In vitro Reconstitution of Peptidoglycan Assembly ... - ACS Publications

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In vitro Reconstitution of Peptidoglycan Assembly from the GramPositive Pathogen Streptococcus pneumoniae André Zapun,*,†,‡,§ Jules Philippe,†,‡,§ Katherine A. Abrahams,∥ Luca Signor,†,‡,§ David I. Roper,∥ Eefjan Breukink,⊥ and Thierry Vernet†,‡,§ †

Université Grenoble Alpes, Institut de Biologie Structurale (IBS), Grenoble F-38027, France CNRS, IBS, UMR 5075, 71 av. des Martyrs, Grenoble F-38027, France § CEA, DSV, IBS, Grenoble F-38027, France ∥ Department of Biological Sciences, University of Warwick, Coventry CV4 7AL, United Kingdom ⊥ Department of Chemical Biology and Organic Chemistry, Institute of Biomembranes, Bijvoet Center for Biomolecular Research, Utrecht University, Utrecht 3584 CH, The Netherlands ‡

S Supporting Information *

ABSTRACT: Understanding the molecular basis of bacterial cell wall assembly is of paramount importance in addressing the threat of increasing antibiotic resistance worldwide. Streptococcus pneumoniae presents a particularly acute problem in this respect, as it is capable of rapid evolution by homologous recombination with related species. Resistant strains selected by treatment with β-lactams express variants of the target enzymes that do not recognize the drugs but retain their activity in cell wall building, despite the antibiotics being mimics of the natural substrate. Until now, the crucial transpeptidase activity that is inhibited by βlactams was not amenable to in vitro investigation with enzymes from Gram-positive organisms, including streptococci, staphylococci, or enterococci pathogens. We report here for the first time the in vitro assembly of peptidoglycan using recombinant penicillin-binding proteins from pneumococcus and the precursor lipid II. The two required enzymatic activities, glycosyl transferase for elongating glycan chains and transpeptidase for cross-linking stem-peptides, were observed. Most importantly, the transpeptidase activity was dependent on the chemical nature of the stem-peptide. Amidation of the second residue glutamate into iso-glutamine by the recently discovered amido-transferase MurT/GatD is required for efficient crosslinking of the peptidoglycan.

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the product of point mutations and homologous recombination between related species. Understanding how these modified enzymes retain the ability to build the cell wall, while they do not react with β-lactams that resemble the natural substrate, is a pressing question. As an essential first step toward an answer, we report here the in vitro reconstitution of peptidoglycan assembly using recombinant enzymes from a drug-susceptible strain of Streptococcus pneumoniae. The enzymes building peptidoglycan are termed penicillinbinding proteins, or PBPs, since they are also the targets of β-

nhibiting bacterial cell wall assembly has saved millions of lives over the past seven decades since the introduction of penicillin.1 The main constituent of the cell wall is the giant single molecule termed peptidoglycan that completely encases the cell in chains of disaccharides cross-linked by short peptide bridges.2 Formation of these peptide cross-links is prevented by β-lactams, as they form a covalent adduct within the active site of the responsible enzymes.3 β-Lactams owe their efficacy to their structural likeness to the peptides that are cross-linked to form the peptidoglycan.4 In pneumococcus, a naturally transformable Gram-positive pathogen and a major bacterial scourge that inflicts over 1.6 million human deaths per year, widespread β-lactam resistance results from the expression of altered target enzymes that harbor multiple amino acid substitutions encoded by mosaic genes.5 These mosaics are © 2013 American Chemical Society

Received: July 31, 2013 Accepted: September 18, 2013 Published: September 18, 2013 2688

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Figure 1. Scheme of the amidation of Lys-containing lipid II by MurT/GatD. The red arrow points to the amidation site.

