APPLIED AND ENVIRONMENTAL MICROBIOLOGY,0099-2240/00/$04.0010

Oct. 2000, p. 4318–4324 Vol. 66, No. 10

Copyright © 2000, American Society for Microbiology. All Rights Reserved.

Use of Two-Dimensional Electrophoresis To Study DifferentialProtein Expression in Divercin V41-Resistant and

Wild-Type Strains of Listeria monocytogenesFREDERIQUE DUFFES,1 PAUL JENOE,2 AND PATRICK BOYAVAL1*

Laboratoire de Recherches de Technologie Laitiere, Institut National de la Recherche Agronomique,35042 Rennes Cedex, France,1 and Department of Biochemistry, Biozentrum of

the University of Basel, CH-4056 Basel, Switzerland2

Received 3 April 2000/Accepted 28 July 2000

The use of bacteriocins from food-grade lactic acid bacteria to fight against the food-borne pathogen Listeriamonocytogenes has been gaining interest. However, the emergence of resistant cells is frequently reported whenListeria is exposed to such antibacterials. A two-dimensional electrophoresis study of whole-cell protein ex-pression of Listeria monocytogenes variants sensitive or resistant to the action of a bacteriocin produced byCarnobacterium divergens V41, divercin V41, is reported in this paper. The resistant variant obtained from thesensitive strain of L. monocytogenes P was also resistant to piscicocins V1 and SF668, but remained sensitiveto nisin. Its growth rate was 50% less than the sensitive strain, and the MIC for it was 104 times higher. Noreversion of the resistance was observed after 20 successive cultures in the absence of divercin V41. Compar-ison of the protein patterns by two-dimensional gel electrophoresis analysis showed clear differences. In theresistant variant pattern, at least nine spots had disappeared and eight new ones were observed. One of thenewly synthesized proteins was identified as a flagellin of L. monocytogenes. Direct interaction between flagellinand divercin V41 was not evidenced. Intracellular synthesis of flagellin is probably an indirect effect of a mod-ification in transcriptional regulation with widespread effects through a sigma factor. An intense protein, onlypresent in the sensitive strain, was identified as a non-heme iron-binding ferritin displaying strong similaritiesto Dps proteins. Common modifications in the transcriptional regulation for these two proteins are discussed.

During the past decade, Listeria monocytogenes has beenincriminated in numerous food-borne outbreaks and severalsporadic episodes of listeric illness (25). The emergence andpersistence of L. monocytogenes on a large variety of dairy,ready-to-eat, and processed foods has led to enhanced interestin antimicrobials for its control. In addition to conventionalantimicrobials (organic acids, radiation, packaging, etc.), inter-est in the use of bacteriocins from food-grade lactic acid bac-teria (LAB) has increased. Bacteriocins were defined as ribo-somally produced precursor polypeptides or proteins that, intheir mature (active) form, exert an antibacterial effect againsta narrow spectrum of closely related bacteria. Most of thereported bacteriocins are produced by LAB, which are natu-rally present in a lot of food products or are added for theirtechnological and preserving characteristics (40).

However, in most studies, when Listeria is exposed to suchantibacterial activity, emergence of resistant cells is frequent-ly reported (35). The mechanisms underlying the bacteriocinresistance phenomenon are largely unknown. Because bac-teriocin acts mainly in the cytoplasmic membrane, potentialmodifications of bilayer lipid content and quality have beeninvestigated. Resistance to nisin has been correlated with bothmodified fatty acid and phospholipid composition (27).

Even if differences in protein expression between sensitivetarget cells and resistant cells are potentially numerous, theroles of proteins in bacteriocin resistance are unclear. In sometarget cell species, specific membrane-located bacteriocin re-ceptors of a proteic nature have been identified (42). Modifi-

cations or the absence of such receptors could lead to resis-tance. Some killer toxin-resistant mutants of Saccharomycescerevisiae expressed much smaller amounts of a protein whichacts as a docking protein, facilitating toxin binding to the mem-brane, where it forms lethal ion channels, like bacteriocins do(38). Synthesis of new membrane proteins could interfere withbacteriocin anchorage on the receptor or in the membrane. InLactococcus lactis subsp. lactis biovar diacetylactis, the nisinresistance gene nsr encodes a putative protein with a molecularmass of 35 kDa. A strongly hydrophobic region supports theprediction that this protein is an integral membrane proteinwhich could decrease bacteriocin activity. Decreased bacterio-cin penetration could also appear to result from membraneprotein oversynthesis, as observed by Koch et al. (22) in mul-tidrug-resistant mouse and hamster cells. Synthesis of an en-zyme able to degrade the bacteriocin is also a potential efficientresistance mechanism. Jarvis (20) described a nisinase whichinactivated nisin. Moreover, cell wall proteins could play acrucial role in resistance, as clearly demonstrated by Dielband-hoesing et al. (10) for two cell wall proteins in nisin resistanceof yeast cells. Knowledge of the involvement of proteins inbacteriocin resistance, even if studied in gram-positive bacte-ria, could also highlight the role of outer membrane proteins ingram-negative resistance, which is probably essential, as dem-onstrated for Omp4 for the bacteriocin 28b resistance pheno-type in Escherichia coli (18).

