Laboratoire de Microbiologie Fondamentale et Appliquée, CNRS ESA 6031, IBMIG, UFR Sciences, 40 avenue du Recteur Pineau, 86022 Poitiers Cedex, France1
Unité des Interactions Bactéries-Cellules, Institut Pasteur, 28 rue du Dr Roux, 75724 Paris Cedex 15, France2
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: bacteriocin, receptor, helicase, transport, sugar
Abbreviations: PTS; phosphotransferase system
a The European Genome Consortium is composed of Philippe Glaser, Alexandra Amend, Fernando Baquero-Mochales, Patrick Berche, Helmut Bloecker, Petra Brandt, Carmen Buchrieser, Trinad Chakraborty, Alain Charbit, Elisabeth Couvé, Antoine de Daruvar, Pierre Dehoux, Eugen Domann, Gustavo Dominguez-Bernal, Lionel Durant, Karl-Dieter Entian, Lionel Frangeul, Hafida Fsihi, Francisco Garcia del Portillo, Patricia Garrido, Werner Goebel, Nuria Gomez-Lopez, Torsten Hain, Joerg Hauf, David Jackson, Jurgen Kreft, Frank Kunst, Jorge Mata-Vicente, Eva Ng, Gabriele Nordsiek, Jose Claudio Perez-Diaz, Bettina Remmel, Matthias Rose, Christophe Rusniok, Thomas Schlueter, Jose-Antonio Vazquez-Boland, Harmut Voss, Jurgen Wehland and Pascale Cossart.
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INTRODUCTION |
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To find a link between 54 and sensitivity of L. monocytogenes to mesentericin Y105, we searched for
54-associated activators and
54-dependent genes in the L. monocytogenes EGDe genome. We found an activator and a PTS permease of the mannose family that are required for sensitivity of L. monocytogenes 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 by Sambrook et al. (1989) . Restriction and modification enzymes purchased from Life Technologies were used as recommended by the manufacturer. DNA fragments, used for gene interruption experiments, were amplified by PCR using Taq polymerase and specific primers bearing a HindIII site, as follows: manR primers, 5'-CTGCCAAGCTTGGAAGAACG-3' and 5'-CATCATCTTCCAAAGCTTGATCC-3'; mptA primers, 5'-GCTGAAGCTTTTTTGCAGTCCG-3' and 5'-GATGAGAAAGCTTCAATCAACATTGG-3'; mptD primers, 5'-CATCTCCAAAGCTTGGGGTAAC-3' and 5'-GGCGCAAGCTTGATATTTACCC-3'. A 1559 bp HindIII fragment, corresponding to the Tn917lac/rpoN junction of pRT758 (Robichon et al., 1997
), was used for rpoN interruption. The latter DNA fragment and the PCR products were digested with HindIII and ligated at the same site in the erythromycin-resistant pHV1248
Tn10 plasmid (Petit et al., 1990
), giving rise to plasmids pLMK50 (rpoN), pLMK51 (manR), pLMK54 (mptA) and pLMK55 (mptD). These plasmids were used to create independent knockouts in rpoN, manR, mptA and mptD by homologous recombination with the L. monocytogenes EGDe chromosome, as previously described (Kocks et al., 1992
). Several mutants from each transformation were analysed by Southern blotting of chromosomal DNA digested by HindIII and hybridized with probe labelled by random priming from the PCR products described above (Sambrook et al., 1989
).
Allelic exchange of mptD.
Allelic exchange of mptD was achieved by integration-excision of a plasmid bearing a deleted fragment of mptD. This fragment was obtained by PCR as follows. One gene fragment from each side of the additional domain we wanted to delete was amplified with the following primers: Del1 (5'-ATGAAGCTTTTCAAGGGGTTAAAGT TGG-3') and Del2 (5'-GCAAGGTTGTTACTTTAATTTCCGCACCTTCATCAAGTTTAACTTTCG-3') together and Del3 (5'-CGAAAGTTAAACTTGATGAAGGTGCGGAAATTAAAGTAACAACCTTGC-3') and Del4 (5'-ACGTAAGCTTTAAGTCCAGTATACGC-3') together. The resulting PCR products, overlapping by 25 bp, were then used in a PCR reaction, leading to a 84 bp deleted fragment of mptD (655738mptD). This fragment was first cloned in pGEM-T (Promega), confirmed by sequencing and then subcloned as a HindIII fragment in pHV1248
Tn10. The resulting plasmid, pLMY2, was used to achieve integration in mptD by homologous recombination. Excision events were then screened by their erythromycin sensitivity. Finally, erythromycin-sensitive clones were tested for the presence of the required deletion by PCR with primers Del1 and Del4 and sequencing. This gave rise to the strain EGY2 (EGDe-
655738mptD).
