UDP-N-acetylmuramic acid (UDP-MurNAc) is a potent inhibitor of MurA (enolpyruvyl-UDP-GlcNAc synthase)

Purified recombinant MurA (enolpyruvyl-UDP-GlcNAc synthase) overexpressed in Escherichia coli had significant amounts of UDP-MurNAc (UDP-N-acetylmuramic acid) bound after purification. UDP-MurNAc is the product of MurB, the next enzyme in peptidoglycan biosynthesis. About 25% of MurA was complexed with UDP-MurNAc after five steps during purification that should have removed it. UDP-MurNAc isolated from MurA was identified by mass spectrometry, NMR analysis, and comparison with authentic UDP-MurNAc. Subsequent investigation showed that UDP-MurNAc bound to MurA tightly, with K(d,UDP)(-)(MurNAc) = 0.94 +/- 0.04 microM, as determined by fluorescence titrations using ANS (8-anilino-1-naphthalenesulfonate) as an exogenous fluorophore. UDP-MurNAc binding was competitive with ANS and phosphate, the second product of MurA, and it inhibited MurA. The inhibition patterns were somewhat ambiguous, likely being competitive with the substrate PEP (phosphoenolpyruvate) and either competitive or noncompetitive with respect to the substrate UDP-GlcNAc (UDP-N-acetylglucosamine). These results indicate a possible role for UDP-MurNAc in regulating the biosynthesis of nucleotide precursors of peptidoglycan through feedback inhibition. Previous studies indicated that UDP-MurNAc binding to MurA was not tight enough to be physiologically relevant; however, this was likely an artifact of the assay conditions.     

Study of the formation of N-glycolylmuramic acid from Nocardia asteroides (author's transl)]

Nocardia asteroides was grown in Sauton medium containing sodium [carboxy-14C]acetate. The biosynthesis of the peptidoglycan was inhibited by adding penicillin or phosphonomycin to the growth medium. These antibiotics give an accumulation of radioactive nucleotidic precursors of the peptidoglycan. In the presence of penicillin, there was an accumulation of uridine diphosphate-N-glycolylmuramyl peptide (UDP-MurNGlyc peptide) and of a mixture of uridine diphosphate-N-acetyl and N-glycolylmuramic acid (UDP-MurNAc) and UDP-MurNGlyc). In the presence of phosphonomycin, the biosynthesis of muramic acid was blocked and there was an accumulation of uridine diphosphate-N-acetylglucosamine (UDP-GlcNAc) and uridine diphosphate-N-glycolyglucosamine (UDP-GlcNGlyc). Thus the formation of a N-glycolyl group can be performed upon the neucleotidic derivatives of glucosamine and muramic acid. However in the peptidoglycan synthesized in vivo in the absence of antibiotic, only muramic acid was glycolyated. So, glycolylation seems to take place essentially on UDP-MurNAc. When the binding of peptide chain to muramic acid is achieved, all the muramic acid is glycolylated, then the polymerisation of glycan and peptidoglycan units by the mean of particulate enzymes is carried out on the N-glycolylated derivative of muramic acid. A cell-free preparation from Nocardia asteroides was obtained which can hydroxylate the acetyl group of UDP-MurNAc. The activity was localised in the soluble fraction. This system acts as a hydroxylase and requires the presence of NADPH.     

The MurC ligase essential for peptidoglycan biosynthesis is regulated by the serine/threonine protein kinase PknA in Corynebacterium glutamicum

The Mur ligases play an essential role in the biosynthesis of bacterial cell-wall peptidoglycan and thus represent attractive targets for the design of novel antibacterials. These enzymes catalyze the stepwise formation of the peptide moiety of the peptidoglycan disaccharide peptide monomer unit. MurC is responsible of the addition of the first residue (L-alanine) onto the nucleotide precursor UDP-MurNAc. Phosphorylation of proteins by Ser/Thr protein kinases has recently emerged as a major physiological mechanism of regulation in prokaryotes. Herein, the hypothesis of a phosphorylation-dependent mechanism of regulation of the MurC activity was investigated in Corynebacterium glutamicum. We showed that MurC was phosphorylated in vitro by the PknA protein kinase. An analysis of the phosphoamino acid content indicated that phosphorylation exclusively occurred on threonine residues. Six phosphoacceptor residues were identified by mass spectrometry analysis, and we confirmed that mutagenesis to alanine residues totally abolished PknA-dependent phosphorylation of MurC. In vitro and in vivo ligase activity assays showed that the catalytic activity of MurC was impaired following mutation of these threonine residues. Further in vitro assays revealed that the activity of the MurC-phosphorylated isoform was severely decreased compared with the non-phosphorylated protein. To our knowledge, this is the first demonstration of a MurC ligase phosphorylation in vitro. The finding that phosphorylation is correlated with a decrease in MurC enzymatic activity could have significant consequences in the regulation of peptidoglycan biosynthesis.     

