Distinct pathways for modification of the bacterial cell wall by non-canonical D -amino acids
2011; Springer Nature; Volume: 30; Issue: 16 Linguagem: Inglês
10.1038/emboj.2011.246
ISSN1460-2075
AutoresFelipe Cava, Miguel A. de Pedro, Hubert Lam, Brigid M. Davis, Matthew K. Waldor,
Tópico(s)Amino Acid Enzymes and Metabolism
ResumoArticle26 July 2011free access Distinct pathways for modification of the bacterial cell wall by non-canonical D-amino acids Felipe Cava Felipe Cava Department of Microbiology and Molecular Genetics, Channing Laboratory, Brigham and Women's Hospital, Harvard Medical School and HHMI, Boston, MA, USA Centro de Biología Molecular ‘Severo Ochoa’, Universidad Autónoma de Madrid-Consejo Superior de Investigaciones Científicas, Madrid, Spain Search for more papers by this author Miguel A de Pedro Miguel A de Pedro Centro de Biología Molecular ‘Severo Ochoa’, Universidad Autónoma de Madrid-Consejo Superior de Investigaciones Científicas, Madrid, Spain Search for more papers by this author Hubert Lam Hubert Lam Department of Microbiology and Molecular Genetics, Channing Laboratory, Brigham and Women's Hospital, Harvard Medical School and HHMI, Boston, MA, USA Search for more papers by this author Brigid M Davis Brigid M Davis Department of Microbiology and Molecular Genetics, Channing Laboratory, Brigham and Women's Hospital, Harvard Medical School and HHMI, Boston, MA, USA Search for more papers by this author Matthew K Waldor Corresponding Author Matthew K Waldor Department of Microbiology and Molecular Genetics, Channing Laboratory, Brigham and Women's Hospital, Harvard Medical School and HHMI, Boston, MA, USA Search for more papers by this author Felipe Cava Felipe Cava Department of Microbiology and Molecular Genetics, Channing Laboratory, Brigham and Women's Hospital, Harvard Medical School and HHMI, Boston, MA, USA Centro de Biología Molecular ‘Severo Ochoa’, Universidad Autónoma de Madrid-Consejo Superior de Investigaciones Científicas, Madrid, Spain Search for more papers by this author Miguel A de Pedro Miguel A de Pedro Centro de Biología Molecular ‘Severo Ochoa’, Universidad Autónoma de Madrid-Consejo Superior de Investigaciones Científicas, Madrid, Spain Search for more papers by this author Hubert Lam Hubert Lam Department of Microbiology and Molecular Genetics, Channing Laboratory, Brigham and Women's Hospital, Harvard Medical School and HHMI, Boston, MA, USA Search for more papers by this author Brigid M Davis Brigid M Davis Department of Microbiology and Molecular Genetics, Channing Laboratory, Brigham and Women's Hospital, Harvard Medical School and HHMI, Boston, MA, USA Search for more papers by this author Matthew K Waldor Corresponding Author Matthew K Waldor Department of Microbiology and Molecular Genetics, Channing Laboratory, Brigham and Women's Hospital, Harvard Medical School and HHMI, Boston, MA, USA Search for more papers by this author Author Information Felipe Cava1,2, Miguel A de Pedro2, Hubert Lam1, Brigid M Davis1 and Matthew K Waldor 1 1Department of Microbiology and Molecular Genetics, Channing Laboratory, Brigham and Women's Hospital, Harvard Medical School and HHMI, Boston, MA, USA 2Centro de Biología Molecular ‘Severo Ochoa’, Universidad Autónoma de Madrid-Consejo Superior de Investigaciones Científicas, Madrid, Spain *Corresponding author. Department of Microbiology and Molecular Genetics, Channing Laboratory, Brigham and Women's Hospital, Harvard Medical School and HHMI, 181 Longwood Avenue, Boston, MA 02115, USA. Tel.: +1 617 525 4646; Fax: +1 617 525 4660; E-mail: [email protected] The EMBO Journal (2011)30:3442-3453https://doi.org/10.1038/emboj.2011.246 PDFDownload PDF of article text and main figures. Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Production of non-canonical D-amino acids (NCDAAs) in stationary phase promotes remodelling of peptidoglycan (PG), the polymer that comprises the bacterial cell wall. Impairment of NCDAAs production leads to excessive accumulation of PG and hypersensitivity to osmotic shock; however, the mechanistic bases for these phenotypes were not previously determined. Here, we show that incorporation of NCDAAs into PG is a critical means by which NCDAAs control PG abundance and strength. We identified and reconstituted in vitro two (of at least three) distinct processes that mediate NCDAA incorporation. Diverse bacterial phyla incorporate NCDAAs into their cell walls, either through periplasmic editing of the mature PG or via incorporation into PG precursor subunits in the cytosol. Production of NCDAAs in Vibrio cholerae requires the stress response sigma factor RpoS, suggesting that NCDAAs may aid bacteria in responding to varied environmental challenges. The widespread capacity of diverse bacteria, including non-producers, to incorporate NCDAAs suggests that these amino acids may serve as both autocrine- and paracrine-like regulators of chemical and physical properties of the cell wall in microbial communities. Introduction Nearly all bacteria synthesize a cell wall that is found outside of the cell membrane. This strong yet elastic network counteracts osmotic pressure, maintains cell shape and serves as a protective barrier against physical, chemical and biological threats (Holtje, 1998; Vollmer et al, 2008a). In both gram-positive and gram-negative bacteria, the cell wall is composed of a peptidoglycan (PG) polymer (also known as murein) that consists of glycan chains (alternating N-acetylglucosamine (GlcNAc) and N-acetylmuramic acid (MurNAc)) crosslinked by short peptides. The peptide chains vary somewhat among bacterial species, but normally include the D-amino acids D-Ala and D-Glu (or its amidated form, D-Gln). Unlike L-amino acids, D-amino acids are not used for ribosomal synthesis of proteins; in bacteria, their principal role is as cell wall constituents (Holtje, 1998; Vollmer et al, 2008a). Synthesis of PG requires many enzymes and takes place in two cellular compartments—the cytosol and the periplasm/extracytoplasm. D-Ala and D-Glu are made by racemases from the corresponding L-amino acids, and the D-alanyl-D-alanine dipeptide (D-Ala-D-Ala) is subsequently produced by D-alanyl-D-alanine ligase (Ddl) (Walsh, 1989; Duncan et al, 1990; Choi et al, 1992). UDP-muramyl-L-Ala-D-γ-Glu-meso-diaminopimelate (UDP-M3) is formed in sequential steps requiring MurA-MurE; MurF then adds the D-Ala-D-Ala to generate UDP-MurNAc-pentapeptide (UDP-M5) (Barreteau et al, 2008). UDP-M5 is transferred to a C55 lipid phosphate (bactoprenyl phosphate) on the cytoplasmic face of the cell membrane generating lipid I; finally, UDP-GlcNAc is added to lipid I, yielding lipid II, the lipid-disaccharyl pentapeptide. All steps up to this point occur within the cytosol, and many require ATP. Lipid II is ‘flipped’ from the cytoplasmic to the periplasmic face of cell membrane for PG assembly and further modification (Bouhss et al, 2008; Mohammadi et al, 2011). In the periplasmic space of gram-negative bacteria (or extracytoplasmic space of gram-positive organisms), membrane-bound transglycosylases and transpeptidases, such as high molecular weight penicillin-binding proteins (PBPs), assemble disaccharyl pentapeptide subunits into the PG polymer (Young, 2001; Scheffers and Pinho, 2005). Peptide chains are linked and modified, typically by D,D-transpeptidation reactions that link the D-Ala at position 4 in one peptide to the D-center of the meso-diaminopimelate residue [DAP] of a second peptide, either directly or through a short peptide bridge (Holtje, 1998; Vollmer et al, 2008a). As such transpeptidation reactions transfer energy from one peptide bond to another, they proceed in the absence of ATP, which is not present in the periplasm. The D,D-transpeptidases that generate these 4 → 3 crosslinks contain active site serines. Recently, a new class of cysteine transpeptidases, which are able to transfer L,D-peptide bonds to appropriate acceptors, have been identified and characterized. These L,D-transpeptidases (Ldts) can be subdivided in two types: those that mediate formation of DAP-DAP (3 → 3) crosslinks (Mainardi et al, 2005; Biarrotte-Sorin et al, 2006; Magnet et al, 2008) and those that attach lipoproteins to DAP (only reported in some gram-negatives species) (Magnet et al, 2007b). PG is a dynamic polymer. Glycan chains and peptide crosslinks are cleaved by glycosylases and peptidases in order to permit expansion during cell growth (Vollmer et al, 2008b). Conversely, PG is often modified as cells enter stationary