Characterization and Purification of Helveticin J and Evidence for aChromosomally Determined Bacteriocin Produced by Lactobacillus
helveticus 481tMELISSA C. JOERGER AND TODD R. KLAENHAMMER*
Department ofFood Science, North Carolina State University, Raleigh, North Carolina 27695-7624
Received 3 February 1986/Accepted 25 April 1986
Lactobacillus helveticus 481 produced an antimicrobial agent active against five closely related species. Thesensitive indicators included L. helveticus 1846 and 1244, L. bulgaricus 1373 and 1489, and L. lactis 970. Theantimicrobial compound was active at neutral pH under aerobic or anaerobic conditions, was sensitive toproteolytic enzymes and heat (30 min at 100°C), and demonstrated a bactericidal mode of action againstsensitive indicators. These data confirmed that antimicrobial activity of L. helveticus 481 was mediated by abacteriocin, designated helveticin J. Production of helveticin J was maximized in an anaerobic fermentor heldlit a constant pH'of 5.5. Ultrafiltration experiments on cWuture supernatants containing the bacteriocin revealedthat helveticin J was present as an aggregate with a molecular weight in excess of 300,000. Sodium dodecylsulfate-polyacrylamide gel electrophoresis of helveticin J purified through Sephadex chromatography resolveda 37,000-dalton p,rotein band with bacteriocin activity. L. helveticus 481 was shown to harbor a single8-megadalton plasmid (pMJ1008). Isolates cured of pMJ1008 were phenotypically identical to plasmid-bearingcells in fermentation patterns, helveticin J activity, and unmunity spectra. The data provided evidence for achromosomal location of helveticin J and host immunity determinants.
Lactobacillus helveticus and other lactobacilli have beenshown to inhibit only closely related species (3, 5,10, 25). Anarrow inhibitory spectrum, proteinaceous nature, and bac-tericidal mode of action are typical characteristics of antimi-crobial agents classified as *bacteriocins (23). Very fewlactobacifli have been confirmed to produce true bacte-riocins within these criteria (3, 4, 6, 25, 26). Only a singlebacteriocin has been identified and characterized within theL. helveticus species. Upreti and Hinsdill (25, 26) confirmedthe production of a bacteriogcin, designated lactocin LP27, byL. helveticus LP27. Lactocin LP27 exhibited a narrowactivity spectrum by inhibiting strains of L. acidophilus andL. helveticus. A proteinaceous nature was demonstrated byinactivation of the bacteriocin with trypsin and pronase. Incontrast to a bactericidal mode of action typical of bacte-riocins, lactocin LP27 had a bacteriostatic effect on theindicator, L. helveticus LS18. Protein synthesis was termi-nated by lactocin LP27, but DNA and RNA synthesis andATP levels were not affected.
Bacteriocin production and resistance phenotypes offerexcellent markers for study of genetic linkages and transfersystems. However, there is little information available onbacteriocins, genetic linkages, and gene transfer systems inLactobacillus species. Gram-negative and gram-positivebacteria commonly harbor plasmid-borne genetic determi-nants of bacteriocin production and of host cell bacteriocinimmunity (17, 19, 20, 23, 27). However, all attempts to locateplasmid determinants responsible for bacteriocin productionand host immunity in lactobacilli have been unsuccessful.Difficulties in curing lactobacilli of bacteriocin phenotypesand plasmids have been reported (3, 26) and suggest thatchromosomal determinants may be involved.
* Corresponding author.t Paper 10300 of the Journal Series of the North Carolina Agri-
cultural Research Service.
In the present report, a unique bacteriocin produced by L.helveticus 481 was isolated and characterized. The bacte-riocin, designated helveticin J, has a narrow inhibitoryspectrum, proteinaceous nature, and bactericidal mode ofaction. It was demonstrated that genetic determinants ofhelveticin J production and of host cell bacteriocin immunityare not plasmid borne.
MATERIALS AND METHOD$Bacterial cultures and media. All Lactobacillus cultures
were maintained as frozen stocks at -20°C in MRS broth(Difco Laboratories, Detroit, Mich.) plus 10% glycerol andpropagated as described previously (3). MRS tubes wereheld in a boiling water bath for 5 min to drive off excess 02and then cooled before inoculation (1%) with L. helveticus481. Agar medium was prepared by adding 1.5% granulatedagar to MRS broth. Overlay agar was prepared with 0.75%agar.
