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1 UMR-INRA 1014 SECALIM, ENITIAA, Rue de la Géraudière, BP 82225, Nantes Cedex 3, France
2 Laboratoire de Microbiologie de l'Environnement, EA 956, USC INRA 2017, IRBA, Université de Caen, Caen Cedex, France
Correspondence
Djamel Drider
drider{at}enitiaa-nantes.fr
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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54 factor and the ManR activator. In this investigation, three genes associated with the resistance of Ent. faecalis JH2-2 to divercin V41, a pediocin-like bacteriocin from Carnobacterium divergens V41, were clearly identified by screening an insertional mutant library of Ent. faecalis JH2-2. These genes correspond to the well-known rpoN gene, which encodes 54 factor, and to genes encoding a glycerophosphoryl diester phosphodiesterase (GlpQ) and a protein with a putative phosphodiesterase function (PDE). Resistance of the three mutants defective in the aforementioned genes appeared to be graduated: the rpoN mutant was more resistant than the glpQ mutant, which was more resistant than the pde mutant. Moreover, this resistance was specific to class IIa bacteriocins.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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) and in medicine as antiviral agents (Wachsman et al., 1999, 2003). Class IIa bacteriocins, referred to as pediocin-like bacteriocins, are listericidal, small (<10 kDa), heat stable, non-lanthionine-containing peptides of 37 to 48 amino acids with a consensus sequence YGNGVXCX4(C)XVX4A (X denotes any amino acid) in their charged N-terminal region (Drider et al., 2006). These bacteriocins also have a similar tertiary structure (Frégeau-Gallagher et al., 1997; Uteng et al., 2003) and mode of action (Ennahar et al., 2000). A promising method of food protection by using class IIa bacteriocins could be compromised by the development of resistant strains. This drawback could be overcome by using combinations of bacteriocins (Bouttefroy & Millière, 2000), or bacteriocins combined with other sublethal treatments such as sodium diacetate and sodium lactate (Uhart et al., 2004). These combinations appear to be more efficient in controlling the growth of the foodborne pathogen Listeria monocytogenes. Resistance to class IIa bacteriocins has been studied but remains poorly understood overall. Studies aimed at characterizing the resistance mechanisms of bacterial targets have revealed the stability of this phenomenon (Rekhif et al., 1994; Dykes & Hastings, 1998), which occurs at either a low or a high level. In L. monocytogenes and Enterococcus faecalis, low-level resistance is attributed to alterations in membrane lipid composition (Vadyvaloo et al., 2002, 2004; Naghmouchi et al., 2006) and high-level resistance results from the inactivation of the mptACD operon, which encodes the 
mannose permease of the phosphotransferase system (PTS) (Dalet et al., 2001; Héchard et al., 2001). The aim of this study was to gain more insights into the molecular resistance mechanism developed by Ent. faecalis JH2-2 in response to pediocin-like bacteriocins. In the food quality area, Ent. faecalis is used as an indicator of faecal contamination and has been shown to be implicated in outbreaks of foodborne illness, while in the clinical area this bacterium has become a serious nosocomially transmitted pathogen. Our data strongly suggest that inactivation of genes encoding a glycerophosphoryl diester phosphodiesterase (GlpQ) and a protein with a putative phosphodiesterase function (PDE) confer a resistant character to Ent. faecalis JH2-2 towards divercin V41. |
METHODS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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) and its mutant derivatives were grown without shaking in M17 medium (Biokar) supplemented with glucose at 0.5 % (w/v) (Terzaghi & Sandine, 1975). When necessary, erythromycin (150 µg ml–1) or tetracycline (15 µg ml–1) was added to the growth medium. Carnobacterium divergens V41 (Pilet et al., 1995), Leuconostoc mesenteroides Y105 (Héchard et al., 1992) and Pediococcus acidilactici B5627 (University of Poitiers, France) were grown in MRS (de Man-Rogosa-Sharpe) medium (Biokar). Escherichia coli Origami, harbouring plasmid pCR03, which confers the ability to synthesize recombinant divercin V41 (Richard et al., 2004), was grown at 37 °C with shaking in LB medium containing ampicillin (100 µg ml–1) and chloramphenicol (30 µg ml–1) (Richard et al., 2004). Listeria innocua F (ENITIAA) was grown in Elliker medium (Biokar) at 30 °C, without shaking.
Production of pediocin-like bacteriocins.
