Lait (1997) 77, 267-278© Elsevier/INRA

267

Original article

Purification and characterization of a proteinaceouscompound from Pseudomonas fluorescens ATCC 948with inhibitory activity against sorne Gram-positive

and Gram-negative bacteria of dairy interest

M Gobbetti *, A Corsetti, E Smacchi, J Rossi

Institute of Dairy Microbiology. Agriculture Faculty, S Costanzo 06126, Perugia, Italy

(Received 21 May 1996; accepted 16 September 1996)

Summary - A proteinaceous antimicrobial compound from Pseudomonas fluorescens ATCC 948was purified to homogeneity by chromatography steps and characterized. The compound showed alarge spectrum of activity. Inhibition of thermophilic Lactobacillus, Listeria monocytogenes, Enter-obacteriaceae and P fluorescens strains was mainly detected. The antimicrobial compound pro-duced in skim milk had an apparent molecular mass of 67 kDa, was quite heat-stable (80 "C for15 min), insensitive to lipase and a-amylase, and completely inactivated by treatment with proteolyticenzymes. A bactericidal mode of action was detected against Lactobacillusfermentum D 13 chosenas indicator strain. Pfluorescens ATCC 948 was not sensitive to the antimicrobial compound producedby itself. The production of the proteinaceous substance started in the early exponential phase ofgrowth, and was highest after 72 h of incubation at 20 "C and pH 6.5. Sorne considerations about thenature of the antimicrobial compound are given.

Pseudomonas fluorescens / proteinaceous antimicrobial compound / thermophilic Lactobacillus

Résumé - Purification et caractérisation d'une substance protéinique par Pseudomonas fluo-rescens ATCC 948, avec activité d'inhibition contre certaines bactéries Gram positives et Gramnégatives d'intérêt laitier. Une protéine présentant une activité antimicrobienne a été produite parPseudomonasfluorescens ATCC 948. Elle a été purifiée jusqu'à homogénéité par chromatographieet caractérisée. Elle présente un large spectre d'activité et une inhibition contre certaines souches deLactobacillus thermophiles, contre Listeria monocytogenes, Enterobacteriaceae et des souches dePfluorescens. Le composé antimicrobien produit dans du lait écrémé présente un poids moléculaireapparent de 67 kDa, est suffisamment thermostable (80 "C pendant 15 minutes), pas sensible à la lipaseet à l'a-amylase, mais est complètement inactivé par traitement aux enzymes protéolytiques. Son moded'action bactéricide a été determiné contre Lactobacillus fermentum D 13, choisi comme souche-témoin. Pfluorescens ATCC 948 n'est pas sensible au composé antimicrobien produit par lui-même.

* Correspondence and reprints

268 M Gobbetti et al

La production de cette substance protéinique a commencé pendant la première phase de développe-ment exponentiel, et a été la plus importante après 72 heures d'incubation à 20 "C et pH 6,5. Quelquesconsidérations concernant la nature du composé protéinique ont été proposées.

Pseudomonas fluorescens 1composé antimicrobien protéinique 1Lactobacillus thermophile

INTRODUCTION

Psychrotrophic pseudomonads are the mostcommon microorganisms that grow in rawmilk during cold storage (Cousin, 1982).Pseudomonas fluorescens has been shown tosecrete extracellular lipases, and especiallyproteinases, which are resistant to both pas-teurization and UHT treatments (Griffithset al, 1981). Proteinases cause bitterness inUHT milk (Gobbetti, 1993) and contribute,together with plasmin, to age gelation ofunconcentrated UHT milk (de Koning et al,1985).

Several studies have been conducted onthe inhibitory effect of lactic acid bacteriaagainst Pseudomonas strains. Inhibition ofpsychrotrophic pseudomonads, either bylowering pH, producing lactic acid andhydrogen peroxide or by more complexmicrobial interactions, has been shown withLeuconostoc mesenteroides subsp dextran-icum (Mans De Marion et al, 1995); com-mercial lactic cultures (Champagne et al,1990) inoculated in raw milk stored at lowtempe rature; lactic acid bacteria starters usedin cultured milks (Balasubramanyam andVaradaraj, 1994); culture supernatants ofstrains of Bifidobacterium cultivated in skimmilk (Mitsuhashi and Murata, 1991) andwith neutralized extracellular culture fil-trates of Lactobacillus and Lactococcusspecies (Varadaraj et al, 1993). On the con-trary, psychrotrophic pseudomonads whichare initially not antagonized can promotethe lactic acid bacteria metabolism by theirproteolytic products (Koburger and Clay-don, 1961; Tayfour et al, 1981) and by ure-ase activity, which produces a decrease in

the amount of native urea of milk andincreases in the ammonia concentration(Jaspe et al, 1995).