Bacteriocin assays. L. helveticus 481 was examined forbacteriocin production by direct (3, 23) and deferred (3, 9)methods. An adaptation of the critical dilution assay (16) wasused for titration of helveticin J activity. Serial twofolddilutions of bacteriocin were spotted (10 ,ul) onto freshindicator lawns of L. bulgaricus 1489, and the plates wereincubated for 16 to 18 h at 37°C under anaerobic conditions.The titer was defined as the reciprocal of the highest dilutionexhibiting complete inhibition of the indicator lawn and wasexpressed in activity units (AU) per milliliter.
Bactericidal action. Culture extract containing helveticin Jwas dialyzed against 0.1 M sodium acetate buffer (pH 5.3),filter sterilized, and diluted to obtain 0, 3.2, and 160 AU/ml.A 10-ml sample of each preparation was added to a separatesterile cuvette. Log-phase L. bulgaricus 1489 indicator cells,washed with sterile 0.1 M sodium acetate buffer (pH 5.3),were added to each cuvette to yield 107 CFU/ml. Opticaldensity at 590 nm and CFU per milliliter were determined
immediately after the indicator cells were added and after 3h of incubation at 37°C.
Production studies at controlled pH. Sterile MRS broth (1liter) was aseptically transferred to a sterile Multigen bench-top fermentor connected to an automatic pH controller (NewBrunswick Scientific Co., Inc., Edison, N.J.). The pH of thebroth was adjusted initially to pH 5.5, 6.0, or 7.0 andmaintained during fermentation with 12% ammonium hy-droxide. Cells from an overnight culture of L. helveticus 481grown in MRS broth (pH 6.0) were centrifuged, washed, andresuspended at a 5 x concentration in fresh MRS broth. Thiscell preparation (1 ml) was used for inoculation of thefermentor culture. Throughout the fermentation, the vesselwas held at 37°C, purged with a mixture of 5% C02, 10% H2,and 85% N2 and agitated continuously. Over a 24-h period,10-ml samples were aseptically removed to determine opticaldensity at 590 nm and AU of helveticin J per ml. Samplesassayed for helveticin J activity were filter sterilized.
Ultrafiltration and dissociation of CHJ. To determine thesize of crude helveticin J (CHJ) fermentation supernatant(250 ml) was subjected to ultrafiltration through a series ofDiaflo membranes (Amicon Corp., Lexington, Mass.).Diaflo membranes employed included XM300, XM100A,XM50A, and YM10. Filtrate from the preceding larger-poremolecular exclusion filter was subjected to ultrafiltrationthrough the next smaller-pore molecular exclusion filter andso on. Volumes of retentates and filtrates were recorded.Titers of retentates and filtrates were then determined forhelveticin J activity.For dissociation experiments, bacteriocin extract was
repeatedly washed with 0.1 M sodium acetate buffer (pH 5.3)as the extract was forced through an XM300 Diaflo mem-brane to remove low-molecular-weight helveticin J mole-cules. The ultrafiltration was continued until no helveticin Jwas detected in the filtrate. Helveticin J with a molecularweight of >300,000 remained in the retentate. The retentatewas designated high-molecular-weight helveticin J (largehelveticin J [LHJ]).LHJ was treated with 1% sodium dodecyl sulfate (SDS),
0.2% P-mercaptoethanol and 1% SDS (100°C, 5 min), 8 Murea, 1 mM dithiothreitol, 0.1% Triton X-100, and 6 Mguanidine-hydrochloride. A control of LHJ and sodiumacetate buffer was used. The control and the treated LHJwere passed through XM300 Diaflo membranes. Titers ofretentate and filtrate were taken to determine whetherhelveticin J had dissociated into lower-molecular-weightunits. To detect possible inhibition of the indicator lawn,titers were determined for controls consisting of dissociatingagents suspended in 0.1 M sodium acetate buffer (pH 5.3) atthe same concentrations as used to treat LHJ.