Divercin V41 (DvnV41), mesentericin Y105 (MesY105) and pediocin PA-1/AcH (Ped PA-1/AcH) were prepared from C. divergens V41, Leuconostoc mesenteroides Y105 and P. acidilactici B5627, respectively. Samples (1 ml) of cell cultures of each strain were centrifuged (6 min, 5000 g, 4 °C), then the recovered supernatant was heated for 10 min at 100 °C and kept at –20 °C until use. Recombinant divercin V41 (DvnRV41) was obtained from the heterologous host E. coli Origami/pCR03 and purified as described previously (Richard et al., 2004). Recombinant divercin V41 differs from natural divercin V41 by one amino acid in the N-terminal sequence, P-dvnV41 (Richard et al., 2004). DvnV41 supernatant was used for established screening conditions and for screening the mutant library, then purified DvnRV41 was used for determining the MIC.
The anti-Listeria activity of DvnV41, DvnRV41, MesY105 and Ped PA-1/AcH against L. innocua F was verified as previously described (Pilet et al., 1995). Briefly, 10 µl samples of a twofold serial dilution in Elliker broth for each bacteriocin studied were spotted on an Elliker agar (1 %) plate inoculated with the indicator strain, L. innocua F, at 107 c.f.u. ml–1. Plates were incubated at 30 °C for 16 h. The bacteriocin activity was expressed in arbitrary units per ml (AU ml–1) and was defined as the reciprocal of the lowest dilution that did not show growth inhibition of L. innocua F. Bacteriocin activity was found to be 820 000 AU ml–1 for DvnV41, 3 300 000 AU ml–1 for DvnRV41, and 256 000 AU ml–1 for MesY105 and Ped PA-1/AcH.
Isolation of Ent. faecalis JH2-2 divercin V41-resistant mutants.
The library of insertional mutants of Ent. faecalis JH2-2 used in this study was constructed by Le Breton et al. (2002) as follows. DNA fragments of 200 bp to 1.5 kb were generated by partial digestion of chromosomal DNA from Ent. faecalis JH2-2 with AluI then cloned into pORI19 (pWV01-derived Ori+ RepA–) using the RepA+ helper E. coli strain EC101 (Law et al., 1995) to obtain a bank of approximately 37 200 recombinant plasmids. A mixture of these recombinant plasmids was then transferred into Ent. faecalis JH2-2 that had previously received the pWV01-derived Ori+ RepATS pG+host3 plasmid (pVE6007) (Maguin et al., 1992). Clones were grown at 30 °C in GM17 medium containing erythromycin and chloramphenicol (the thermosensitive RepATS protein is active at 30 °C and allowed replication of pG+host3 and pORI19 recombinant plasmids). Cells were transferred into GM17 containing erythromycin (150 µg ml–1) and the incubation temperature was shifted to 42 °C to inactivate the RepATS protein and consequently to provoke the loss of pG+host3 and the integration of the pORI19 recombinant plasmid by homologous recombination. Mutants (9600) were then screened for resistance to DvnV41 as follows: individual clones were grown at 37 °C in 96-well microtitre plates containing GM17 broth with 150 µg erythromycin ml–1 then replica plated in GM17 broth containing 5 % (v/v) DvnV41 (820 000 AU ml–1). Integrated plasmids from all selected DvnV41-resistant mutants were excised as previously described by Le Breton et al. (2002) and used to identify the insertion loci. After sequencing the DNA insert carried by the pORI19 recombinant plasmid from each mutant, the nucleotide sequence was compared to that of the Ent. faecalis (V583) genomic sequence available at The Institute for Genomic Research (http://www.tigr.org/).
Analysis of mRNAs by RT-PCR.
Total RNA of Ent. faecalis JH2-2 was isolated from exponentially growing cells in GM17 broth using the RNeasy Midi kit (Qiagen). For reverse transcriptions 10 µg of the RNA preparation was treated for 30 min at 37 °C with 20 U RNase-free DNase I (Roche Molecular Biochemicals) followed by a phenol-acid extraction at 65 °C. Reverse transcriptase reactions were performed on 2 µg of the RNA at 37 °C using the Omniscript RT kit (Qiagen) with 20 pmol random hexamer primers. cDNA was then purified using the QIAquick kit (Qiagen) and PCRs were performed with purified cDNA as template and 20 pmol primers listed in Table 1 using 2.5 U Taq DNA polymerase (Q-BIOgene). Amplifications were carried out for 30 cycles consisting of 94 °C for 30 s, 56 °C for 30 s, and 72 °C for 1 min, then the samples were analysed on a 1 % agarose gel in TAE (40 mM Tris-acetate pH 8.0, 1 mM EDTA) buffer. The absence of contaminating genomic DNA was checked by regular PCR performed under the same conditions, except that the reverse transcriptase was replaced by H2O. A positive control was also performed by replacing cDNA with genomic DNA (data not shown).