To our knowledge, no reports have beenmade on the antagonistic activity of psy-chrotrophic pseudomonads towards lacticacid bacteria. Siderophores, pterines,indoles, phenazynes, lipids, peptides andbacteriocins are produced by Pseudomonasspp strains and have been extensively stud-ied for their antimicrobial activity in the bio-control of plant diseases (Dowling and 0'Gara, 1994). Bacteriocins from P glycinea,P phaseolicola (Vidaver et al, 1972),P syringae pv syringae (Smidt and Vidaver,1986), P syring ae subsp savastanoi(Iacobellis et al, 1995), P solanacearum(Cuppels et al, 1978) and P fluorescens(Reeves, 1965) have been isolated and char-acterized. A narrow spectrum of activity, inparticular shown against important plantpathogens, is a corn mon feature of pseu-domonad bacteriocins: several studies (Cup-pels et al, 1978; Iacobellis et al, 1995) haveshown a species-specific inhibition. Thereare sorne strains, however, that have showna wider spectrum of antagonism, includingunrelated Gram-negative and Gram-posi-tive species.

Analogous interactions have beenreported in foods. Inhibition of foodbornebacteria by Pseudomonas spp has beenobserved and attributed to siderophores orbacteriocin-like substances (Freedman et al,1989; Charnpomier- Vergès and Richard,1994) but inhibition of unrelated Bacilluscereus and Listeria spp strains has only beendetected in a few strains (Freedman et al,1989).

A proteinaceous antimicrobial compound from P fluorescens

In this study, a proteinaceous compoundwith inhibitory activity mainly towards lac-tic acid bacteria has been purified and char-acterized from P fluorescens A Tee 948.

MATERIALS AND METHODS

Strains and culture conditions

Pseudomonas fluorescens ATCC 948 and theother pseudomonad strains isolated from rawmilk (Gobbetti and Rossi, 1992), were aerobi-cally cultivated in nutrient broth (BN) (DifcoLaboratories, Detroit, MI, USA) at 28 "C for24 h. Mesophilic and thermophilic Lactobacil-lus and Enterococcus strains were propagated inMRS (De Man et al, 1960), streptococci and lac-tococci in M 17 (Difco) with 0.5% glucose(GM 17); Enterobacteriaceae in McConkeymedium (Difco) with 0.5% glucose; Listeriamonocytogenes in TSY (Difco) and Bacillusstrains in BN (Difco) media.

Production of culture supernatants. and detection of antagonistic activity

Pseudomonas fluorescens ATCC 948 and 20other strains were grown at 28 "C for 48 h in BNor skim milk, the cells were harvested by cen-trifugation (7000 g for 10 min), and the super-natants adjusted to pH 6.5 and filtered through a0.22 um pore size filter (Nucleopore; CostarCorp, Cambridge, MA, USA). Due to the pro-teolytic activity of Pfluorescens ATCC 948 dur-ing the 48-h growth period, a limpid whey wasalso harvested by the centrifugation of cellsgrown in skim milk. To detect antagonistic activ-ity, a weil-diffusion assay was used (Schillingerand Lücke, 1989). The assays were conductedin different agar media (specifie for each indi-cator strain) overlaid with 7 mL of different softagar media which contained 4% inoculum of anovernight culture of the indicator strain. Wells,3 mm in diameter, were eut into these agar platesand filled with 300 ul, of the culture supernatantof the potential producer strain. The plates wereincubated for 24 h at 28 or 37 "C and subse-quently examined for zones of inhibition. Inhi-bition was scored positive if the width of theclear zone around the wells was 1.0 mm or larger.