Two-dimensional electrophoresis (2DE) of proteins is cur-rently the highest-resolution analytical technique available forthe study of protein expression patterns. This technique hasalready been used for studying minocycline-susceptible and-resistant Mycobacterium smegmatis (44). Comparative pro-teome analysis of Mycobacterium tuberculosis virulent and non-virulent vaccine strains was carried out with the help of 2DE

* Corresponding author. Mailing address: INRA, Laboratoire deRecherches de Technologie Laitiere, 65 Rue de St Brieuc, 35042Rennes Cedex, France. Phone: 33-(0) 2 23 48 53 39. Fax: 33-(0) 2 23 4853 50. E-mail: boyaval@labtechno.roazhon.inra.fr.

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(21). 2DE can be an important resource in identifying proteinsinvolved in bacteriocin resistance. Thus, 2DE is a powerful toolto highlight the biochemical mechanisms governing develop-ment of cell resistance and then will help in the design of newefficient molecules or mixing of molecules with different celltargets.

In this paper, we report physiological and metabolic differ-ences between Listeria monocytogenes variants sensitive (wildtype) and resistant to the action of divercin V41, a bacteriocinproduced by the LAB Carnobacterium divergens V41. More-over, 2DE was carried out to study differential protein expres-sion in these two characterized variants.

MATERIALS AND METHODS

Bacterial strains, culture conditions, and bacteriocin production. Experi-ments were carried out with Listeria monocytogenes P (serotype 4b), a wild-typesensitive strain isolated from vacuum-packed cold-smoked salmon (Escola Su-perior de Biotechnologia, Porto, Portugal) and its bacteriocin-resistant variant(RV41) obtained as described below. The bacteriocin used for resistance studieswas divercin V41, the bacteriocin produced by Carnobacterium divergens V41(34).

Bacteria were subcultured and cultured overnight aerobically at 37°C in Ellikerbroth (Biokar, Bauvais, France). Growth was determined by optical density at550 nm (OD550) measurements and by enumeration on Elliker agar after incu-bation for 24 h at 37°C.

Divercin V41 was purified as described by Metivier et al. (29).Divercin MIC determination. The MIC of divercin V41 was determined after

growth on Elliker broth or the simulated cold smoked fish system (SCSFS) (12)at 37°C in microtiter plates containing 100 ml of 1% glucose-supplementedMuller-Hinton broth (Biokar) in each well. The total protein content of thepurified bacteriocin stock solution was found to be 700 mg/ml as determined bythe bicinchoninic acid assay (Pierce, Rockford, Ill.). The divercin V41 was seri-ally diluted in Mueller-Hinton broth (1:1). Each well received 100 ml of inoculumsuspension (105 cells per well), and the microtiter plate was incubated overnightat 37°C. After incubation, yellow wells were positive for growth and red wellswere negative. The MIC was arbitrarily defined as the amount of total bacteri-ocin in the highest dilution which inhibited L. monocytogenes growth.

Isolation of a divercin V41-resistant variant of L. monocytogenes. After MICexperiments, L. monocytogenes P cells were picked from the well containing 87.5mg of divercin per ml and grown in divercin-free Elliker medium. One of theresistant variants, Listeria monocytogenes RV41, was isolated on a Palcam plate(selective agar and supplement; Merck, Nogent sur Marne, France), and divercinsensitivity and resistance were verified according to the spotting method de-scribed by Pilet et al. (34). The frequency of isolation of resistant variants wasdetermined according to the method of Dykes and Hastings (13), in the presenceof divercin V41 at five times the MIC.

Antibiogram of the two L. monocytogenes variants. To determine if the resis-tance to divercin confers a particular antibiotic phenotype to the resistant vari-ant, 19 antibiotics (Table 1) acting on different cell targets were tested. Themethod used was based on the disk technique using Mueller-Hinton medium (3).

