1 National Food Research Institute, 2-1-12 Kannondai, Tsukuba, Ibaraki 305-8642, Japan
2 Research Institute for Biological Resources and Functions, National Institute of Advanced Industrial Science and Technology (AIST), 1-1-1 Higashi, Tsukuba, Ibaraki, 305-8566, Japan
Correspondence
Jun Shima
shimaj{at}nfri.affrc.go.jp
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ABSTRACT |
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INTRODUCTION |
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In Listeria monocytogenes, resistance to nisin correlated with changes in membrane fatty acid composition, phospholipid composition and the cell wall, and required divalent cations (Davies & Adams, 1994; Davies et al., 1996
; Mazzotta & Montville, 1997
; Verheul et al., 1997
; Crandall & Montville, 1998
; Mantovani & Russell, 2001
). Although physiological and molecular biological studies of nisin resistance have been undertaken, there have been few studies of resistance to class IIa bacteriocins (Ennahar et al., 2000
; Gravesen et al., 2002
). The aim of our study was to isolate mutants resistant to class IIa bacteriocins and to determine the characteristics of these resistant mutants and their resistance mechanisms.
Class II bacteriocins, including subclasses IIa and IIb, are low-molecular-mass, heat-stable, nonlanthionine peptides consisting of 3060 amino acid residues (Klaenhammer, 1988; Jack et al., 1995
; Ennahar et al., 2000
). Class IIa bacteriocins contain peptides with the consensus sequence YGNGV near the N terminus (Eguchi et al., 2001
; Kawamoto et al., 2002
). In general, class IIa bacteriocins are strongly active against strains that are primarily close relatives of their producer strains (Ennahar et al., 2000
). Almost all class IIa bacteriocins are active against food-borne bacteria, including Listeria and Clostridium strains (Jack et al., 1995
; Eguchi et al., 2001
; Kawamoto et al., 2002
). Mundticin KS is a 43 amino acid peptide produced by Enterococcus mundtii NFRI 7393 and is active against E. mundtii, Enterococcus faecium, Enterococcus faecalis and L. monocytogenes (Kawamoto et al., 2002
). Enterocin SE-K4 is a class IIa bacteriocin that has a YGNGV consensus motif and is active against E. faecium, E. faecalis and L. monocytogenes (Eguchi et al., 2001
).
In this study, we have characterized mutants resistant to mundticin KS as a model of class IIa bacteriocins. We used E. faecium as the parent strain for deriving resistant mutants because E. faecium, which is closely related to E. mundtii, is highly sensitive to class IIa bacteriocins, including mundticin KS and enterocin SE-K4. The cross-resistance of these mutants to various antimicrobial agents, including six antibiotics and three bacteriocins, was investigated. We also determined the effects of magnesium on mundticin KS resistance and the membrane composition of fatty acids and phospholipids in the mutants.
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METHODS |
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Chemicals.
Nisin was purchased from Asama Kasei (Japan). Erythromycin, kanamycin and tetracycline were obtained from Nakarai Tesque (Japan). Streptomycin and ampicillin were purchased from SigmaAldrich. Hygromycin B was purchased from Wako (Japan). Plates for TLC were purchased from Merck. Phospholipid standards were obtained from Funakoshi (Japan). Other chemicals used in this study were purchased from Nakarai Tesque.
Bacteriocin preparation.
A crude preparation of mundticin KS was incorporated into agar medium for isolation of resistant mutants. To produce mundticin KS, E. mundtii cells were cultivated in MRS medium at 37 °C for 16 h. The culture supernatant was concentrated tenfold by ultrafiltration (Viva Cell System; Sartorius). Purified mundticin KS and enterocin SE-K4 were used in the characterization of the mundticin-resistant mutants. Mundticin KS and enterocin SE-K4 were purified by a previously described method (Eguchi et al., 2001; Kawamoto et al., 2002
).
Isolation of mundticin-resistant mutants from E. faecium.
E. faecium IFO13712 was utilized as parent strain to isolate mundticin-resistant mutants. Bacteria were grown for 16 h in MRS medium and portions containing 106108 cells were plated onto MRS agar containing crude mundticin KS (0·12·5 µg ml-1) and cultivated at 37 °C or room temperature.
Assay for sensitivity to antimicrobial substances.
Cells of wild-type or resistant mutants were grown to OD600=1·0 and then washed with MES-buffered saline (0·9 % NaCl, pH 6·5). The cells were resuspended in 50 mM MES buffer (pH 6·5) at a cell density of approximately 5x108 cells ml-1 (OD600=1·0). The cell suspension was divided into 1 ml fractions and antibiotics or bacteriocin were added to the fractions at various concentrations. After addition of enterocin SE-K4, mundticin KS or kanamycin, the cell suspension was incubated at 37 °C for 20 min without shaking. After the addition of nisin, the cell suspension was supplemented with 10 mM MgSO4 and then incubated at 37 °C for 60 min with shaking. After treatment with antibiotic or bacteriocin, cell suspensions were centrifuged, the pellet was resuspended in MES buffer and portions were plated onto MRS agar and incubated at 37 °C for 16 h for determination of c.f.u.
