increased level of food for homogeneity of colicin ECL12 was provided by the safety. Additional genetic characterization and subsequent absence of the contaminating protein on SDS-PAGE. The cloning of colicin gene(s) into plasmids that are expressed molecular weight of colicin ECL12 (Table 5) is similar to by food-grade bacteria (e.g., lactic acid bacteria) is required that of colicins A (62,989), K (69,000), El (57,279), E2 for safe utilization of this bacteriocin in foods.

(61,561), E3 (58,000), and DF13 (59,283) (12, 18). How- Downloaded from http://meridian.allenpress.com/jfp/article-pdf/60/12/1520/2301394/0362-028x-60_12_1520.pdf by guest on 01 October 2021 ever, these colicins are considerably different from colicin ACKNOWLEDGMENTS ECL12 in regard to their antimicrobial spectra, especially their ability to inhibit E. coli 0157:H7, mode of action, This research was funded by the Food Safety Consortium at Iowa N-terminal sequence, and receptor usage (Table 5) (2, 20). State University. The authors would like to thank William C. Cray (National The outer membrane proteins of E. coli are known to Animal Disease Center; Agriculture Research Service; U. S. Department of function as receptors for many bacteriophages and colicins. Agriculture; Ames, IA) for providing E. coli 0157:H7 strains. Bradley and Howard (2) showed that rough strains of E. coli K-12, those strains lacking the O-antigen portion of the REFERENCES lipopolysaccharide (LPS) moiety, are more susceptible to 1. Ausubel, F. M. 1989. Media preparation and bacteriological tools, p. phage and colicin absorption (2, 20). Bradley and Howard 3. In F. M. Ausubel, R. Brent, R. E. Kingston, D. D. Moore, J. G. (2) concluded that the 0 antigen in most cases blocks the Seidman, J. A. Smith, and K. Struhl (ed.), Short protocols in colicin receptor thereby preventing effective binding of molecular biology: a compendium of methods from Current Protocols colicin onto cell receptors of E. coli 0157:H7 cells. Bradley in Molecular Biology. John Wiley & Sons, New York. and Howard showed that colicin E2 was relatively ineffec- 2. Bradley, D. E., and S. P. Howard. 1990. Colicinogeny of 0157:H7 enterohemorrhagic Escherichia coli and the shielding of colicin and tive in inhibiting E. coli 0l57:H7 with only 30% of the phage receptors by their O-antigenic side chains. Can. J. Microbiol. strains inhibited, while colicins V, G, and H inhibited nearly 37:97-104. 100% of the tested strains (2). The reported MW for colicin 3. Brenner, D. J. 1984. Gram-negative facultatively anaerobic rod, p. 408-423. In N. R. Krieg, and J. G. Holt (ed.), Bergey's manual of V is 5,800 (12) and the MWs of colicins G and H are systematic bacteriology. Williams & Wilkins, Baltimore, MD. unknown (7,12,18). 4. Cole, S. T., B. Saint-Joanis, and P. Pugsley. 1985. Molecular The MW of purified colicin ECL12 was further verified characterization of the colicin E2 operon and identification of its by applying purified colicin obtained by ion exchange to a products. Mol. Gen. Genet. 148:465-472. 5. Degnan, A., and J. B. Luchansky. 1992. Influence of beef tallow and gel filtration column. A single peak was eluted that contained muscle on the antilisterial activity of pediocin AcH and liposome- colicin activity, and the MW of the substance was deter- encapsulated pediocin AcH. J. Food Prot. 55(7):552-554. mined to be approximately 65,000 by comparison of its 6. Doyle, M. P. 1991. Escherichia coli 0157:H7 and its significance in elution volume to that of MW standards. Inconsistencies in foods. Int. J. Food Microbiol. 12:289-302. 7. Fath, M. J., L. H. Zhang, J. Rush, and R. Kolter. Purification and the MW determined by gel filtration and SDS-PAGE can be characterization of colicins V from Escherichia coli culture superna- reconciled by the observation that bacteriocins readily tants. Biochemistry 33:6911-6917. associate with nonbacteriocin components which are then 8. Griffin, P. M., and R. V. Tauxe. 