Serotyping and lysotyping of the two L. monocytogenes variants. Serotyping ofListeria monocytogenes strains was performed as described previously (39). Phagetyping was carried out according to reference 37 with 29 well-characterizedphages isolated from lysogenic strains.

Two-dimensional PAGE. 2D polyacrylamide gel electrophoresis (PAGE) ex-periments were performed essentially according to the method of Gormond andPhan-Thanh (16). When not indicated, chemicals and materials were from Phar-macia-Biotech (Orsay, France).

Sample preparation. Sensitive Listeria monocytogenes and RV41 cells weregrown at 37°C until the middle exponential phase (OD550 5 0.5, which corre-sponds to 109 CFU/ml) in 10 ml of Elliker broth. Cells were harvested bycentrifugation (4,000 3 g, at 25°C for 30 min). Pellets were washed three timesin 10 ml of physiological water (8.5 g of NaCl per liter) and recovered in 0.2 mlof Tris (2-amino-2-hydroxymethyl-1,3-propanediol) buffer (10 mM, pH 7.2) con-taining 5 mM Mg21, 5 ml of a cocktail of protease inhibitors containing leu-peptin, pepstatin, and PEFABLOC [4-(2-aminoethyl)-benzenesulfonyl fluo-ride hydrochloride; Boehringer, Mannheim, Germany] at 2 mg/ml, 5 ml ofdeoxyribonuclease and ribonuclease (100 mg/ml; Boehringer), and 20 ml of asolubilization solution containing 20% CHAPS {3-[(3-cholamidopropyl)-dimeth-ylammonio]propane sulfonate}, 10% DTT (dithiothreitol) and 20% IPG (im-mobilized pH gradient) 4/7 buffer. Cells were sonicated (Vibra cell; Bioblock,Illkirch, France) three times for 1 min at a power setting of 5 and 50% pulse at4°C. The mixture was incubated at room temperature for 30 min. Urea (7 M) wasadded, samples were vigorously agitated (Vortex level 5) at room temperaturefor 15 min, and the mixture was finally centrifuged (20,000 3 g at room temper-ature for 20 min). Supernatants could be stored at 220°C. Proteins were quan-tified by the Bradford method (5).

IEF. The 1D separation was carried out on immobilized pH gradients (4/7Immobilin dry strips of 18 cm) as described by Gorg et al. (15) with Multiphor IIapparatus. The following voltage gradient was applied: from 0 to 50 V in 0.02 h;50 V for 1 h; from 50 to 150 V in 0.02 h; 150 V for 1 h; from 150 to 300 V in0.02 h; 300 V for 2 h; from 300 to 3,500 V in 5 h; and 3,500 V for 11 h. Proteinsamples (100 mg) were loaded into cups at the anode end. After isoelectricfocusing (IEF), strips were equilibrated in a solution containing 50 mM Tris (pH6.2), 6 M urea, 30% glycerol (vol/vol), 2% (wt/vol) sodium dodecyl sulfate (SDS),and 0.3% (wt/vol) DTT followed by a second bath with the same solution, butwith 4.5% (wt/vol) iodoacetamide in place of DTT.

SDS-PAGE. The 2D separation was performed, essentially according to themethod of Laemmli (24), in an IsoDalt apparatus (Hoeffer, San Francisco, Calif.)with a 14% acrylamide separating gel, but without a stacking gel and at constantvoltage (below 180 V). Large plate gels (200 by 250 by 1 mm) were used toimprove resolution. Ten gels were run simultaneously in one tank to improvereproducibility. A low-molecular-weight electrophoresis calibration kit (Amer-sham-Pharmacia-Biotech, Buckinghamshire, England) was used for protein mo-lecular mass (daltons) reference standards (phosphorylase b, 94,000; bovineserum albumin, 67,000; ovalbumin, 43,000; carbonic anhydrase, 30,000; soybeantrypsin inhibitor, 20,100; and a-lactalbumin, 14,400).

1D SDS-PAGE was also performed (Protean II or Mini-Protean; Bio-Rad,Paris, France). Samples were diluted twice in a mixture of 62.5 M Tris-HCl (pH6.8), 10% glycerol, 2% SDS, and 5% b-mercaptoethanol and heat treated at100°C for 2 min before loading (20 ml) on wells of SDS-PAGE gels at a 14%polyacrylamide concentration.