lactams. PBPs come in different families.6 Class A PBPs are bifunctional enzymes that carry out both the polymerization of the glycan chains, through their glycosyl transferase (GT) domain, and the peptide cross-linking with their transpeptidase (TP) domain. Class B PBPs are monofunctional with a TP module as sole enzymatic domain. Class C PBPs have a recognizable TP domain, sequence-wise, but these enzymes are carboxypeptidases that release the fifth residue of the stempeptides by hydrolysis, instead of catalyzing a transpeptidation. Pneumococcus has 6 PBPs, including the three class A PBP1a, PBP2a, and PBP1b; the two class B PBP2b and PBP2x; and the single class C PBP3.5 Of these, PBP2x and 2b are essential,7 and either PBP1a or PBP2a must be functional for viability.8,9 The coordinated function of the various PBPs in the life cycle of S. pneumoniae remains largely unknown. Ovococci such as pneumococcus are thought to build cell wall by two distinct processes: the formation of the septal cross-wall and a cylindrical elongation.10,11 PBP2x and PBP2b are the class B PBPs participating in the septal and cylindrical synthesis. No class A PBP could be attributed clearly to either process. PBP1a and PBP2a are the most important, as one of them must be present for growth.9 PBP1b appears to have a minor role in laboratory cultures, as no detectable phenotype results from its absence. Our efforts to reconstitute peptidoglycan synthesis have therefore been focused on the four main synthetic PBPs: PBP1a, PBP2a, PBP2b, and PBP2x. Variants of these four PBPs are responsible for β-lactam resistance, although the involvement of PBP2a remains exceptional.5 Although the reaction of PBPs with β-lactams has been thoroughly probed for decades, in vitro studies of the physiological reactions catalyzed by recombinant PBPs were made possible relatively recently with advances in the synthesis of large amounts of the membrane-linked precursor.12−15 The substrate of PBPs, termed lipid II, consists of a GlcNAcMurNAc disaccharide, with a pentapeptide assembled on the MurNAc and a pyrophosphate-attached undecaprenyl hydrophobic tail, also linked to the MurNAc.16 Glycan chains are formed by successive attacks of the growing chain (donor) at the reducing end of the lipid II (acceptor), a process catalyzed by the GT domains of class A PBPs.17 This reaction was achieved in vitro with various PBPs from Gram-positive and Gram-negative organisms, including Staphylococcus aureus, S. pneumoniae, or Escherichia coli (e.g., refs 17−21). In vitro bridging of glycan chains by transpeptidation between stempeptides, i.e., completion of the process of PG assembly, has

been reported only with enzymes from Gram-negative E. coli, both class A and B PBPs.22−25 In contrast to that of Gram-negative organisms, complete PG assembly from Gram-positive bacteria has been more difficult to reconstitute in vitro. This difficulty was already apparent in the early studies when cell extracts or purified native proteins were used to synthesize PG from radiolabeled precursors. Both GT and TP activities were detected with membranes or purified PBPs of Bacillus megaterium26,27 or Bacillus stearothermophilus.28,29 However, the numerous reports of failures to observe the TP activity, including in studies from the same laboratories with closely related organisms, attest to this difficulty.28,30−32 The difficulty to observe in vitro the TP activity with PBPs from Gram-positive bacteria may be due to the nature of the precursor. The composition of the stem-peptides that adorn the glycan chains shows some variation between species. The third residue, for example, is a meso-diaminopimelate in E. coli and B. subtilis, whereas it is a lysine in S. aureus and S. pneumoniae. The second residue can be a D-glutamate or can be amidated into a D -iso-glutamine in species such as staphylococci and streptococci (Figure 1). This amidation activity was found in cell extracts of S. aureus and shown to act on lipid II.33 The responsible amidotransferase was recently identified in S. aureus and consists of a complex of two proteins, MurT and GatD, expressed from an operon.34,35 That second residue amidation of lipid II could be required for efficient peptidoglycan cross-linking in some Gram-positive bacteria was suggested by the detailed analysis of the pneumococcal cell wall.36 As expected, non-amidated glutamate-containing peptides were found to be scarce in S. pneumoniae. Most of them were detected in non-cross-linked peptide (12.6% of monomers), whereas glutamate was nearly absent in cross-linked peptides (1.8% in dimers, undetected in trimmers). To test the importance of the amidation of the lipid II for the TP activity of pneumococcal PBPs, we purified the recombinant MurT/GatD amidotransferase from S. pneumoniae and modified Lys-containing lipid II in vitro. As a result, we present here the first reconstitution of peptidoglycan synthesis using recombinant PBPs from a Gram-positive organism and demonstrate that the transpeptidase activity is favored by the amidation of the stem-peptides.

