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Applied and Environmental Microbiology, June 2002, p. 2910-2916, Vol. 68, No. 6
0099-2240/02/$04.00+0     DOI: 10.1128/AEM.68.6.2910-2916.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.

Variations in the Membrane Fatty Acid Composition of Resistant or Susceptible Leuconostoc or Weissella Strains in the Presence or Absence of Mesenterocin 52A and Mesenterocin 52B Produced by Leuconostoc mesenteroides subsp. mesenteroides FR52

Maxime Limonet,1 Anne-Marie Revol-Junelles,1* and Jean-Bernard Millière1,,2

Laboratoire Bioprocédés Agro-Alimentaires, Ecole Nationale Supérieure d'Agronomie et des Industries Alimentaires, Institut National Polytechnique de Lorraine (ENSAIA-INPL), F-54505, Vandoeuvre-lès-Nancy Cedex,1 Institut Universitaire de Technologie (IUT) de Nancy-Brabois, Le Montet, F-54600 Villers-lès-Nancy, France2

Received 6 December 2001/ Accepted 4 March 2002


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ABSTRACT
 
Mesenterocins 52A (Mes52A) and 52B (Mes52B) are antimicrobial peptides produced by Leuconostoc mesenteroides subsp. mesenteroides FR 52. Mes52A is a class IIa bacteriocin of lactic acid bacteria with a broad spectrum of activity. Mes52B is an atypical class II bacteriocin with a narrow spectrum of activity. Four Leuconostoc and Weissella wild-type strains were selected for their susceptibility or insensitivity to these mesenterocins. Four strains resistant to Mes52A or Mes52B were generated from the three susceptible wild-type strains by increasing bacteriocin concentrations in culture media. These resistant strains were at least 30 times more resistant than the wild-type strains. No cross-resistance to Mes52A and Mes52B was observed in these strains. No significant differences in membrane fatty acid composition were observed among the three susceptible wild-type strains and the four resistant strains cultured in MRS broth. Thus, the mesenterocin resistance is unlikely to be due to changes in membrane fatty acid composition. When cultured with Mes52A or Mes52B, the membranes of insensitive and resistant strains contained more saturated fatty acids (1 to 10% more) and less unsaturated fatty acids (3 to 6% less), resulting in a more rigid membrane. Thus, the presence of mesenterocin in the culture media of insensitive or resistant strains induced a significant increase in saturated fatty acid contents and a decrease in unsaturated fatty acid contents. Weissella paramesenteroides DSM 20288BR, resistant to Mes52B, responded atypically, probably due to the production of an inhibitor.


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INTRODUCTION
 
Some lactic acid bacteria, defined to be generally recognized as safe, produce bacteriocins that are industrially interesting antimicrobial substances. These small peptides have inhibitory activity against related bacterial species. In 1993, Klaenhammer (14) proposed a classification of lactic acid bacterial bacteriocins based on their modes of action and their structures. In this classification, class II bacteriocins are small, heat-stable, nonlanthionine peptides consisting of 30 to 60 amino acids (<10 kDa). Subclass IIa contains peptides with the consensus sequence YGNGV near the N terminus that are active against Listeria strains.

The lethal activity of bacteriocins involves three steps (peptide binding, [ii] peptide insertion and association, and [iii] pore formation) leading to intracellular compound efflux (1, 7, 8, 19). To investigate this mechanism of action, resistant strains were generated from susceptible wild-type strains by two methods: (i) culture of the wild-type strain in the presence of a large amount of bacteriocin (4, 18) and (ii) culture in media with increasing bacteriocin concentrations (11, 16, 28). Some induced resistance provides cross-resistance to other bacteriocins (4, 21). Some authors have therefore concluded that resistance to a class IIa bacteriocin protects against the lethal activities of other bacteriocins of this class (6, 22, 27). The resistance mechanism seems to be complex. In 1998, Crandall and Montville (4) put forward a model for the nisin resistance of Listeria monocytogenes ATCC 700302 which included three factors: (i) variation of peptidoglycan composition (15), which should make it possible to increase the binding of divalent cations that should interact with the cationic peptide; (ii) modification of the electric charge of the membrane by phospholipid content changes, thereby preventing pore formation (4, 18, 26); and (iii) increase in membrane rigidity, preventing peptide insertion and association (18).

