Laboratoire de Microbiologie Fondamentale et Appliquée, CNRS FRE 2224, IBMIG, UFR Sciences, 40 avenue du Recteur Pineau, 86022 Poitiers Cedex, France1
Author for correspondence: Yann Héchard. Tel: +33 5 49 45 40 07. Fax: +33 5 49 45 35 03. e-mail: yann.hechard{at}univ-poitiers.fr
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ABSTRACT |
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Keywords: antagonism, subclass IIa, phosphotransferase, sugar, helicase
Abbreviations: 2DG, 2-deoxyglucose; PTS, phosphotransferase system
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INTRODUCTION |
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Interestingly, most of the subclass IIa bacteriocins were described to be also active against another pathogenic species, E. faecalis, in which the 54 factor is involved in sensitivity (Dalet et al., 2001
). Consequently,
54-associated activators and
54-dependent genes were sought and analysed. Knockout of these different genes was then achieved to study their involvement in E. faecalis sensitivity to mesentericin Y105.
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METHODS |
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DNA manipulations and gene interruption.
Molecular cloning and DNA manipulations were performed as described previously (Sambrook et al., 1989 ). Restriction and modification enzymes purchased from Life technologies were used as recommended by the manufacturer. Internal gene fragments, used for knockout experiments, were amplified by PCR with the following primers bearing a HindIII site: mpoR primers, GTCAAGCTTCTGGCTATACCCG and TTTTAAGCTTCGCCCATCCGTG; mptR primers, GTAAAGCTTTGAATCAACTGGTTCG and CAGAAAGCTTCCATCTGCTTCATC; mphR primers, TCAAAGCTTCCAAGAAATGATCGATG and TGGCAAAGCTTAACCGACACG; lpoR primers, AGTAAGCTTCGGACAAGTCAGCG and TCCAAGCTTAATGGCAAAAGCAGATG; mptB primers, CGTTAAGCTTGAATTGATGATCG and AATAAGCTTGGCTGTCTGCTGC; mptD primers, AGCTGAAGCTTGGCGTTCAAC and AGAACAAGCTTTGTAACCAAACTC. The resulting PCR fragments were digested with HindIII and ligated at the same site in pUCB300 (Frère et al., 1993
), a non-replicative plasmid bearing an erythromycin-resistance gene. It gave rise to the pEF17 (mpoR), pEF16 (mptR), pEF23 (mphR), pEF5 (lpoR), pEF18 (mptB) and pEF19 (mptD) plasmids. They were then used to transform E. faecalis JH2-2 as described by Wyckoff et al. (1991)
to achieve independent gene knockout by homologous recombination with the E. faecalis JH2-2 chromosome. The resulting interrupted mutants (erythromycin-resistant) from each experiment were analysed by Southern blotting of chromosomal DNA (Sambrook et al., 1989
), previously digested by HindIII. The DNA probes used for hybridization were synthesized by random priming from the PCR fragments described above.
2-Deoxyglucose-resistant strain selection.
E. faecalis JH2-2 was grown on LB agar plates supplemented with fructose at 2 g l-1, as a carbon source, and 2-deoxyglucose (2DG), a non-metabolizable analogue of glucose, at 10 mM. The resulting colonies, corresponding to the growth of 2DG-resistant mutants, were isolated twice on the same medium. 2-DG is a toxic molecule that enters bacteria via a PTS permease of the mannose family (Bond et al., 1999 ). Consequently, this permease is not usually expressed in 2DG-resistant mutants.
Bacteriocin purification and assays.
Mesentericin Y105, produced by Leuconostoc mesenteroides Y105, was purified as previously described (Guyonnet et al., 2000 ). E. faecalis susceptibility was assayed by spot on lawn or microtitre plate tests. The former was achieved by overlaying a BHI agar plate (1·5%) with a BHI agar lawn (0·7%) inoculated at 1% with an E. faecalis culture. Purified mesentericin Y105 (5 µl) was spotted on the lawn and the plate was then incubated overnight at 37 °C before inhibition zones were noted. The microtitre plate assay was carried out by inoculating 200 µl BHI or LB supplemented with various sugars at 2 g l-1. Bacterial growth was monitored by measurement of the OD620 and 5 µl purified mesentericin Y105 was added when the OD620 reached 0·1.
DNA sequencing.
Cycle sequencing was achieved with the ABI Prism BigDye Terminator Cycle Sequencing Ready Reaction kit (Perkin-Elmer) and analysed with the ABI Prism 310 genetic analyser. Sequence data from E. faecalis V583 were obtained from the Institute for Genomic Research through the website at http://www.tigr.org/.
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RESULTS |
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Thus, we first used an amino acid sequence of the central domain to screen the E. faecalis V583 genome. BLAST results displayed five high scores (>45% identities), corresponding to five ORFs, named mpoR, mptR, mphR, lpoR and xpoR. These genes potentially encode 54-associated activators of 937, 961, 923, 901 and 403 amino acids, respectively. The latter, xpoR, is interrupted by a sequence which is identical to the transposon Tn4001 from Staphylococcus aureus (Byrne et al., 1989
). Interestingly, all these activators share highest identities, about 30%, with an unusual member of the
54-associated activator family, LevR of B. subtilis (Débarbouillé et al., 1991a
). Besides the classical motifs usually described in
54-associated activators, the central domain of all these activators displayed a DEAH motif, according to Prosite (Fig. 1
), usually found in helicases. Such similarities with helicases have already been looked for in activators to explain their involvement in DNA conformational changes (Buck et al., 2000
), but none have been found. We suggest that, owing to the presence of the DEAH motifs, these activators could be directly responsible for ATP-dependent DNA unwinding, allowing initiation of transcription. In addition, we found degenerate DEAH motifs in all the other
54-associated activators so far described in the database (data not shown), indicating that they could also bear a helicase activity.