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RESULTS AND DISCUSSION Lipid II Amidotransferase from Pneumococcus. In replicating the pioneering experiment of Strominger33 with “particulate enzymes” from S. pneumoniae, we observed that 2689

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Figure 2. Activity of MutT/GatD in vitro and depletion of MurT/GatD in R6 S. pneumoniae. (a) Dansylated glutamate-containing lipid II was incubated with pneumococcal particular extract in the absence (CTL) or presence of ammonium and ATP, prior to thin layer chromatography (TLC) and visualization under UV illumination. (b) The murT/gatD operon was introduced at the bgaA locus of the R6 strain under the control of a zinc-inducible promoter to create the spnLR29 strain. The endogenous murT/gatD operon was then disrupted to create the spnLR30 strain. The three strains grown in Todd−Hewitt broth supplemented with 150 μM ZnCl2 were streaked on Columbia blood agar plates with or without the same concentration of ZnCl2. (c) The three strains washed in broth without added zinc were inoculated in Todd−Hewitt broth without (solid lines) or with 150 μM ZnCl2 (broken lines). R6 is in black, spnLR29 is in red, and spnLR30 is in blue. (d) Iodine-colored TLC of non-amidated lipid II (LII(Glu)) and lipid II that has been amidated by the action of MurT/GatD in the presence of Gln and ATP to yield LII(iGln). A mixture of both forms was analyzed in the central sample.

Figure 3. Assembly of peptidoglycan by pneumococcal PBP2a and dependence on the amidation of lipid II. (a) A mixture of 50 μM amidated lipid II and 5 μM non-amidated dansylated lipid II was incubated with 1 μM PBP2a, or its S410A transpeptidase-inactivated variant, in the presence or absence of moenomycin (Moe) or penicillin G (Pen). (b) Mixtures of 50 μM amidated (LII(iGln)) or non-amidated lipid II (LII(Glu)) and 5 μM nonamidated dansylated lipid II were incubated with 1 μM PBP2a, in the absence or presence of penicillin G. (c) A mixture of 50 μM amidated lipid II and 5 μM non-amidated dansylated lipid II was incubated with 1 μM transpeptidase-inactivated PBP2a-S410A, 1 μM glycosyl transferase-inactivated PBP2a-E110Q, or both, in the absence or presence of penicillin G. (d) A mixture of 90 μM lipid II and 9 μM dansylated lipid II was incubated with about 0.3 μM MurT/GatD and 10 mM L-glutamine, in the absence or presence of 10 mM ATP, for 3 h at 37 °C prior to the addition of PBP2a to 1 μM and DMSO to 25% (v/v) and further incubation with or without 1 mM penicillin G (Pen). All PG assembly reactions were overnight at 30 °C. Samples were analyzed by SDS-PAGE, and the dansyl fluorescence was imaged by blue or UV trans-illumination.

identified spr1443/1444 as the orthologous genes in the S. pneumoniae R6 strain. The two genes form an operon with a one base pair overlap between the stop codon of murT and the start codon of gatD. The operon was introduced at the bgaA locus under the control of a zinc-inducible promoter.37 The endogenous murT/gatD locus was then disrupted while

dansylated lipid II was modified to a species of greater mobility in thin layer chromatography, in a manner dependent on the presence of ATP and ammonium (Figure 2a). This finding indicated the likely presence of a lipid II amidotransferase in pneumococcus. Following the identification of the murT/gatD genes encoding the amidotransferase in S. aureus,34,35 we 2690