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), itself cut with the same restriction enzymes. The recombinant plasmids were directly transferred into the Ent. faecalis mutant strain by electroporation using the following conditions: 25 µF, 400 
and 2500 V in a 0.2 cm cuvette (Pulse Controller Plus; Bio-Rad). The resulting cells were plated onto GM17 plates containing chloramphenicol (10 µg ml–1) and erythromycin (10 µg ml–1). Plates were incubated at 37 °C for 48 h. As a negative control, we used cells harbouring only plasmid pAM401.
Susceptibility/resistance test to divercin V41.
Susceptibilities of Ent. faecalis JH2-2 wild-type, mutant strains 35A1 (pde), 36H4 (glpxQ) and transformants 35A1+pde, 36H4+glpQ toward DvnV41 were tested. Cultures were diluted (1/100) and plated onto GM17 medium containing 1 % agar; 10 µl samples of DvnV41 were spotted and the plates were incubated at 37 °C for 16 h. Inhibition haloes were observed and compared between the different strains.
Identification of Ent. faecalis JH2-2 divercin V41-resistant mutants.
Identification of the mutants was performed by phenotypic and molecular methods. After Gram staining, each mutant strain was checked for its catalase and oxidase activities. Identification was completed by PCR-RAPD (random amplified polymorphic DNA) using the following conditions: 1 µl (50 ng) chromosomal DNA, 5 µl 10x reaction buffer (New England Biolabs), 5 mM MgCl2 (Sigma), 1 mM dNTPs (New England Biolabs), 0.5 µM primer M13V (5'-GTTTTCCCAGTCACGAC-3') and 0.75 U Taq DNA polymerase (New England Biolabs). The PCR reactions were carried out on a PTC-100 thermocycler (MJ Research). The cycling programme was: 3 min at 94 °C, 5 min at 40 °C, 5 min at 72 °C for 3 cycles; 1 min at 94 °C, 2 min at 60 °C, 3 min at 72 °C for 32 cycles. Thereafter, 5 µl of each PCR product was separated on a 1.5 % agarose gel run at 100 V for 20 min and visualized by UV after ethidium bromide staining. A 100 bp ladder (New England Biolabs) was used as a reference for molecular size.
MIC determination.
Twofold serial dilutions of DvnRV41 in GM17 medium were placed in microplate wells (Nunc). Each well was inoculated with 50 µl of Ent. faecalis JH2-2 wild-type or mutant cultures at 106 c.f.u. ml–1. The microplate was incubated aerobically for 18 h at 30 °C then OD600 was measured hourly using an UltraMicroplate Reader (Bio-Tek Instruments). Sterile medium incubated under the same conditions was used as a blank. The MIC was calculated from the highest dilution showing complete inhibition of the tested strain (OD600 equals OD600 of the blank). The MIC determinations were repeated independently three times and the mean value is presented in the Results.
Cross-resistance to bacteriocins and antibiotics.
The resistance level of Ent. faecalis JH2-2 and mutants resistant to DvnV41 was tested against class IIa bacteriocins (DvnV41, MesY105 and Ped PA-1/AcH), the class I bacteriocin nisin (Sigma) and the following antibiotics: cephalothin (0.01 mg ml–1) (Sigma), penicillin G (10 IU) (Biomérieux), and streptomycin (10 IU) (Bio-Rad). Bacteriocin activity was assessed by the agar diffusion test (ADT) against L. innocua F as indicator strain (Pilet et al., 1995). Briefly, brain heart infusion (BHI; Biokar) agar plates were inoculated with 1 ml of Ent. faecalis JH2-2 wild-type or mutant strains at 107 c.f.u. ml–1. Next, 10 µl samples of a supernatant culture containing any of the class IIa bacteriocins cited above or nisin at 10 mg ml–1 were spotted onto the agar plate and incubated at 30 °C for 16 h. Cephalothin, or a disk of penicillin G or streptomycin, was applied in a similar way and plates were incubated at 30 °C for 16 h. After this, the plates were inspected for the formation of inhibition zones by measuring the diameter of each halo.
