Department of Dairy and Food Science, Centre for Advanced Food Studies, LMC, The Royal Veterinary and Agricultural University, Rolighedsvej 30, DK-1958 Frederiksberg C, Denmark1
Department of Biochemistry, University of Stellenbosch, Private Bag X1, 7602 Matieland, South Africa2
German Research Centre for Biotechnology, Department of Cell Biology, Mascheroder Weg 1, Braunschweig D-38124, Germany3
Laboratoire de Microbiologie Fondamentale et Appliquée, CNRS FRE 2224, IBMIG, UFR Sciences, 40 Avenue du Recteur Pineau, 86022 Poitiers Cedex, France4
Author for correspondence: Anne Gravesen. Tel: +45 3528 3272. Fax: +45 3528 3231. e-mail: alg{at}kvl.dk
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ABSTRACT |
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Keywords: pediocin, PTS, mannose, ß-glucoside, sigma-54
Abbreviations: 2D, two-dimensional; PFK, 6-phosphofructokinase; PTS, phosphoenolpyruvate-dependent phosphotransferase system
a The first two authors contributed equally to this work.
b Present address: Foss Electric A/S, Slangerupgade 69, DK-3400 Hillerød, Denmark.
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INTRODUCTION |
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Cross-resistance between different class IIa bacteriocins has frequently been reported (Dykes & Hastings, 1998 ; Ramnath et al., 2000
; Rasch & Knøchel, 1998
), indicating an identical or similar resistance mechanism. Previous studies aimed at characterizing mechanisms of class IIa resistance in L. monocytogenes have, however, reported seemingly varying results. Spontaneous resistance development resulted in an increase and a decrease of two different phosphoenolpyruvate-dependent phosphotransferase systems (PTSs), which are responsible for the uptake and concomitant phosphorylation of a number of sugars in both Gram-negative and Gram-positive bacteria (for a review, see Postma et al., 1993
). A leucocin A-resistant mutant of L. monocytogenes B73 no longer synthesized the IIAB subunit of a mannose-specific PTS (Ramnath et al., 2000
), and 12 independent mutants of L. monocytogenes 412 overexpressed two ß-glucoside-specific PTS genes (Gravesen et al., 2000
). A mutant with resistance to divercin V41 had several changes in protein synthesis, which was suggested to be due to a mutation in a sigma transcription factor (Duffes et al., 2000
). This suggestion was partly based on the fact that transposon mutagenesis of rpoN, encoding the
54 transcription factor, conferred resistance to the IIa bacteriocin mesentericin Y105 (Robichon et al., 1997
). Recently, the mannose-specific PTS,
, was shown by the construction of defined mutants to be directly involved in sensitivity to mesentericin Y105, and a specific domain of the MptD subunit was suggested to be involved in target recognition by the bacteriocin (Dalet et al., 2001
).
One explanation for the apparently diverging results may be that the specific strain in each study developed resistance by different mechanisms. Alternatively, the diverse observations could be windows to the same, complex mechanism, revealed by the different experimental approaches. If different mechanisms do exist, it is conceivable that resistance developed in foods would differ from that developed under standard laboratory conditions.
The aim of the present study is to resolve the differences observed in previous work regarding class IIa resistance in L. monocytogenes, and to further the understanding of the underlying mechanisms, focusing on food relevance. We have compared IIa resistance developed in a panel of listerial wild-type strains following exposure to different IIa bacteriocins in a range of systems, including food. Our results indicate that one general mechanism is responsible for spontaneous class IIa resistance development in L. monocytogenes, and possible models incorporating the different observations are discussed.
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METHODS |
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MIC of leucocin A.
The MICs were determined by a spot-on-lawn assay, essentially as described previously (Ramnath et al., 2000 ). Five-microlitre spots of a twofold serial dilution of 4 mg synthetic leucocin A ml-1 in 0·1% trifluoroacetic acid was spotted onto BHI agar lawns (0·7% agar, 0·1% Tween 80) containing approx. 107 c.f.u. listerial cells ml-1. The MIC was determined as the minimal concentration giving a visible zone of inhibition after 20 h at 37 °C, as the median of three to four independent experiments.