In the same way, delay gels were performed with flagellin extracted from cellsgrown at 20 and 37°C (see below) and purified divercin V41. Interaction studieswere performed with flagellin extract of RV41 (200 mg) and purified divercinV41 (3.5 mg) in phosphate-buffered saline (PBS) (0.01 M sodium phosphate with0.15 M sodium chloride [pH 7.6]) for 10 min at room temperature. Separationswere carried out in either the presence or absence of SDS to visualize interac-tions between the two proteins.

Gels were stained either with silver (41) or with Coomassie brilliant blue(PhastGel Blue R; Amersham-Pharmacia-Biotech) and stored in a 20% ethanolsolution at 4°C for several weeks.

Analysis of protein spots on 2D gels. Images were scanned (CanoScan FB620P, Canon, France). Protein gel analysis was performed with Melanie II 2D PAGEsoftware (release 2.2; Bio-Rad, Ivry sur Seine, France). Reference points (land-marks) were marked on images to align and match gels. After gel alignment andmatching, pairs (spots present in several gels) could be highlighted. The reversefunction evidenced the differences between each gel. Ten gels per sample wereanalyzed and compared. Spots present in at least nine gels were considered to beconsistent spots and were taken into account.

Protein identification. Coomassie blue-stained proteins which had been sep-arated by 2D gel electrophoresis were excised and washed five times with 30 mleach of 40% n-propanol, followed by five washes with 30 ml each of 0.2 Mammonium carbonate containing 50% acetonitrile. The gel pieces were com-pletely dried under reduced pressure (Speed-Vac; Savant). To the dried gel

TABLE 1. Antibiogram spectrum of L. monocytogenessensitive and resistant to divercin V41

Antibiotic Target

L. monocytogenesresponse to

divercin V41a

Sensitive Resistant

Penicillin Peptidoglycan S SAmoxicillin Peptidoglycan S SImipenem Peptidoglycan S SOxacillin Peptidoglycan R RCeftriaxone Peptidoglycan R RCefoxitin Peptidoglycan R RAztreonam Peptidoglycan R RVancomycin Peptidoglycan S STeicoplanin Peptidoglycan S SFosfomycin Peptidoglycan S SColistin Outer membrane of gram-

negative isolateR R

Pefloxacin Gyrase inhibitor S SRifampin ARN polymerase inhibitor S SChloramphenicol Protein synthesis inhibitor S SErythromycin Protein synthesis inhibitor S SKanamycin Protein synthesis inhibitor S SGentamicin Protein synthesis inhibitor S STetracycline Protein synthesis inhibitor S SMinocycline Protein synthesis inhibitor S S

a R, resistant; S, sensitive.

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pieces, 0.5 mg of trypsin in 10 ml of 100 mM ammonium carbonate (digestionbuffer) was added to allow reswelling of the gel pieces. About 15 ml of digestionbuffer was added to completely immerse the gel pieces. Digestion was carried outat 37°C for 2 h. The supernatant was collected, and the gel pieces were extractedwith 15 ml of 0.1% formic acid, followed by 15 ml of acetonitrile. Extraction wasrepeated twice, and all supernatants were pooled and dried in a Speed Vac. Tobe able to remove supernatants from the gel pieces, two 500-ml Eppendorf tubesarranged concentrically were used. The tube containing gel pieces was piercedwith a hypodermic needle to generate a hole large enough to allow the liquid tobe centrifuged into the lower tube, but to retain the gel pieces. Also in order toavoid possible cross-contamination, all buffers and wash solutions were pipettedwith clean Hamilton syringes, which were reserved solely for the handling of thesame solutions, making sure that the needle never touched the gel pieces.

For mass spectral analysis, the peptides were dissolved in 10 ml of 0.1%trifluoroacetic acid, and 5 ml was used for identification. Separation of the digestswas carried out on 100-mm capillary columns which were packed with POROSR2 material (PerSeptive Biosystems, Framingham, Mass.). In short, fused silicacapillaries (100 by 280-mm LC Packings; Polymer Laboratories, Marseille,France) were drawn to an aperture of 1 to 2 mm on a laser puller (SutterInstruments, Science Products AG, Geneva, Switzerland). A column frit wasconstructed by introducing a few grains of 5-mm-diameter silica beads. ThePOROS material was then packed into the capillary with the aid of a stainlesssteel reservoir connected to a high-performance liquid chromatography pumpcapable of delivering pressures up to 6,000 lb/in2. After the packing process, thecolumns were cut to approximately 2.5 cm and inserted into a microsource. Themicrocolumns were developed with a linear gradient of 0.02% acetonitrile to80% methanol containing 0.02% acetic acid in 15 min at a flow rate of approx-imately 200 nl/min. Mass spectral data were acquired on a TSQ7000 triplequadrupole instrument (Finnigan, San Jose, Calif.) with data-controlled switch-ing between precursor ions and daughter ions during a single chromatographicrun. For precursor ion scanning, the resolution of the instrument was set to 1 Da.For operation in the MS/MS mode, the resolution of Q1 was set to transmit awindow of 4 Da, and the resolution of Q3 was adjusted to 1.5 Da. Daughter ionscanning was performed between 50 and 2,000 Da in 3.5 s. Argon was used as thecollision gas at a pressure of 3.0 Torr. The collision energy was kept constant at232 eV during individual runs. The sequences of trypsic peptides were comparedto known Listeria peptides on the sequence database SwissProt.