All these hypotheses were essentially developed with nisin (class I) and Listeria monocytogenes as the target strain. These studies were performed with only one susceptible wild-type strain, one or more resistant strains generated from this wild-type strain, and only one bacteriocin. None included an insensitive strain, precluding comparison of the mechanisms of natural and induced resistance. The absence of such a strain prevented studies of the effects of bacteriocin independently of resistance.

Mesenterocins 52A and 52B (Mes52A and Mes52B) are produced by Leuconostoc mesenteroides subsp. mesenteroides FR52 (23). Mes52A is a 37-residue class IIa peptide, identical to mesentericin Y105, produced by L. mesenteroides Y105 (13). Mes52B is a 32-amino-acid class II peptide, identical to dextranicin J 24 produced by Leuconostoc mesenteroides subsp. dextranicum J 24 (22), and to mesentericin B105 from L. mesenteroides Y105 (12). This peptide is atypical, because no class II subclass corresponds to it. The amphipathic and cationic characters and {alpha}-helix conformation of these peptides are related to their mechanism of action, with the cell membrane as the primary target (3, 9, 10).

The aim of this work was to study the effects of both natural and induced resistance to bacteriocins from different classes on the fatty acid contents of cell membranes. The model was made with two bacteriocins (Mes52A and Mes52B) and eight target strains from the genera Leuconostoc and Weissella which were insensitive or had been made resistant to one mesenterocin.


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MATERIALS AND METHODS
 
Bacterial strains and culture conditions.
The 35 wild-type strains used were obtained from public collections (Collection de l'Institut Pasteur [CIP], Paris, France; Deutsche Sammlung von Mikro-Organismen und Zellkulturen [DSM], Göttingen, Germany; and Institut National de la Recherche Agronomique [INRA], Jouy-en-Josas, France) and from our collection (Laboratoire de Microbiologie Alimentaire [LMA], Vandoeuvre-lès-Nancy, France) (Table 1). Lactobacillus, Leuconostoc, and Weissella strains were grown in MRS broth (Biokar, Beauvais, France), and Listeria spp. were grown in Trypticase soy broth (BioMérieux, Marcy l'Etoile, France) supplemented with 6 g of yeast extract (Biokar) · liter-1. These strains were stored at -24°C without prior growth. The four resistant strains (designated AR or BR for Mes52A- or Mes52B-resistant strains, respectively) were usually grown in 90% MRS broth (concentrated by a factor of 1.11) supplemented with 10% (vol/vol) phosphate buffer (5 mM; pH 6.5; Labosi, Elancourt, France) containing the bacteriocin. Mes52A and Mes52B were prepared at concentrations equal to the MIC for the wild-type strain. They were stored in MRS broth with 30% (vol/vol) glycerol (Prolabo, Fontenay, France). Before use, the 39 strains were cultured for two consecutive periods of 16 h in 10 ml of adapted broth at 30°C.


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  		TABLE 1. Activity spectra of mesenterocins 52A and 52B

Mesenterocin activity.
Bacteriocin activity was determined by the agar well diffusion method against the 35 target strains (Table 1), as previously described (16). The activities of mesenterocins 52A and 52B were quantified using Listeria ivanovii LMA 94 and Weissella paramesenteroides DSM 20288T, respectively, as target strains (23). For MIC determinations, 20 µl of a 1:2 dilution series of a bacteriocin solution was placed in wells. The MIC (in arbitrary units [AU] · milliliter-1) was defined as the lowest concentration of bacteriocin that induced an inhibition zone.