Insertional inactivation of the putative ß-glucoside-specific PTS genes.
The putative ß-glucoside-specific PTS enzyme II (EIIBgl, annotated as lmo00027) and the phospho-ß-glucosidase (lmo00319), which were overexpressed following pediocin resistance development in L. monocytogenes 412 (Gravesen et al., 2000 ), were inactivated in L. monocytogenes 412 and 412P using the 9 kb temperature-sensitive integration vector pAUL-A (Chakraborty et al., 1992
). For EIIBgl, a 1306 bp PCR fragment was made from chromosomal DNA of L. monocytogenes 412P using primers P1 (5'-CATCTGCTAAAGTTACGATTTCGCC-3') and X2 (5'-AAYCAYGTNCCNGAYGT-3', where the mixed bases N and Y correspond to ACGT and CT, respectively). P1 was designed from the previously found C-terminal gene fragment (Gravesen et al., 2000
), and the degenerate primer X2 was designed from an N-terminal conserved region in ß-glucoside-specific EII permeases. An internal 674 bp EcoRI restriction fragment of the PCR product was cloned in pAUL-A, resulting in pAG540. This plasmid was transformed to L. monocytogenes 412 and 412P by electroporation (Park & Stewart, 1990
) and integrated into the chromosome by propagation at 42 °C in the presence of 5 µg erythromycin ml-1 (Chakraborty et al., 1992
), resulting in strains L. monocytogenes AG122 and AG119, respectively. For inactivation of the phospho-ß-glucosidase, a 991 bp PCR product made with primers P3 (5'-GGACTTTCCGTTCAAGATG-3') and P4 (5'-GTGGTTTTTGGTATCTATCC-3') was cloned in pAUL-A, giving plasmid pAG538. The two primers were designed from the two previously found fragments of the gene (Gravesen et al., 2000
). Chromosomal integration of pAG538 in L. monocytogenes 412 and 412P resulted in strains L. monocytogenes AG115 and AG117, respectively. Correct integration was verified by PCR using standard primers complementary to the vector sequence combined with a primer recognizing a chromosomal region adjacent to the segment employed for insertion. For the EII gene, primer P1 was used. For the phospho-ß-glucosidase, primer P4X (5'-ATAGCGCCAAGTCCGTTCTC-3'), situated 20 bp downstream of P4, was used. The class IIa bacteriocin sensitivity of the insertional mutants was tested by plating on TSA supplemented with 30% pediocin PA-1 fermentate, and by determining the MIC for leucocin A.
Northern hybridization.
Expression analysis of the putative ß-glucoside-specific PTS genes was modified after Gravesen et al. (2000) . Cells were harvested from late-exponential cultures (OD600 approx. 0·40·6, depending on the strain) grown in BHI, pH 7·2, at 30 °C without selection, and lysed with a FastPrep FP 120 instrument (Bio 101) at a power setting of 6·0 m s-1 for 45 s. For analysis of insertional and deletion mutants, these strains and the corresponding wild-types were cultured at 37 °C. RNA was purified with the RNeasy Mini kit (Qiagen). The probe for EIIBgl was the fragment A RFDD (restriction fragment differential display) PCR product (Gravesen et al., 2000
) encompassing 292 bp of the permease. For the putative phospho-ß-glucosidase, the 991 bp P3P4 PCR product was used. The probes were labelled with [
-33P]dATP, and hybridization was visualized with a STORM 840 PhosphorImager (Molecular Dynamics).
Protein extraction for two-dimensional (2D) gel electrophoresis.
L. monocytogenes strains were grown until late-exponential phase (OD600 0·450·5) in BHI, pH 7·2, 37 °C. Chloramphenicol (Sigma) was added at a final concentration of 20 µg ml-1 to halt protein synthesis. Bacterial cells were harvested, washed, resuspended in buffer containing Complete Mini tablets (protease inhibitors; Roche), and stored at -80 °C. Once thawed, cell suspensions were disrupted using the FastPrep FP 120 instrument at a power setting of 6·5 for 45 s. This cycle was repeated five times, with chilling of the tubes on ice between cycles. The cell lysate was treated with DNase I and RNase I, and, subsequently, 9·5 M urea, 100 mM DTT (Sigma), 4% (w/v) CHAPS and 2% (v/v) Pharmalyte (pH 310) were added. The supernatant was clarified by centrifugation and stored at -80 °C. All reagents for the protein isolation and 2D gel electrophoresis were from Amersham Pharmacia Biotech, unless stated otherwise.