Preparation of crude flagella. One liter of culture was harvested by centrifu-gation at 5,000 3 g, washed twice in a 0.01 M sodium phosphate–0.15 M sodiumchloride (pH 7.6) (PBS) buffer, and resuspended at a ratio of 5 ml of PBS/literof broth culture. Samples, in bottles containing glass beads, were shaken vigor-ously for 30 min at 20°C. The suspension was centrifuged at 5,000 3 g for 15 min,and the supernatant was retained. The pellet was washed twice with PBS byvigorous pipetting to remove sheared flagella trapped within the cell mass. Thesupernatant and cell washing were pooled and centrifuged at 14,000 3 g for 40min to clear the remaining bacteria, and the resulting supernatant was centri-fuged at 200,000 3 g for 90 min to harvest crude flagella (33). One milligram ofcrude flagellar protein was recovered in each experiment and solubilized in 50 mlof PBS.

Sample preparation for scanning electron microscopy. Five milliliters of thecultures was filtered over membranes (0.2-mm-pore-diameter GTTP 01300; Mil-lipore Polycarbonate, Molsheim, France). Membranes were loaded on the sur-face of a 0.1 M sodium cacodylate (pH 7.2) buffer containing 2.5% glutaralde-hyde. Cells or flagella were fixed for at least 48 h at room temperature.Membranes were dehydrated in successive baths of ethanol (10, 25, 50, 75, 95,and 100%) for 10 to 20 min. Samples were desiccated by introducing them intoa pressurized enclosure where ethanol was replaced by liquid CO2 (10°C). Sam-ples were desiccated by heating until the critical point was reached (31°C and73.8 bars) without deterioration and then were metalized with gold and observed.

RESULTS AND DISCUSSION

Physiological comparison of Listeria monocytogenes sensitiveand RV41 variants. Sensitive and resistant variants present thesame phenotypic characters, the same serotype (4b), and thesame lysotype (1444, 1317, 3274, 2671, and 340) (data notshown). Thus, no drastic surface modifications could be pos-tulated on these bases. The MICs for the sensitive and RV41strains were determined after growth on Elliker and SCSFSbroths. In Elliker broth, the MIC for the resistant variant wasat least 104 times higher than that for the sensitive strain(higher than 104 and 0.01 mg/ml21, respectively). This resultwas in accordance with that obtained by Metivier (28) withL. monocytogenes Scott A. In the SCSFS medium, the MIC forRV41 was 106 times higher than that for the sensitive strain(higher than 104 and 5.7 1025 mg/ml21, respectively). For thelatter strain, the MIC was 175 times higher on Elliker broththan on SCSFS. These results could be explained by two facts.

(i) SCSFS is a less nutritive medium in which L. monocytogeneshad difficulty in growing, and thus it was more sensitive tobacteriocin activity. (ii) Elliker broth contains many more mol-ecules which are able to interfere with the bacteriocin andartificially decrease the number of molecules free to interactwith the target cells. These observations underlined the impactof environmental conditions on bacterial growth and bacterialsensitivity to antibacterial agents.

The frequency of appearance of resistance was 3.5 3 1025 atfive times the MIC. The stability of the resistant variant wastested against divercin V41, and no reversion of the resistancewas observed after 20 successive cultures in the absence ofdivercin V41 as observed by Rekhif et al. (35) for mesentericin52, curvaticin 13, and plantaricin C19. On the contrary, Dykesand Hastings (13) found a reversion frequency within the rangeof 1024 to 1025 with leucocins A, B, and E and sakacin A. Thestability or instability of the resistant phenotype remained un-explained, but it is reasonable to argue for several resistancemechanisms among the different species, and possibly withinthe same species, leading to different mechanisms and thusdifferent frequencies of reversibility.