Database searches and sequence analyses.
Searches for promoter locations and prediction of the pde and glpQ transcription start point were performed with the neural network program (http://www.fruitfly.org/seq_tools/promoter.html). Ent. faecalis genome analyses were performed using the Ent. faecalis (V583) genomic sequence available at The Institute for Genomic Research (http://www.tigr.org/).
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RESULTS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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DNA sequence analysis
The Ent. faecalis (V583) genomic sequence in the region corresponding to the insertion locus in mutant 35A1 provided evidence for the presence of a ribosome-binding site sequence (GGAGG) located 6 nt upstream of the EF0011 ORF initiation codon (ATG). Translation of this ORF should allow synthesis of a 658 amino acid protein sharing homology with a putative phosphoesterase of the desert hedgehog (DHH) protein family, enzymes that hydrolyse polyphosphates into Pi. A prediction of transmembrane topology with the TMpred program (Hofmann & Stoffel, 1993) revealed that amino acid regions 10–28 and 34–53 could be membrane-spanning segments. In Ent. faecalis (V583), the EF0011 ORF is flanked by two genes, rpsR and rpII, encoding ribosomal protein S18 and ribosomal protein L9, respectively (Fig. 1). In this study, we refer to EF0011 ORF as the pde gene.
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54 factor (Dalet et al., 2000). It is preceded by a gene encoding a tRNA (tRNA-Arg2) oriented in the opposite direction and followed by the EF0783 ORF, which encodes a 625 amino acid putative acetyl transferase (Fig. 1
). Immediately downstream of the last codon of the rpoN gene, there is a 201 bp intergenic region, which contains an inverted repeat (5'-AAACAACCAAAGCTTATGA(N7)TCATAAGCTTTGGTTGTTT-3'; 
G=–30.2 kcal mol–1; –126.4 kJ mol–1), which could act as a rho-independent terminator (Fig. 1).
The EF2163 ORF (insertion site in mutant 36H4) encodes a 248 amino acid protein (28.4 kDa) sharing high similarity with putative glycerophosphoryl diester phosphodiesterase enzymes, which are known to hydrolyse deacylated phospholipids into glycerol 3-phosphate and the corresponding alcohols. In this study, this ORF is named the glpQ gene. The entire amino acid sequence, deduced from the glpQ gene, was highly hydrophobic. Moreover, the glpQ gene is preceded by the EF2164 ORF, which encodes a putative membrane protein of 603 amino acids with an unknown function. Immediately downstream of the termination codon of the glpQ gene, the EF2162 ORF corresponds to the miaA gene, which encodes a 309 amino acid protein sharing high similarity with tRNA 2-isopentenylpyrophosphate transferase (tRNA and rRNA base modification). Four base pairs downstream of the miaA gene, the EF2161 ORF encodes a 413 amino-acid putative GTP-binding protein. This latter is followed by an inverted repeat (5'-TAGAAATGATTTGT(N4)ACAAATCATTTCTA-3'; G=–16.4 kcal mol–1; –68.6 kJ mol–1), which could act as a rho-independent terminator. Further experiments were performed only with mutants 35A1 (pde inactivated), 36H4 (glpQ inactivated) and 35H1 (rpoN inactivated).
mRNA transcriptional analysis and complementation test
To identify accurately genes associated with resistance of Ent. faecalis JH2-2 to DvnV41, RT-PCR analyses were performed in order to study the mode of transcription of the inactivated genes as well as those located downstream of them. Thus, in mutant 36H4, defective in the putative GlpQ protein, the glpQ gene (EF2163 ORF) was shown to be cotranscribed with the miaA gene (EF2162 ORF), and the gene encoding a putative GTP-binding protein (EF2161 ORF) was itself cotranscribed with the glnR gene (EF2160 ORF) (Figs 1a and 2a). Regarding these RT-PCR results, we cannot discard a polar mutational effect resulting from the inactivation of the target glpQ gene. This inactivation could lead to either down- or up-regulation of miaA (EF2162 ORF), the gene encoding a putative GTP-binding protein (EF2161 ORF), and even glnR (EF2160 ORF) located downstream of the putative rho-independent terminator, as a band corresponding to a read-through transcript was obtained with primers J2161F and J2160R (Fig. 1). The same is true for the gene encoding a putative phosphoesterase (EF0011 ORF), which was found to be cotranscribed with the rpII gene (EF0012 ORF), which was itself cotranscribed with the dnaB gene (EF0013 ORF) (Figs 1b and 2b). However, as no RT-PCR product was detected with primers J0782F and J0783R (Fig. 2c), the rpoN gene (EF0782 ORF) appeared to be expressed as a monocistronic mRNA. To clearly ascribe resistance of Ent. faecalis 35A1 and 36H4 to inactivation of pde and glpQ genes rather than a polar effect of genes located in the neighbourhood, we performed a complementation test by incorporation in trans in each mutant pAM401 plasmids containing either the pde or glpQ gene. The new strains (transformants) 35A1+pde and 36H4+glpQ exhibited similar phenotypes to wild-type Ent. faecalis JH2-2 (Fig. 3a), whilst transformants harbouring only pAM401 vector remained resistant to DvnV41 (data not shown).