269

A quantitative determination based on a crit-ical dilution assay (Barefoot and Klaenhammer,1983) was used to express the arbitrary units(AU) of antimicrobial activity. An AU wasdefined as the maximum dilution of the antimi-crobial compound preparation which inhibitedLb fermentum 0) 3 after 24 h of incubation at37 oc.

Purification of the antimicrobialcompound

Five L of the supematant culture of PfluorescensATCC 948 in BN medium were concentrated30-fold by freeze-drying (Edwards MaO EIPTB;Edwards, Milan, Italy), resuspended in50 mmol/L phosphate buffer, pH 7.0 and crys-talline ammonium sulphate was gradually addedwith slow stirring until 50% saturation wasreached. The mixture was kept overnight at 4 "Cfor complete precipitation and then centrifuged at15 000 g for 15 min. The precipitate was dis-solved in 20 mmol/L Na-acetate buffer, pH 5.0,dialyzed for 24 h at 4 "C and applied to aS-Sepharose HR 16/50 column (Pharmacia FineChemicals, Uppsala, Sweden), equilibrated withthe same buffer. After washing, elution was car-ried out at a flow rate of 35 mL /h with a linearNaCI gradient, 0.1 to 0.5 mollL. As for the otherpurification steps, the collected fractions (7 mL)were tested for inhibition against Lb fermentum013 by the weil-diffusion method. This bac-terium was chosen as indicator bec au se, amongthe lactic acid bacteria, it had shown an inter-mediate sensitivity to the antimicrobial com-pound. Fractions with strong inhibitory activitywere pooled, dialyzed for 24 h at 4 "C against5 mmol/L phosphate buffer, pH 7.0 and concen-trated 1O-fold by freeze-drying.

Inhibitory fractions from the previous stepwere redissolved in a small volume of 50 mmollLphosphate buffer, pH 7.0, and further purifiedby gel-filtration on Superose 12 HR 10130 col-umn (Pharrnacia), equilibrated with 50 mmollLphosphate buffer, pH 7.0, containing 0.15 mollLof NaCI. Elution with the same buffer was at aflow rate of 40 mL /h and l-mL fractions werecollected and assayed for inhibition.

The pooled, dialyzed and 10-fold concentratedinhibitory fractions from gel-filtration were finallypurified on a FPLC-Mono-Q HR 5/5 column(Pharmacia). Elution was made with 20 mmol/LBis-Tris propane buffer, pH 6.5, by a linear NaCI

270 M Gobbetti et al

gradient (0 to 0.3 mol/L) at a flow rate of45 mL th; 0.5-mL fractions were coIlected.

Protein determination

The protein concentration in the solutions con-taining the antimicrobial compound was deter-mined by the method of Bradford (1976), usingbovine serum albumin as standard. The proteinconcentration during the purification steps wasmonitored by measuring absorbance at 280 nm.

Electrophoresis

Purity and molecular mass of the proteinaceouscompound were estimated by SDS-PAGE.p-Galactosidase (116.4 kDa), fructose-6-phos-phate kinase (85.2 kDa), glutamate dehydroge-nase (55.6 kDa), aldolase (39.2 kDa) andtriosephosphate isomerase (26.6 kDa) were usedas molecular mass markers (BoehringerMannheim GmbH, Germany). The SDS-PAGEwas conducted according to Lacrnmli's proce-dure (1970); gels contained 10% acrylamide (sep-aration distance, 10 cm; gel thickness, 1 mm).The protein sampIes were mixed 1:1 with samplebuffer (0.18 mol/L Tris-HCI, pH 6.8, 0.3% SDS,8.6% glycerol, 1% mercaptoethanol and 0.07%bromophenol blue) and applied to the gels. Elec-trophoresis was performed at 200 V for approx-imately 45 min. Gels were stained withCoomassie briIliant blue R250 (Sigma Chemi-cal Co, St Louis, MO, USA).

Characterization of the antimicrobialcompound

Sensitivity to heat and hydrolytie enzymes

Solutions of the purified antimicrobial compoundin 50 mmollL phosphate buffer, pH 7.0, wereheated at 80 to 100 "C for 5 to 20 min, and theremaining activity was determined by weIl-dif-fusion assay.

