Minimal exposure of lipid II cycle intermediates triggers cell wall antibiotic resistance.

Cell wall antibiotics are crucial for combatting the emerging wave of resistant bacteria. Yet, our understanding of antibiotic action is limited, as many strains devoid of all resistance determinants display far higher antibiotic tolerance in vivo than suggested by the antibiotic-target binding affinity in vitro. To resolve this conflict, here we develop a comprehensive theory for the bacterial cell wall biosynthetic pathway and study its perturbation by antibiotics. We find that the closed-loop architecture of the lipid II cycle of wall biosynthesis features a highly asymmetric distribution of pathway intermediates, and show that antibiotic tolerance scales inversely with the abundance of the targeted pathway intermediate. We formalize this principle of minimal target exposure as intrinsic resistance mechanism and predict how cooperative drug-target interactions can mitigate resistance. The theory accurately predicts the in vivo efficacy for various cell wall antibiotics in different Gram-positive bacteria and contributes to a systems-level understanding of antibiotic action.

Cardiolipin Alters Rhodobacter sphaeroides Cell Shape by Affecting Peptidoglycan Precursor Biosynthesis.

Cardiolipin (CL) is an anionic phospholipid that plays an important role in regulating protein biochemistry in bacteria and mitochondria. Deleting the CL synthase gene (Δcls) in Rhodobacter sphaeroides depletes CL and decreases cell length by 20%. Using a chemical biology approach, we found that a CL deficiency does not impair the function of the cell wall elongasome in R. sphaeroides; instead, biosynthesis of the peptidoglycan (PG) precursor lipid II is decreased. Treating R. sphaeroides cells with fosfomycin and d-cycloserine inhibits lipid II biosynthesis and creates phenotypes in cell shape, PG composition, and spatial PG assembly that are strikingly similar to those seen with R. sphaeroides Δcls cells, suggesting that CL deficiency alters the elongation of R. sphaeroides cells by reducing lipid II biosynthesis. We found that MurG-a glycosyltransferase that performs the last step of lipid II biosynthesis-interacts with anionic phospholipids in native (i.e., R. sphaeroides) and artificial membranes. Lipid II production decreases 25% in R. sphaeroides Δcls cells compared to wild-type cells, and overexpression of MurG in R. sphaeroides Δcls cells restores their rod shape, indicating that CL deficiency decreases MurG activity and alters cell shape. The R. sphaeroides Δcls mutant is more sensitive than the wild-type strain to antibiotics targeting PG synthesis, including fosfomycin, d-cycloserine, S-(3,4-dichlorobenzyl)isothiourea (A22), mecillinam, and ampicillin, suggesting that CL biosynthesis may be a potential target for combination chemotherapies that block the bacterial cell wall.IMPORTANCE The phospholipid composition of the cell membrane influences the spatial and temporal biochemistry of cells. We studied molecular mechanisms connecting membrane composition to cell morphology in the model bacterium Rhodobacter sphaeroides The peptidoglycan (PG) layer of the cell wall is a dominant component of cell mechanical properties; consequently, it has been an important antibiotic target. We found that the anionic phospholipid cardiolipin (CL) plays a role in determination of the shape of R. sphaeroides cells by affecting PG precursor biosynthesis. Removing CL in R. sphaeroides alters cell morphology and increases its sensitivity to antibiotics targeting proteins synthesizing PG. These studies provide a connection to spatial biochemical control in mitochondria, which contain an inner membrane with topological features in common with R. sphaeroides.

Enzyme structures of the bacterial peptidoglycan and wall teichoic acid biogenesis pathways.

