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Clostridium difficile Has an Original Peptidoglycan Structure with a High Level of N-Acetylglucosamine Deacetylation and Mainly 3-3 Cross-links*

Open AccessPublished:June 17, 2011DOI:https://doi.org/10.1074/jbc.M111.259150
      The structure of the vegetative cell wall peptidoglycan of Clostridium difficile was determined by analysis of its constituent muropeptides with a combination of reverse-phase high pressure liquid chromatography separation of muropeptides, amino acid analysis, mass spectrometry and tandem mass spectrometry. The structures assigned to 36 muropeptides evidenced several original features in C. difficile vegetative cell peptidoglycan. First, it is characterized by a strikingly high level of N-acetylglucosamine deacetylation. In addition, the majority of dimers (around 75%) contains A2pm3 → A2pm3 (A2pm, 2,6-diaminopimelic acid) cross-links and only a minority of the more classical Ala4 → A2pm3 cross-links. Moreover, a significant amount of muropeptides contains a modified tetrapeptide stem ending in Gly instead of d-Ala4. Two l,d-transpeptidases homologues encoding genes present in the genome of C. difficile 630 and named ldtcd1 and ldtcd2, were inactivated. The inactivation of either ldtcd1 or ldtcd2 significantly decreased the abundance of 3-3 cross-links, leading to a marked decrease of peptidoglycan reticulation and demonstrating that both ldtcd1-and ldtcd2-encoded proteins have a redundant l,d-transpeptidase activity. The contribution of 3-3 cross-links to peptidoglycan synthesis increased in the presence of ampicillin, indicating that this drug does not inhibit the l,d-transpeptidation pathway in C. difficile.

      Introduction

      Clostridium difficile, a Gram-positive spore-forming bacterium, is the major cause of intestinal diseases associated with antibiotic therapy such as ampicillin, clindamycin, and cephalosporins, which disrupt the barrier intestinal flora and allow C. difficile colonization (
      • Viswanathan V.K.
      • Mallozzi M.J.
      • Vedantam G.
      ,
      • Finegold S.M.
      ). Clinical manifestations range from asymptomatic colonization or mild diarrhea to pseudomembranous colitis (
      • Kelly C.P.
      • LaMont J.T.
      ). The main virulence factors have been identified as toxin A and B (
      • Voth D.E.
      • Ballard J.D.
      ). Recent outbreaks have led to increasing morbidity and mortality and have been associated with a new highly virulent strain (BI/NAP1/027) of C. difficile. Antibiotic treatment of C. difficile-associated disease requires metronidazole or vancomycin therapy.
      Peptidoglycan (PG)
      The abbreviations used are: PG
      peptidoglycan
      RAM
      retrotransposition-activated marker
      A2pm
      2,6-diaminopimelic acid
      GlcNH2
      N-deacetylated GlcNAc
      MurOHNAc
      N-acetylmuramicitol
      MurNac
      N-acetylmuramic acid
      GlcNac
      N-acetylglucosamine.
      of Gram-positive bacteria usually consists of long linear glycan strands cross-linked by short stem peptides (
      • Höltje J.V.
      ,
      • Scheffers D.J.
      • Pinho M.G.
      ). PG is usually connected by 4-3 cross-links catalyzed by d,d-transpeptidases, which belong to the penicillin-binding proteins and whose substrate is the peptidyl d-Ala4-d-Ala5 extremity of PG precursors. d,d-Transpeptidases are the essential targets of β-lactams antibiotics, which are structural analogues of the d-Ala4-d-Ala5 terminus precursor molecule (
      • Sauvage E.
      • Kerff F.
      • Terrak M.
      • Ayala J.A.
      • Charlier P.
      ,
      • Zapun A.
      • Contreras-Martel C.
      • Vernet T.
      ). Alternatively, PG can be connected by 3-3 cross-links generated by l,d-transpeptidases (
      • Mainardi J.L.
      • Villet R.
      • Bugg T.D.
      • Mayer C.
      • Arthur M.