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Using nonsaturated fluorescence images of gels to analyze products of reactions catalyzed by PBP2a in the absence and presence of penicillin, we quantified the amount of labeled material along the electrophoretic migration path (Figure 4). In

expression from the newly introduced copy was induced with zinc. The resulting strain formed colonies on agar plates and grew in liquid medium in the presence of zinc but failed to grow in the absence of inducer (Figure 2b and c). The lipid II amidotransferase is therefore essential in R6 pneumococcus, as found previously in a high-throughput gene disruption study of the TIGR4 strain.38 In order to amidate glutamate lipid II into iso-glutamine lipid II in vitro, we have produced recombinant pneumococcal MurT/GatD. The whole operon was introduced in a vector for expression in E. coli with a poly histidine tag in N-terminus of MurT. The amidotransferase complex was purified to homogeneity by a succession of Ni2+-affinity and anionexchange chromatography. The preparation was used to fully amidate Lys-containing lipid II in the presence of ATP and Lglutamine as monitored by thin layer chromatography (Figure 2d). The replacement of a hydroxyl by an amino group was confirmed by mass spectrometry, which measured the 1 Da mass loss, from 1875 to 1874 Da. Peptidoglycan Assembly Activities of S. pneumoniae PBP2a. We had shown previously that full length PBP2a exhibits GT activity in vitro with a glutamate-containing lipid II and a dansylated-lysine in position 3.20 It has also been shown with E. coli PBP1b that transpeptidation can occur when only 10% of the precursor molecules are dansylated, thus generating high mass products that do not migrate in a gel electrophoresis system. Un-cross-linked chains, in contrast, can be observed as a smear under near-ultraviolet illumination, whereas unused precursors appear in the migration front.20,39 Using this qualitative analysis system, we found that PBP2a does catalyze the cross-linking of glycan chains in addition to their polymerization from iso-glutamine- and lysine-containing lipid II (Figure 3a). The elongation of the glycan chains was inhibited by moenomycin, a known inhibitor of GT activity, whereas the formation of the high mass products was prevented by penicillin G, demonstrating that products resulted from the TP activity of PBP2a. When lipid II contained glutamate instead of iso-glutamine, no transpeptidation occurred as shown by the absence of high mass products and effect of penicillin (Figure 3b). As the glutamate and iso-glutamine lipid II were of different batches, the possibility remained that the difference in TP activity was due to a factor other than the amidation. To rule out this possibility, glutamate-containing lipid II was incubated with MurT/GatD and glutamine, in the absence and presence of ATP, prior to the addition of PBP2a. PBP2a polymerized glycan chains in all cases, but the TP activity was observed only if ATP had been included, demonstrating the dependence on the amidation of lipid II (Figure 3d). When the GT inactivated variant PBP2a-E131Q was used, no glycan chains were assembled (Figure 3c), as observed previously.20 In contrast, glycan chains were assembled by the GT activity of the PBP2a-S410A variant with the TP active site serine replaced by an alanine residue (Figure 3c). No high molecular mass products accumulated with this PBP2a-S410A variant, providing further evidence that they result from TP activity. To test whether the TP activity is coupled to the GT activity of the same PBP2a molecule, both variants were incubated with amidated lipid II and 10% dansylated lipid II. The appearance of high mass material, which was prevented by penicillin G, showed that the TP activity of PBP2a can process glycan chains assembled by other PBP2a molecules (Figure 3c).