The bacterial cell wall is a complex polymeric structure with essential roles in defence, survival and pathogenesis. Common to both Gram-positive and Gram-negative bacteria is the mesh-like peptidoglycan sacculus that surrounds the outer leaflet of the cytoplasmic membrane. Recent crystallographic studies of enzymes that comprise the peptidoglycan biosynthetic pathway have led to significant new understanding of all stages. These include initial multi-step cytosolic formation of sugar-pentapeptide precursors, transfer of the precursors to activated polyprenyl lipids at the membrane inner leaflet and flippase mediated relocalization of the resulting lipid II precursors to the outer leaflet where glycopolymerization and subsequent peptide crosslinking are finalized. Additional, species-specific enzymes allow customized peptidoglycan modifications and biosynthetic regulation that are important to bacterial virulence and survival. These studies have reinforced the unique and specific catalytic mechanisms at play in cell wall biogenesis and expanded the atomic foundation to develop novel, structure guided, antibacterial agents.

Lipid Requirements for the Enzymatic Activity of MraY Translocases and in Vitro Reconstitution of the Lipid II Synthesis Pathway.

Screening of new compounds directed against key protein targets must continually keep pace with emerging antibiotic resistances. Although periplasmic enzymes of bacterial cell wall biosynthesis have been among the first drug targets, compounds directed against the membrane-integrated catalysts are hardly available. A promising future target is the integral membrane protein MraY catalyzing the first membrane associated step within the cytoplasmic pathway of bacterial peptidoglycan biosynthesis. However, the expression of most MraY homologues in cellular expression systems is challenging and limits biochemical analysis. We report the efficient production of MraY homologues from various human pathogens by synthetic cell-free expression approaches and their subsequent characterization. MraY homologues originating from Bordetella pertussis, Helicobacter pylori, Chlamydia pneumoniae, Borrelia burgdorferi, and Escherichia coli as well as Bacillus subtilis were co-translationally solubilized using either detergent micelles or preformed nanodiscs assembled with defined membranes. All MraY enzymes originating from Gram-negative bacteria were sensitive to detergents and required nanodiscs containing negatively charged lipids for obtaining a stable and functionally folded conformation. In contrast, the Gram-positive B. subtilis MraY not only tolerates detergent but is also less specific for its lipid environment. The MraY·nanodisc complexes were able to reconstitute a complete in vitro lipid I and lipid II forming pipeline in combination with the cell-free expressed soluble enzymes MurA-F and with the membrane-associated protein MurG. As a proof of principle for future screening platforms, we demonstrate the inhibition of the in vitro lipid II biosynthesis with the specific inhibitors fosfomycin, feglymycin, and tunicamycin.

Crystal Structure of the N-Acetylmuramic Acid α-1-Phosphate (MurNAc-α1-P) Uridylyltransferase MurU, a Minimal Sugar Nucleotidyltransferase and Potential Drug Target Enzyme in Gram-negative Pathogens.

The N-acetylmuramic acid α-1-phosphate (MurNAc-α1-P) uridylyltransferase MurU catalyzes the synthesis of uridine diphosphate (UDP)-MurNAc, a crucial precursor of the bacterial peptidoglycan cell wall. MurU is part of a recently identified cell wall recycling pathway in Gram-negative bacteria that bypasses the general de novo biosynthesis of UDP-MurNAc and contributes to high intrinsic resistance to the antibiotic fosfomycin, which targets UDP-MurNAc de novo biosynthesis. To provide insights into substrate binding and specificity, we solved crystal structures of MurU of Pseudomonas putida in native and ligand-bound states at high resolution. With the help of these structures, critical enzyme-substrate interactions were identified that enable tight binding of MurNAc-α1-P to the active site of MurU. The MurU structures define a "minimal domain" required for general nucleotidyltransferase activity. They furthermore provide a structural basis for the chemical design of inhibitors of MurU that could serve as novel drugs in combination therapy against multidrug-resistant Gram-negative pathogens.

Characterization of a UDP-N-acetylglucosamine biosynthetic pathway encoded by the giant DNA virus Mimivirus.