The foIlowing enzymes (Sigma Chemical Co,St Louis, MO, USA) were tested for theirhydrolytic activity on the purified antimicrobialcompound: proteinase K (2.6 U/mg), trypsin(22 U/mg), proteinase B500 (45 Ulmg), lipase(50 U/mg) and a-amylase (15 U/mg). The assays

were performed at a final concentration of0.5 mg/mL and at pH 6.5, except for pepsin(pH 3.0). Samples with and without enzymeswere held at 35 "C for 6 h and the remainingactivity was determined by weIl-diffusion assay.

Mode of action

To study the effect of the antimicrobial compoundfrom P fluorescens ATCC 948 on sensitive cells,2 mL of a 30-fold concentrated (under vacuum),adjusted to pH 6.5 and filtered supematant (spe-cific activity, 37.5 AU/mg) were added to 10 mLof MRS broth containing Lb fermentum DI3 ina cell concentration of log 5.2 cfu/mL. For com-parison, a concentrated supematant from P jluo-rescens M8, which is not a producer of antimi-crobial compound, was used. At 3-h intervals, thenumber of viable cells was determined on MRSagar plates after incubation at 37 "C for 48 h.

To determine whether the compound was bac-teriostatic or bactericidal, the Lb fermentum D 13cells were cultivated for 9 h in MRS broth con-taining the lowest inhibitory concentration of thecompound (5 AU/mg), the broth was then diluted5-fold by the addition of MRS and the growthof cells was allowed to continue for 48 h at 37 "Cbefore plating.

Phase of growth, pH and temperatureoptima

P fluorescens ATCC 948 cells were cultivatedon BN medium and skim milk at 28 "C for 144 hand samples were taken at different periods oftime to determine the inhibitory activity onLbfermentum D13.

The influence of the pH and temperature onthe production of the proteinaceus antimicrobialcompound was assayed by growing PfluorescensATCC 948 in BN medium, pH 4.0-8.0, and in BNmedium, pH 7.0, and at temperatures of 5 to 40 oc.

RESULTSInhibitory spectrum of the antimicrobialcompound

The inhibitory spectrum of the antimicro-bial compound contained in the extracellu-lar supernatant of Pfluorescens ATee 948culture in BN broth is shown in table 1. No

A proteinaceous antimicrobial compound from P fluorescens 271

Table I. Inhibitory spectrum of the antimicrobial compound produced by P fluorescensATCC 948.Spectre d'inhibition du composé antimicrobien produit par P f1uorescens ATCC 948.

Indicator species Origin" Inhibition bStrain no

Gram-positive bacteriaBacillus subtilisB cereusEnterococcus faecalisEfaecalisEfaeciumLactobacillus plantarum!JJ plantarum!JJ pseudoplantarumLb pseudoplantarumLb caseiLb casei!JJ curvatusLb curvatusLb bulgaricusStreptococcus thermophilusLb acidophilusLb acidophilusLb fermentumLb fermentum!JJ helveticus!JJ lactis!JJ reuteriLactococcus lactis ssp lactisListeria monocytogenesStaphylococcus aureus

Gram-negative bacteriaPseudomonas fluorescensP fluorescensP fluorescensP fluorescensP fluorescensP fluorescensP fluorescensP fluorescensP fluorescensP fluorescensP fluorescensProteus vulgarisSalmonella sspSerratia liquefaciensShigella

5217464E58E71E3427412739274227452752275620812042B397F142A2N2DT41013PR4DT4220016MG1363X88M35

948M4M8M20M22M30M31M40M44M45M47X31X14X53X23

Our strain collectionNCTCOur strain collectionOur strain collectionOur strain collectionUCCUCCUCCUCCUCCUCCUCCUCCOur strain collectionOur strain collectionOur strain collectionOur strain collectionOur strain collectionOur strain collectionOur strain collectionOur strain collectionOur strain collectionOur strain collectionOur strain collectionOur strain collection

(+)

(+)

+++

(+)(+)(+)+

+++

(+)

+(+)