Bacteriocin production and purification.
As indicated above, L. mesenteroides subsp. mesenteroides strain FR 52 produces Mes52A and Mes52B. However, as Mes52B extraction from the culture supernatant was difficult, L. mesenteroides subsp. mesenteroides strain J 24 was used for Mes52B production. These two producer strains were grown overnight at 25°C in flasks containing 2 liters of MRS broth. Bacteriocins were extracted from producer cells and culture broths by the method of Yang et al. (28) as modified by Revol-Junelles et al. (23). After extraction from the L. mesenteroides subsp. mesenteroides FR 52 culture supernatant, 1.5 ml of sample was purified by high-performance liquid chromatography, as described by Revol-Junelles et al. (23). Fractions (1 ml) were collected and tested for their biological inhibitory activity. Fractions active only against Listeria ivanovii LMA 94 were collected and concentrated by a factor of 10 under vacuum (Büchi 461; Rungis, France) at 30°C to obtain an activity of about 7,000 AU · ml-1. After extraction from L. mesenteroides subsp. dextranicum J 24 culture supernatant, samples (90 ml) were freeze-dried (Leybold-Heraeus, Koln, France) to obtain approximately 600 mg of powder containing Mes52B at a concentration of 180 AU · mg-1. The samples were stored at -24°C until they were used.

Selection of mesenterocin-resistant strains.
To obtain resistant strains, three wild-type strains, L. mesenteroides subsp. mesenteroides LMA 7, W. paramesenteroides DSM 20288T, and Leuconostoc pseudomesenteroides CIP 103316T, were cultured in MRS broth with increasing concentrations of bacteriocin corresponding to 1:2 and then to one-, three-, and sixfold the MIC. The stability of these resistances in cultures without mesenterocin was checked and determined by MICs.

Cross spectrum of activity.
The four wild-type strains (L. mesenteroides subsp. mesenteroides LMA 7, W. paramesenteroides DSM 20288T, L. pseudomesenteroides CIP 103316T, and Leuconostoc citreum CIP 103405), the two bacteriocin-producing strains (L. mesenteroides subsp. mesenteroides FR 52 and L. mesenteroides subsp. dextranicum J 24), and the target strain, Listeria ivanovii LMA 94, were cultured in 10 ml of adapted broth at 30°C for 16 h. One milliliter of culture was centrifuged (10,000 x g; 10 min; 4°C), and the inhibitory activity of the supernatant was assessed as previously described against these seven strains. The diameters of inhibition zones were measured.

Cellular fatty acid determination.
Wild-type strains were subcultured twice in MRS broth at 30°C, and resistant strains were subcultured in the presence of bacteriocin. Broth (300 ml) consisting of 270 ml of MRS concentrated by a factor of 1.11 and 30 ml of phosphate buffer (5 mM; pH 6.5), containing bacteriocin as indicated, was inoculated with the appropriate strain. To ensure the stability of membrane fatty acid composition, cultures were stopped in the stationary phase, after 24 h of incubation. The cells were harvested by centrifugation (17,700 x g; 10 min; 4°C) and washed twice in phosphate buffer (5 mM; pH 6.5). The cells were added to 100 µg of glass beads (diameter, 100 to 250 µm; Sigma, St. Quentin Fallavière, France) and 2 ml of phosphate buffer (5 mM; pH 6.5) and shaken for 10 min (bead meal; Bioblock, Illkirch, France). Cellular lipid extraction and methylation were performed using the method of Bligh and Dyer (2) as modified by Rementzis and Samelis (21). Samples were then stored under nitrogen at -24°C until they were analyzed.

Methyl esters were analyzed on a Peri 2000 gas chromatograph (Perichrome, Saulx-lès-Chartreux, France) equipped with a hydrogen flame ionization detector and connected to a Winilab II integrator (Perichrome). Samples were analyzed on a silica-coated capillary column (25 m by 0.32 mm) containing polyethylene glycol and terephthalic acid under the following conditions: injection and detector temperatures, 260°C; column temperature, 70°C, increased by 39.9°C · min-1 until 180°C and then increased by 3°C · min-1 to 220°C and kept at 220°C for 20 min; carrier gas, nitrogen at a flow rate of 60 ml · min-1; injection volume, 1.0 µl. Peaks were identified by comparing retention times with those of methyl ester standards (Supelco [Sigma] and PUFA No. 1 [marine origin] and PUFA No. 2 [animal origin] [Interchrom, Montluçon, France]) and C19cy (9) (from the Laboratory of Agronomy and Environment of the Ecole Nationale Supérieure d'Agronomie et des Industries Alimentaires). Each sample was injected three times. Unknown fatty acid peaks were identified by gas chromatography-mass spectrometry (GC-MS). GC was carried out on an HP 6890 (Hewlett-Packard, Evry, France) chromatograph equipped with an hp5 capillary column (Hewlett-Packard) and with a mass spectrometer (Hewlett-Packard 5973). The temperature gradient was the same as that used with the GC-flame ionization detector, and the helium flow rate was 1.5 ml · min-1. Integration was performed with HP Chem software (Hewlett-Packard).