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.     

Biochemical characterization and physiological properties of Escherichia coli UDP-N-acetylmuramate:L-alanyl-gamma-D-glutamyl-meso-diaminopimelate ligase

The UDP-N-acetylmuramate:L-alanyl-gamma-D-glutamyl-meso-diaminopimelate ligase (murein peptide ligase [Mpl]) is known to be a recycling enzyme allowing reincorporation into peptidoglycan (murein) of the tripeptide L-alanyl-gamma-D-glutamyl-meso-diaminopimelate released during the maturation and constant remodeling of this bacterial cell wall polymer that occur during cell growth and division. Mpl adds this peptide to UDP-N-acetylmuramic acid, thereby providing an economical additional source of UDP-MurNAc-tripeptide available for de novo peptidoglycan biosynthesis. The Mpl enzyme from Escherichia coli was purified to homogeneity as a His-tagged form, and its kinetic properties and parameters were determined. Mpl was found to accept tri-, tetra-, and pentapeptides as substrates in vitro with similar efficiencies, but it accepted the dipeptide L-Ala-D-Glu and L-Ala very poorly. Replacement of meso-diaminopimelic acid by L-Lys resulted in a significant decrease in the catalytic efficacy. The effects of disruption of the E. coli mpl gene and/or the ldcA gene encoding the LD-carboxypeptidase on peptidoglycan metabolism were investigated. The differences in the pools of UDP-MurNAc peptides and of free peptides between the wild-type and mutant strains demonstrated that the recycling activity of Mpl is not restricted to the tripeptide and that tetra- and pentapeptides are also directly reused by this process in vivo. The relatively broad substrate specificity of the Mpl ligase indicates that it is an interesting potential target for antibacterial compounds.     

Mode of action of glycine on the biosynthesis of peptidoglycan

The mechanism of glycine action in growth inhibition was studied on eight different species of bacteria of various genera representing the four most common peptidoglycan types. To inhibit the growth of the different organisms to 80%, glycine concentrations from 0.05 to 1.33 M had to be applied. The inhibited cells showed morphological aberrations. It has been demonstrated that glycine is incorporated into the nucleotide-activated peptidoglycan precursors. The amount of incorporated glycine was equivalent to the decrease in the amount of alanine. With one exception glycine is also incorporated into the peptidoglycan. Studies on the primary structure of both the peptidoglycan precursors and the corresponding peptidoglycan have revealed that glycine can replace l-alanine in position 1 and d-alanine residues in positions 4 and 5 of the peptide subunit. Replacement of l-alanine in position 1 of the peptide subunit together with an accumulation of uridine diphosphate-muramic acid (UDP-MurNAc), indicating an inhibition of the UDP-MurNAc:l-Ala ligase, has been found in three bacteria (Staphylococcus aureus, Lactobacillus cellobiosus and L. plantarum). However, discrimination against precursors with glycine in position 1 in peptidoglycan synthesis has been observed only in S. aureus. Replacement of d-alanine residues was most common. It occurred in the peptidoglycan with one exception in all strains studied. In Corynebacterium sp., C. callunae, L. plantarum, and L. cellobiosus most of the d-alanine replacing glycine occurs C-terminal in position 4, and in C. insidiosum and S. aureus glycine is found C-terminal in position 5. It is suggested that the modified peptidoglycan precursors are accumulated by being poor substrates for some of the enzymes involved in peptidoglycan synthesis. Two mechanisms leading to a more loosely cross-linked peptidoglycan and to morphological changes of the cells are considered. First, the accumulation of glycine-containing precursors may lead to a disrupture of the normal balance between peptidoglycan synthesis and controlled enzymatic hydrolysis during growth. Second, the modified glycine-containing precursors may be incorporated. Since these are poor substrates in the transpeptidation reaction, a high percentage of muropeptides remains uncross-linked. The second mechanism may be the more significant in most cases.     