The resistant variant obtained from the wild-type strain ofL. monocytogenes P was resistant to divercin V41, piscicocinV1, and piscicocin SF668, but kept its sensitivity to nisin (datanot shown). The cross-resistance between nonlantibiotic bac-teriocins has been already observed by Rekhif et al. (35) andMetivier (28). The difference in sensitivity of RV41 to divercinV41 and nisin has been correlated with a potential difference inthe mode of action of lantibiotics and nonlantibiotic bacterio-cins.

The resistance or sensitivity of both strains to several anti-biotics, acting on different cell targets was investigated to ob-serve potential differences or similarities between bacteriocinand antibiotic resistance. The results are presented in Table 1and show, first, that there are no differences between the twospectra and, second, that the resistances or sensitivities mea-sured correspond to the common phenotype of L. monocyto-genes 4b. These results show that resistance to divercin doesnot confer any resistance to the antibiotics tested. This pointhad already been observed for the nisin resistance phenotype(8).

No clear morphological difference was observed between thesensitive and RV41 variants either by optical or by scanningelectron microscopy (data not shown). The absence of mor-phological differences between sensitive and divercin-resistantcells was in accordance with the results of Crandall andMontville (8) on L. monocytogenes ATCC 700302 resistant tonisin.

Comparative growth of the L. monocytogenes sensitive andresistant variants in Elliker broth at 37°C is represented in Fig.1. The sensitive strain had a 4-h lag phase, followed by a rapidexponential phase (m 5 0.13 h21) up to the 10 hours of growth.For the resistant variant, the lag phase was shorter (2 h), butthe growth rate was twofold lower (m 5 0.07 h21). The de-crease in the growth rate could be explained by the energy costof the potential resistance metabolic pathway(s) which reducesthe fitness for growth (13). However, this rather importantdifference in growth efficiency could be the result of a morewide alteration of the cell metabolism than only a difference inbacteriocin resistance, which is the unique phenotypic charac-teristic observed here.

Comparison of the protein patterns of L. monocytogenes sen-sitive and RV41 variants. No significant difference was ob-served between the two 1DE patterns of the total proteinsfrom the two variants of L. monocytogenes P (data not shown).

The protein extractability was similar for both, as demon-

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strated by the protein content of preparations (2.10 6 0.12 and2.14 6 0.11 mg/ml for RV41 and the sensitive strain, respec-tively). When minor differences between sample preparationsoccurred, standardization of the amount of protein of the sam-ples was performed before 2DE experiments in order to allowdirect comparison of the patterns.

Ten 2D gels were run for each L. monocytogenes variant.Figure 2 shows two typical patterns of the L. monocytogenesstrain sensitive to divercin V41 (Fig. 2a) and the resistantvariant (Fig. 2b). 211 and 278 spots could be detected on eachgel, respectively. Silver staining is not quantitative; only pro-teins present or absent on nine of the two patterns were con-sidered. Arrows indicate the main differences between the twopatterns and were determined after computer-assisted analysiswith Melanie II software.

Nine intense spots, lacking in the resistant pattern, werechosen in the sensitive strain pattern (Fig. 2a). Three of themare small (,20 kDa) and acidic (pI of ,5.20) proteins; fourhave average size (about 30 kDa) proteins with a pI rangingfrom 4.6 to 5.8; two are bigger proteins (,35 kDa) with pIshigher than 5.4 and 5.85. In parallel, in the protein patternderived from the resistant strain, we selected eight new andintense protein spots absent in the sensitive one. All of theseproteins have a molecular mass ranging from 25 to 65 kDa anda pI between 4.5 and 5.8 (Fig. 2b).

Among the spots analyzed by mass spectroscopy, only spotsR1 and S86 were identified as L. monocytogenes or L. innocuaproteins, respectively (Table 2). The masses and the segmentsequences of the other spots did not share any homology withidentified proteins of Listeria spp. (available in the SWISSPROT database). The protein R1, present only in the RV41variant, has been identified as a flagellin of L. monocytogenescharacterized by Dons et al. (11). Flagellin is the main com-ponent of the flagellar filament. This is the engine of mobilebacteria which allows movement to high-nutrient-concentratedzones or away from toxic substances. How could flagellin beinvolved in the bacteriocin resistance phenomenon? Two hy-potheses are presented: the flagella could act directly in theresistance phenomenon. It could play the role of a biologicalmagnet, attracting, by electrostatic forces (14), molecules ofdivercin which then become unavailable to interact withL. monocytogenes membrane. A second hypothesis is that inRV41 cells, flagellin synthesis is indirectly affected by modifi-cation of gene control expression with widespread effects.