Statistical analysis.
Fatty acid data were analyzed using ANOVA (StatBox version 2.5; Grimmer Logiciel, Clignancourt, France) with a model including one effect (type of strain or culture condition). The model included three modalities (L. citreum CIP 103405 cultured without bacteriocin, with Mes52A, and with Mes52B), four modalities (L. mesenteroides subsp. mesenteroides LMA 7 and LMA 7AR cultured with and without bacteriocin and W. paramesenteroides DSM 20288T and DSM 20288BR cultured with and without bacteriocin), or five modalities (L. pseudomesenteroides CIP 103316T cultured without bacteriocin and CIP 103316AR and CIP 103316BR cultured with and without bacteriocin). The comparison of means was performed using the Newman-Keuls test.


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RESULTS
 
Model characterization. (i) Activity spectrum.
Both purified mesenterocins were tested against the 35 indicator strains (Table 1). Mes52A inhibited 16 strains of Leuconostoc and Listeria and two strains each of Lactobacillus and Weissella. Mes52B inhibited 12 Weissella and Leuconostoc strains. The three wild-type strains most susceptible to Mes52A and/or Mes52B were chosen for studies of mechanisms of resistance to mesenterocins: L. mesenteroides subsp. mesenteroides LMA 7, susceptible only to Mes52A; W. paramesenteroides DSM 20288T, susceptible only to Mes52B; and L. pseudomesenteroides CIP 103316T, susceptible to both mesenterocins. L. citreum CIP 103405, the only Leuconostoc strain insensitive to both bacteriocins, was also selected.

(ii) Cross spectrum of activity.
The L. mesenteroides subsp. mesenteroides FR 52 culture supernatant, containing Mes52A and Mes52B, inhibited L. mesenteroides subsp. dextranicum J 24, Listeria ivanovii LMA 94, L. mesenteroides subsp. mesenteroides LMA 7, W. paramesenteroides DSM 20288T, and L. pseudomesenteroides CIP 103316T (Table 2). The L. mesenteroides subsp. dextranicum J 24 culture supernatant, containing only Mes52B, inhibited W. paramesenteroides DSM 20288T and L. pseudomesenteroides CIP 103316T, susceptible to Mes52B. W. paramesenteroides DSM 20288T, insensitive to Mes52A, had activity against Listeria ivanovii LMA 94. The culture supernatant of this strain, tested against the 35 target strains, also showed biological activity against Listeria grayi CIP 6818T, Lactobacillus casei DSM 20211T, and Lactobacillus büchneri DSM 20257T (data not shown).


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  		TABLE 2. Inhibition zone diameters of cross activity spectra between producer and target strainsa

(iii) Characterization of resistant strains.
The MICs of Mes52A and Mes52B for the three susceptible wild-type strains selected were 100 AU · ml-1, corresponding to about 2 to 10% of an L. mesenteroides subsp. mesenteroides FR 52 culture supernatant (Table 3). Four resistant strains were obtained from the three susceptible wild-type strains: L. mesenteroides subsp. mesenteroides LMA 7AR, resistant to Mes52A, from L. mesenteroides subsp. mesenteroides LMA 7; W. paramesenteroides DSM 20288BR, resistant to Mes52B, from W. paramesenteroides DSM 20288T; and L. pseudomesenteroides CIP 103316AR and CIP 103316BR, resistant to Mes52A and Mes52B, respectively, from L. pseudomesenteroides CIP 103316T. These resistant strains were at least 30-fold more resistant to mesenterocins than the respective wild-type strains. The resistance phenotype was stable during at least eight successive cultures without bacteriocin, except for that of W. paramesenteroides DSM 20288BR, which was stable during only one culture. No cross-resistance was observed. Indeed, L. pseudomesenteroides CIP 103316AR, resistant to Mes52A, was susceptible to Mes52B, and L. pseudomesenteroides CIP 103316BR, resistant to Mes52B, was susceptible to Mes52A.