Purification of helveticin J. L. helveticus 481 was inocu-lated (0.1%) into a Microferm fermentor (New BrunswickScientific) containing 20 liters of a semidefined medium (4)modified for the following components: Casitone (DifcoLaboratories, Detroit, Mich.), 5 g/liter; yeast extract (BBLMicrobiology Systems, Cockeysville, Md.), 2.5 g/liter;MnC12, 1.2 mg/liter; xanthine, 5 mg/liter; adenine sulfate, 5mg/liter; guanine-hydrochloride, 5 mg/liter; uracil, 5 mg/liter;calcium panthothenate, 1 mg/liter; nicotinic acid, 1 mg/liter;and biotin, 0.01 mg/liter. The fermentor was maintained at37°C and pH 5.5 for 22 h. Helveticin J crude extract washarvested as previously described (4).
(i) Ammnonium sulfate precipitation. The crude extract wasadjusted to pH 3, and 50% (NH4)2SO4 was added. Afterfiltration through cheesecloth, the extract was centrifuged at20,200 x g. The pellet was suspended in 42 ml of 0.1 M
sodium acetate buffer (pH 5.3) and dialyzed against the samebuffer. The dialyzed sample was centrifuged at 27,000 x gfor 30 min, and the pellet was resuspended in 30 ml ofsodium acetate buffer. SDS (6% wt/vol) was added to thesuspension to solubilize helveticin J. The supernatant con-taining helveticin J activity was collected after 10 min ofcentrifugation at 12,100 x g.
(ii) Gel chromatography. Helveticin J concentrate wasapplied to a column containing Sephadex G-200-120 (SigmaChemical Co., St. Louis, Mo.) and maintained at 30°C witha thermocirculator. The column was packed and equilibratedwith 0.1 M sodium acetate (pH 5.3) containing 0.1% SDS and0.02% sodium azide. Activity was eluted with the samebuffer, and the eluent was monitored for A280 and helveticinJ activity. Active fractions were pooled, concentrated, andthen applied to a Sephadex G-200-50 (Sigma) column. Chro-matography proceeded as described above. After this pro-cedure, partially purified helveticin J was obtained.
(iii) PAGE. Discontinuous polyacrylamide gel electropho-resis (disc-PAGE) in the presence of 0.1% SDS was carriedout as described by Laemmli (13). Polyacrylamide concen-trations in the stacking gel and separating gel were 4.75 and10%, respectively. Disc-PAGE was conducted at a constantcurrent of 1 mA per gel for 16 h. Gels were stained withCoomassie brilliant blue-R (Sigma). Protein standards andtheir molecular weights included the following: phosphory-lase b, 94,000; albumin, 67,000; ovalbumin, 43,000; carbonicanhydrase, 30,000; trypsin inhibitor, 20,100; and oa-lactalbumin, 14,400 (Pharmacia Fine Chemicals, Pisca-taway, N.J.). Unstained gels were sliced into 2-mm sections.Each section was placed into a tube containing 0.5 ml of 0.1M sodium acetate buffer (pH 5.3). Slices were crushed andheld at 4°C overnight to allow elution of helveticin J activityinto the buffer. Buffer (0.3 ml) from each fraction wasremoved, and titers were determined for activity. Gradient(10 to 20%) PAGE in the presence of 0.1% SDS wasperformed with precast slab gels (Integrated SeparationSystems, Newton, Mass.). PAGE was conducted at a con-stant current of 8 mA per gel for 18 h. Staining proceduresand protein standards were employed as described above.
Sensitivity to heat and proteolytic enzymes. CHJ (1 ml;1,600 AU/ml) was heated in a boiling water bath. Sampleswere removed at 0, 30, and 60 min, and titers were deter-mined to detect activity. Helveticin J obtained after gelchromatography was dialyzed against saline (pH 7) andadded to buffer-enzyme suspensions to yield a final concen-tration of 25,600 to 51,200 AU/ml. The following enzymes (1mg/ml) and respective buffers were employed: ficin (EC3.4.22.3), 0.02 M cysteine-hydrochloride, 0.01 M disodiumEDTA, 0.15 M NaCI (pH 7.0); pronase (type VI; Sigma), 0.1M sodium borate, 0.05 M HCl, 5 mM CaC12, 1 mM CoC12(pH 7.5); trypsin (EC 3.4.21.4), 0.04 M Tris-hydrochloride,0.01 M CaCl2 (pH 8.1); pepsin (EC 3.4.4.1), 0.02 N HCl;proteinase K (type XI; Sigma), 0.1 M sodium acetate, 0.005M calcium acetate (pH 7.5); subtilisin (type VIII; Sigma), 0.1M potassium phosphate (pH 7.0); lysozyme (grade 1;Sigma), 0.066 M potassium phosphate (pH 6.24); and lipase(EC 3.1.1.3), 0.1 M potassium phosphate (pH 6.0). Controlsincluded buffers; buffers and respective enzymes; buffersand respective heat-inactivated enzymes; and buffers andhelveticin J. Buffer-enzyme-helveticin J reaction mixturesand all controls were incubated for 1 h at 37°C, and titerswere determined for helveticin J activity. Pepsin reactionswere neutralized to pH 6.0 before titration.