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). This resulted in the restoration of the DvnV41-sensitive phenotype, proving that plasmid insertion was responsible for this resistant phenotype (data not shown). Further experiments, based on phenotypic and molecular tests, were performed to confirm the enterococcal identity of each strain. As a result, all the strains were shown to be Gram-positive bacteria, cocci associated in pairs, and devoid of both catalase and oxidase activities. The identification procedure was completed by PCR-RAPD using the oligonucleotide primer M13V (Muller et al., 2001). The PCR-RAPD profiles of the wild-type and mutant strains were identical (data not shown).
Growth characteristics of Ent. faecalis JH2-2 and its mutant derivatives were determined and compared. In the absence of DvnV41, the growth rates of wild-type and mutant strains were not significantly different. Addition of 5 µl of DvnV41, at 820 000 AU ml–1, to 150 µl cultures, did not affect the growth rate of the mutant strains but extended the lag period of the wild-type strain by 4 h, or even 8 h when the amount of DvnV41 was doubled (data not shown). Characterization of the sensitivity of wild-type and mutant strains was carried out by measuring the MIC of DvnRV41, which ranged between 0.22 and 112 µg ml–1 (Table 2). The lowest value was obtained for the wild-type strain, which was 100 times more sensitive than mutants 35A1 (defective phenotype in putative PDE) and 36H4 (defective phenotype in putative GlpQ) and 500 times more sensitive than mutant 35H1 (defective phenotype in 54 factor RNA polymerase).
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summarizes the related cross-resistance obtained with class IIa bacteriocins (DvnV41, Ped PA-1/AcH and MesY105), class I bacteriocin (nisin), and antibiotics. In direct comparison, the cross-resistance exhibited by the mutant strains to class IIa bacteriocins (Table 3; Fig. 3b) is in agreement with that formerly reported (Dalet et al., 2000; Ramnath et al., 2000; Gravesen et al., 2002). Notably, the resistance was evaluated by two independent experiments including ADT and MIC (Tables 2 and 3). Both tests indicated that the resistance to DvnV41 developed by the wild-type and mutant strains was graduated since mutant 35H1 (defective in 54 factor) was more resistant than mutant 36H4 (defective in putative GlpQ), which was itself slightly more resistant than the mutant 35A1 (defective in putative PDE) (Table 3). In contrast, no difference was revealed between the wild-type and mutant strains in terms of resistance to nisin (Table 3). The partial cross-resistance to class IIa bacteriocins, but not to the class I bacteriocin nisin, is consistent with the results of Dalet et al. (2000). Concerning resistance to antibiotics, there was no difference between the phenotype of Ent. faecalis JH2-2 wild-type and the three mutants. In summary, we suggest that a cross-resistance exists to class IIa bacteriocins but not to nisin and antibiotics.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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). The mpt operon encodes a mannose permease (
) which belongs to the (PTS. The level of mpt expression correlates with the level of sensitivity (Dalet et al., 2001; Héchard et al., 2001), which implies that 
permease might be a target molecule for class IIa bacteriocins. Given that the IIC and IID subunits are probably present in the membrane, they are potential targets of class IIa bacteriocins. The mptACD operon of L. monocytogenes heterologously expressed in an insensitive species, such as Lactococcus lactis (Ramnath et al., 2004), renders this strain sensitive to various class IIa bacteriocins. Each gene of the mptACD operon was expressed independently in Lac. lactis and the expression of mptC alone was found to be sufficient to confer sensitivity. The IIC subunit has therefore been proposed as the target molecule of the class IIa bacteriocins (Ramnath et al., 2004). Spontaneous resistance of L. monocytogenes is accompanied by a respective increase and decrease of two phosphoenolpyruvate-dependent PTSs, which are responsible for sugar uptake and phosphorylation in Gram-negative and Gram-positive bacteria (Postma et al., 1993).