In order to assess if flagella were directly implicated inbacteriocin resistance, the MICs for sensitive, RV41, andRV41 flagellum-free (obtained by vigorous shaking and cen-

trifugation) strains were determined at 20 and 37°C, temper-atures at which cells are mobile and immobile, respectively(33). No change in MICs was evidenced between the two tem-peratures and between RV41 and flagellum-free RV41 (datanot shown).

The physical interaction between flagellin and divercin V41was explored. Figure 3 shows that no migration delay wasobserved when flagellin and divercin V41 were comigrated inelectrophoresis experiments under native and SDS conditions.This suggests that, under our experimental conditions, there isno interaction between these two molecules. The presence offlagella on the RV41 cell surface was investigated. Opticalmicroscopy showed that, for whichever variant considered,L. monocytogenes cells were mobile at 20°C and not at 37°C.Scanning electron microscopy confirmed that both L. monocy-togenes sensitive and RV41 cells possessed flagella on theirsurface at 20°C and not at 37°C. Thus it could be postulatedthat flagellin is present in a large amount in the intracellularfraction of the RV41 cells, cultivated at 37°C, but flagellinexport at the cell surface does not occur. Thus direct involve-ment of flagellin in the resistance phenomenon is doubtful,except, maybe, it plays a potential unknown role at the internalface of the cytoplasmic membrane.

Variation in the level of flagellin synthesis in RV41 is prob-ably the result of modification(s) in the transcriptional regula-tion of this protein. This hypothesis is based first on the factthat flagellin synthesis is thermoregulated at the transcriptionallevel (35). In our experiments, flagellin synthesis is repressed at37°C in the sensitive variant and seems derepressed in the re-sistant one. A second element of response is based on theresults obtained by Robichon et al. (36). They found a mesen-tericin Y105-resistant phenotype of L. monocytogenes obtainedby transposition insertion. The insertion of the transposon wasin the rpoN gene encoding an alternative transcriptional s54

factor. s54 is known to be involved in the control of manygenes, including some genes of flagellar synthesis (36).

The flagellin role is of particular interest, because thisprotein has been hypothesized to increase the virulence ofL. monocytogenes (9). PrfA, the transcriptional activator ofvirulence genes, which is maximally expressed at 37°C, downregulates motility genes in Listeria (31). If alteration(s) oc-curred in the transcriptional regulation of these genes, leadingto flagellin synthesis as observed in our variant, we supposethat other genes encoding proteins directly involved in sensi-tivity and resistance phenomena are also deregulated. More-over, such genes encoding entry and belonging to a multigenefamily have been observed near the virulence genes, on thesame notA fragment of the physical map of L. monocytogenes(30). The expression of the gene encoding flagellin is fre-quently reported as being not only temperature regulated, butalso influenced by stresses such as osmotic stress (19). In E.coli, the RNA polymerase sF subunit, involved in the transcrip-tion of the flagellar and chemotaxis genes, possesses a strictpromoter recognition property as found for minor sigma sub-units involved in stress response. The transcription efficiency issalt dependent (23). Could we postulate that bacteriocin isconsidered by the target cell as a stress, like heat or osmoticstress, and that the cell response then uses the same kinds ofmechanisms? This is an open question. This comparison wasalso suggested by O’Connor et al. (32), studying the responseof Salmonella enterica serovar Typhimurium to deleteriousconditions, including, besides oxidative and osmotic stresses,exposure to toxic cationic peptides. The regulation of the pro-teins involved in these resistance mechanisms is complex andoverlapping. Moreover, a recent report (43) demonstrated thatacid-adapted L. monocytogenes cells exhibit increased toler-

FIG. 1. Growth curves of the L. monocytogenes wild type (solid symbols) andthe resistant variant (open symbols) on Elliker broth at 37°C.

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ance toward nisin and lacticin 3147. These results suggest com-mon cell responses toward both types of attack.

Spot S86, only present in the sensitive strain, has been iden-tified as a DNA-binding protein already described by Bozzi etal. (4) in Listeria innocua. This non-heme iron-binding ferritinis able to sequester many iron atoms inside the protein cage.Bacteriocin activity leads to intracellular ion leakage throughthe altered membrane. The absence of such iron-chelating

intracellular systems, as observed in the resistant variant, couldbe a major problem for the cell attacked by bacteriocin mole-cules, but the mechanisms are unknown. Divalent cations(Mg21, Ca21, Mn21, and Ba21) increased the resistance of anisin-resistant strain of L. monocytogenes Scott A in a concen-tration-dependent manner (8). Iron was not tested. In theirdiscussion, the authors described a model in which cations mayinterfere with the lipids of the membrane and the cell wall.