      ), which were originally detected in Enterococcus faecium (Ldtfm) (
      • Mainardi J.L.
      • Fourgeaud M.
      • Hugonnet J.E.
      • Dubost L.
      • Brouard J.P.
      • Ouazzani J.
      • Rice L.B.
      • Gutmann L.
      • Arthur M.
      ) and then in other Gram-positive bacteria(
      • Lavollay M.
      • Arthur M.
      • Fourgeaud M.
      • Dubost L.
      • Marie A.
      • Riegel P.
      • Gutmann L.
      • Mainardi J.L.
      ,
      • Magnet S.
      • Arbeloa A.
      • Mainardi J.L.
      • Hugonnet J.E.
      • Fourgeaud M.
      • Dubost L.
      • Marie A.
      • Delfosse V.
      • Mayer C.
      • Rice L.B.
      • Arthur M.
      ), in mycobacteria (
      • Lavollay M.
      • Arthur M.
      • Fourgeaud M.
      • Dubost L.
      • Marie A.
      • Veziris N.
      • Blanot D.
      • Gutmann L.
      • Mainardi J.L.
      ,
      • Lavollay M.
      • Fourgeaud M.
      • Herrmann J.L.
      • Dubost L.
      • Marie A.
      • Gutmann L.
      • Arthur M.
      • Mainardi J.L.
      ,
      • Gupta R.
      • Lavollay M.
      • Mainardi J.L.
      • Arthur M.
      • Bishai W.R.
      • Lamichhane G.
      ), and in Escherichia coli (
      • Magnet S.
      • Bellais S.
      • Dubost L.
      • Fourgeaud M.
      • Mainardi J.L.
      • Petit-Frère S.
      • Marie A.
      • Mengin-Lecreulx D.
      • Arthur M.
      • Gutmann L.
      ,
      • Magnet S.
      • Dubost L.
      • Marie A.
      • Arthur M.
      • Gutmann L.
      ). Ldts use acyl donors containing a tetrapeptide stem (
      • Mainardi J.L.
      • Villet R.
      • Bugg T.D.
      • Mayer C.
      • Arthur M.
      ) and were consequently expected to confer resistance to β-lactams (
      • Mainardi J.L.
      • Fourgeaud M.
      • Hugonnet J.E.
      • Dubost L.
      • Brouard J.P.
      • Ouazzani J.
      • Rice L.B.
      • Gutmann L.
      • Arthur M.
      ,
      • Mainardi J.L.
      • Legrand R.
      • Arthur M.
      • Schoot B.
      • van Heijenoort J.
      • Gutmann L.
      ).
      Another possible variation of the PG structure is the occurrence of N-deacetylation or O-acetylation of glycan strands, either on GlcNAc or on MurNAc residues (
      • Vollmer W.
      ,
      • Bernard E.
      • Rolain T.
      • Courtin P.
      • Guillot A.
      • Langella P.
      • Hols P.
      • Chapot-Chartier M.P.
      ). N-Deacetylation in Listeria monocytogenes (
      • Boneca I.G.
      • Dussurget O.
      • Cabanes D.
      • Nahori M.A.
      • Sousa S.
      • Lecuit M.
      • Psylinakis E.
      • Bouriotis V.
      • Hugot J.P.
      • Giovannini M.
      • Coyle A.
      • Bertin J.
      • Namane A.
      • Rousselle J.C.
      • Cayet N.
      • Prévost M.C.
      • Balloy V.
      • Chignard M.
      • Philpott D.J.
      • Cossart P.
      • Girardin S.E.
      ) or Streptococcus pneumoniae (
      • Vollmer W.
      • Tomasz A.
      ) and O-acetylation in Staphylococcus aureus have been linked to lysozyme resistance (
      • Bera A.
      • Herbert S.
      • Jakob A.
      • Vollmer W.
      • Götz F.
      ).
      The PGs of C. difficile should have some specificities regarding the effect of antibiotics inhibiting PG biosynthesis; C. difficile, although susceptible to β-lactams, exhibits higher minimal inhibitory concentrations than in other Clostridium species such as Clostridium perfringens (
      • Citron D.M.