How is resistance to DvnV41 mediated in L. monocytogenes and Ent. faecalis JH2-2?
Earlier attempts to unravel the resistance of L. monocytogenes to DvnV41 involved a proteomic approach, comparing protein profiles of L. monocytogenes wild-type and a mutant resistant to DvnV41. L. monocytogenes with a DvnV41-resistant phenotype displayed differential protein synthesis (Duffes et al., 2000). This mutant strain lacked at least nine protein spots, two of which have, interestingly, a molecular mass and pI matching those of the mptA cluster, which is controlled by the 54 transcription factor in coordination with the ManR regulator (Dalet et al., 2001). Undoubtedly, the identification of the proteins present within the two spots will be of major interest in understanding the resistance of L. monocytogenes to DvnV41. In the present study, we have identified three genes associated with the resistance of Ent. faecalis JH2-2 to DvnV41. The first one is the rpoN gene, which encodes the 54 factor, an alternative subunit of RNA polymerase responsible for the transcription of a specific set of genes. The rpoN gene may or may not be involved directly in a general mechanism of sensitivity to class IIa bacteriocins. It has been postulated that rpoN is involved in the expression of a target molecule for class IIa bacteriocins, loss of whose expression leads to resistance (Drider et al., 2006). The second gene identified encodes a putative glycerophosphoryl diester phosphoesterase (GlpQ). GlpQ is an exoprotein found to participate in fatty acid and phospholipid degradation in many bacteria (Antelmann et al., 2000). A hypothesis that could be drawn from our data is that the absence of GlpQ activity in mutant 36H4 leads to an intact fatty acid and phospholipid composition of the cell membrane, which should contribute to the resistant phenotype of this mutant. Moreover, the GlpQ protein was originally suggested to belong to the Pho regulon in Bacillus subtilis (Antelmann et al., 2000), and to be governed by a pleiotropic two-component regulatory system PhoP-PhoR (Groisman, 2001). Interestingly, PhoP and Mg2+ also control the resistance of many Gram-negative bacteria to antimicrobial peptides, as supported by a number of lines of evidence (Groisman et al., 1992; Moss et al., 2001). Similarly, in Gram-positive bacteria, different studies have highlighted the role of a two-component regulatory system in the resistance of L. monocytogenes (Cotter et al., 2002), Ent. faecalis (Comenge et al., 2003) and Staphylococcus aureus (Kuroda et al., 2003) to inhibiting substances such as antimicrobial peptides and antibiotics. At present, it is unclear what contribution a two-component regulatory system makes to Ent. faecalis JH2-2 under harsh environmental conditions with a significant amount of DvnV41. To answer this question, and in order to gain a more comprehensive view of the role of a two-component signal transduction system pathway in resistance to pediocin-like bacteriocins, we examined the resistance of each insertional mutant characterized so far in Ent. faecalis JH2-2 (Le Breton et al., 2003) and Ent. faecalis V583 (Hancock & Perego, 2004). The results obtained reject any relationship between a two-component regulatory system and resistance to DvnV41. Finally, the third gene identified encodes a putative phosphoesterase (PDE), which belongs to the DHH family. At this stage of investigation, it is quite difficult to unravel the role of phosphoesterase in resistance mechanism of Ent. faecalis JH2-2 to divercin V41. The resistance measured by the ADT (Table 3) showed that the size of the inhibition halo is pediocin-like bacteriocin dependent. Interestingly, the resistance of mutant 36H4 to MesY105 was higher than that observed to DvnV41; this could be explained by the activity and/or potency of each bacteriocin.
Overall, it is clear that the rpoN gene is associated with the high level resistance and the newly identified genes with the intermediate resistance of Ent. faecalis JH2-2 to DvnV41, MesY105 and Ped PA-1/Ach.
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ACKNOWLEDGEMENTS |
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Edited by: G. M. Dunny
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Received 1 December 2006;
revised 15 January 2007;
accepted 19 January 2007.
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