Plasmid analysis. Large-scale plasmid isolations followedthe method of Anderson and McKay (1) as modified by
Steenson and Klaenhammer (22). L. helveticus 481 cells forplasmid DNA isolation were prepared in 600 ml of MRSbroth (4.5% inoculum) and incubated at 37°C for 4 h. Duringthe alkaline step of the lysis protocol, NaOH (3 N) wasadded until a pH of 12.0 to 12.2 was obtained. Plasmid DNAwas purified through cesium chloride-ethidium bromide den-sity gradients as previously described (12). Agarose gelelectrophoresis was conducted as described previously (11).
Hybridization. Total DNA preparations were obtainedfrom wild-type L. htlveticus 481 and presumptive pMJ1008-cured isolates. MRS broth (1 liter) was inoculated (0.1%) andincubated at 37°C for 24 h. The lysis procedure of Klaenham-mer et al. (12) was used except that a final concentration of20 mg of lysozyme per ml was employed, and 5 M sodiumperchlorate was added in place of 1 M NaCl to a finalconcentration of 1 M. The ethanol-precipitated DNA wasdissolved in dilute 0.1x SSC (NaCl, 0.877 g/liter; sodiumcitrate, 0.441 g/liter; pH 7.0) (lx SSC is 0.15 M NaCl plus0.015 M sodium citrate). A 1/10 volume of 1Ox SSC was thenadded. Heat-treated (100°C, 10 min) RNase (Sigma; 0.5 mg)was added to the DNA preparation, followed by incubationat 37°C for 30 min. Pronase (Sigma; 0.001 g) was added, andincubation at 37°C continued for another 45 min. Phenol andchloroform extractions were performed, and the DNA wasprecipitated with ethanol. The Southern blot technique (21)was used to transfer EcoRI (Bethesda Research Laborato-ries, Gaithersburg, Md.)-digested chromosomal and plasmidDNAs from agarose gels to nitrocellulose filters (BA85,0.45-,um pores; Schleicher & Schuell, Inc., Keene, N.H.).pMJ1008 was cut with EcoRI and labeled with [32P]CTP by amodification of the "random primer" method (8). EcoRIdigestions were performed according to the instructions ofthe manufacturer. Hybridization reactions and autoradiogra-phy procedures were carried out as described previously (8),except that hybridizations were performed at 65°C.
RESULTS
Inhibitory spectrum. L. helveticus 481 exhibited bacte-riocin activity against closely related species and one otherL. helveticus strain. Sensitive strains of the species includedL. bulgaricus 1373 and 1489, L. lactis 970, and L. helveticus1244 and 1846. Other strains of L. helveticus (13), L.bulgaricus (7), L. lactis (5), and all strains of L. acidophilus(21), L. plantarum (18), L. fermentum (1), L. casei (4), andL. leichmannii (1) tested were insensitive to CHJ. Theproducer culture, L. helveticus 481, was also insensitive toCHJ. Antimicrobial activity of CHJ against L. bulgaricus1489 was demonstrated (Fig. 1) by the two assay methods.CHJ activity was confirmed via a well diffusion assay (24) byusing a pH-neutralized, catalase-treated extract to eliminatethe possibility of either hydrogen peroxide or lactic acidinhibition (Fig. 1). CHJ activity was nondialyzable against0.1 M sodium acetate buffer (pH 5.3) in membrane tubingwith molecular exclusion limits of 12,000 to 14,000 daltons.These observations substantiated that the antimicrobialcompound was not a low-molecular-weight substance, suchas hydrogen peroxide or lactic acid, and suggested involve-ment of a bacteriocin.Mode of action. To determine whether helveticin J had a
bactericidal or bacteriolytic mode of action, viability andlysis of L. bulgaricus 1489 were monitored in the presence ofhelveticin J. The addition of increasing amounts of helveticinJ resulted in proportional increases in cell death (data notshown). Helveticin J added at 3.2 AU/ml resulted in a 3-logreduction of the viable cell population after 3 h. The highest
Bf
FIG. 1. (A) Inhibition of L. bulgaricus 1489 by L. helveticus 481culture supernatant via the agar well diffusion method of Tagg andMcGiven (24). Culture supernatants were neutralized to pH 6.0 andtreated with 68 U of catalase per ml. (B) Deferred antagonismshowing zones of inhibition around L. helveticus 481 coloniesoverlaid with a lawn of L. bulgaricus 1489.