Mimivirus is a giant DNA virus belonging to the Megaviridae family and infecting unicellular Eukaryotes of the genus Acanthamoeba. The viral particles are characterized by heavily glycosylated surface fibers. Several experiments suggest that Mimivirus and other related viruses encode an autonomous glycosylation system, forming viral glycoproteins independently of their host. In this study, we have characterized three Mimivirus proteins involved in the de novo uridine diphosphate-N-acetylglucosamine (UDP-GlcNAc) production: a glutamine-fructose-6-phosphate transaminase (CDS L619), a glucosamine-6-phosphate N-acetyltransferase (CDS L316) and a UDP-GlcNAc pyrophosphorylase (CDS R689). Sequence and enzymatic analyses have revealed some unique features of the viral pathway. While it follows the eukaryotic-like strategy, it also shares some properties of the prokaryotic pathway. Phylogenetic analyses revealed that the Megaviridae enzymes cluster in monophyletic groups, indicating that they share common ancestors, but did not support the hypothesis of recent acquisitions from one of the known hosts. Rather, viral clades branched at deep nodes in phylogenetic trees, forming independent clades outside sequenced cellular organisms. The intermediate properties between the eukaryotic and prokaryotic pathways, the phylogenetic analyses and the fact that these enzymes are shared between most of the known members of the Megaviridae family altogether suggest that the viral pathway has an ancient origin, resulting from lateral transfers of cellular genes early in the Megaviridae evolution, or from vertical inheritance from a more complex cellular ancestor (reductive evolution hypothesis). The identification of a virus-encoded UDP-GlcNAc pathway reinforces the concept that GlcNAc is a ubiquitous sugar representing a universal and fundamental process in all organisms.

Requirement of lipid II biosynthesis for cell division in cell wall-less Wolbachia, endobacteria of arthropods and filarial nematodes.

Obligate Wolbachia endobacteria have a reduced genome and retained genes are hypothesized to be crucial for survival. Although intracellular bacteria do not need a stress-bearing peptidoglycan cell wall, Wolbachia encode proteins necessary to synthesize the peptidoglycan precursor lipid II. The activity of the enzymes catalyzing the last two steps of this pathway was previously shown, and Wolbachia are sensitive to inhibition of lipid II synthesis. A puzzling characteristic of Wolbachia is the lack of genes for l-amino acid racemases essential for lipid II synthesis. Transcription analysis showed the expression of a possible alternative racemase metC, and recombinant Wolbachia MetC indeed had racemase activity that may substitute for the absent l-Ala racemase. However, enzymes needed to form mature peptidoglycan are absent and the function of Wolbachia lipid II is unknown. Inhibition of lipid II biosynthesis resulted in enlargement of Wolbachia cells and redistribution of Wolbachia peptidoglycan-associated lipoprotein, demonstrating that lipid II is required for coordinated cell division and may interact with the lipoprotein. We conclude that lipid II is essential for Wolbachia cell division and that this function is potentially conserved in the Gram-negative bacteria.

Lipodepsipeptide empedopeptin inhibits cell wall biosynthesis through Ca2+-dependent complex formation with peptidoglycan precursors.

Empedopeptin is a natural lipodepsipeptide antibiotic with potent antibacterial activity against multiresistant Gram-positive bacteria including methicillin-resistant Staphylococcus aureus and penicillin-resistant Streptococcus pneumoniae in vitro and in animal models of bacterial infection. Here, we describe its so far elusive mechanism of antibacterial action. Empedopeptin selectively interferes with late stages of cell wall biosynthesis in intact bacterial cells as demonstrated by inhibition of N-acetylglucosamine incorporation into polymeric cell wall and the accumulation of the ultimate soluble peptidoglycan precursor UDP-N-acetylmuramic acid-pentapeptide in the cytoplasm. Using membrane preparations and the complete cascade of purified, recombinant late stage peptidoglycan biosynthetic enzymes and their respective purified substrates, we show that empedopeptin forms complexes with undecaprenyl pyrophosphate containing peptidoglycan precursors. The primary physiological target of empedopeptin is undecaprenyl pyrophosphate-N-acetylmuramic acid(pentapeptide)-N-acetylglucosamine (lipid II), which is readily accessible at the outside of the cell and which forms a complex with the antibiotic in a 1:2 molar stoichiometry. Lipid II is bound in a region that involves at least the pyrophosphate group, the first sugar, and the proximal parts of stem peptide and undecaprenyl chain. Undecaprenyl pyrophosphate and also teichoic acid precursors are bound with lower affinity and constitute additional targets. Calcium ions are crucial for the antibacterial activity of empedopeptin as they promote stronger interaction with its targets and with negatively charged phospholipids in the membrane. Based on the high structural similarity of empedopeptin to the tripropeptins and plusbacins, we propose this mechanism of action for the whole compound class.