Bacteriocin purification and assays.
Mesentericin Y105 was purified as reported (Guyonnet et al., 2000 ). Nisin, a class I bacteriocin, was purchased from Sigma. L. monocytogenes sensitivity was assayed by spot-on-lawn or microtitre plate tests. The former was achieved by overlaying a BHI agar (1·5%) plate with a BHI agar lawn (0·7%) previously inoculated with 1% L. monocytogenes. Purified bacteriocin was then spotted on the lawn, the plate was incubated overnight at 37 °C and zones of inhibition were recorded. The microtitre plate tests were conducted as follows. Bacteria were grown overnight in LB medium and inoculated in 1 ml fresh LB medium to a final OD620 between 0·01 and 0·03. The culture was supplemented or not with either fructose, glucose, mannose or cellobiose at 2 g l-1. Four aliquots of 200 µl from each sample were then distributed in a microtitre plate. Plates were incubated at 37 °C with agitation at 120 r.p.m. and bacterial growth was monitored by measurement of the OD620. Purified mesentericin Y105 (100 ng) was added after 2 h, when the culture had reached an OD620 between 0·05 and 0·1.
Transcription analysis.
L. monocytogenes EGDe and its derivatives were grown in 3 ml LB medium supplemented or not with glucose, mannose, fructose or cellobiose (2 g l-1) to an OD600 of 0·6. The bacterial pellets collected by centrifugation were resuspended in 100 µl lysis buffer (10 mg lysozyme ml-1, 10 mM Tris, 1 mM EDTA, pH 7·5) and incubated for 1 h at 37 °C. Total RNA was then extracted with the RNAwiz reagent (Ambion) and treated as recommended. The RNA pellets were finally resuspended in 50 µl diethylpyrocarbonate-treated water containing 20 U RNase-free DNase I. Slot-blot hybridization was performed as described by Sambrook et al. (1989) . A 32P-labelled mptD probe was prepared by random priming from the PCR product obtained with the mptD primers described above. Radioactivity was measured with an Instant Imager apparatus (Packard).
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.
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RESULTS |
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MptD plays a particular role in sensitivity
The distal gene of the mpt operon, mptD, seems to play a particular role in sensitivity since its interruption led to resistance. Interestingly, MptD bears an additional domain compared to three other IIDMan subunits found in the L. monocytogenes EGDe genome and to most of IIDMan subunits described in the literature except E. faecalis (Héchard et al., 2001 ) and Streptococcus salivarius (Lortie et al., 2000
) or found in GenBank (Fig. 3
). Among 22 bacterial sequenced genomes (finished or unfinished) found to possess at least one orthologue of IIDMan, only several Gram-positive bacteria (i.e. E. faecalis, Streptococcus spp. (5 examples), Lactococcus lactis and Clostridium acetobutylicum) have a IIDMan with an additional domain. Interestingly, these Gram-positive bacteria have sometimes been described to be sensitive to subclass IIa bacteriocins. The mutant strain EGY2 has an 84 bp in-frame deletion of mptD (
655738mptD), created by allelic exchange (see Methods). It likely encodes the
219246MptD protein with a 28 aa deletion in the additional domain (bold characters in Fig. 3
). Strain EGY2 was tested for sensitivity to mesentericin Y105, as described above. It was fully resistant, indicating that the presence of the additional domain is of primary importance for sensitivity.
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Mannose and glucose influence sensitivity of L. monocytogenes to mesentericin Y105
L. monocytogenes EGDe was grown in LB medium supplemented with glucose, mannose, fructose or cellobiose at 2 g l-1. Fig. 5(a) shows that, in the absence of mesentericin Y105, no significant difference in L. monocytogenes EGDe growth curves could be observed, whereas the presence of mesentericin Y105 affected L. monocytogenes growth in a medium supplemented with mannose or glucose but not with cellobiose or fructose. These results show that glucose and mannose specifically induce sensitivity of L. monocytogenes to mesentericin Y105.