      • Merriam C.V.
      • Tyrrell K.L.
      • Warren Y.A.
      • Fernandez H.
      • Goldstein E.J.
      ), and it also displays a preserved susceptibility to vancomycin despite the presence of a vanG-like operon (
      • Sebaihia M.
      • Wren B.W.
      • Mullany P.
      • Fairweather N.F.
      • Minton N.
      • Stabler R.
      • Thomson N.R.
      • Roberts A.P.
      • Cerdeño-Tárraga A.M.
      • Wang H.
      • Holden M.T.
      • Wright A.
      • Churcher C.
      • Quail M.A.
      • Baker S.
      • Bason N.
      • Brooks K.
      • Chillingworth T.
      • Cronin A.
      • Davis P.
      • Dowd L.
      • Fraser A.
      • Feltwell T.
      • Hance Z.
      • Holroyd S.
      • Jagels K.
      • Moule S.
      • Mungall K.
      • Price C.
      • Rabbinowitsch E.
      • Sharp S.
      • Simmonds M.
      • Stevens K.
      • Unwin L.
      • Whithead S.
      • Dupuy B.
      • Dougan G.
      • Barrell B.
      • Parkhill J.
      ). However, little is known about the PG structure and biosynthesis in C. difficile. In the present work, we report the fine structure of C. difficile vegetative PG. This structure reveals the presence of a high proportion of non-acetylated glucosamine residues on the glycan strands and the unusual abundance of 3 → 3 peptide cross-links generated by l,d-transpeptidation. Mutations of two putative l,d-transpeptidase genes demonstrate the role of the corresponding proteins in the formation of 3 → 3 cross-links. The participation of the l,d-transpeptidases to peptidoglycan cross-linking increases in the presence of ampicillin, indicating that this drug does not inhibit the l,d-transpeptidation pathway in C. difficile.

      DISCUSSION

      This work reports, for the first time to our knowledge, the composition and structure of PG of C. difficile. The most prevalent muropeptide monomer (peak 7, 26.2% of all peaks) (Fig. 1 and Table 1) represents the basic disaccharide tetrapeptide subunit, whose tetrapeptide stem consists of the usual l-Ala-d-Glu-A2pm-d-Ala. Of note, some muropeptides (monomer 2 and monomer 10, Table 1) lacked a GlcNAc residue. They could result from cleavage by the previously described Acd glucosaminidase (
      • Dhalluin A.
      • Bourgeois I.
      • Pestel-Caron M.
      • Camiade E.
      • Raux G.
      • Courtin P.
      • Chapot-Chartier M.P.
      • Pons J.L.
      ). Schleifer and Kandler (
      • Schleifer K.H.
      • Kandler O.
      ) previously reported the amino acid composition of PG of many species of the genus Clostridium. Most species contained only meso-A2pm, Ala, and Glu, although C. perfringens revealed l,l-A2pm instead of meso-A2pm and additional Gly in the interpeptide bridge, and Clostridium innocuum contained l-Lys instead of A2pm. C. difficile was not reported in this previous work, but based on our amino acid content detection and on the phylogenetic link of C. difficile with Clostridium bifermentans and Clostridium sordellii (
      • Collins M.D.
      • Lawson P.A.
      • Willems A.
      • Cordoba J.J.
      • Fernandez-Garayzabal J.
      • Garcia P.
      • Cai J.
      • Hippe H.
      • Farrow J.A.
      ), which contains meso-A2pm (
      • Schleifer K.H.
      • Kandler O.
      ), PG of C. difficile very likely contains meso-A2pm.
      A high proportion of N-deacetylation of the glycan strands was observed in C. difficile (Table 1). This structural variation occurred only on GlcNAc residues (Fig. 2), whose 93% were N-deacetylated, whereas MurNAc residues remained fully acetylated. Nonacetylated glucosamine (GlcN) or muramic acid (MurN) residues have already been reported, but at a lower rate, in some Gram-positive bacteria (
      • Vollmer W.
      • Blanot D.
      • de Pedro M.A.