Effect of divalent metal ions on bacteriocin resistance.
Cells of wild-type or resistant mutants were grown in Lactobacilli MRS broth to OD600=1·0. The cells were then washed with MES-buffered saline (pH 6·5) and resuspended at a cell density of approximately 5x108 cells ml-1 (OD600=1·0) in 50 mM MES buffer (pH 6·5) containing either divalent cations (10 mM final concn), divalent cations plus 20 mM EDTA to chelate divalent cations, or MES buffer only. Mundticin KS was added to the cell suspension at a concentration of 3 µg ml-1. The cell suspensions were incubated at 37 °C for 20 min and then plated onto MRS agar and incubated at 37 °C for 16 h for determination of c.f.u.
Measurement of membrane fatty acids.
Wild-type or resistant mutants were grown in MRS medium with or without 0·2 µg mundticin KS ml-1 to early stationary phase (OD600=1·7). Samples were collected from five cultures incubated separately under each condition. Cellular fatty acids were converted to methyl esters by using anhydrous methanolic HCl. The fatty acid methyl esters were analysed by using a Hitachi M7200A GC/3DQM system equipped with a DB-5ms capillary column (30 mx0·25 mm; J&W Scientific) coated with (5 %-phenyl)-methylpolysiloxane. The column temperature profile was initially maintained at 100 °C for 1 min and then gradually increased to 280 °C at a rate of 10 °C min-1, and finally held at 280 °C for 12 min. In all samples, the measurements with GC/MS were run in triplicate.
Measurement of cellular phospholipids.
The analysis of phospholipids was done as follows. The wild-type or mundticin-KS-resistant mutants were grown in MRS medium with or without 0·2 µg mundticin KS ml-1 to early stationary phase (OD600=1·7). The cells were harvested, washed and then resuspended in 0·9 % NaCl. Total phospholipids were extracted from the cells by the method of Bligh & Dyer (1959). Individual lipids were separated from the total lipid extracts by one- or two-dimensional TLC. Individual lipids on TLC were visualized using the following stains: iodine vapour (general lipid detection), ninhydrin (amino group) and DittmerLester reagents (phosphate group). Major components of phospholipids were visualized by iodine vapour and then extracted from TLC by using chloroform. Phospholipids in the extracts were measured as described by Bartlett et al. (1959)
.
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RESULTS |
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First, we did a morphological analysis of the two resistant mutants (MunrS and MunrF) by using phase-contrast microscopy. No significant differences in cell shape, cell size and degree of cell aggregation were observed between wild-type and the two mutants (data not shown). Then, we determined the growth characteristics of wild-type, MunrS and MunrF in MRS liquid medium containing mundticin KS (Fig. 1). The wild-type or the two resistant mutants were inoculated into MRS medium at a concentration of approximately 4·5x105 cells ml-1, incubated at 37 °C and growth was monitored by measuring OD600 every 1·5 h. The growth characteristics of wild-type and Munr mutants were nearly identical in the absence of mundticin KS (Fig. 1a
). In contrast, in the presence of 5 µg mundticin KS ml-1 growth of wild-type cells was inhibited (Fig. 1b
), whereas growth of the mundticin KS mutants was relatively unaffected.
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To determine resistance to these antimicrobial agents in more detail, we measured numbers of viable cells (c.f.u.) after treating cell suspensions with these antimicrobial reagents. At first, we assessed resistance to class IIa bacteriocins. As shown in Fig. 2(a), the MunrF and MunrS mutants showed higher resistance to mundticin KS compared with the wild-type strain. These mutants were not inhibited even at a mundticin KS concentration of >50 µg ml-1 (data not shown). Both Munr mutants also showed strong resistance to enterocin SE-K4 (Fig. 2b
). We next assessed resistance to nisin (Fig. 3
a). Because nisin is not highly active against E. faecium, we could not detect differences between wild-type and Munr mutants at low nisin concentrations (<100 µg ml-1). However, at higher nisin concentrations, Munr mutants showed less sensitivity to nisin compared with wild-type. In particular, the MunrF mutant was 101000 times more resistant to nisin than wild-type at concentrations of 200500 µg ml-1. As shown in Fig. 3(b)
the MunrF mutant showed sensitivity to kanamycin similar to that of the wild-type strain. However, the MunrS mutant was hypersensitive to >150 µg kanamycin ml-1 Fig. 3(b)
. These results suggest that Munr mutations cause pleiotropic changes in resistance to different antimicrobial agents.
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Fig. 5 shows the TLC analysis of phospholipids extracted from wild-type and Munr mutants. Three major components of phospholipids are clearly evident. By comparing the mobilities of the phospholipids on TLC with those of standards and those extracted from Escherichia coli DH5
, we tentatively identified two of the three as phosphatidylglycerol (PG) and cardiolipin (CL), respectively. The third could not be identified. We designated this third component amino-containing phospholipid (ACP) because the unidentified spot in TLC was reactive with DittmerLester reagents and ninhydrin. We compared phospholipid composition between the wild-type and Munr mutants grown in the presence or absence of mundticin KS. The amounts of PG and CL in both Munr mutants were significantly less than that in the wild-type (Table 2
), whereas the amount of ACP was significantly higher. Addition of mundticin KS to the medium did not affect phospholipid composition. These results suggest that the decrease in PG and CL and/or increase in ACP contribute to mundticin KS resistance in Munr mutants.