Functional analysis of the cytoskeleton protein MreB from Chlamydophila pneumoniae.

In rod-shaped bacteria, the bacterial actin ortholog MreB is considered to organize the incorporation of cell wall precursors into the side-wall, whereas the tubulin homologue FtsZ is known to tether incorporation of cell wall building blocks at the developing septum. For intracellular bacteria, there is no need to compensate osmotic pressure by means of a cell wall, and peptidoglycan has not been reliably detected in Chlamydiaceae. Surprisingly, a nearly complete pathway for the biosynthesis of the cell wall building block lipid II has been found in the genomes of Chlamydiaceae. In a previous study, we discussed the hypothesis that conservation of lipid II biosynthesis in cell wall-lacking bacteria may reflect the intimate molecular linkage of cell wall biosynthesis and cell division and thus an essential role of the precursor in cell division. Here, we investigate why spherical-shaped chlamydiae harbor MreB which is almost exclusively found in elongated bacteria (i.e. rods, vibrios, spirilla) whereas they lack the otherwise essential division protein FtsZ. We demonstrate that chlamydial MreB polymerizes in vitro and that polymerization is not inhibited by the blocking agent A22. As observed for MreB from Bacillus subtilis, chlamydial MreB does not require ATP for polymerization but is capable of ATP hydrolysis in phosphate release assays. Co-pelleting and bacterial two-hybrid experiments indicate that MreB from Chlamydophila (Chlamydia) pneumoniae interacts with MurF, MraY and MurG, three key components in lipid II biosynthesis. In addition, MreB polymerization is improved in the presence of MurF. Our findings suggest that MreB is involved in tethering biosynthesis of lipid II and as such may be necessary for maintaining a functional divisome machinery in Chlamydiaceae.

Tripropeptin C blocks the lipid cycle of cell wall biosynthesis by complex formation with undecaprenyl pyrophosphate.

Tripropeptin C (TPPC) is a naturally occurring cyclic lipodepsipeptide antibiotic produced by a Lysobacter sp. TPPC exhibits potent antibacterial activity against methicillin-resistant Staphylococcus aureus (MRSA), vancomycin-resistant enterococci (VRE), and penicillin-resistant Streptococcus pneumoniae. This antibiotic also inhibits the incorporation of N-acetylglucosamine into the peptidoglycan of S. aureus at a 50% inhibitory concentration (IC(50)) of 0.7 µM, which is proportional to its MIC (0.87 µM; equivalent to 1.0 µg/ml). Treatment of exponential-phase S. aureus cells with TPPC resulted in accumulation of UDP-MurNAc-pentapeptide in the cytoplasm. The antimicrobial activity of TPPC was weakened by the addition of prenyl pyrophosphates but not by prenyl phosphates, UDP-linked sugars, or the pentapeptide of peptidoglycan. The direct interaction between TPPC and undecaprenyl pyrophosphate (C(55)-PP) was observed by mass spectrometry and thin-layer chromatography analysis, indicating that TPPC can potentially inhibit C(55)-PP phosphatase activity, which plays a crucial role in the lipid cycle of peptidoglycan synthesis. As expected, TPPC inhibits this enzymatic reaction at an IC(50) of 0.03 to 0.1 µM in vitro, as does bacitracin. From the analysis of accumulation of lipid carrier-related compounds, TPPC was found to cause the accumulation of C(55)-PP in situ, leading to the accumulation of a glycine-containing lipid intermediate. This suggested that the TPPC/C(55)-PP complex also inhibits the transglycosylation step or flippase activity, adding to the inhibition of C(55)-PP dephosphorylation. This mode of action is different from that of currently available drugs such as vancomycin, daptomycin, and bacitracin.