phase. Quiescent cells are often smaller than their rapidly growing counterparts (Nystrom, 2004). However, there is relatively scant knowledge of the factors and mechanisms that regulate the composition, architecture and amount of PG. Recently, we found that stationary phase bacteria from diverse phyla release distinct sets of D-amino acids other than those classically thought to be part of PG (‘non-canonical’ D-amino acids, in short NCDAAs). Furthermore, such D-amino acids, which were not previously known to be synthesized by bacteria, regulate the chemistry, amount and strength of PG. In Vibrio cholerae, NCDAAs (principally D-Met and D-Leu) are produced by BsrV, a periplasmic broad spectrum racemase (Lam et al, 2009). Chromosome sequence analyses suggest that other bacterial species may also encode amino acid racemases that enable synthesis of NCDAAs (Lam et al, 2009). D-Met was previously shown to be incorporated into V. cholerae PG during stationary phase; however, the mechanism and physiologic consequences of such incorporation were not determined. It has been hypothesized that NCDAAs alter cell wall properties, particularly its strength, via their incorporation into the PG polymer and/or via altering the activity of PBPs, key enzymes for PG synthesis (Lam et al, 2009). Here, we explored the sites, mechanisms and consequences of incorporation of NCDAAs into bacterial cell walls. All bacterial species tested can incorporate NCDAAs into their PG; however, the sites and mechanisms utilized vary among species. Incorporation can be mediated both by periplasmic enzymes, predominantly Ldts, that ‘edit’ polymerized PG and by cytoplasmic enzymes that incorporate NCDAAs into PG precursors. In V. cholerae, interference with NCDAA incorporation leads to hypersensitivity to osmotic shock and excess accumulation of PG in the cell wall. NCDAA production is dependent upon the alternative sigma factor RpoS, a stress-associated regulator, suggesting that D-amino acid synthesis and incorporation into the bacterial exoskeleton may promote bacterial resistance to diverse environmental threats. Results Bacteria from diverse phyla incorporate NCDAAs into their cell walls Diverse gram-negative and gram-positive bacteria produce NCDAAs and release them into the extracellular media (Lam et al, 2009). For example, supernatants from stationary phase V. cholerae cultures contain ∼1 mM of D-amino acids, primarily D-Met and D-Leu. HPLC analyses revealed that these unusual D-amino acids can become incorporated into PG (Lam et al, 2009). Such incorporation is not directly coupled to D-amino acid production; strains that fail to produce D-amino acids can nonetheless incorporate them into PG. For example, when bsrV V. cholerae (a non-producer; Lam et al, 2009) was grown for 16 h in a culture chamber linked to another chamber containing wild-type (WT) V. cholerae (a D-amino acid producer), using an apparatus that allows for the passage of small molecules but not cells, 10% of muropeptides isolated from the bsrV strain contained D-Met (Figure 1A). In contrast, no D-Met incorporation was detected when both chambers of the co-culture apparatus contained the bsrV mutant. Addition of supra-physiological (5 mM) D-Met to cultures of the bsrV mutant also resulted in its incorporation to the PG, at a level (19%) comparable to that observed with WT V. cholerae (17%) under equivalent conditions (Figure 1B). Exogenously supplied D-Met (5 mM) was likewise incorporated into the cell wall muropeptides of all bacterial species assayed, both those that synthesize NCDAAs, although not necessarily D-Met (e.g., Pseudomonas aeruginosa, Bacillus subtilis, Enterococcus faecalis and Staphylococcus aureus) and those do not (e.g., Escherichia coli and Caulobacter crescentus) (Figure 1B; Supplementary Figure S1). The extent of incorporation did not correlate with the capacity to synthesize these molecules. These observations suggest that the ability to incorporate NCDAAs into PG is widespread throughout the bacterial kingdom, and is mechanistically independent of the synthesis of such molecules. Figure 1.Incorporation of NCDAAs into PG muropeptides of diverse bacteria is not linked to their synthesis. The percentage of D-Met-containing peptides was calculated