ATCCOur strain collectionOur strain collectionOur strain collectionOur strain collectionOur strain collectionOur strain collectionOur strain collectionOur strain collectionOur strain collectionOur strain collectionOur strain collectionOur strain collectionOur strain collectionOur strain collection

+

+

(+)

(+)(+)(+)+

a Abbreviations: NCTC: National Collection of Type Cultures (London); ATCC: American Type CultureCollection (Rockville, MD); UCC: Culture Collection of the Department of Food Chemistry of UniversityCollege Cork, Ireland; b Symbols: ++: very large inhibition zone (> 5.0 mm); +: large inhibition zone (> 2.0mm); (+): small inhibition zone (2.0- 1.0 mm); -: no inhibition zone.

272

SDS-P AGE showed that the enzymepreparation was homogeneous, with anapparent molecular mass of 67 kDa (fig 2).

M Gobbetti et al

differences were detected by cultivating thestrain in skim milk. The large spectrum ofinhibition included Gram-positive andGram-negative species. Thermophilic Lac-tobacillus, L monocytogenes, sorne P jluo-rescens strains and Enterobacteriaceae weremarkedly inhibited. ln particular, Lb del-brueckii subsp bulgaricus B397 and Lb hel-veticus PR4 showed a zone of inhibitionlarger than 5.0 mm. P fluorescensATCC 948 was not sensitive to the antimi-crobial compound produced by itself.

Similar results were obtained by usingthe culture supematant of P fluorescens M22and M40 strains, but only a narrow spec-trum of inhibition (including a few Pseu-domonas strains and Proteus vulgaris) wasdetermined with the other 18 pseudomonadstrains tested (data not shown).

Purification of the antimicrobialcompound

The antimicrobial compound produced byP fluorescens ATCC 948 in BN broth waspurified by precipitation with ammoniumsulphate and subsequent chromatographysteps (table II). On the S-Sepharose column,the most inhibitory fractions were elutednear the start of the salt gradient (fig 1a)and were separated from most of the pro-tein contained in the ammonium sulphateprecipitated. Chromatography on Super-ose 12 (fig 1b) resol ved the fractions withinhibitory activity into one major peak cor-responding to the lowest protein peak. Thefinal purification was achieved on a Mono-Qanion exchange column (fig le); the peakof activity was superimposed on the secondprotein peak. The antimicrobial compoundproduced from P fluorescens ATCC 948was purified 260-fold with a final yield of6% and specifie activity of 9750 AU/mg(table II).

Characterization of the antimicrobialcompound

The purified antimicrobial compound pro-duced by P fluorescens ATCC 948 wasassayed for heat sensitivity and activity ofhydrolytic enzymes. It was resistant to treat-ment at 80 "C for 15 min, but the inhibitoryactivity was completely abolished by treat-ment at 100 "C for 5 min. The antimicro-bial compound was insensitive to a-amy-lase and lipase enzymes and was inactivatedby ail the proteolytic enzymes used. A rep-resentative plate with inactivation by trypsin,proteinase K and heat treatment at 100 "Cfor 5 min is shown in figure 3.

The addition of the concentrated culturesupematant of P fluorescens ATCC 948 toa culture of Lb fermentum D 13 in MRSbroth rapidly inactivated the sensitive cells(fig 4). After 6 h of incubation, the numberof viable cells decreased from log 5.2 to log2.6 cfu/mL and to log 1.2 cfu/mL after 9 h.No growth was observed after exposure(9 h) of Lb fermentum D 13 to the lowestinhibitory concentration of the compoundand subsequent cultivation (48 h) in MRScontaining the 1:5 diluted antimicrobial sub-stance. This result indicated that thecompound produced by P fluorescensATCC 948 seems to have a bactericidalaction.

The production of the antimicrobial com-pound did not begin before the early expo-nential phase of growth (- log 4.8 cfu/mL)and rapidly increased during the last stagesof growth (fig 5). The highest productionof the antimicrobial compound was detectedafter 72 h of incubation at 28 "C followed bya decrease, probably due to the partialhydrolysis of the proteinaceous compoundby extracellular proteinases (data not

A proteinaceous antimicrobial compound from P fluorescens 273

Table II. Purification of the proteinaceous compound from the culture supernatant ofP fluorescens ATCC 948.Purification du composé protéinique présent dans le surnageant de la culture de P fluorescensATCC948.