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DISCUSSION |
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In this study, we successfully isolated two types of mundticin-resistant mutants (MunrS and MunrF). Although no genetical analysis of these two mutants was performed, the two mutants apparently have different mutations that determine their mundticin resistance based on their differential sensitivities to a range of antimicrobial agents. MunrS mutants showed hypersensitivity to kanamycin, which is an aminoglycoside antibiotic and inhibits protein translation. The mechanism for this hypersensitivity to kanamycin remains unclear.
Resistance to bacteriocins has been widely studied. Almost all those studies have involved nisin (class I) with L. monocytogenes as the target strain (Mazzotta & Montville, 1997; Verheul et al., 1997
; Crandall & Montville, 1998
). Recently, resistance to class IIa bacteriocins has been reported. For example, Limonet et al. (2002)
reported variations in fatty acid composition of mutants of Leuconostoc and Weissela strains resistant to the class IIa bacteriocin mesenterocin 52A (Mes 52A). They showed that resistant strains contained more saturated fatty acids when cultured with Mes 52A. Vadyvaloo et al. (2002)
reported that L. monocytogenes strains resistant to class IIa bacteriocins showed increased levels of desaturated PG.
Models of modes of class IIa bacteriocin activity have been proposed (Ennahar et al., 2000). In brief, the modes of bacteriocins consist of at least three steps: (1) electrostatic interaction between the N terminus of bacteriocin and membrane phospholipids, (2) reorientation and insertion into the membrane, and (3) pore formation. At present, no evidence for a protein receptor for binding of class IIa bacteriocins has been reported (Chen et al., 1997b
). In our study, we did not detect significant differences in the mundticin-binding affinity between Munr mutants and wild-type cells, even in the presence of Mg2+ ions (data not shown). An affinity assay suggests that mutation in Munr mutants does not occur in genes that are directly involved in bacteriocin-binding, such as genes for protein receptors.
Recently, Limonet et al. (2002) reported variations in fatty acid composition of mutants of Leuconostoc and Weissela strains resistant to mesenterocin 52A (Mes 52A). We have also found differences in fatty acid (Table 1
) and phospholipid contents (Table 2
). Based on these observations, the composition of membrane fatty acids appears to affect resistance to class IIa bacteriocins. In particular, an increase in saturated fatty acids seems to correlate with resistance, possibly because saturated fatty acids might change the physical properties of the membrane, making the membrane more rigid.
We also showed that, in the two Munr mutants, the amount of ACP was higher, whereas amounts of PG and CL were lower, compared to amounts in the wild-type strain. We speculate that the chemical structure of ACP might be lysyl PG, because Kocun (1970) reported that lysyl PG is one of the major components of the phospholipids of E. faecalis IoCI, a strain closely related to E. faecium. Conditions favourable to electrostatic interactions in the membrane are important for the binding class IIa bacteriocins to phospholipid vesicles (Chen et al., 1997a
). Because ACP could have zwitterionic properties, an increased level of ACP might inhibit binding of bacteriocin by positively charging the membrane. It has been reported that changes in the phospholipid composition occur in nisin-resistant strains of L. monocytogenes Scott A (Verheul et al., 1997
). Specific changes include more zwitterionic phosphatidylethanolamine and less anionic PG and CL (Verheul et al., 1997
; Crandall & Montville, 1998
). Changes in the composition of phospholipids in Munr mutants that we observed are similar to these changes in phospholipids in L. monocytogenes. Thus the level of zwitterionic phospholipids is a critical determinant in the resistance to both class I and class IIa bacteriocins. Vadyvaloo et al. (2002)
reported that class-IIa-resistant L. monocytogenes strains showed increased levels of desaturated PG. In our study, we did not determine the composition of fatty acids specifically binding to PG.
The changes in membrane components in the two Munr mutants were similar to those in nisin-resistant strains (Tables 1 and 2). Munr mutants showed cross-resistance to nisin (Fig. 3
), suggesting that the resistance mechanisms to class IIa and class I bacteriocins are similar. The relationship between changes in membrane components, such as fatty acids and phospholipids, and the molecular mechanisms of resistance remains unclear for class IIa bacteriocins. At present, we do not know whether a specific gene determines resistance to mundticin KS in Munr mutants. Gene cloning of determinants of mundticin KS resistance might help to clarify the mechanisms of bacteriocin resistance, if a specific resistance gene is present. Cross-resistance between class I and class IIa bacteriocins might be a critical problem when bacteriocins are used as biopreservatives. To overcome this problem, characterization of resistance mechanisms to bacteriocins at the molecular level is crucial.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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Received 23 April 2003;
revised 18 June 2003;
accepted 1 July 2003.
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