Figure 4. Quantification of the peptidoglycan cross-linked by the activity of PBP2a. (a) A mixture of 50 μM amidated lipid II and 5 μM non-amidated dansylated lipid II was incubated with 1 μM PBP2a, in the presence or absence of penicillin G (Pen). Duplicates are shown. Reactions were overnight at 30 °C. Analysis was as in Figure 3. Sections of the gel are numbered on the left. (b) Quantification of the dansyl fluorescence in the different sections of the gel shown in panel a. Black and gray bars are in absence and presence of penicillin G, respectively. Bars are the mean of the duplicates shown in panel a.

the absence of penicillin, over 20% of the polymerized material was found in high mass products, compared to 5% in its absence, the latter presumably being long chains. Interestingly, in the absence of penicillin, it is the longest chains that have been immobilized into large mass products, the amount of shorter chains being unaffected. This observation could mean that PBP2a exerts its TP activity on long chains only or that glycan chain cross-linking precludes their elongation. Better quantitation and chain length determination might be possible using radiolabeled precursors with the same electrophoretic system.39 Ideally, full characterization and quantitation of the products might be achieved by HPLC analysis.22,24,25,36 Peptidoglycan Assembly Activities of S. pneumoniae PBP1a. Recombinant full length PBP1a was also found to exhibit GT and TP activities using amidated lipid II as precursor (Figure 5a and b). A time course of the reactions catalyzed by PBP1a showed that cross-linking by TP activity occurs rapidly after elongation of the glycan chains has started (Figure 5c). Mass spectrometry analysis of the products following digestion with muramidase yielded monoprotonated species with masses of 966.330 and 1842.743 Da, corresponding to the expected masses of the non-cross-linked monomer disaccharide-pentapeptide and the cross-linked dimer tetra-pentapeptide, respectively (Figure 5d). The dimer species was not found when penicillin was present during the reaction. The activity of PBP1a was subject to inhibition by GT and TP inhibitors in a similar manner to PBP2a (Figure 5a), but unlike PBP2a, PBP1a exhibited residual TP activity with glutamate-containing stem-peptides (Figure 5b). A limited 2691

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Figure 5. continued spectra of reaction samples. PBP1a (1 μM) was incubated for 6 h at 30 °C with 50 μM amidated lipid II, in the absence or the presence of 1 mM penicillin G. After digestion with 0.1 mg mL‑1 of muramidase at 37 °C for 3 h, samples were analyzed by MALDI-TOF mass spectrometry. Monoisotopic masses of monoprotonated [M + H]+ and monosodiated ions [M + Na]+ are given on the spectra. The expected mass for the non-protonated monomer disaccharide pentapeptide is 965.46 Da. The expected mass for the dimer tetrapentapeptide is 1842.86 Da.

three points time-course comparison of PBP1a and 2a showed the latter to be over 10-fold slower. It is therefore possible that both enzymes have a similar preference for amidated stempeptides, but that reactions catalyzed by PBP2a are too slow to permit detection of TP activity with non-amidated lipid II. Transpeptidase Activities of PBP2b and PBP2x. Both full-length class B PBPs from S. pneumoniae were found to exhibit TP activity, provided the precursor was amidated by the action of MurT/GatD (Figure 6). No cross-linking of glycan chains was observed when glutamate-containing lipid II was used as precursor (Figure 6b and Supplementary Figure 1). In order to reveal the activity of the class B PBPs, glycan chains were prepared in situ using the PBP2a-S410A variant that lacks TP activity. The TP activity of PBP2b was robust, with most of the material found in high mass cross-linked products after overnight incubation (Figure 6a). In contrast, the TP activity of PBP2x was very weak, with a limited amount of material found in high mass products in the absence of penicillin G (Figure 6c). Mechanistic Insights. Transpeptidation catalyzed by PBPs whereby the terminal D-Ala residue of the stem-peptides in polymerized glycan chains is replaced by another D-amino acid has been reported using E. coli PBP1a.40 Also, diverse D-amino acids added to cell cultures can be incorporated in the peptidoglycan of a variety of bacteria.41−43 These observations suggest that the first step of the transpeptidation catalysis, i.e., the formation of a covalent acyl-enzyme intermediate, is reversible (Figure 7). Furthermore, the backward aminolysis of this intermediate by a D-amino acid can compete with the forward completion of the transpeptidation with the ε-amino group of the third residue of an acceptor stem-peptide. This reaction scheme predicts that an excess of D-amino acid should inhibit the TP activity of PBPs. Indeed when PBP2a was incubated with amidated lipid II in the presence of various amounts of D-Ala, the TP activity was inhibited by the highest concentration (5 mM), whereas the GT activity was not hampered (Figure 8). We attempted to observe transpeptidation using glycan chains preassembled by PBP2a-S410A, adding subsequently PBP2a-E131Q or PBP2b to the reaction mix. No TP activity of either PBP2a-E131Q or PBP2b could be recorded in these conditions (Supplementray Figure 2). This observation indicates that the TP reaction may require ongoing GT activity, even though the latter can be performed by another protein. An early publication on the hydrolytic activity of PBP2x on the S2d thioesther analogue indicated an optimal activity at pH 5.5, two units lower than the pH of our assay.44 This report prompted us to test the activity of PBP2a at pH 5.5. Although the GT activity was not affected, at least in terms of end point of an overnight reaction, the TP activity was markedly inhibited