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The sensitivity of these knockout strains was then tested by a spot on lawn assay with purified mesentericin Y105 (Table 1). E. faecalis JH3, JH8 and JH11 remained sensitive to mesentericin Y105, similarly to the wild-type JH2-2 strain. In contrast, the JH7 strain (knockout of mptR) became fully resistant to purified mesentericin Y105, as was the previously described JH1 strain (
54-deficient mutant) (Dalet et al., 2001
). Consequently, MptR is the only E. faecalis
54-associated activator presumably involved in sensitivity to mesentericin Y105. This suggests that MptR and
54 control the expression of proteins involved in E. faecalis sensitivity to mesentericin Y105.
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Sugar effects on E. faecalis JH2-2 sensitivity to mesentericin Y105
Since E. faecalis JH2-2 sensitivity to mesentericin Y105 seems to be linked to a specific PTS mannose permease, the effect of various sugars on sensitivity was tested. Indeed, expression of PTS permeases is specifically induced by the transported sugar (Saier & Reizer, 1994 ). E. faecalis JH2-2 was grown in LB medium supplemented independently with the following sugars at 2 g l-1: fructose, cellobiose, glucose and mannose. Fig. 4
shows that the sensitivity of E. faecalis was highly increased in the presence of glucose or mannose, compared to cellobiose or fructose. E. faecalis was also weakly sensitive to mesentericin Y105 in LB medium without any added sugar (data not shown). This suggests that glucose and mannose activate the expression of a protein involved in sensitivity to mesentericin Y105, probably the
PTS permease.
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DISCUSSION |
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According to the acquired resistance phenotype displayed by knockout mutants, MptR appears as the only one, among the five activators, to be involved in sensitivity to mesentericin Y105. MptR probably activates the transcription, together with 54, of the flanking downstream operon, which encodes a mannose PTS permease,
. In addition, knockout within this operon also led to resistance, which supports
being involved in resistance and being dependent on MptR. We have also recently shown that knockout within an operon encoding a
54-dependent PTS permease of the mannose family leads to L. monocytogenes resistance (K. Dalet, Y. Cenatiempo, P. Cossart, The European Listeria Genome Consortium and Y. Héchard, unpublished results). Moreover, the IIAB subunit of a mannose PTS permease was shown to be absent in a spontaneous mutant of L. monocytogenes resistant to leucocin A (Ramnath et al., 2000
), favouring the lack of EIIMan expression. Another spontaneous mutant of L. monocytogenes, resistant to pediocin PA-1 (a subclass IIa bacteriocin), has been reported by others to overexpress a PTS permease from the ß-glucoside family (Gravesen et al., 2000
). However, the same group found out that knockout of the ß-glucoside operon did not modify the sensitivity to pediocin PA-1 and that this PTS is also overexpressed in our L. monocytogenes rpoN mutant, lacking
54 (A. L. Gravesen, personal communication). These results on both E. faecalis and L. monocytogenes point towards an essential role of an EIIMan permease in sensitivity to mesentericin Y105 and related subclass IIa bacteriocins. Our current hypothesis is that EIIMan is probably a receptor for these bacteriocins. Finally, we showed that the presence of glucose and mannose in the culture medium greatly increases E. faecalis sensitivity to mesentericin Y105. Since PTS permease expression has been reported to be specifically induced by transported sugars, we hypothesize that glucose and mannose induce the expression of
, thereby leading to an increase in the number of potential protein receptors for mesentericin Y105.
Further data favour the above hypotheses and focus on a particular component of the permease. Knockout of mptD, the distal gene of this operon, led to resistance and thus the corresponding MptD subunit seems essential in E. faecalis sensitivity to mesentericin Y105. In L. monocytogenes, knockout or in-frame deletion of a gene, encoding a IIDMan subunit, also led to resistance (K. Dalet, Y. Cenatiempo, P. Cossart, The European Listeria Genome Consortium and Y. Héchard, unpublished results). Moreover, this IIDMan subunit of L. monocytogenes harbours an additional motif, as found here in E. faecalis MptD, and the above-mentioned in-frame deletion removed this domain. Thus, in these two organisms, the IID subunit of the EIIMan involved in sensitivity bears an additional motif (a putative external loop) not found within the other IID subunits described, except ManN of S. salivarius. The additional domain of E. faecalis shares 64% similarity with that of L. monocytogenes and only 39% with that of S. salivarius. Moreover, IID subunits are integral membrane proteins and could therefore interact directly with mesentericin Y105. Accordingly, we propose that an EIIMan is a receptor for various subclass IIa bacteriocins in L. monocytogenes and E. faecalis and that its IIDMan component plays a central role in bacteriocin action, probably by a direct proteinprotein interaction involving the additional domain.
Whether an EIIMan is also implicated in sensitivity to subclass IIa bacteriocins when present in other bacteria is being analysed. Direct proteinprotein interaction between subclass IIa bacteriocins and the permease will be further investigated, together with the implication of the additional domain found in their IID components.
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ACKNOWLEDGEMENTS |
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Received 1 November 2000;
revised 6 January 2001;
accepted 11 January 2001.