relative to total muropeptide isolated. (A) Co-culture system that permits chemical communication between two strains while they are kept physically apart (top). Percentage of D-Met-containing muropeptides in PG from strain B (bsrV V. cholerae) following co-culture with either WT or bsrV V. cholerae (bottom); note that the strain A culture was inoculated 2 h before the strain B culture to permit higher NCDAAs accumulation and hence better yields of incorporation into the PG. (B) Percentage of D-Met-containing muropeptides in PG from diverse bacteria following growth in the presence of exogenous 2 mM (C. crescentus) or 5 mM (remaining species) D-Met. The data shown are representative of two independent experiments. Download figure Download PowerPoint D-Met is incorporated in the fourth and fifth positions of the peptide moiety of V. cholerae cell wall muropeptides HPLC analyses revealed that D-Met is incorporated at two locations within PG subunits from stationary phase V. cholerae (Figure 2). Most of the D-Met replaces D-Ala in the fourth position of muropeptides ([muro4M]; Figure 2A and B; Supplementary Figure S2A), either within monomers [mono4M] or within 4 → 3 crosslinked dimers [di4,4M], as previously reported (Lam et al, 2009). However, further analyses of minor PG components from stationary phase samples revealed that D-Met can also replace D-Ala in the fifth position of muropeptides [muro5M], either within monomers [mono5M] or 4 → 3 crosslinked dimers [di4,5M] (Figure 2A and B; Supplementary Figure S2A). As expected, when exogenous D-Met is not supplied, all incorporation of D-Met into the cell wall is dependent upon its production by the BsrV racemase (Figure 2B). Analyses of exponential phase V. cholerae grown with non-deleterious supra-physiological concentrations (up to 10 mM) of D-Met did not lead to identification of additional D-Met-containing muropeptides, suggesting that incorporation is restricted to these sites within PG (muro4M and muro5M; Figure 2C; Supplementary Figure S2A). Figure 2.L,D-transpeptidases incorporate non-canonical D-amino acids into tetrapeptides of V. cholerae PG. Muropeptides within purified PG were identified and quantified using HPLC. (A) Schematic representation of muropeptide structures, illustrating amino acid content and peptide chain length. The canonical pentapeptide structure is shown on the left. (B) Percentage of muro4M and muro5M peptides, relative to total muropeptides, in PG purified from the indicated V. cholerae strains at stationary phase following growth in LB. (C) Percentage of muro4M and muro5M peptides, relative to total muropeptides, in PG purified from WT V. cholerae following the exponential phase growth in LB supplemented with the indicated concentration of D-Met. (D) Abundance of DAP-DAP muropeptides (muro-DAP-DAP), Lpp muropeptides (muro-Lpp) and D-Met muropeptides (muro-D-Met), relative to total amount of muropeptides from the indicated strains. Muro-Lpp includes the lipoprotein-attached muropeptides mono3-Lpp (GlcNAc-MurNAc-L-Ala-D-Glu-γ-meso-DAP-ε-L-Lys-L-Thr) and the crosslinked dimer GlcNAc-MurNAc-L-Ala-D-Glu-γ-meso-DAP-D-Ala-meso-DAP-(ε-L-Lys-L-Thr)-γ-D-Glu-L-Ala-MurNAc-GlcNAc. The Thr-Lys dipeptide corresponds to the C-terminal dipeptide of the V. cholerae homologue for E. coli Braun's lipoprotein. Digestion of sacculi with pronase E, one step of HPLC sample processing, degrades the covalently bound lipoprotein molecules leaving the C-terminal dipeptide bound to the connecting muropeptide which can therefore be easily differentiated. (E) Immunodetection of D-cysteine-labelled murein in sacculi from the indicated strains of V. cholerae. For each strain, the inset box was magnified to generate the subpanel on the right. The data shown are representative of three independent experiments. Download figure Download PowerPoint Multifunctional Ldts incorporate D-amino acids into the cell wall The most likely pathway for the generation of muro4M is an amino acid exchange reaction mediated by an Ldt in which the L,D-peptide bond from the L-center of DAP is transferred to the amino group of the acceptor D-amino acid, as previously hypothesized (Caparros et al, 1992). Recent studies have revealed that Ldts catalyse the formation of DAP-DAP