Figure 5. Assembly of peptidoglycan by pneumococcal PBP1a. (a) A mixture of 50 μM amidated lipid II and 5 μM non-amidated dansylated lipid II was incubated with 1 μM PBP1a, in the presence or absence of moenomycin (Moe) or penicillin G (Pen). (b) Mixtures of 50 μM amidated (LII(iGln)) or non-amidated lipid II (LII(Glu)) and 5 μM non-amidated dansylated lipid II were incubated with 1 μM PBP1a, in the absence of presence of penicillin G. All reactions were overnight at 30 °C. (c) Time course. A mixture as in panel a was incubated with PBP1a at 30 °C. Aliquots were withdrawn after various time intervals, and the reaction was stopped by the addition of moenomycin and penicillin G. Analysis was as in Figure 3. (d) Mass 2692

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Figure 6. Cross-linking of peptidoglycan by PBP2b and PBP2x. (a) A mixture of 50 μM amidated lipid II and 5 μM non-amidated dansylated lipid II was incubated with 1 μM transpeptidase-inactivated PBP2a-S410A, 1 μM PBP2b, or both, in the absence or presence of penicillin G. (b) Mixtures of 50 μM amidated (LII(iGln)) or non-amidated lipid II (LII(Glu)) and 5 μM non-amidated dansylated lipid II were incubated with 1 μM of transpeptidase-inactivated PBP2a-S410A and 1 μM of PBP2b in the absence or presence of penicillin G. (c) As in panel a but with PBP2x. All reactions were overnight at 30 °C. Analysis was as in Figure 3

species that use lipid II with a negatively charged second residue appears to be more positively charged than that of PBPs from Gram-positive species (Supplementary Figure 4). In trying to answer this question by preparing glycan-chains with various stem-peptides prior to cross-linking by transpeptidation, we found that PBP2a or 2b did not exhibit TP activity between any preformed chains. This is surprising as the TP domain of both PBP2a and 2b was found to be active if the GT activity is carried out by another protein. This observation suggests that PBPs interact with themselves or with each other and that the TP activity might function on glycan chains being elongated by a partner PBP, as was proposed on the basis of observation with E. coli PBPs.23,24 This model could explain why PBP2x was found to have only marginal TP activity, compared to PBP2b, using PBP2a-S410A as glycosyltransferase. PBP2a and 2b might be natural partners, whereas PBP2x could work optimally with PBP1a. These functional associations should be probed in the future to finally attribute a function to the different class A PBPs of ovococcal bacteria.