helveticin J concentration (160.0 AU/ml) reduced the viablecell population by 3-log cycles immediately upon addition ofthe bacteriocin to the cells. After 3 h, there were <10 viablecells per ml. A significant decrease in optical density was notobserved over the 3-h period, indicating that cell lysis hadnot occurred. Helveticin J exhibited a bactericidal mode ofaction, killing L. bulgaricus 1489 without concomitant celllysis.
Effect of pH on helveticin J production. Production ofhelveticin J was evaluated during anaerobic growth of L.helveticus 481 in MRS broth under constant pH conditions(Fig. 2). At pH 7.0, 800 AU/ml was detected at 12 h, afterwhich the activity gradually declined. Growth at pH 6.0yielded a higher titer of 3,200 AU/ml after 5 h. The level ofactivity remained stable over several hours and thendropped off sharply. Maximum production of helveticin Jwas observed at pH 5.5. After 11 h of growth, a titer of 6,400AU/ml was detected. No decline in activity was observedover a 24-h period. Accumulation of helveticin J was de-tected between the late-log and stationary phases of growthof L. helveticus 481.
Dissociation of helveticin J. During preliminary ultrafiltra-tion studies of CHJ, 64% of helveticin J activity was retainedby an XM300 Diaflo membrane, 14% by an XM100A Diaflomembrane, 9% by an XM50A Diaflo membrane, and 3% byan YM10 Diaflo membrane (Table 1). Molecular exclusionlimits for the membranes were 3 x l05, 1 x 105, 5 x 104, and1 x 104, respectively. Retention of helveticin J activity bythe different membranes suggested that aggregates of varioussizes were present, with the majority of the aggregatesexceeding 3 x i05 M, A 10% activity loss accompanied theultrafiltrations, which may have resulted from protein aggre-
a 400,000 AU of CHJ was subjected to ultrafiltration.
all LHJ activity, and the 8 M urea treatment resulted in a50% reduction of activity. Dissociating agents that wereunsuccessful in breaking up LHJ were 1 mM dithiothreitoland 0.1% Triton X-100.
Purification of helveticin J. Helveticin J was purified fromsupematants of broth cultures prepared in semidefined me-dium to minimize the level of contaminating proteins andpeptides. Growth and helveticin J production at pH 5.5 in thesemidefined medium were comparable to those in MRScultures (data not shown). After an ammonium sulfate pelletwas obtained by using a 50% ammonium sulfate saturationlevel, difficulty was experienced in solubilizing helveticin Jin 0.1 M sodium acetate buffer (pH 5.3). The addition of 6%SDS (wt/vol) to the suspension aided dissolution, and 100%of the helveticin J activity was recovered (data not shown).Throughout the remainder of the purification steps, hel-veticin J was maintained in the presence of 0.1% SDS.Chromatographs of helveticin J on a Sephadex G-200-120
column, with a fractionation range of 5,000 to 600,000daltons, resulted in a peak of activity between two absor-bance peaks of contaminating proteins (Fig. 3). Subsequentapplication of concentrated, active fractions to a SephadexG-200-50 column, with a fractionation range of 5,000 to250,000 daltons, yielded a large peak of activity correspond-ing to the largest absorbance peak in the elution profile (Fig.3). This peak was flanked by four smaller peaks representingcontaminating proteins.A scaled-down purification of helveticin J with approxi-
mately 2 liters of culture supernatant as starting material isrepresented in Table 3. An increase in the amount of proteinwas detected after the last chromatographic step on Sepha-dex G-200-50. This aberration is most likely due to the limitsof the assay employed in detecting low concentrations ofprotein, combined with the possible introduction of UV-absorbing substances during the last purification step.Disc-SDS-PAGE of helveticin J (Fig. 4) resulted in three
main protein bands; the middle band was the largest andmost distinct. Helveticin J activity was eluted from a gel
Time (h)
FIG. 2. Growth of L. helveticus 481 (0) and production ofhelveticin J (A) in fermentors held constant at pH 5.5 (A), 6.0 (B),and 7.0 (C).