Purification step Total protein Total activity Specifie activity Yield Purification(mg) (AU) (AU/mg) (%) (-fold)

Culture supernatant 1652 6.2 x 104 37.5 100 1(NH4)2S04 617 4.7x104 76.2 76 2precipitationS-Sepharose 52 1.9 x 104 365.4 30 10Superose 12 4 8.8 x 103 2200 14 58Mono-Q 0.4 3.9 x 103 9750 6 260

Total activity is expressed as arbitrary units (AU) of inhibitory activity (Barefoot andKlaenhammer, 1983). Specifie activity was defined as AU per mg protein.

a

60 0.5

60O.4~

0.31.40 ~

0.2 g,20 01 ~

010 20 30 40 50 60

b

0.6 60

Fig 1. Purification of the proteina-'?

0.6 60 2:ceous antimicrobial compound from ~

40 :~P fluorescens ATCC 948: protein 0.4 1i

~concentration (0), inhibitory activ- 0.2 20 ~ity (.Â.) and NaCI gradient (---). ~a. Cation-exchange chromatogra- ."" 0

phy on S-Sepharose. b. Gel filtra- 10 15 20 25 30

tion on Superose 12. c. Anion-exchange chromatography onMono-Q. cPurification du composé protéinique

0.3 40 0.3à activité antimicrobienne produitpar P f1uorescens ATCC 948. 30 ~concentration protéique (0). acti- 0.2 021.

20 ]vité inhibitrice (~) et gradient de '5

NaCI (---). a. Chromatographie 0.1 0.1 g,10 sà échange cationique sur

S-Sepharose. b. Gel filtration sur 0

Sephacryl200. c. Chromatographie 10 15 20 25 30

à échange an ionique sur Mono-Q. Fraction numbers

274 M Gobbetti et al

1 2

116.4 kDa

85.2kDa

55.6 k Da

39.2 k Da

26.6 k Da

Fig 3. Inhibition of Lb fennentum DI 3 by Pfluo-rescens ATee 948 proteinaceous antimicrobialcompound by the agar weil-diffusion assay.Wells contained the filtered (0.22 urn) antimi-crobial compound solution, which was treatedby trypsin (0.5 mg/mL) (weil 1), proteinase K(0.5 mg/mL) (weil 2), a-amylase (0.5 mg/mL)(weil 3), lipase (0.5 mg/mL) (weil 4) and by treat-ment for 5 min at 100 "C (weil 5).

Inhibition de Lb fermentum D 13 par le composéprotéinique à activité antimicrobienne produitpar P tluorescens ATCC 948 mise en évidencepar la technique de diffusion en puits. Les puitscontenaient la solution du composé antimicro-bien filtré (0,22 p m}, traitée à la trypsine(0,5 mg/ml.) (puits 1), à la protéinase K(0,5 mg/ml.) (puits 2), à l'a-amylase (0,5 mg/ml.)(puits 3), à la lipase (0,5 mg/ml.) (puits 4) et partraitement pendant 5 min à 100 oC (puits 5).

Fig 2. The SOS-PAGE of the purified proteina-ceous antimicrobial compound l'rom P fluo-rescens ATee 948. Lane 1, reference proteins;lane 2, purified proteinaceous antimicrobialcompound.SDS-PAGE du composé protéinique à activitéantimicrobienne produit par P tluorescens ATCC948. Ligne l, protéines de référence; ligne 2,composé protéinique purifié à activitéantimicrobienne.

10 ~--------.--.

0-1--+--+-->-->----<>----<----<--<o 12 15 18 21

Tfme Ih)

24

Fig 4. Bactericidal effect of the proteinaceousantimicrobial compound produced by P fluo-rescens ATee 948 on a sensitive strain (Lb fer-mentum D 13). (Â), addition of a concentratedculture supernatant from P fluorescens ATee948 in MRS containing log 5.2 cfu/mL of Lb fer-mentum D 13; (_), addition of a concentratedculture supernatant from P fluorescens M8 not aproducer of proteinaceous antimicrobial com-pound.Effet bactéricide du composé protéinique à acti-vité antimicrobienne produit par P tluorescensATCC 948 contre une souche sensible (Lb fer-mentum DI3); (""), adjonction d'un surnageantde culture concentré de P fluorescens ATCC 948dans MRS contenant log 5,2 cfu/mL de Lb fer-mentum D13; (.), adjonction d'un surnageantde culture concentré produit par P tluorescensM8 non producteur de composé protéinique àactivité antimicrobienne.