crosslinks between muropeptides as well as the linkage between DAP and Braun's lipoprotein (Lpp) in some Gram-negative bacteria (Magnet et al, 2007a, 2007b, 2008; Supplementary Figure S2B). However, to date they have not been shown to catalyse amino-acid exchanges in vivo. We found two homologues of the five recently characterized E. coli Ldts (Magnet et al, 2007b, 2008) in the genome of V. cholerae N16961. One locus, vc1268, encodes a protein homologous to the Ldts YcbB and YnhG, whereas the other, vca0058, encodes a protein homologous to the Ldts ErfK, YcfS and YbiS (Supplementary Figure S2C). Cell fractionation and immunoblotting experiments revealed that, like other characterized Ldts, the two putative V. cholerae Ldts are membrane proteins (Supplementary Figure S2D). Furthermore, topologic predictions suggest that both VC1268 and VCA0058 are primarily periplasmic proteins anchored to the inner membrane by a single transmembrane helix (Supplementary Figure S2E). Analysis of mutants lacking these putative transpeptidases revealed that V. cholerae lacking vc1268 (here renamed ldtA) is devoid of DAP-DAP crosslinks, while it maintains WT levels of Braun's Lpp-linked muropeptides (muro-Lpp: mono3-Lpp and di4,3-Lpp; Figure 2D). In contrast, V. cholerae lacking vca0058 (here renamed ldtB) has normal levels of DAP-DAP-linked muropeptides, but lacks Lpp covalently bound to the PG (Figure 2D). Thus, in these assays, LdtA and LdtB appear to have mutually exclusive roles. Neither class of crosslink was detected in PG from V. cholerae lacking both ldtA and ldtB. However, each could be restored in this background by expression of the relevant gene, thereby confirming the roles inferred from analysis of the individual mutants (Figure 2D). Further analyses revealed that both LdtA and LdtB contribute to incorporation of NCDAAs, such as D-Met, at the fourth position within muropeptides. Deletion of either ldtA or ldtB significantly reduced the percentage of muro4M peptides in PG from stationary phase V. cholerae, and deletion of both genes rendered muro4M peptides undetectable, suggesting that they account for most, if not all, of the DAP-D-Met linkages in V. cholerae PG (Figure 2B; Supplementary Figure S3A). Complementation analyses confirmed the roles of LdtA and LdtB in production of muro4M (Figure 2B). In contrast, neither LdtA nor LdtB appears important for generation of muro5M peptides, as the level of such muropeptides in PG from the ldtA and ldtB mutants was equivalent to that of the WT strain (Figure 2B; Supplementary Figure S3A). This result is consistent with the fact that muro5M peptides contain a D,D-peptide bond between D-Met and the adjacent D-Ala, rather than the L,D-bond typically synthesized by Ldts. LdtA and LdtB were also shown to be required for incorporation of D-Met into muro4M peptides by exponential phase cultures of V. cholerae grown in the presence of exogenous D-Met (Supplementary Figure S3B). D-Cysteine labelling of sacculi is mediated by Ldts Almost two decades ago, de Pedro et al (1997) demonstrated that D-Cys could be incorporated into the murein of several bacterial species, but the mechanism by which this occurs has not been elucidated. Nevertheless, D-Cys-mediated labelling of PG has facilitated important studies of the dynamics of PG synthesis in diverse organisms (de Pedro et al, 2003; Aaron et al, 2007). Based on our observation that Ldts mediate incorporation of D-Met into V. cholerae PG, we explored whether these enzymes are also responsible for incorporation of D-Cys (another NCDAA). We found that D-Cys is incorporated into the V. cholerae sacculus via a process that is largely dependent on ldtA and ldtB, since virtually no D-Cys incorporation was detected in the ldtA ldtB double mutant (Figure 2E). D-Cys labelling of the ldtA ldtB mutant was at least partially restored by expression of plasmid-encoded ldtA or ldtB, providing further evidence that these genes contribute to the labelling process in V. cholerae (Figure 2E). Similarly, deletion of Ldts that crosslink DAP residues in E. coli (YnhG and YcbB) greatly reduced immunodetection of D-Cys in the sacculi (Supplementary Figure S3C). However, it is likely that processes other than L,D-transpeptidation contribute to