Correlation of daptomycin resistance in a clinical Staphylococcus aureus strain with increased cell wall teichoic acid production and D-alanylation.

Cell wall thickening is a common feature among daptomycin-resistant Staphylococcus aureus strains. However, the mechanism(s) leading to this phenotype is unknown. We examined a number of cell wall synthesis pathway parameters in an isogenic strain set of S. aureus bloodstream isolates obtained from a patient with recalcitrant endocarditis who failed daptomycin therapy, including the initial daptomycin-susceptible parental strain (strain 616) and two daptomycin-resistant strains (strains 701 and 703) isolated during daptomycin therapy. Transmission electron microscopy demonstrated significantly thicker cell walls in the daptomycin-resistant strains than in the daptomycin-susceptible strain, a finding which was compatible with significant differences in dry cell weight of strain 616 versus strains 701 to 703 (P < 0.05). Results of detailed analysis of cell wall muropeptide composition, the degree of peptide side chain cross-linkage, and the amount of the peptidoglycan precursor, UDP-MurNAc-pentapeptide, were similar in the daptomycin-susceptible and daptomycin-resistant isolates. In contrast, the daptomycin-resistant strains contained less O-acetylated peptidoglycan. Importantly, both daptomycin-resistant strains synthesized significantly more wall teichoic acid (WTA) than the parental strain (P < 0.001). Moreover, the proportion of D-alanylated WTA species was substantially higher in the daptomycin-resistant strains than in the daptomycin-susceptible parental strain (P < 0.05 in comparing strain 616 versus strain 701). The latter phenotypic findings correlated with (i) enhanced tagA and dltA gene expression, respectively, and (ii) an increase in surface positive charge observed in the daptomycin-resistant versus daptomycin-susceptible isolates. Collectively, these data suggest that increases in WTA synthesis and the degree of its D-alanylation may play a major role in the daptomycin-resistant phenotype in some S. aureus strains.

Mechanism of action and limited cross-resistance of new lipopeptide MX-2401.

MX-2401 is a semisynthetic calcium-dependent lipopeptide antibiotic (analogue of amphomycin) in preclinical development for the treatment of serious Gram-positive infections. In vitro and in vivo, MX-2401 demonstrates broad-spectrum bactericidal activity against Gram-positive organisms, including antibiotic-resistant strains. The objective of this study was to investigate the mechanism of action of MX-2401 and compare it with that of the lipopeptide daptomycin. The results indicated that although both daptomycin and MX-2401 are in the structural class of Ca²âº-dependent lipopeptide antibiotics, the latter has a different mechanism of action. Specifically, MX-2401 inhibits peptidoglycan synthesis by binding to the substrate undecaprenylphosphate (C55-P), the universal carbohydrate carrier involved in several biosynthetic pathways. This interaction resulted in inhibition, in a dose-dependent manner, of the biosynthesis of the cell wall precursors lipids I and II and the wall teichoic acid precursor lipid III, while daptomycin had no significant effect on these processes. MX-2401 induced very slow membrane depolarization that was observed only at high concentrations. Unlike daptomycin, membrane depolarization by MX-2401 did not correlate with its bactericidal activity and did not affect general membrane permeability. In contrast to daptomycin, MX-2401 had no effect on lipid flip-flop, calcein release, or membrane fusion with 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC)/1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-(1'-rac-glycerol) (sodium salt) (POPG) liposomes. MX-2401 adopts a more defined structure than daptomycin, presumably to facilitate interaction with C55-P. Mutants resistant to MX-2401 demonstrated low cross-resistance to other antibiotics. Overall, these results provided strong evidence that the mode of action of MX-2401 is unique and different from that of any of the approved antibiotics, including daptomycin.

Phosphorylated hydroxyethylamines as novel inhibitors of the bacterial cell wall biosynthesis enzymes MurC to MurF.