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  		TABLE 3. Mes52A and Mes52B MICs for wild-type and resistant strains

Membrane fatty acid analysis. (i) Membrane fatty acid compositions of wild-type strains.
The membranes of the four wild-type strains were composed principally of five fatty acids, C14, C16, C18:1{Delta}9, C18:1{Delta}7, and C19cy, accounting for 95.6 to 98.0% of the fatty acids present (Table 4). Fatty acids C18, C18:2 (data not shown), and C17 were also present, but in minor proportions. The position of the C19cy cyclopropane was not determined, even by GC-MS. The L. citreum CIP 103405 membrane contained 45.5% C19cy and 7.8% C18:1, values significantly different from those of the other strains. The other three strains, phenotypically more similar, also had more similar fatty acid profiles. However, the L. mesenteroides subsp. mesenteroides LMA 7 membrane had the highest content of C14 plus C16 (39.2%). The W. paramesenteroides DSM 20288T membrane contained more C19cy and fewer saturated fatty acids (Fig. 1). The L. pseudomesenteroides CIP 103316T membrane displayed the highest C18:1 content (26.8%). Thus, membrane fatty acid composition differed significantly among strains, especially for C14 plus C16, C18:1, and C19cy.


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  		TABLE 4. Membrane fatty acid compositions of two cultures of wild-type and resistant strains under different culture conditions



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  		FIG. 1. (A) Membrane fatty acid composition of L. mesenteroides subsp. mesenteroides. Open bars, LMA 7; hatched bars, LMA 7AR; stippled bars, LMA 7 cultured with 100 AU of Mes52B · ml-1; solid bars, LMA 7AR cultured with 500 AU of Mes52A · ml-1. (B) Membrane fatty acid composition of W. paramesenteroides. Open bars, DSM 20288T; hatched bars, DSM 20288BR; stippled bars, DSM 20288T cultured with 100 AU of Mes52A · ml-1; solid bars, DSM 20288BR cultured with 100 AU of Mes52B · ml-1. (C) Membrane fatty acid composition of L. pseudomesenteroides. Open bars, CIP 103316T; stippled bars, CIP 103316AR; hatched bars, CIP 103316BR; shaded bars, CIP 103316AR cultured with 100 AU of Mes52A · ml-1; solid bars, CIP 103316BR cultured with 100 AU of Mes52B · ml-1. (D) Membrane fatty acid composition of L. citreum. Open bars, CIP 103405; hatched bars, CIP 103405 cultured with 100 AU of Mes52A · ml-1; solid bars, CIP 103405 cultured with 100 AU of Mes52B · ml-1. A, B, and C, mean per fatty acid (and strain group) significantly different (P < 0.05); the error bars indicate the standard errors of the mean.

(ii) Comparison between wild-type strains and resistant strains.
No significant differences in membrane fatty acid composition were found between W. paramesenteroides DSM 20288T and DSM 20288BR and between L. pseudomesenteroides CIP 103316T, CIP 103316AR, and CIP 103316BR (Table 4 and Fig. 1). On the other hand, differences were noted between strains L. mesenteroides subsp. mesenteroides LMA 7 and LMA 7AR; the latter strain showed C18:1{Delta}9 and C18:1{Delta}7 contents 1.3 and 1.4% lower, respectively. These differences led to a lower content of unsaturated fatty acids (3.5% less). Nevertheless, these weak modifications concerned only one pair of strains and two fatty acids.

(iii) Comparison between insensitive wild-type strains and resistant strains in the absence or in the presence of mesenterocin.
Changes were observed between insensitive wild-type strains cultured with and without bacteriocin: an increase in saturated fatty acid content in L. mesenteroides subsp. mesenteroides LMA 7 and L. citreum CIP 103405 cultured with Mes52B (7.5 and 10.4% more, respectively) and a decrease in unsaturated fatty acid content in L. mesenteroides subsp. mesenteroides LMA 7 and L. citreum CIP 103405 cultured with Mes52A or Mes52B (3.7 to 5.5% less). W. paramesenteroides DSM 20288T displayed an atypical content of fatty acids when cultured in the presence of Mes52A only by a decrease in saturated fatty acid content (6.6% less). Except for W. paramesenteroides DSM 20288T, the insensitive wild-type strains cultured with mesenterocin displayed increases in the level of saturation (Fig. 1).