      ) such as Bacillus anthracis (
      • Zipperle Jr., G.F.
      • Ezzell Jr., J.W.
      • Doyle R.J.
      ), Bacillus subtilis (
      • Atrih A.
      • Bacher G.
      • Allmaier G.
      • Williamson M.P.
      • Foster S.J.
      ), S. pneumoniae (
      • Vollmer W.
      • Tomasz A.
      ), or L. monocytogenes (
      • Boneca I.G.
      • Dussurget O.
      • Cabanes D.
      • Nahori M.A.
      • Sousa S.
      • Lecuit M.
      • Psylinakis E.
      • Bouriotis V.
      • Hugot J.P.
      • Giovannini M.
      • Coyle A.
      • Bertin J.
      • Namane A.
      • Rousselle J.C.
      • Cayet N.
      • Prévost M.C.
      • Balloy V.
      • Chignard M.
      • Philpott D.J.
      • Cossart P.
      • Girardin S.E.
      ). The presence of these non-acetylated amino sugars confers resistance to lysozyme, an exogenous muramidase, which normally cleaves PG between the glycosidic β1–4-linked residues of GlcNAc and MurNAc. The N-deacetylation of the GlcNAc residues is achieved by PgdA deacetylases, which have been shown to provide a protective role against host defenses in L. monocytogenes (
      • Boneca I.G.
      • Dussurget O.
      • Cabanes D.
      • Nahori M.A.
      • Sousa S.
      • Lecuit M.
      • Psylinakis E.
      • Bouriotis V.
      • Hugot J.P.
      • Giovannini M.
      • Coyle A.
      • Bertin J.
      • Namane A.
      • Rousselle J.C.
      • Cayet N.
      • Prévost M.C.
      • Balloy V.
      • Chignard M.
      • Philpott D.J.
      • Cossart P.
      • Girardin S.E.
      ) and S. pneumoniae (
      • Vollmer W.
      • Tomasz A.
      ). Further studies should be performed to examine the impact of GlcNAc N-deacetylation on lysozyme resistance in C. difficile. Our in silico analysis of the genome sequence of C. difficile 630 revealed the presence of 10 putative polysaccharide deacetylases encoding genes belonging to the carbohydrate esterase family CE4. Ten putative polysaccharide deacetylase-encoding genes were also identified in the B. anthracis and Bacillus cereus genomes (
      • Psylinakis E.
      • Boneca I.G.
      • Mavromatis K.
      • Deli A.
      • Hayhurst E.
      • Foster S.J.
      • Vårum K.M.
      • Bouriotis V.
      ), which exhibit important N-deacetylation levels (
      • Vollmer W.
      • Blanot D.
      • de Pedro M.A.
      ,
      • Zipperle Jr., G.F.
      • Ezzell Jr., J.W.
      • Doyle R.J.
      ).
      An important and unexpected feature of the composition of C. difficile PG is the abundance of A2pm3 → A2pm3 cross-links generated by l,d-transpeptidation. This unusual type of cross-link was originally detected in E. coli, in which it represents about 5 and 12% of the total muropeptide content in the exponential and stationary growth phases, respectively (
      • Pisabarro A.G.
      • de Pedro M.A.
      • Vázquez D.
      ) and more recently in several Gram-positive bacteria (
      • Lavollay M.
      • Arthur M.
      • Fourgeaud M.
      • Dubost L.
      • Marie A.
      • Riegel P.
      • Gutmann L.
      • Mainardi J.L.
      ,
      • Lavollay M.
      • Arthur M.
      • Fourgeaud M.
      • Dubost L.
      • Marie A.
      • Veziris N.
      • Blanot D.
      • Gutmann L.
      • Mainardi J.L.
      ,
      • Lavollay M.
      • Fourgeaud M.
      • Herrmann J.L.
      • Dubost L.
      • Marie A.
      • Gutmann L.
      • Arthur M.
      • Mainardi J.L.
      ,
      • Mainardi J.L.
      • Legrand R.
      • Arthur M.
      • Schoot B.