gation or irreversible adsorption to the Diaflo membranes.LHJ (>3 x 105 daltons) was treated with dissociating agentsand subjected to ultrafiltration through a filter with a molec-ular exclusion limit of 3 x 10 daltons. The filtrates weremonitored for helveticin J activity to detect dissociation ofLHJ (Table 2). LHJ was dissociated with the use of 1% SDS,and 0.2% ,-mercaptoethanol plus 1% SDS. An increase inactivity was also noted upon the dissociation. This suggestedthe formation of small active subunits upon dissociation of alarger active unit resulting in more lethal hits. ,B-Mercap-toethanol (0.2%) and 6 M guanidine-hydrochloride destroyed
TABLE 2. Dissociation of LHJAU of helveticin J/ml with
Dissociating agenta a mol wt of:>3 x 105 <3 x 105
0.1 M sodium acetate buffer 400 01 mM dithiothreitol 400 00.1% Triton X-100 400 08 M urea 200 01% SDS 800 8000.2% P-mercaptoethanol + 1% SDS 400 8000.2% ,B-mercaptoethanol 0 06 M guanidine hydrochloride 0 0
a Dissociating agents did not inhibit the indicator lawn, with the exceptionof 6 M guanidine hydrochloride, which had an inhibitory titer of 200 AU/ml.
FIG. 3. Elution profiles of partially purified helveticin J in thepresence of 0.1% SDS on Sephadex G-200-120 (A) and G-200-50 (B)columns. Symbols: 0, A2N; 0, helveticin J titer.
slice corresponding to the middle protein band (Fig. 4). Anaverage molecular weight of 37,000 was obtained from fourtrials (sy = 1,062). The eluted active component was con-centrated and applied to a polyacrylamide-gradient (10 to20%) SDS gel alongside helveticin J eluted from the Sepha-dex columns. A single protein band migrated exactly thesame distance as the middle protein band previously identi-fied as helveticin J (data not shown). These results confirmedthat completely dissociated, active helveticin J has a molec-
la
.......-
... .,
A... _
10,000AU/ml
Helveticin J TiterFIG. 4. Lanes: A, disc-SDS-PAGE of partially purified
helveticin J (M,, 37,000); B, molecular weight markers (phosphor-ylase b [MW, 94,000], albumin [Mw, 67,000], ovalbumin [Mw,43,000], carbonic anhydrase [MW, 30,000], trypsin inhibitor [Mw,20,100], and a-lactalbumin [Mw, 14,400], from top to bottom). Thebar graph indicates the helveticin J titer as eluted from 2-mm slicesof a complementary polyacrylamide gel.
ular weight of 37,000 which could be resolved as a singleprotein band. The inhibitory spectrum of purified helveticinJ was in accordance with the inhibitory spectrum of CHJ(data not shown). Therefore, the antimicrobial agent whichhad been originally detected in L. helveticus 481 culturesupematants was purified.Enzyme and heat inactivation. Helveticin J activity was
completely eliminated upon treatment with all proteolyticenzymes tested (data not shown). The proteolytic enzymesemployed included ficin, pronase, trypsin, pepsin, protein-ase K, and subtilisin. Lysozyme or lipase had no effect onhelveticin J. After subjecting CHJ (1,600 AU/ml) to 100°C for30 min, the bacteriocin activity was reduced to 100 AU/ml.The sensitivity of helveticin J to heat and proteolytic en-zymes demonstrated the proteinaceous nature of the antimi-crobial compound.