Similar differences were observed when the four resistant strains were cultured in the presence of a bacteriocin: they displayed a decrease in C18:1 (2.7 to 6.1% less). L. mesenteroides subsp. mesenteroides LMA 7AR and W. paramesenteroides DSM 20288BR displayed increases in C14 contents of 2.5 and 2.4%, respectively. Moreover, W. paramesenteroides DSM 20288BR displayed increases in C16 and C16:1 contents of 5.6 and 1.8%. These modifications led to increases of the saturated fatty acid contents in L. mesenteroides subsp. mesenteroides LMA 7AR, W. paramesenteroides DSM 20288BR, and L. pseudomesenteroides CIP 103316AR and decreases of the unsaturated fatty acid contents in L. mesenteroides subsp. mesenteroides LMA 7AR and L. pseudomesenteroides CIP 103316BR. Thus, in all resistant strains cultured with mesenterocin, these variations led to an increase of the level of saturation, either by an increase in saturated fatty acid content or by a decrease in unsaturated fatty acid content (Fig. 1).


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DISCUSSION
 
The first step of this work was to choose an original model that permits the study of natural and induced resistances to two bacteriocins from different classes produced by the same strain and the effects of bacteriocins on the membrane fatty acids of insensitive and resistant strains. The activity spectrum against closely related strains permitted us to select four wild-type strains: three hypersusceptible to only one or to both mesenterocins and one insensitive to both. This choice of strains was made in order to work with strains requiring similar culture conditions (broth and temperature) and having only minor genotypic and phenotypic differences. The cross spectrum of activity between producer and target strains indicated that W. paramesenteroides DSM 20288T produced an antibacterial factor. The eventual associated immunity peptide of that strain could interact in the strain resistance against Mes52A. Therefore, the results obtained with this strain must be interpreted with care.

The four resistant strains, obtained by successive cultures of three susceptible wild-type strains in media with increasing bacteriocin concentrations, were resistant to concentrations of bacteriocin at least 30 times the MIC for the wild-type strain. This resistance phenotype was stable for at least eight cultures without mesenterocin, except for W. paramesenteroides DSM 20288BR, in which it was stable for only one culture. The bacteriocin resistance phenotype seems to be bacteriocin and strain dependent. The nisin resistances of Listeria monocytogenes Scott A (17) and Pediococcus acidilactici UL5 (11) were stable after 20 and 60 cultures without bacteriocin, respectively. Resistance to class IIa bacteriocins may be stable (20) or unstable (6). To minimize the loss of resistance, all subcultures of resistant strains were performed in the presence of mesenterocin. As no cross-induced resistance was observed between Mes52A- and Mes52B-resistant strains, it is therefore possible that the two bacteriocins have different mechanisms of inhibition or induce different resistance mechanisms.

The membrane fatty acid contents of the four wild-type strains used in this study (95.6 to 98.0% C14, C16, C16:1, C18:1, and C19cy) were consistent with the results reported by Tracey and Britz (24) and Dykes et al. (5) with Leuconostoc and Weissella strains (between 90 and 99%). Although their fatty acid compositions were similar, there were significant differences, especially in C14, C16, C18:1, and C19cy. The presence of a high proportion of C19cy in L. citreum CIP 103405 (designated DSM 20188) was also reported by Dykes et al. (5), who found a content of 41.5%. This strain had the lowest content of unsaturated fatty acids and the highest content of C19cy, resulting in a more rigid membrane; its insensitivity to both mesenterocins seems to be due to its membrane rigidity. Ming and Daeschel (18), working with a nisin-resistant strain of Listeria monocytogenes Scott A, suggested that membrane rigidity would act in opposition to the insertion and/or association of peptides in the membrane; nevertheless, these authors did not work with insensitive strains. To confirm this hypothesis, it would therefore be interesting to extend our study to a larger panel of susceptible or insensitive wild-type strains.