(Supplementary Figure 3). This observation points to the role of an ionizable group with a pKa between 5.5 and 7.5 participating to the TP reaction. Conclusion. Full synthesis of peptidoglycan of the Grampositive human pathogen S. pneumoniae was achieved using recombinant enzymes. We report here the first in vitro full assembly of peptidoglycan from a Gram-positive organism, following the 1970s pioneering works with cell extracts from Bacillus species.26−29 Both class A PBP1a and 2a exhibited GT activity to polymerize glycan chains from the lipid II precursor. Of the four PBPs tested that are absolutely required in combination or isolation for pneumococcal viability, we showed for the first time that the bifunctional PBP1a and 2a and the class B PBP2b and 2x demonstrated TP activity in cross-linking peptide-stems of polymerized glycan chains. Most importantly, the transpeptidation efficiency is dependent on the chemical nature of the stem-peptide. Specifically, amidation of the second residue of the lipid II peptide from a Glu into an iso-Gln is preferred for the TP activity of PBP2a, 2b, and 2x. PBP1a demonstrated a higher TP activity on glycan chains prepared from iso-Glncontaining lipid II, although it retained some activity with Glucontaining chains. The prior step of glycan chain elongation by PBP2a and 1a is not dependent on the amidation. Amidation of lipid II was performed in vitro with recombinant pneumococcal amido transferase MurT/GatD. Importantly this new methodology provides a way forward for biochemical studies of the mechanisms selected in the evolution of pneumococcal PBPs to evade β-lactam inhibition of the TP activity. Among the remaining questions, it is of interest to determine whether amidation of the second residue is required on either the donor or the acceptor stem-peptides or both. The nature of the stem-peptide may be more important for the donor, as it is known that the acceptor can be diverse, as exemplified by the branched-peptides in pneumococcus,36 or the incorporation of various D-amino acids.43 In addition, the exact mechanism by which PBPs recognize amidation of the stem-peptide and why this should be important in Gram-positive organisms generally can now be addressed. Preliminary examination of the available crystal structures of TP domains of synthetic PBPs from Grampositive and Gram-negative organisms did not allow the identification of a defined feature that could discriminate the amidation of the second residue. However, the surface surrounding the TP active site of PBPs from Gram-negative

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METHODS

Pneumococcal Strains. The wild type strain was R6. Derivative spnLR29 was constructed by introducing at the bgaA locus the murT/ gatD operon (spr1443/4) under the control of a zinc-inducible promoter. The endogenous murT/gatD operon was then deleted to create strain spnLR30. Transformants were selected in the presence of 150 μM ZnCl2. Full details are given in the Supporting Information. Proteins. All pneumococcal proteins were produced in E. coli. MurT/GatD with an N-ter His-tag on MurT was purified by Niaffinity and anion-exchange chromatography. Full-length PBPs were purified from Triton X-100 solubilized membranes. PBP2a without tag was purified by successive cation- and anion-exchange chromatography.20 PBP1a with an N-terminal His-tag was purified by Ni-affinity and size-exclusion chromatography. PBP2b and PBP2x with a C-ter Strep-tag were purified by Strep-Tactin affinity and anion-exchange chromatography. Full details are given in the Supporting Information. Preparation of Glutamate- and Iso-glutamine-Containing Lipid II. Lys-containing lipid II and dansylated lipid II were prepared as described previously.13,20 Amidation was performed in vitro by incubation for 4 h at 30 °C of 100 μM lipid II in 150 mM Tris, pH 8, 5 mM KCl, 40 mM MgCl2, 0.5% (w/v) Triton X-100, 10 mM Lglutamine, and 10 mM ATP, in the presence of 5 μM MurT/GatD. Amidated lipid II was then extracted and purified as previously.13 Reactions of Peptidoglycan Synthesis. Unlabeled (glutamateor iso-glutamine-containing) lipid II and dansylated lipid II in organic solvent were mixed in a 10:1 ratio and dried under nitrogen flow. The dried lipid II mix was then redissolved in the reaction mix typically containing 50 mM HEPES, pH 7.5, 150 mM NaCl, 10 mM MgCl2, 2693