FIG. 2. Silver-stained 2D gels of 100 mg of total proteins of L. monocytogenes strains sensitive (a) and resistant (b) to the divercin V41. Arrows indicate noncommonproteins. Mr, molecular mass.

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However the decreased bactericidal activity of lactostrepcin 5on Streptococcus cremoris in the presence of Mg21 and Ca21

was attributed to their stimulative role on membrane-boundATPase (45).

Moreover, this ferritin shows strong similarities to Dps pro-teins. These stress-induced widespread conserved polypep-tides, present in diverse groups of bacteria, are involved inDNA protection during oxidative stress (26). Proteins inducedby stress are considered to be members of global regulatorynetworks which comprise multiple unlinked genes and operonscoordinately controlled by a common regulatory signal. InEscherichia coli, mutant cells lacking Dps show dramaticchanges in the pattern of proteins synthesized during starva-tion. This result prompted Almiron et al. (1) to postulate thatDps plays a role in gene expression.

Altuvia et al. (2) found that Dps mRNA levels were con-trolled by RpoS and s70 factors. These data had to be linkedwith the conclusions of Robichon et al. (36) on s54 involve-ment in mesentericin resistance and confirm that genes respon-sible for divercin resistance are controlled by sigma factors.The role of Dps in bacteriocin sensitivity remains unexplored,

but is of special concern because of the importance of iron inthe infection process caused in human cells by L. monocyto-genes (17). In Bacillus subtilis, mrgA, encoding a Dps protein, isa gene repressed by metal irons (6). Expression of virulencegenes (inl) is positively iron regulated at the transcriptionallevel in L. monocytogenes (7). It could be interesting to test theinfluence of iron on L. monocytogenes sensitivity or resistanceto bacteriocins and strain virulence.

We are currently trying to establish a relationship betweenvariants exhibiting different levels of resistance and a quanti-tative evolution of the observed differences between sensitiveand resistant variants. Experiments to determine the role oftranscriptional regulation with respect to resistance acquisitionare under way, and the results will be reported later.

We will also investigate the resistance phenotype through,on one hand, identification of all the proteins detected ashighly repressed or oversynthesized in the resistant clone, and,on the other hand, 2DE of specially extracted membrane pro-teins from purified membrane fractions of wild and resistantvariants.

ACKNOWLEDGMENTS

We thank Jane Hall for improving the English of this paper; J.Rocourt for serotyping and lysotyping of strains; G. Jan, M. Bossis, andL. Phan-Than for help with 2D gel electrophoresis training; ThierryMini for mass spectroscopy analyses; and J. Berrier for expertise inelectron microscope studies.

F.D. was given a grant by the European Community (FAIR CT95-1207).

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TABLE 2. Characterization of R1 and S86 proteic spots

Spot Mol mass(Da) Amino acid sequencea

R1 30,445 MKVNTNIISLKTQEYLRKNNEGMTQAQERLASGKRINSSLDDAAGLAVVTRMNVKSTGLDAASKNSSMGIDLLQTADSALSSMSSILQRMRQLAVQSSNGSFSDEDRKQYTAEFGSLIKELDHVADTTNYNNIKLLDQTATGAATQVSIQASDKANDLINIDLFNAKGLSAGTITLGSGSTVAGYSALSVADADSSQEATEAIDELINNISNGRALLGAGMSRLSYNVSNVNNQSIATKASASSIEDADMAAEMSEMTKYKILTQTSISMLSQANQTPQMLTQLINS

S86 18,048 MKTINSVDTKEFLNHQVANLNVFTVKIHQIHWYMRGHNFFTLHEKMDDLYSEFGEQMDEVAERLLAIGGSPFSTLKEFLENASVEEAPYTKPKTMDQLMEDLVGTLELLRDEYKQGIELTDKEGDDVTNDMLIAFKASIDKHIWMFKAFLGKAPLE

a Boldface letters represent sequence obtained by mass sequence analysis oftryptic peptides.

FIG. 3. Flagellin expression and interaction with divercin V41 of sensitiveand RV41 samples cultivated at 37°C. Lanes: 1, flagellin extract of the sensitivevariant; 2, flagellin extract of sensitive strain plus divercin V41 (3.5 mg); 3,molecular mass standards (kilodaltons); 4, divercin V41 (3.5 mg); 5, flagellinextract of RV41 (200 mg) plus divercin V41 (3.5 mg); 6, flagellin extract (200 mg)of RV41. Mr, molecular mass.

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