      • van Heijenoort J.
      • Gutmann L.
      ). In Mycobacterium tuberculosis, the majority of the cross-links are generated by d,d-transpeptidation during exponential growth, whereas 80% of the cross-links are generated by l,d-transpeptidation during the stationary growth phase (
      • Lavollay M.
      • Arthur M.
      • Fourgeaud M.
      • Dubost L.
      • Marie A.
      • Veziris N.
      • Blanot D.
      • Gutmann L.
      • Mainardi J.L.
      ). In the present work the PG structure of C. difficile from the stationary and exponential growth phases revealed a similar profile, characterized by a large proportion of 3 → 3 cross-links, suggesting that the l,d-transpeptidases of C. difficile constitutively contribute to PG cross-linking. To our knowledge, this predominant contribution of l,d-transpeptidases to PG cross-linking has never been previously reported in low GC% Gram-positive bacteria.
      We identified three putative l,d-transpeptidases encoding genes, named ldtcd1, ldtcd2, and CD3007 in the genome of C. difficile 630 and obtained ldtcd1, ldtcd2 single and double mutants strains. These genes are significantly transcribed during exponential growth in the wild type strain but are not transcriptionally up-regulated in response to loss of one or more l,d-transpeptidase-encoding genes in the different mutant strains (data not shown). The PG profiles of the wild type and different l,d-transpeptidase mutant strains showed significant differences. The mutant strains show a marked decrease in the abundance of dimers to the partial benefice of monomers. Among the dimers, the proportion of muropeptides cross-linked by d,d-transpeptidation is slightly affected, whereas the proportion of muropeptides generated by l,d-transpeptidation is reduced by about one-half. These modifications lead to a marked decrease of the cross-linking index. Thus, both Ldtcd1 and Ldtcd2 constitute functional l,d-transpeptidases in C. difficile. Of note, mutation of either ldtcd1 or ldtcd2 reduces but does not abolish the formation of A2pm3 → A2pm3 cross-links, and the muropeptide profile of a double mutant strain lacking both Ldtcd1 and Ldtcd2 is similar to that of the single mutant strains. These data indicate the presence of at least a third functional l,d-transpeptidase (possibly CD3007) that could partially compensate for the loss of Ldtcd1 and Ldtcd2 encoding genes.
      Another notable feature of the PG structure of Ldtcd1 and Ldtcd2 mutants is the presence of three new muropeptides. Two of them have a dimeric structure with one tetrapeptide and one tripeptide side chain or two tetrapeptide side chains, whereas the third has a trimeric structure with one tripeptide and two tetrapeptide side chains. Interestingly, MS-MS sequencing of the new muropeptide dimers revealed the absence of a peptide cross-link but the presence of a bond between the MurNAc residue of the first disaccharide and the GlcNAc residue of the second disaccharide. It appears that the glycosidic bond between the MurNAc and GlcNH2 residues of the new muropeptides are insensitive to the mutanolysin activity. It was reported in B. subtilis that the glycosidic bond adjacent to a muramic δ-lactam in endospore PG is resistant to the action of muramidases (
      • Warth A.D.
      • Strominger J.L.
      ,
      • Atrih A.
      • Zöllner P.
      • Allmaier G.
      • Foster S.J.
      ). However, in C. difficile, the mechanism of the mutanolysin resistance remains unclear and could result from a change in the MurNAc residue or from the formation of an unusual glycosidic bond.
      The relative contribution of d,d-transpeptidation and l,d-transpeptidation for cross-linking has been related to ampicillin resistance in E. faecium (
      • Mainardi J.L.
      • Morel V.
      • Fourgeaud M.
      • Cremniter J.
      • Blanot D.
      • Legrand R.
      • Frehel C.
      • Arthur M.
      • Van Heijenoort J.
      • Gutmann L.
      ). Partial inhibition of the d,d-transpeptidases by ampicillin increased the proportion of the dimeric muropeptides containing an A2pm3 → A2pm3 cross-link, suggesting that the l,d-transpeptidation pathway is insensitive to ampicillin in C. difficile. However, despite a predominant amount of 3-3 cross-links, C. difficile remains susceptible to ampicillin (although minimal inhibitory concentration values are higher than in species such as C. perfringens) (
      • Citron D.M.