A proteinaceous antimicrobial compound from P fluorescens

12 16 20 24

Time (hours)

Fig S. Production of the proteinaceous antimi-crobial compound from P fluorescens ATee 948(_) during the growth period (0).Production du composé protéinique à activitéantimicrobienne par P fluorescens ATCC 948(_) pendant la période de développement (0).

by extracellular proteinases (data notshown).

The optimum pH for producing theantimicrobial compound was 6.5 (fig 6a).The compound produced showed stabilityfrom pH 3.0 to 8.0 (data not shown).

Optimal production of the proteinaceouscompound was at 20 "C (fig 6b), which didnot coincide with the optimal growth ofP fluorescens ATee 948 (data not shown).A relatively high production was alsoobserved at low temperatures (5 to 15 "C),The antimicrobial compound was stableafter 45 days at - 15 "C, but a decrease inactivity was observed on the freeze-driedcompound after 15 days of storage at-20 oc.

Similar results conceming the character-ization of the antimicrobial compound wereobtained by cultivating P fluorescensATee 948 in BN medium or in skim milk.

DISCUSSION AND CONCLUSION

A 67-kDa proteinaceous compound withinhibitory activity against unrelated Gram-negative and Gram-positive species has been

275

25 a

4,5 7,55,5 6 6,5pH

b

'"~

8~60

i-;§ 20~

o5 10 15 20 25 3D 35 40

Temperature (oC)

Fig 6. Effect of pH (a) and temperature (b) on theproduction of the proteinaceous antimicrobialcompound from P fluorescens ATee 948.Effet du pH (a) et de la température (b) sur laproduction du composé protéinique à activitéantimicrobienne produit par P fluorescensATCC948.

extracellularly produced and purified fromP fluorescens .vrcc 948. The inhibitionwas shown to be particularly marked onthermophilic Lactobacillus, and the strainof L monocytogenes tested was alsoinhibited.

Psychrotrophic pseudomonads have beenextensively studied for their production ofantimicrobial compounds, which play animportant role in the biocontrol of plant dis-eases (Dowling and 0' Gara, 1994). Most ofthem (indoles, phenazines, lipids, antibi-otics and pterines) are not of protein natureor represent complex substances in whichthe protein is not the only biologically activemoiety. Siderophores, produced by pseu-domonads, are high-affinity iron transportmolecules which could sequester available

276 M Gobbetti et al

iron and reduce the activity of the electrontransfer proteins, of the enzyme involved inhydrogen peroxide and oxygen metabolism,as weil as the activity of the DNA biosyn-thetic enzymes (Freedman et al, 1989).Weil-diffusion assays conducted in the pres-ence of iron-supplemented media showedinhibitory activity similar to that determinedwithout integration (data not shown), exclud-ing the role of siderophores in the antimi-crobial activity of Pfluorescens ATCC 948.An antagonism linked to bacterial virusescontained in the extracellular culture filtratehas been shown in pseudomonads (Vidaver,1983; Smidt and Vidaver, 1986) and othermicrobial species (De Klerk and Coetzee1961; Betz and Anderson, 1964; Hamadaand Ooshima, 1975). Dilutions of the antimi-crobial preparation from P fluorescensATCC 948 produced smaller zones of inhi-bition and did not increase plaque forma-tion (data not shown), thus also excludingthe presence of viruses in the culture super-natant. Antibiotics and complex toxins(syringomycin and syringotoxin) are alsoproduced by Pseudomonas strains (Iaco-bellis et al, 1992; Dowling and O'Gara1994). The proteinaceous nature of theantimicrobial compound purified in thisstudy, together with the high molecular massand the absence of carbohydrate and lipidmoities (treatments with acetone and n-butanol did not modify the inhibitory activ-ity of P fluorescens ATCC 948 supematant)(Iacobellis et al, 1992) seem to exclude thecompound from this category. Bacteriocinswith features quite similar to those deter-mined for the antimicrobial compound pro-duced by P fluorescens ATCC 948 (activeprotein moiety, molecular mass of - 60 kDa,optimal production at 20 "C and a certaindegree ofthermostability) have been shownin P syringae pv syringae (Smidtand Vidaver, 1986), P syringae susbpsavastanoi (lacobellis et al, 1995),P solanacearum (Cuppels et al, 1978),P phaseolicola and P glycinea (Vidaver etal, 1972). By comparing these studies, the