The fatty acid contents of the resistant strains cultured without bacteriocin were similar to those of the wild-type strains, except for L. mesenteroides subsp. mesenteroides LMA 7AR, which displayed 2.7% more C18:1 than its susceptible counterpart. Since these differences were weak, isolated, and not counteracted by an increase of other fatty acid content, they are probably due to experiment and/or analysis but not to resistance. Thus, the resistance to the two types of bacteriocins did not seem to be related to changes in fatty acid content. Verheul et al. (26) and Van Schaik et al. (25) drew similar conclusions for nisin-resistant strains of Listeria monocytogenes Scott A. Mazzotta and Montville (17) suggested that induced resistance makes possible the adaptation of a strain during its culture with bacteriocin. These authors drew a parallel between the homeoviscous adaptation of the resistant strain to the presence of bacteriocin and temperature variations. In our study, the four resistant strains cultured with bacteriocins differed in membrane fatty acid content from the wild-type strains, with higher saturated fatty acid contents and lower unsaturated fatty acid contents, resulting in a more rigid membrane. Alternatively, Mes52A and Mes52B may directly or indirectly induce modifications in membrane fatty acid content in the resistant strains. Insensitive wild-type strains cultured in the presence of bacteriocin presented modifications in fatty acid content: an increase in saturated fatty acid content and a decrease in unsaturated fatty acid content. Thus, Mes52A and Mes52B induced changes in the membrane fatty acid compositions of insensitive and resistant strains. W. paramesenteroides DSM 20288T displayed an atypical response in the presence of Mes52A. This was probably due to the production by this strain of a substance active against Listeria ivanovii LMA 94. One possible reason for these modifications in membrane fatty acid content is that mesenterocins, by binding to membranes, induce an increase in membrane fluidity that is counteracted by an increase in the proportion of fatty acids, leading to a more rigid membrane. This implies that mesenterocins reach the membranes of insensitive and resistant strains and that the cell wall does not prevent diffusion of the peptide to contact the membrane. Nevertheless, Maisnier-Patin and Richard (15) showed, in a nisin-resistant strain of Listeria monocytogenes Scott A, that the cell wall was involved in nisin resistance. Similarly, Crandall and Montville (4) proposed a model in which cell wall characteristics were involved in the nisin resistance of strains of Listeria monocytogenes ATCC 700032.

We studied one factor described by Crandall and Montville (4) as being responsible for resistance to bacteriocins: membrane fatty acid content seems not to be related to resistance but may be related to insensitivity. Moreover, mesenterocins induce several changes in the fatty acid contents of insensitive and resistant strains. Future research will focus on the further characterization of membrane phospholipid content and cell wall characteristics and on the purification of the antimicrobial factor produced by W. paramesenteroides DSM 20288T.


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ACKNOWLEDGMENTS
 
GC coupled with flame ionization detection was carried out in the Laboratoire de Physico-Chimie de l'ENSAIA-INPL, Vandoeuvre-lès-Nancy Cedex, France. We thank M.-N. Maucourt and C. Pierret for helping us with this technique. GC coupled with MS was carried out in the Laboratoire de Biologie Forestière of the Université Henri Poincaré, Nancy I, France, directed by B. Botton. We thank R. Belleville for his help.


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FOOTNOTES
 
* Corresponding author. Mailing address: Laboratoire Bioprocédés Agro-Alimentaires, Ecole Nationale Supérieure d'Agronomie et des Industries Alimentaires, Institut National Polytechnique de Lorraine (ENSAIA-INPL), 2 Ave. de la Forêt de Haye, BP 172, F-54505 Vandoeuvre-lès-Nancy Cedex, France. Phone: 33 3 83 59 58 84. Fax: 33 3 83 59 58 04. E-mail: anne-marie.revol{at}ensaia.inpl-nancy.fr. Back


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Applied and Environmental Microbiology, June 2002, p. 2910-2916, Vol. 68, No. 6
0099-2240/02/$04.00+0     DOI: 10.1128/AEM.68.6.2910-2916.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.



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