Plasmid involvement. L. helveticus 481 was found toharbor an 8-megadalton plasmid, designated pMJ1008. Ef-forts were made to determine whether pMJ1008 carriedgenetic determinants responsible for helveticin J production
FIG. 5. Agarose gel electrophoresis of cesium chlori(bromide-purified plasmid DNA from E. coli V517 (32,2.2, 1.7, 1.5, and 1.2 megadaltons [15]), L. helveticus 481and six presumptive pMJ1008-cured L. helveticus 481 isOpen circular plasmid forms; Chr, chromosomal DNA.
and host bacteriocin immunity. No mutants d4helveticin J production (Hlv-) were detected durination of 5,349 single colony isolates for loss of ttype. Ethidium bromide (0.6 ,ug/ml), acriflavine, (Cand novobiocin (20 to 200 ng/ml) were ineffectiveating Hlv- variants.
Plasmid DNA isolations were performed oncolonies isolated after growth of L. helveticus 48broth containing 200 ng of novobiocin per ml.Hlv+ isolates were found to be cured of pMJ1008 (Iexclude the possibility of novobiocin-induced chriintegration of pMJ1008, total DNA from four (
presumptively cured isolates was probed for the pthe plasmid (Fig. 6). 32P-labeled pMJ1008 hybricwith purified pMJ1008 and total DNA from the wihelveticus 481. No hybridization of pMJ1008 withfrom the four plasmid-cured isolates was detecte(
L. helveticus 481(pMJ1008) and the pMJ1008-lates were examined for phenotypic differences.tion patterns, arginine and esculin hydrolysis, spbacteriocin activity, bacteriocin sensitivity, and bimmunity were identical for the parent and pMJIvariants (data not shown).
DISCUSSION
Characterization of helveticin J confirmed that tcrobial agent produced by L. helveticus 481 wariocin according to the criteria outlined by Tagg 4Helveticin J has a narrow spectrum of inhibitoiwithin the Lactobacillaceae family. Only speciirelated to the producer, L. helveticus 481, were inthe bacteriocin. Sensitive species included L. helibulgaricus, and L. lactis. These Lactobacillus spbeen shown to have a high degree of relatednerstudies of percent guanine-plus-cytosine content,mology, and protein structure via immunodiffusi(helveticus 481 demonstrated host immunity to he
Producer cell immunity, although poorly understood, isbelieved to result from synthesis of a specific immunitysubstance (23).
Helveticin J was a protein that exhibited bactericidalactivity against sensitive indicators. Inactivation of thebacteriocin occurred during heat and proteolytic enzymetreatments. CHJ with a molecular weight of >300,000 wasdissociated with SDS, purified, and resolved by using SDS-PAGE as a single protein band with a molecular weight of37,000. Aggregations of crude bacteriocins have been ob-
---Chr served previously during studies of other bacteriocins in-cluding Lactacin B and Lactocin 27, produced by L.acidophilus N2 and L. helveticus LP27, respectively (4, 7,25). Bacteriocins have also been reported to bind to cellularcomponents such as membrane vesicles, thus conferring anapparent large molecular weight to the antimicrobial agent(2). However, CHJ was probably not bound to cellularcomponents, since an increase in activity and smallerhelveticin J molecules were detected upon treatment with1% SDS and 0.2% 3-mercaptoethanol plus 1% SDS. Thesephenomena indicated the presence of an aggregate.
de-ethidium Helveticin J differed from previously characterized5.2, 3.5, 3, Lactobacillus bacteriocins (3-6, 25, 26). First, the inhibitoryL(pMJ1008), spectra of helveticin J, lactacin B, lactocin 27, and the L.olates. OC, fermentum bacteriocin include different species and strains
of lactobacilli. Helveticin J inhibited L. helveticus 1244 and1846, L. bulgaricus 1373 and 1489, and L. lactis 970; lactacinB repressed growth of L. leichmannii 4797, L. bulgaricus
eficient in 1489, L. helveticus 87, and L. lactis 970 (3); lactocin 27ing exami- inhibited L. acidophilus, L. helveticus, and one naturallyhe pheno- isolated Lactobacillus species (25); and the L. fermentum).6 ,ug/ml), bacteriocin was antagonistic towards other L. fermentumin gener- strains and L. acidophilus (5). Second, the molecular
weights of the purified Lactobacillus bacteriocins varied;i 17 Hlv+ helveticin J was the largest, at 37,000. Lactacin B and31 in MRS lactocin 27 had respective molecular weights of 6,000 andSix of the 12,400 (3, 25).Fig. 5). To Helveticin J was heat sensitive, whereas lactacin B andomosomal lactocin 27 were heat stable when held for 1 h at 100°C (3,of the sixgresence ofdized onlyild-type L.l the DNAd.cured iso-Fermenta-)ectrum ofiacteriocin1008-cured
the antimi-Ls a bacte-et al. (23).ry activityies closelyihibited byveticus, L.ecies havess throughDNA ho-
cn (14). L.-lveticin J.