Enzymes involved in the biosynthesis of bacterial peptidoglycan represent important targets for development of new antibacterial drugs. Among them, Mur ligases (MurC to MurF) catalyze the formation of the final cytoplasmic precursor UDP-N-acetylmuramyl-pentapeptide from UDP-N-acetylmuramic acid. We present the design, synthesis and biological evaluation of a series of phosphorylated hydroxyethylamines as new type of small-molecule inhibitors of Mur ligases. We show that the phosphate group attached to the hydroxyl moiety of the hydroxyethylamine core is essential for good inhibitory activity. The IC(50) values of these inhibitors were in the micromolar range, which makes them a promising starting point for the development of multiple inhibitors of Mur ligases as potential antibacterial agents. In addition, 1-(4-methoxyphenylsulfonamido)-3-morpholinopropan-2-yl dihydrogen phosphate 7a was discovered as one of the best inhibitors of MurE described so far.

Functional conservation of the lipid II biosynthesis pathway in the cell wall-less bacteria Chlamydia and Wolbachia: why is lipid II needed?

Cell division and cell wall biosynthesis in prokaryotes are driven by partially overlapping multiprotein machineries whose activities are tightly controlled and co-ordinated. So far, a number of protein components have been identified and acknowledged as essential for both fundamental cellular processes. Genes for enzymes of both machineries have been found in the genomes of the cell wall-less genera Chlamydia and Wolbachia, raising questions as to the functionality of the lipid II biosynthesis pathway and reasons for its conservation. We provide evidence on three levels that the lipid II biosynthesis pathway is indeed functional and essential in both genera: (i) fosfomycin, an inhibitor of MurA, catalysing the initial reaction in lipid II biosynthesis, has a detrimental effect on growth of Wolbachia cells; (ii) isolated cytoplasmic membranes from Wolbachia synthesize lipid II ex vivo; and (iii) recombinant MraY and MurG from Chlamydia and Wolbachia exhibit in vitro activity, synthesizing lipid I and lipid II respectively. We discuss the hypothesis that the necessity for maintaining lipid II biosynthesis in cell wall-lacking bacteria reflects an essential role of the precursor in prokaryotic cell division. Our results also indicate that the lipid II pathway may be exploited as an antibacterial target for chlamydial and filarial infections.

Cell wall growth during elongation and division: one ring to bind them?

The role of the cell division protein FtsZ in bacterial cell wall (CW) synthesis is believed to be restricted to localizing proteins involved in the synthesis of the septal wall. In this issue of Molecular Microbiology, the groups of Christine Jacobs-Wagner and Waldemar Vollmer provide compelling evidence that in Caulobacter crescentus, FtsZ plays an additional role in CW synthesis in non-dividing cells. During elongation (cell growth) FtsZ is responsible for the incorporation of CW material in a zone at the midcell by recruiting MurG, a protein involved in peptidoglycan (PG) precursor synthesis. This resembles earlier findings of FtsZ mediated PG synthesis activity in Escherichia coli. A role of FtsZ in PG synthesis during elongation forces a rethink of the current model of CW synthesis in rod-shaped bacteria.

The tubulin homologue FtsZ contributes to cell elongation by guiding cell wall precursor synthesis in Caulobacter crescentus.

The tubulin homologue FtsZ is well known for its essential function in bacterial cell division. Here, we show that in Caulobacter crescentus, FtsZ also plays a major role in cell elongation by spatially regulating the location of MurG, which produces the essential lipid II peptidoglycan cell wall precursor. The early assembly of FtsZ into a highly mobile ring-like structure during cell elongation is quickly followed by the recruitment of MurG and a major redirection of peptidoglycan precursor synthesis to the midcell region. These FtsZ-dependent events occur well before cell constriction and contribute to cell elongation. In the absence of FtsZ, MurG fails to accumulate near midcell and cell elongation proceeds unperturbed in appearance by insertion of peptidoglycan material along the entire sidewalls. Evidence suggests that bacteria use both a FtsZ-independent and a FtsZ-dependent mode of peptidoglycan synthesis to elongate, the importance of each mode depending on the timing of FtsZ assembly during elongation.

Insights into in vivo activities of lantibiotics from gallidermin and epidermin mode-of-action studies.