D-Cys-mediated labelling in other bacterial species (discussed later). LdtA substrate specificity His-tagged LdtA and LdtB were purified to enable in vitro characterization of these proteins' biochemical properties. Purified LdtB showed no activity in D-amino-acid incorporation assays; consequently, we focused on elucidating LdtA's activity. Initially, we tested whether sacculi from exponential phase V. cholerae, which contain no D-Met (Lam et al, 2009), could serve as a substrate for LdtA. After purified sacculi were incubated with LdtA and D-Met for 2 h, 75% of the dimer and monomer tetrapeptides (muro4) contained D-Met rather than D-Ala in position 4 (muro4M; Figure 3A; Supplementary Figure S4A). No incorporation of D-Met was found in position 5, consistent with the in vivo observations presented above that the Ldts only incorporate D-Met at position 4. Thus, LdtA can recognize and modify native V. cholerae PG in the absence of additional cofactors. Figure 3.In vitro characterization of LdtA. Muropeptides were identified and quantified using HPLC; no reaction products other than those shown in the figures were detected. (A) The percentage of substrate converted into product via in vitro L,D-transpeptidation by LdtA is shown for various muropeptides. Reactions were incubated for 30 min (purified muropeptides) or 2 h (sacculi). (B) The percentage of mono4 altered by in vitro LdtA transpeptidation to have a different terminal amino acid when incubated (2 h) in the presence of the indicated L- or D-amino acid is shown. The data shown in (A) and (B) are representative of three and two independent experiments, respectively. Download figure Download PowerPoint We then used crosslinked dimer (di) and monomer (mono) muropeptides purified from PG to further assess the substrate specificity of LdtA. Assay conditions (see Materials and methods) were optimized with mono4 and D-Met as substrates (Supplementary Figure S4B). Under our standard conditions, about 10% of mono4 was converted into mono4M in 30 min at 37°C, a value corresponding to 10 μg of muropeptide converted per minute and per microgram of enzyme. Assays with di4,4 as the donor substrate revealed that LdtA acted far more efficiently on this muropeptide than on mono4, with a conversion into di4,4M of 96% under otherwise identical conditions. Neither mono5 nor di4,5 was accepted as substrates for the exchange reaction even after an extended (2 h) incubation period (Figure 3A; Supplementary Figure S4C). Since D-Ala is present in the periplasm as a consequence of PG remodelling and turnover (Park and Uehara, 2008), we tested whether LdtA could catalyse the reverse reaction, with D-Met muropeptides (mono4M and di4,4M) as donor substrates and D-Ala as acceptor. LdtA was indeed able to do so, although at significantly lower rates than the forward (D-Ala → D-Met) reaction and, as before, the crosslinked muropeptide di4,4M was a better substrate than the monomer, showing 38 and 4.5% conversion, respectively (Figure 3A; Supplementary Figure S4C). The difference between efficiencies for the forward (D-Ala → D-Met) and reverse (D-Met → D-Ala) reactions suggests that accumulation of D-Met muropeptides would be favoured in vivo, even if both D-Met and D-Ala were simultaneously present at similar concentrations. Therefore, in stationary phase, when D-Met is present, LdtA is likely to promote accumulation of muro4M peptides, rather than a continual exchange of the C-terminal amino acid within tetrapeptides. In natural environments, bacterial communities typically contain multiple species (Straight and Kolter, 2009), which could produce a variety of D-amino acids (Lam et al, 2009). Therefore, we tested whether LdtA could incorporate other amino acids besides D-Met or D-Cys into PG. Indeed, LdtA accepted, at varying efficiencies, all the D-amino acids tested as acceptors for the exchange reaction using mono4 as the donor substrate (Figure 3B; Supplementary Figure S4D). However, none of the corresponding L-amino acids was accepted, as previously reported for the E. faecium Ldt (Ldtfm) (Mainardi et al, 2005). Interestingly, both D-Pro, which has a secondary amino group, and Gly, which lacks a chiral centre, were accepted as substrates for the in vitro reaction (Figure 