main difference lies in a narrower spectrumof activity, in sorne cases species-specific,which characterizes the isolated bacteri-ocins. A bacteriocin-like compound recentlycharacterized for P fluorescens from fish(Champomier- Vergès and Richard, 1994),has shown inhibition against related pseu-domonad pathogens. Nevertheless, inhibi-tion against sorne unrelated Gram-negative(Escherichia coli and Erwinia), and Gram-positive (Bacillus cereus and Listeria spp)species and fungi has been shown by a fewstrains of P syring ae, P phaseolicola,P glycinea (Vidaver et al, 1972), P cepacia(Abe and Nakazawa, 1994), P aeruginosa(Rasool and Khan, 1993) and by fluocinsfrom Pfluorescens (Hamon et al, 1961), notexcluding the synthesis of broad range bac-teriocins. Only three strains (ATCC 948,M22 and M40) among the 21 Pfluorescensstrains tested in this study have shown asimilar wide spectrum of activity.

Based on extensive studies of the col-icins produced by Gram-negative bacteria,Tagg et al (1976), and subsequently Jack etal (1995) suggested six criteria for charac-terizing bacteriocins mainly produced byGram-positive microorganisms, and pro-posed the definition 'bacteriocin-likeinhibitory substance' for proteins which arenot widely accepted or genetically charac-terized. Exceptions to these six criteria havebeen noted. Montville and Kaiser (1993)hold that there are only two requisites forbacteriocins: their proteinaceous nature andtheir lack of lethality to cells which producethem. The antimicrobial compound pro-duced by Pfluorescens ATCC 948 is of pro-tein nature, not lethal for the producingstrain, with a bactericidal mode of actionbut which shows a broad spectrum of activ-ity. As a consequence of these features wedecided, before further characterization(mass spectrometry and genetic studies),not to include it in the bacteriocins but tosimply define it as a 'proteinaceous com-pound with a broad range of inhibitoryactivity' .

A proteinaceous antimicrobial compound From P fluorescens

Psychrotrophic pseudomonads have beenextensively studied for their proteolyticenzymes which cause spoilage in dairy prod-ucts (de Koning et al, 1985; Gobbetti, 1993).A correlation between cell growth duringthe cold storage of raw milk and the pro-duction of extracellular proteinases has beenshown (Mottar, 1989). This study showsthat P fluorescens ATCC 948 when grownin skim milk produces an antimicrobial com-pound with inhibitory activity against ther-mophilic Lactobacillus (especially one strainof Lb delbrueckii subsp bulgaricus 8397and of Lb helveticus PR4 were inhibited).Mesophilic Lactobacillus and other lacticacid bacteria strains have a limited sensi-tivity or are insensitive. The proteinaceouscompound, optimally produced at pH 6.5,shows the highest rate of production within72 h of incubation (the synthesis began dur-ing the early exponential phase of growth,log 4.8 cfu/mL), is resistant to pasteuriza-tion, is stable at 15 "C for 45 days and main-tains more than 50% of the maximuminhibitory activity in the temperature rangeof 5 to 10°C, which coincides with a limitedgrowth of the psychrotrophic strain. Eventhough the microbial relationships have to beconsidered in a complex interactivedynamic, these features raise another prob-lem linked to the excessive growth of psy-chrotrophic pseudomonads in raw milk.

Considering its importance in the dairyfield and, in food microbiology, in general(inhibition of foodborne bacteria), massspectrometry and genetic studies of thisantimicrobial compound from PfluorescensATCC 948 are warranted.

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