      • Merriam C.V.
      • Tyrrell K.L.
      • Warren Y.A.
      • Fernandez H.
      • Goldstein E.J.
      ). These observations suggest that the residual d,d-transpeptidase activity of penicillin-binding proteins detected in the presence of ampicillin could be essential to the PG assembly of C. difficile.
      The activity of the Ldtfm l,d-transpeptidase in E. faecium is limited by the production of its tetrapeptide substrate (
      • Mainardi J.L.
      • Fourgeaud M.
      • Hugonnet J.E.
      • Dubost L.
      • Brouard J.P.
      • Ouazzani J.
      • Rice L.B.
      • Gutmann L.
      • Arthur M.
      ), which results from the activity of a β-lactam insensitive metallo-d,d-carboxypeptidase named DdcY and belonging to the VanY superfamily (
      • Sacco E.
      • Hugonnet J.E.
      • Josseaume N.
      • Cremniter J.
      • Dubost L.
      • Marie A.
      • Patin D.
      • Blanot D.
      • Rice L.B.
      • Mainardi J.L.
      • Arthur M.
      ). In C. difficile, the impact of ampicillin on the PG structure suggests the presence of a homologous gene to the DdcY encoding gene responsible for the tetrapeptide substrate production. The genome of C. difficile harbors three putative β-lactams-insensitive d,d-carboxypeptidases, generating tetrapeptide substrate. Among them, VanXYG is encoded by a member of the vanG-like operon that does not confer resistance to vancomycin. Further studies should be considered to investigate the involvement of the different d,d-carboxypeptidases in the tetrapeptide substrate production, which is critical for the main 3-3 cross-linking in C. difficile. This study shows that C. difficile displays an original PG structure including a high level of N-deacetylated GlcNAc and a predominant proportion of 3 → 3 cross-links generated by at least two l,d-transpeptidases.

      Acknowledgments

      We thank Nigel P. Minton and John T. Heap as creators of the ClosTron gene knockout system and Bruno Dupuy for the gift of the strain 630Δerm of C. difficile.

      REFERENCES

        • Viswanathan V.K.
        • Mallozzi M.J.
        • Vedantam G.
        Gut Microbes. 2010; 1: 234-242
        • Finegold S.M.
        Scand. J. Infect Dis. Suppl. 1986; 49: 160-164
        • Kelly C.P.
        • LaMont J.T.
        Annu. Rev. Med. 1998; 49: 375-390
        • Voth D.E.
        • Ballard J.D.
        Clin. Microbiol. Rev. 2005; 18: 247-263
        • Höltje J.V.
        Microbiol. Mol. Biol. Rev. 1998; 62: 181-203
        • Scheffers D.J.
        • Pinho M.G.
        Microbiol. Mol. Biol. Rev. 2005; 69: 585-607
        • Sauvage E.
        • Kerff F.
        • Terrak M.
        • Ayala J.A.
        • Charlier P.
        FEMS Microbiol. Rev. 2008; 32: 234-258
        • Zapun A.
        • Contreras-Martel C.
        • Vernet T.
        FEMS Microbiol. Rev. 2008; 32: 361-385
        • Mainardi J.L.
        • Villet R.
        • Bugg T.D.
        • Mayer C.
        • Arthur M.
        FEMS Microbiol. Rev. 2008; 32: 386-408
        • Mainardi J.L.
        • Fourgeaud M.
        • Hugonnet J.E.
        • Dubost L.
        • Brouard J.P.
        • Ouazzani J.
        • Rice L.B.
        • Gutmann L.
        • Arthur M.
        J. Biol. Chem. 2005; 280: 38146-38152
        • Lavollay M.
        • Arthur M.
        • Fourgeaud M.
        • Dubost L.
        • Marie A.
        • Riegel P.
        • Gutmann L.
        • Mainardi J.L.