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DISCUSSION |
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We demonstrate here that the 54 regulon of L. monocytogenes is clearly involved in sensitivity to mesentericin Y105, bringing experimental support to assessments arising from our earlier work on E. faecalis (Héchard et al., 2001
). Interruption of either rpoN, manR, mptA or mptD (encoding, respectively,
54, a
54-associated activator and two subunits of the
permease) led to resistance of L. monocytogenes EGDe.
, a PTS permease of the mannose family, is encoded by the mpt operon. This operon, which bears a -24/-12 promoter, was not expressed in the rpoN and manR mutants, showing that its expression is positively controlled by
54 and ManR. In E. faecalis, the mpt operon also bears a putative -24/-12 promoter although it was not experimentally demonstrated to be regulated by MptR or
54 (Héchard et al., 2001
). The localization of the mpo operon immediately downstream from manR suggests that it could also be controlled by ManR and preliminary results indicate a possible cross-regulation between the mpo and mpt operons. Accordingly, ManR likely controls these two operons whereas MptR of E. faecalis presumably controls expression of mpt only. Relationships between these operons would not be surprising according to the known regulation of carbohydrate metabolism.
The presence of glucose or mannose induced sensitivity of L. monocytogenes and E. faecalis to mesentericin Y105. These sugars also induced expression of the mpt operon of L. monocytogenes (not shown in E. faecalis), indicating that transports glucose and mannose in accordance with previous observations showing a specific inducible effect of the transported sugar on PTS permease expression (Postma et al., 1993
). These correlated results suggest that the level of
expression is directly linked to sensitivity of L. monocytogenes to mesentericin Y105.
Since mesentericin Y105 and related subclass IIa bacteriocins have a narrow spectrum of inhibition, we are wondering about the specificity of the target strains. The IIDMan membrane subunit of , MptD, contains an additional domain compared to most other IIDMan proteins. The mutant EGY2, which putatively encodes a truncated MptD protein, i.e. lacking 28 aa in this additional domain, became resistant to mesentericin Y105. Assuming that the truncated MptD protein is expressed and remains functional, it strongly suggests a primary role of this domain in the sensitivity of L. monocytogenes. The introduction of point mutations in the additional domain constitutes the next step of our work to confirm the role of this domain and to identify the implicated amino acids as well as their possible interaction.
The involvement of in sensitivity to subclass IIa bacteriocins is emphasized by the report of a spontaneous mutant resistant to leucocin A, a subclass IIa bacteriocin (Ramnath et al., 2000
). The authors clearly showed the absence of expression of a IIABMan PTS component in this mutant. Moreover, the N-terminal sequence of the protein shares high identity (17 of 20 residues) with the MptA protein described here. In addition, Gravesen et al. (2000)
recently reported the overexpression of a ß-glucoside PTS in a spontaneous L. monocytogenes mutant resistant to pediocin PA-1, another subclass IIa bacteriocin. This pediocin PA-1 resistant mutant was then shown to be defective in
expression (Y. Héchard, unpublished results) and the ß-glucoside PTS was shown to be overexpressed in our L. monocytogenes LUT758 mutant lacking
54 (A. L. Gravesen, personal communication). In S. salivarius, mutants that lack the synthesis of IIABLMan (similar to MptA) are derepressed for several genes, such as the ß-galactosidase gene (Gauthier et al., 1990
). We thus speculate that overexpression of the ß-glucoside PTS is a consequence of the lack of
expression. Taken together, these results are evidence that
is a key component for sensitivity of L. monocytogenes to different subclass IIa bacteriocins.
In conclusion, we propose that could either influence the expression of an unknown molecule involved in sensitivity or, via its IICManIIDMan membrane complex, be a docking molecule or a receptor for mesentericin Y105 and other subclass IIa bacteriocins. Since deletion of the additional domain of MptD led to resistance, we propose that this domain could directly interact with bacteriocins or that its deletion could change the structure of the permease, leading to a lower affinity for the bacteriocins. Finally, a IICManIIDMan complex has already been described to facilitate penetration of phage lambda DNA across the inner membrane of Escherichia coli (Esquinas-Rychen & Erni, 2001
). It would be interesting to see whether both phage and bacteriocin could interact with cells via a similar mechanism.
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ACKNOWLEDGEMENTS |
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Received 23 April 2001;
revised 3 August 2001;
accepted 23 August 2001.