First-dimension electrophoresis.
Isoelectric focusing was carried out on immobilized pH gradients, pH 47 (11 cm Immobiline Drystrips), on a Multiphor II apparatus according to the manufacturers instructions. The following voltage gradient was applied: from 0 to 300 V in 0·01 h; 300 V for 6·5 h; from 300 to 3500 V in 5 h; and 3500 V for 8 h. Protein samples were cup-loaded at the anodic end. For zoom gels, 18 cm Immobiline Drystrips, pH 56, were used, protein samples were loaded via rehydration, and isoelectric focusing was run according to the manufacturers instructions.
Second-dimension electrophoresis.
Prior to the SDS-PAGE run, isoelectric focusing strips were equilibrated in an SDS equilibration buffer as recommended by the manufacturer, with the following modifications: (1) the concentration of SDS in the SDS equilibration buffer was increased to 4% (w/v); and (2) the concentration of iodoacetamide (Sigma) for the second equilibration step was increased to 4% (w/v). Each equilibration step was carried out for 30 min. The second-dimension run was carried out on pre-cast ExcelGel XL SDS 1214 gels according to the manufacturers instructions. Gels were stained with either Coomassie brilliant blue or silver, and dried as described previously (Rechinger et al., 2000 ). For the extended run of the zoom gel, electrophoresis was stopped after the normal run, the buffer strips were replaced, and the run was resumed for another 2 h.
2D gel analysis.
For each protein sample, at least one Coomassie-stained and one silver-stained gel were examined. Images of Coomassie-stained gels were scanned at a resolution of 200 d.p.i. Quantification and spot matching between gels were done using Z3 Desk Top version 2.0 (Compugen). Differences of at least fourfold up-regulation or 0·3-fold down-regulation were noted by comparison of the 200 most intense spots on the gels of resistant mutants when compared with the corresponding wild-type strain gels. Differences that were found by Z3 analyses were verified by visual examination.
Identification of protein samples by MS.
Protein samples from L. monocytogenes EGDe were excised from Coomassie-stained gels, and in-gel tryptic digestions were carried out overnight. The peptides generated were subjected to electrospray analysis and subsequent peptide sequencing using a quadrupole time-of-flight MS instrument (Q-TOF II; Micromass) equipped with a nanospray ion source. Doubly and triply charged peptides were chosen for collision-induced MS/MS fragmentation experiments, and the corresponding parent ions were selectively transmitted from the quadrupole mass analyser into the collision cell. The resulting daughter ions were separated by an orthogonal time-of-flight mass analyser. Peptide micro-sequencing and protein identification was carried out with the Peptide-Sequencing program within the Biolynx software (version 3.4; Micromass) and with the Sonar program (Proteometrics). The trypsin fragment sequences obtained were compared with the proteins predicted from the L. monocytogenes EGDe genome sequence (http://genolist.pasteur.fr/ListiList/). Predicted molecular mass and pI values were calculated at the Expasy site (http://www.expasy.ch/tools).
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RESULTS |
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Analysis of expression of the putative ß-glucoside-specific PTS genes
The result of the Northern analysis of the spontaneous mutants employing the EIIBgl probe is shown in Fig. 1(a). All eight mutants had a constitutive increase in expression compared to the wild-type strains, which did not have detectable transcription of this gene. The level of the increase varied for the different mutants. There was, however, no correlation between level of increase in expression and level of resistance: the two mutants with low expression, EGDeP4 and DMRICC 4053, had as high resistance to leucocin A as the other mutants. Northern analysis of further 29 high-level-resistant spontaneous mutants of 11 other wild-type strains and of the defined mutants L. monocytogenes EGK51, EGK54 and EGY2 showed that these all had increased expression of the EIIBgl (results not shown).