        Mol. Microbiol. 2009; 74: 650-661
        • Magnet S.
        • Arbeloa A.
        • Mainardi J.L.
        • Hugonnet J.E.
        • Fourgeaud M.
        • Dubost L.
        • Marie A.
        • Delfosse V.
        • Mayer C.
        • Rice L.B.
        • Arthur M.
        J. Biol. Chem. 2007; 282: 13151-13159
        • Lavollay M.
        • Arthur M.
        • Fourgeaud M.
        • Dubost L.
        • Marie A.
        • Veziris N.
        • Blanot D.
        • Gutmann L.
        • Mainardi J.L.
        J. Bacteriol. 2008; 190: 4360-4366
        • Lavollay M.
        • Fourgeaud M.
        • Herrmann J.L.
        • Dubost L.
        • Marie A.
        • Gutmann L.
        • Arthur M.
        • Mainardi J.L.
        J. Bacteriol. 2011; 193: 778-782
        • Gupta R.
        • Lavollay M.
        • Mainardi J.L.
        • Arthur M.
        • Bishai W.R.
        • Lamichhane G.
        Nat. Med. 2010; 16: 466-469
        • Magnet S.
        • Bellais S.
        • Dubost L.
        • Fourgeaud M.
        • Mainardi J.L.
        • Petit-Frère S.
        • Marie A.
        • Mengin-Lecreulx D.
        • Arthur M.
        • Gutmann L.
        J. Bacteriol. 2007; 189: 3927-3931
        • Magnet S.
        • Dubost L.
        • Marie A.
        • Arthur M.
        • Gutmann L.
        J. Bacteriol. 2008; 190: 4782-4785
        • Mainardi J.L.
        • Legrand R.
        • Arthur M.
        • Schoot B.
        • van Heijenoort J.
        • Gutmann L.
        J. Biol. Chem. 2000; 275: 16490-16496
        • Vollmer W.
        FEMS Microbiol. Rev. 2008; 32: 287-306
        • Bernard E.
        • Rolain T.
        • Courtin P.
        • Guillot A.
        • Langella P.
        • Hols P.
        • Chapot-Chartier M.P.
        J. Biol. Chem. 2011; 286: 23950-23958
        • Boneca I.G.
        • Dussurget O.
        • Cabanes D.
        • Nahori M.A.
        • Sousa S.
        • Lecuit M.
        • Psylinakis E.
        • Bouriotis V.
        • Hugot J.P.
        • Giovannini M.
        • Coyle A.
        • Bertin J.
        • Namane A.
        • Rousselle J.C.
        • Cayet N.
        • Prévost M.C.
        • Balloy V.
        • Chignard M.
        • Philpott D.J.
        • Cossart P.
        • Girardin S.E.
        Proc. Natl. Acad. Sci. U.S.A. 2007; 104: 997-1002
        • Vollmer W.
        • Tomasz A.
        Infect Immun. 2002; 70: 7176-7178
        • Bera A.
        • Herbert S.
        • Jakob A.
        • Vollmer W.
        • Götz F.
        Mol. Microbiol. 2005; 55: 778-787
        • Citron D.M.
        • Merriam C.V.
        • Tyrrell K.L.
        • Warren Y.A.
        • Fernandez H.
        • Goldstein E.J.
        Antimicrob. Agents Chemother. 2003; 47: 2334-2338
        • Sebaihia M.
        • Wren B.W.
        • Mullany P.
        • Fairweather N.F.
        • Minton N.
        • Stabler R.
        • Thomson N.R.
        • Roberts A.P.
        • Cerdeño-Tárraga A.M.
        • Wang H.
        • Holden M.T.
        • Wright A.
        • Churcher C.
        • Quail M.A.
        • Baker S.
        • Bason N.
        • Brooks K.
        • Chillingworth T.
        • Cronin A.
        • Davis P.
        • Dowd L.
        • Fraser A.
        • Feltwell T.
        • Hance Z.
        • Holroyd S.
        • Jagels K.
        • Moule S.
        • Mungall K.
        • Price C.