A B C D E F
~~ ~ ~ ~
A B C D E F
FIG. 6. The left panel shows agarose gel electrophoresis ofEcoRI-restricted pMJ1008 (lane A), EcoRI-restricted total DNAisolated from wild-type L. helveticus 481 (lane B), and presumptivepMJ1008-cured L. helveticus 481 isolates (lanes C to F). The rightpanel shows an autoradiogram prepared after hybridization with32P-labeled, EcoRI-restricted pMJ1008. Lanes: A, pMJ1008; B,wild-type L. helveticus 481 DNA; C to F, DNA of pMJ1008-curedisolates.
25). The L. fermentum bacteriocin also did not decrease inactivity when held for 30 min at 96°C (6). The heat sensitivityof helveticin J was consistent with the size and the apparentcomplexity of its protein structure as compared with those ofthe other bacteriocins.
Ficin, pronase, trypsin, pepsin, proteinase K, andsubtilisin successfully inactivated helveticin J after an incu-bation period of 1 h at 37°C. Lactocin 27 was also inactivatedby pronase and trypsin, but not by ficin (25). Trypsininactivated the L. fermentum bacteriocin, but its activitywas decreased only by one-half when incubated at 37°C withpepsin for 24 h (6). The proteolytic enzyme sensitivity oflactacin B was not exhaustively investigated, so differencesbetween it and helveticin J are not evident. Lactacin Bactivity was destroyed by pronase and proteinase K (3, 4).Therefore, based on inhibitory spectra, molecular weight,proteolysis, and heat sensitivity, helveticin J has been con-firmed to be a unique bacteriocin within the Lactobacil-laceae family.
L. helveticus 481 was shown to harbor a single plasmidspecies of 8 megadaltons. However, pMJ1008 did not encodegenetic determinants for helveticin J production or host cellimmunity. Hlv' cells which had been cured of pMJ1008 didnot harbor any other detectable plasmids, and pMJ1008sequences were not found in the bacterial chromosome.These data confirmed that pMJ1008 was not responsible forHlv+ activity and suggested that genetic determinants forbacteriocin production and immunity were localized in thechromosome. The genetic stability of helveticin J productionunder growth conditions that induce plasmid curing providedfurther evidence that chromosomal rather than plasmid-borne determinants were involved. Similar observationshave been reported for lactacin B (3) and lactocin 27 (26).However, until Hlv+ genes are definitively localized in thechromosome, a remote possibility still exists that a stableand undetected plasmid could be responsible for helveticin Jproduction. The colicin-based criteria for bacteriocin identi-fication by Tagg et al. (23) includes a stipulation that geneticdeterminants for bacteriocin production and immunity areplasmid borne. One notable exception reported transforma-tion of chromosomally linked pneumocin genes in Strepto-coccus pneumoniae (18). Determinants for helveticin J pro-duction and immunity in L. helveticus 481 are also notablesince they appear to be chromosomally mediated.
It is important that no phenotypic change in L. helveticus481 was detected upon curing of pMJ1008. Properties exam-ined included bacteriocin production, bacteriocin sensitiv-ity, and carbohydrate fermentation. In the lactic acid bacte-ria, these functions have been routinely associated withplasmid DNA. However, plasmids do not appear to directthese characteristics in L. helveticus 481, and the function ofpMJ1008 remains unknown.
ACKNOWLEDGMENTS
Support for this investigation was provided, in part, by the DairyResearch Foundation, Rosemont, Ill., and by the North CarolinaDairy Foundation.We thank Peter Muriana for sharing preliminary results on
novobiocin-induced curing of plasmids from lactobacilli.This work was submitted to fulfill, in part, the M.S. requirements
of M.C.J.
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