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Effect of inactivation of the putative ß-glucoside-specific PTS genes on IIa sensitivity
EIIBgl and the putative phospho-ß-glucosidase were inactivated in L. monocytogenes 412 and 412P by plasmid integration using a 674 bp and a 991 bp internal gene fragment, respectively, for homologous recombination. The integration in EIIBgl (giving strains AG122 and AG119, respectively) deleted the C-terminal 194 of the predicted 635 amino acids, including the entire catalytic domain IIA and part of the membrane-spanning domain IIC. In the phospho-ß-glucosidase knockouts (strains AG115 and AG117, respectively), the insertion truncated the last 110 of the 474 predicted amino acids, removing the C-terminal glycosylhydrolase motif.
The pediocin sensitivity of the four insertional mutants was determined. All strains had the same phenotype as the respective original strain: L. monocytogenes AG122 and AG115 were pediocin-sensitive, like L. monocytogenes 412, and L. monocytogenes AG117 and AG119 were pediocin-resistant, like L. monocytogenes 412P. In concordance, the MIC for leucocin A was unaffected by the insertion (Table 1).
Analysis of protein expression by 2D gel electrophoresis
Comparison of the protein profiles of the wild-type and spontaneous resistant mutants showed that one protein spot consistently disappeared following class IIa resistance development (Fig. 2). This change was observed for all five wild-type strains employed, and following exposure to each of the four tested class IIa bacteriocins. The protein spot also disappeared in resistant strains that had developed in a food system, either a saveloy-type sausage or salmon juice. Additionally, this spot was not detected in the gels of the deletion and insertional mutants L. monocytogenes EGK51, EGK54 and EGY2 (Fig. 2
).
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No other consistent changes were observed in the protein profiles of all the mutants tested. However, all four mutants of L. monocytogenes EGDe had an increased content of three high-molecular-mass protein spots (molecular mass range 78·584·8 kDa, pI range 4·534·66), which presumably represents a strain-specific effect. In the other strains, no consistent strain-specific changes were found.
A non-haem-iron-binding ferritin was previously observed to be missing in an L. monocytogenes mutant with resistance to divercin V41 (Duffes et al., 2000 ). A protein with similar molecular mass and pI (Fig. 2
) was found in all wild-type and mutant strains studied, showing no apparent change in expression. TOF-MS analysis of the corresponding spot from L. monocytogenes EGDe confirmed its identity as the non-haem-iron-binding ferritin.
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DISCUSSION |
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Expression of mptACD is controlled by the 54 transcription factor and the activator ManR (Dalet et al., 2001
). Knockout mutants of ManR or MptA, which were resistant to mesentericin Y105 (Dalet et al., 2001
) and to all class IIa bacteriocins used in this study, also had increased expression of EIIBgl. This observation corroborates the hypotheses that the mannose and ß-glucoside-specific PTS expression changes are part of the same resistance mechanism, and also indicates that the class IIa resistance mechanism conferring spontaneous, high-level resistance in L. monocytogenes is related to the mannose PTS-mediated effects described by defined mutations (Dalet et al., 2001
).
A divericin 41-resistant mutant of L. monocytogenes P lacked at least nine protein spots (Duffes et al., 2000 ), two of which had a molecular mass and pI very similar to the MptA cluster. It is therefore possible that this mutant also acquired resistance by the same general mechanism; however, verification of this hypothesis would require identification of the proteins in these two spots.
An important aspect of resistance is whether the same mechanism is acquired in different species or genera. Listeria innocua is a non-pathogenic species with high genomic similarity to L. monocytogenes (Glaser et al., 2001 ). Northern analysis with L. monocytogenes probes showed that a class IIa bacteriocin-resistant mutant of each of five L. innocua wild-type strains similarly had increased expression of EIIBgl and the phospho-ß-glucosidase (results not shown). In Enterococcus faecalis, expression of a mannose PTS enzyme II is also involved in class IIa bacteriocin sensitivity (Héchard et al., 2001
). Altogether, the results strongly suggest that class IIa bacteriocin resistance is conferred by the same general mechanism in L. monocytogenes, L. innocua and E. faecalis, and conceivably also in some other Gram-positive organisms.
Direct and indirect effects how is class IIa bacteriocin resistance acquired?