        • Rabbinowitsch E.
        • Sharp S.
        • Simmonds M.
        • Stevens K.
        • Unwin L.
        • Whithead S.
        • Dupuy B.
        • Dougan G.
        • Barrell B.
        • Parkhill J.
        Nat. Genet. 2006; 38: 779-786
        • Hussain H.A.
        • Roberts A.P.
        • Mullany P.
        J. Med. Microbiol. 2005; 54: 137-141
        • Courtin P.
        • Miranda G.
        • Guillot A.
        • Wessner F.
        • Mézange C.
        • Domakova E.
        • Kulakauskas S.
        • Chapot-Chartier M.P.
        J. Bacteriol. 2006; 188: 5293-5298
        • Heap J.T.
        • Pennington O.J.
        • Cartman S.T.
        • Carter G.P.
        • Minton N.P.
        J. Microbiol. Methods. 2007; 70: 452-464
        • Vollmer W.
        • Blanot D.
        • de Pedro M.A.
        FEMS Microbiol. Rev. 2008; 32: 149-167
        • Bielnicki J.
        • Devedjiev Y.
        • Derewenda U.
        • Dauter Z.
        • Joachimiak A.
        • Derewenda Z.S.
        Proteins. 2006; 62: 144-151
        • Depardieu F.
        • Bonora M.G.
        • Reynolds P.E.
        • Courvalin P.
        Mol. Microbiol. 2003; 50: 931-948
        • Dhalluin A.
        • Bourgeois I.
        • Pestel-Caron M.
        • Camiade E.
        • Raux G.
        • Courtin P.
        • Chapot-Chartier M.P.
        • Pons J.L.
        Microbiology. 2005; 151: 2343-2351
        • Schleifer K.H.
        • Kandler O.
        Bacteriol. Rev. 1972; 36: 407-477
        • Collins M.D.
        • Lawson P.A.
        • Willems A.
        • Cordoba J.J.
        • Fernandez-Garayzabal J.
        • Garcia P.
        • Cai J.
        • Hippe H.
        • Farrow J.A.
        Int. J. Syst. Bacteriol. 1994; 44: 812-826
        • Zipperle Jr., G.F.
        • Ezzell Jr., J.W.
        • Doyle R.J.
        Can. J. Microbiol. 1984; 30: 553-559
        • Atrih A.
        • Bacher G.
        • Allmaier G.
        • Williamson M.P.
        • Foster S.J.
        J. Bacteriol. 1999; 181: 3956-3966
        • Vollmer W.
        • Tomasz A.
        J. Biol. Chem. 2000; 275: 20496-20501
        • Psylinakis E.
        • Boneca I.G.
        • Mavromatis K.
        • Deli A.
        • Hayhurst E.
        • Foster S.J.
        • Vårum K.M.
        • Bouriotis V.
        J. Biol. Chem. 2005; 280: 30856-30863
        • Pisabarro A.G.
        • de Pedro M.A.
        • Vázquez D.
        J. Bacteriol. 1985; 161: 238-242
        • Warth A.D.
        • Strominger J.L.
        Biochemistry. 1972; 11: 1389-1396
        • Atrih A.
        • Zöllner P.
        • Allmaier G.
        • Foster S.J.
        J. Bacteriol. 1996; 178: 6173-6183
        • Mainardi J.L.
        • Morel V.
        • Fourgeaud M.
        • Cremniter J.
        • Blanot D.
        • Legrand R.
        • Frehel C.
        • Arthur M.
        • Van Heijenoort J.
        • Gutmann L.
        J. Biol. Chem. 2002; 277: 35801-35807
        • Sacco E.
        • Hugonnet J.E.
        • Josseaume N.
        • Cremniter J.
        • Dubost L.
        • Marie A.
        • Patin D.
        • Blanot D.
        • Rice L.B.
        • Mainardi J.L.
        • Arthur M.
        Mol. Microbiol. 2010; 75: 874-885
        • Glauner B.
        • Höltje J.V.
        • Schwarz U.
        J. Biol. Chem. 1988; 263: 10088-10095