1991. The epidemiology of infections coeluted under the nondenaturing conditions used for gel caused by Escherichia coli 0157:H7, other enterohemorrhagic E. coli, and the associated hemolytic uremic syndrome. Epidemiol. Rev. filtrations (14, 15). Although, colicin ECL12 and other 13:60--98. colicins have similar MWs, it is clear they have distinct 9. Herschman, H. R., and D. R. Helinski. 1967. Purification and biochemical and physical characteristics. Experimental re- characterization of colicin E2 and colicin E3. J. BioI. Chern. sults described in this paper demonstrate that purified colicin 242:5360-5368. ECL12 has DNA endonuclease activity. Colicin E2 also has 10. Laemmli, U. K. 1970. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature (London) 227:680- deoxyribonuclease activity (Table 5) (4, 19); however, its 685. spectra of activity and molecular weight (Table 5) are 11. Lowry, O. H., N. J. Rosebrough, A. L. Farr, and R. J. Randall. 1951. different from colicin ECL12. We are in the process of Protein measurement with Folin phenol reagent. J. BioI. Chern. cloning the gene(s) that encode colicin ECLl2. The se- 193:265-275. 12. Luria, S. E., and J. L. Suit. 1987. Colicins and Col plasmids, p. quence of this gene will allow us to clearly differentiate it 1615-1624. In F. C. Neidhardt, J. L. Ingraham, K. Brooks Low, B. from other colicins that have similar activity spectra and Magasanik, M. Schaechter, and H. E. Umbarger (ed.), Escherichia molecular weights. coli and Salmonella typhimurium: cellular and molecular biology. American Society for Microbiology, Washington D.C. Purified colicin ECL12 added to sensitive cells of strain 13. Lyon, W. J., and B. A. Glatz. 1991. Partial purification and character- 3066 was bactericidal; however, the colicin did not inhibit ization of a bacteriocin produced by Propionibacterium thoenii. Appl. the producer strain ECL12. Colicin ECL12 was similar to Environ. Microbiol. 57:701-706. 1528 LYON AND OLSON

14. Lyon, W. J., and B. A. Glatz. 1993. Isolation and purification of and their plasmids, p. 105-161. In K. G. Hardy (ed.), Plasmids: a propionicin PLG-l, a bacteriocin produced by a strain of Propionibac- practical approach. IRL Press, Washington, D.C. terium thoenii. Appl. Environ. Microbiol. 59:83-88. 19. Schaller, K., and M. Nomura. 1976. Colicin E2 a DNA endonuclease. 15. Lyon, W. J., D. G. Olson, and E. A. Murano. 1995. Isolation and Proc. Nail. Acad. Sci. USA 73:3989-3993. purification of enterocin ELl, a bacteriocin produced by a strain of 20. Van der Ley, P. P. De Graaff, and J. Tommassen. 1986. Shielding of Enterococcusfaecium. J. Food Prot. 58(8):890--898. Escherichia coli outer membrane proteins as receptors for bacte- 16. Merril, C. R., D. Goldman, S. A. Sedman, and M. H. Ebert. 1981. riophages and colicins by O-antigenic chains of lipopolysaccharide. J. Ultrasensitive stain for proteins in polyacrylamide gels shows re- Bacterio1. 168:449-451. gional variation in cerebrospinal fluid proteins. Science 211:1437- 1438. 21. Watson, D. H. 1980. Common inner membrane-specific domains of 17. Padhye, N. v., and M. P. Doyle. 1992. Escherichia coli 0157:H7: colicins El, K, Ia and A. Biochim. Biophys. Acta 622:287-296. epidemiology, pathogenesis, and methods for detection in food. J. 22. Whipp, S. c., M. A. Rasmussen, and W. C. Cray. 1994. Animals as a Food Prot. 55(7):555-565. sources of Escherichia coli pathogenic for human beings. J. Am. Vet. 18. Pugsley, A. P., and B. Oudega. 1987. Methods for studying calicins Med.Assoc.285:1168-1175. Downloaded from http://meridian.allenpress.com/jfp/article-pdf/60/12/1520/2301394/0362-028x-60_12_1520.pdf by guest on 01 October 2021