It was recently reported that the enantiomer of leucocin A was not biologically active (Yan et al., 2000 ), which strongly indicates that activity of class IIa bacteriocins requires chiral interaction with a docking molecule. It is tempting to speculate that the general resistance mechanism involves elimination of this docking molecule.
EIIBgl and the putative phospho-ß-glucosidase were suggested to be encoded in the same operon, based on homology (Gravesen et al., 2000 ). However, according to the recently released L. monocytogenes EGDe genome sequence (Glaser et al., 2001
), they are located at separate positions on the chromosome. Interruption of EIIBgl or the phospho-ß-glucosidase in the resistant mutant L. monocytogenes 412P did not affect IIa resistance. Therefore, the increased expression of either of these genes does not per se cause class IIa bacteriocin resistance, but is presumably a natural regulatory consequence of acquired resistance. Prevention of mptACD expression directly conferred resistance (Dalet et al., 2001
), suggesting that the membrane component, the MptCMptD complex, could function as target for class IIa bacteriocins. Expression of mpt could be prevented through mutation in rpoN, manR or mpt. The multiple possibilities could explain the observation of relatively high IIa resistance frequencies of approximately 10-6 (Gravesen et al., 2002
). The specific location of the mutation in a resistant strain will determine the extent of the changes in the strain, i.e. a mutation in
54 would have more extensive consequences than a mutation in mpt. Abolished mptACD expression could cause up-regulation of EIIBgl and the phospho-ß-glucosidase expression; this is similar to the observation that a mannose PTS regulates expression of other PTS, including ß-glucoside-specific enzymes, as part of the carbon catabolite repression in Streptococcus salivarius and Lactobacillus pentosus (Bourassa & Vadeboncoeur, 1992
; Chaillou et al., 2001
; Gauthier et al., 1990
). Supporting this hypothesis, a sequence with only two mismatches to the cre (catabolite-responsive element; Stülke & Hillen, 1999
) consensus overlaps a putative -35/-10 promoter upstream of the EIIBgl reading frame.
An additional 28 aa domain present in the MptD subunit of compared to other mannose PTS EII sequences was indicated to possibly be involved in IIa sensitivity, since L. monocytogenes EGY2, which has an in-frame deletion of the additional domain, was resistant to mesentericin Y105 (Dalet et al., 2001
). However, the 2D gel analysis showed that L. monocytogenes EGY2 did not produce detectable amounts of MptA, indicating that the mptACD operon is repressed. The repression could be due to the deletion rendering the permease functionally inactive, which in turn could modify the expression through a regulatory cascade.
The two other candidate proteins potentially involved in class IIa bacteriocin resistance, PFK and the non-haem iron-binding ferritin, are apparently not the causative factor. The presence of PFK in two different protein spots could be due to post-translational modifications of the protein, where phosphorylation and/or truncation could result in the lower molecular mass and higher pI observed in the spot adjacent to the consistently disappearing cluster. However, the adjacent PFK spot had similar intensity in wild-type strains and their derived mutants, indicating that there is no overall change in PFK expression related to class IIa bacteriocin resistance. The non-haem iron-binding ferritin, which was missing in one mutant (Duffes et al., 2000 ), did not have any difference in expression in the eight mutants in this study. This observation and the strain-specific changes seen in the L. monocytogenes EGDe mutants underline the importance of analysing several wild-type and mutant strains.
All in all, we suggest that the presently available knowledge allows the following conclusions. Resistance to IIa bacteriocins is acquired through one general mechanism in L. monocytogenes and at least some other Gram-positive organisms. This mechanism is characterized by prevention of synthesis and up-regulation of EIIBgl and the phospho-ß-glucosidase. Up-regulated EIIBgl and phospho-ß-glucosidase expression is not a direct cause of resistance, but is presumably a regulatory consequence of abolished mptACD expression. Prevention of mpt expression directly confers resistance. Although present evidence suggests that the MptCMptD complex interacts as target with class IIa bacteriocins, more work is required to elucidate how shutdown of mpt expression actually causes resistance.
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ACKNOWLEDGEMENTS |
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Received 13 February 2002;
revised 10 April 2002;
accepted 17 April 2002.