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Figure 8. Inhibition of the TP activity of PBP2a by D-alanine. A mixture of 50 μM amidated lipid II and 5 μM non-amidated dansylated lipid II was incubated with 1 μM PBP2a, in the absence or the presence of various concentrations of D-alanine. Reactions were overnight at 30 °C. Analysis was as in Figure 3 Alternatively, a dried mix of unlabeled glutamate-containing lipid II and dansylated lipid II was redissolved in 91 mM HEPES, pH 7.5, 273 mM NaCl, 18 mM MgCl2, 0.036% (w/v) Triton X-100, 10 mM Lglutamine, and about 0.3 μM purified MurT/GatD, with or without 10 mM ATP. After 3 h of incubation at 37 °C, DMSO was added for a final concentration of 25% (v/v), as well as PBP2a with or without penicillin G, to final concentrations of about 1 μM and 1 mM, respectively. The final buffer, salt, and detergent concentrations were as for reactions without MurT/GatD. Incubation was continued overnight at 30 °C. Samples were analyzed by SDS-PAGE with the gel system of Barrett39 visualized with UV or blue trans-illumination using GelDoc EQ or ChemiDoc MP imagers (BioRad).20 Quantifications were performed on unsaturated images using the ImageJ software.45 Mass Spectrometry. After overnight incubation, the buffer of 70 μL reactions was exchanged against 100 mM ammonium acetate on Bio-Spin columns (Bio-Rad). N-Acetylmuramidase (Calbiochem) was added to 0.1 mg mL−1, and the solution was incubated for 3 h at 37 °C, prior to filtration in a Vivaspin 500 device with a 10,000 kDa cutoff. After lyophilization, samples were first resuspended in 10−20 μL of 0.1% (v/v) trifluoroacetic acid in water, then cleaned-up on reversed phase-C18 tips (ZipTip, Millipore), and finally eluted with 2 μL of DHB matrix (10 mg mL−1 in water/acetonitrile/trifluoroacetic acid 50/50/0.1) directly on the target. The analysis was carried out on an Autoflex MALDI mass spectrometer (Bruker Daltonics, Bremen, Germany) in the reflectron positive ion mode detection previously calibrated with standard peptide mixture in the 500−4000 m/z range (peptide calibration II, Bruker Daltonics). The mass spectra were acquired, and the data were processed with Flexcontrol and Flexanalysis softwares (v. 3.0) (Bruker Daltonics).

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Figure 7. Transpeptidation reaction scheme. The first step is reversible.

ASSOCIATED CONTENT

S Supporting Information *

Experimental details regarding protein purification and bacterial strains, as well as additional results. This material is available free of charge via the Internet at http://pubs.acs.org.

25% (v/v) DMSO, and 0.01% or 0.02% (w/v) Triton X-100, and the PBPs were investigated. The concentration of buffer, salts, and especially of detergent contributed by the protein stocks was taken into account. Penicillin G (1 mM, Sigma) or moenomycin (0.5 mM, Flavomycin, Hoechst) were included as required. The reactions were left to proceed overnight at 30 °C unless otherwise stated. For time course experiments, aliquots were withdrawn after various time intervals, and the reaction was stopped by the addition of penicillin G (1 mM) and moenomycin (0.5 mM).

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Notes

The authors declare no competing financial interest. 2694

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ACKNOWLEDGMENTS We thank L. Roux for excellent assistance with pneumococcal transformation and cultures and W. Vollmer and M. NoirclercSavoye for comments and advice. This work was partly funded by the “Coopol innovation France-Chine” program and by Medical Research Council grants G500643 and G0600801. This work used the platforms of the Grenoble Instruct Center (ISBG; UMS 3518 CNRS-CEA-UJF-EMBL) with support from FRISBI (ANR-10-INSB-05-02) and GRAL (ANR-10-LABX49-01) within the Grenoble Partnership for Structural Biology. J.P. is funded by an ADR from the Région Rhône-Alpes (ARC1), and K.A.A. by a University of Warwick Ph.D. studentship.

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