The activity of lanthionine-containing peptide antibiotics (lantibiotics) is based on different killing mechanisms which may be combined in one molecule. The prototype lantibiotic nisin inhibits peptidoglycan synthesis and forms pores through specific interaction with the cell wall precursor lipid II. Gallidermin and epidermin possess the same putative lipid II binding motif as nisin; however, both peptides are considerably shorter (22 amino acids, compared to 34 in nisin). We demonstrate that in model membranes, lipid II-mediated pore formation by gallidermin depends on membrane thickness. With intact cells, pore formation was less pronounced than for nisin and occurred only in some strains. In Lactococcus lactis subsp. cremoris HP, gallidermin was not able to release K+, and a mutant peptide, [A12L]gallidermin, in which the ability to form pores was disrupted, was as potent as wild-type gallidermin, indicating that pore formation does not contribute to killing. In contrast, nisin rapidly formed pores in the L. lactis strain; however, it was approximately 10-fold less effective in killing. The superior activity of gallidermin in a cell wall biosynthesis assay may help to explain this high potency. Generally, it appears that the multiple activities of lantibiotics combine differently for individual target strains.

Scintillation proximity assay for inhibitors of Escherichia coli MurG and, optionally, MraY.

MurG and MraY, essential enzymes involved in the synthesis of bacterial peptidoglycan, are difficult to assay because the substrates are lipidic and hard to prepare in large quantities. Based on the use of Escherichia coli membranes lacking PBP1b, we report a high-throughput method to measure the activity of MurG and, optionally, MraY as well. In these membranes, incubation with the two peptidoglycan sugar precursors results in accumulation of lipid II rather than the peptidoglycan produced by wild-type membranes. MurG was assayed by addition of UDP-[3H]N-acetylglucosamine to membranes in which lipid I was preformed by incubation with UDP-N-acetyl-muramylpentapeptide, and the product was captured by wheat germ agglutinin scintillation proximity assay beads. In a modification of the assay, the activity of MraY was coupled to that of MurG by addition of both sugar precursors together in a single step. This allows simultaneous detection of inhibitors of either enzyme. Both assays could be performed using wild-type membranes by addition of the transglycosylase inhibitor moenomycin. Nisin and vancomycin inhibited the MurG reaction; the MraY-MurG assay was inhibited by tunicamycin as well. Inhibitors of other enzymes of peptidoglycan synthesis--penicillin G, moenomycin, and bacitracin--had no effect. Surprisingly, however, the beta-lactam cephalosporin C inhibited both the MurG and MraY-MurG assays, indicating a secondary mechanism by which this drug inhibits bacterial growth. In addition, it inhibited NADH dehydrogenase in membranes, a hitherto-unreported activity. These assays can be used to screen for novel antibacterial agents.

A method for the enzymatic synthesis and HPLC purification of the peptidoglycan precursor UDP-N-acetylmuramic acid.

UDP-N-acetylmuramic acid (UDP-MurNAc) is a precursor for peptidoglycan biosynthesis in bacteria. A major difficulty in the study of this pathway is that UDP-MurNAc is not commercially available. We have developed an enzymatic synthesis scheme for UDP-MurNAc using two easily purified Escherichia coli polyhistidine tagged peptidoglycan biosynthesis enzymes, MurZ and MurB, followed by a single-step purification of UDP-MurNAc by high-performance liquid chromatography. The identity of the UDP-MurNAc synthesized by our method was confirmed by electrospray ionization mass spectrometry. Furthermore, we show that the UDP-MurNAc can support a UDP-MurNAc-L-alanine ligase reaction.

Substrate analogues to study cell-wall biosynthesis and its inhibition.

It has been known for more than 30 years that Lipid II is an intermediate in peptidoglycan synthesis. Recently, it has become apparent that it is also an important target of numerous antibiotics, including the glycopeptides, the lantibiotics and ramoplanin. It is also utilized by sortases in the construction of Gram-positive cell walls. Recent progress has been made in the synthesis of peptidoglycan intermediates that can be used to study enzymes which make peptidoglycan. These intermediates also enable studies to probe the mechanism of action of a variety of substrate-binding antibiotics.