3B; Supplementary Figure S4D). Simultaneous addition of equal concentrations of D-Met and D-Ala to an LdtA in vitro reaction with mono4 as donor substrate resulted in about a 50% reduction in the incorporation of D-Met (Figure 3B), suggesting that using this substrate LdtA does not preferentially utilize D-Met rather than D-Ala as an acceptor. Finally, in contrast to findings reported in Magnet et al (2007a), LdtA did not use a D-Ala-D-Ala for the exchange reaction with mono4 as the donor substrate, suggesting that there are structural constraints upon its substrates beyond the requirement for a D-amino acid acceptor molecule. Analysis of the LdtA protein sequence revealed a YkuD domain that has been observed in other Ldts (Supplementary Figure S5A; Bielnicki et al, 2006). This domain contains three invariant residues (H, G and C), including the presumed catalytic cysteine residue (Biarrotte-Sorin et al, 2006; Supplementary Figure S5). When all three residues were mutated to Alanine, LdtA activity was abolished (Supplementary Figure S5B), consistent with this domain containing the enzyme's active site and suggesting that the catalytic mechanism of LdtA is similar to previously characterized homologues (Mainardi et al, 2005; Biarrotte-Sorin et al, 2006). Synthesis of muro5M in V. cholerae is dependent on cytoplasmic processes Generation of muro5M could be explained as the result of a D,D-transpeptidase mediated D-amino acid exchange reaction on normal muro5. However, the scarcity of muro5 in V. cholerae PG argues against it. Therefore, we hypothesized that the D-Ala-D-Met-peptide bond in muro5M could be formed via the biosynthetic pathway for generation of cytoplasmic PG precursors. Consistent with this hypothesis, previous studies reported that cytoplasmic Ddl ligases are not highly specific for incorporation of D-Ala at the C-terminal position of dipeptides, at least in unbalanced media (Barreteau et al, 2008). Therefore, we assessed D-Met incorporation into V. cholerae PG in early stationary phase cells treated with sublethal concentrations of D-cycloserine, a D-amino-acid analogue that inhibits both Ddl and the alanine racemase (Barreteau et al, 2008; Figure 4A). D-Cycloserine rendered muro5M peptides undetectable but had a minimal influence on muro4M formation (Figure 4A). The latter observation suggests that cycloserine's effect on muro5M formation is not simply a consequence of reducing D-Met availability, for example, due to inhibition of racemase activity, and is instead likely to ensue from Ddl inhibition. Figure 4.Incorporation of non-canonical D-amino acids into muropeptides occurs via several Ldt-independent pathways. (A) The relative percentage of muro4M and muro5M peptides in PG isolated from stationary phase cultures of V. cholerae grown in the absence (control) or presence of D-cycloserine (100 μg/ml). (B) Schematic representation of the in vitro conversion of D-amino acids (D-Ala and/or D-Met) and UDP-M3 into UDP-M5 and UDP-M5M by Ddl and MurF. (C) Yield, based on HPLC quantification of UDP muropeptides following A262 (UDP detection), of the reaction depicted in (B), when the indicated amino acid substrates are supplied. (D) The percentage of muropeptides that contain D-Met (muro4M or muro5M) in various bacterial species and their ldt mutants following growth in 5 mM D-Met, or 2 mM in the case of C. crescentus. (E, F) The effects of D-cycloserine (100 μg/ml) and/or penicillin G (50 μg/ml) on the relative level of muro5M peptides in PG isolated from C. crescentus (E) or B. subtilis (F) following growth in the presence of 2 mM or 5 mM D-Met, respectively. The data shown are representative of three independent experiments. Download figure Download PowerPoint Cytoplasmic ligases Ddl and MurF catalyse formation of muro5M precursors In vitro analyses provided additional support for the hypothesis that muro5M formation is dependent upon utilization of D-Met by Ddl and MurF, as depicted in Figure 4B. We purified Ddl and MurF from V. cholerae (see Materials and methods) and reconstituted their coupled reaction, which generates UDP-M5, in vitro in the presence of UDP-M3 as substrate. HPLC analysis of the final contents of these reactions s
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