Replacement of diaminopimelic acid by cystathionine or lanthionine in the peptidoglycan of Escherichia coli.

In Escherichia coli, auxotrophy for diaminopimelic acid (A2pm) can be suppressed by growth with exogenous cystathionine or lanthionine. The incorporation of cystathionine into peptidoglycan metabolism was examined with a dapA metC mutant, whereas for lanthionine, a dapA metA mutant strain was used. Analysis of peptidoglycan precursors and sacculi isolated from cells grown with epimeric cystathionine or lanthionine showed that meso-A2pm was totally replaced in the same position by either sulfur-containing amino acid. Moreover, mainly L-allo-cystathionine (95%) or meso-lanthionine (93%) was incorporated into the precursors and sacculi. For this purpose, a new, efficient high-pressure liquid chromatography (HPLC) technique for analysis of the cystathionine isomers was developed. The formation of the UDP-MurNAc tripeptide appeared to be a critical step, since the MurE synthetase accepted meso-lanthionine or D-allo- or L-allo-cystathionine in vitro as good substrates, although with higher Km values. Presumably, the 10-fold-higher UDP-MurNAc-L-Ala-D-Glu pool of cells grown with cystathionine or lanthionine ensured a normal rate of synthesis. The kinetic parameters of the MurF synthetase catalyzing the addition of D-alanyl-D-alanine were very similar for the meso-A2pm-,L-allo-cystathionine-, and meso-lanthionine-containing UDP-MurNAc tripeptides. HPLC analysis of the soluble fragments resulting from 95% digestion by Chalaropsis N-acetylmuramidase of the peptidoglycan material in isolated sacculi revealed that the proportion of the main dimer was far lower in cystathionine and lanthionine sacculi.     

Functional and biochemical analysis of Chlamydia trachomatis MurC, an enzyme displaying UDP-N-acetylmuramate:amino acid ligase activity

Chlamydiae are unusual obligate intracellular bacteria that cause serious infections in humans. Chlamydiae contain genes that appear to encode products with peptidoglycan biosynthetic activity. The organisms are also susceptible to antibiotics that inhibit peptidoglycan synthesis. However, chlamydiae do not synthesize detectable peptidoglycan. The paradox created by these observations is known as the chlamydial anomaly. The MurC enzyme of chlamydiae, which is synthesized as a bifunctional MurC-Ddl product, is expected to possess UDP-N-acetylmuramate (UDP-MurNAc):L-alanine ligase activity. In this paper we demonstrate that the MurC domain of the Chlamydia trachomatis bifunctional protein is functionally expressed in Escherichia coli, since it complements a conditional lethal E. coli mutant possessing a temperature-sensitive lesion in MurC. The recombinant MurC domain was overexpressed in and purified from E. coli. It displayed in vitro ATP-dependent UDP-MurNAc:L-alanine ligase activity, with a pH optimum of 8.0 and dependence upon magnesium ions (optimum concentration, 20 mM). Its substrate specificity was studied with three amino acids (L-alanine, L-serine, and glycine); comparable Vmax/Km values were obtained. Our results are consistent with the synthesis of a muramic acid-containing polymer in chlamydiae with UDP-MurNAc-pentapeptide as a precursor molecule. However, due to the lack of specificity of MurC activity in vitro, it is not obvious which amino acid is present in the first position of the pentapeptide.     

A microplate assay for the coupled transglycosylase–transpeptidase activity of the penicillin binding proteins; a vancomycin-neutralizing tripeptide combination prevents penicillin inhibition of peptidoglycan synthesis

A microplate, scintillation proximity assay to measure the coupled transglycosylase–transpeptidase activity of the penicillin binding proteins in Escherichia coli membranes was developed. Membranes were incubated with the two peptidoglycan sugar precursors UDP-N-acetyl muramylpentapeptide (UDP-MurNAc(pp)) and UDP-[³H]N-acetylglucosamine in the presence of 40 μM vancomycin to allow in situ accumulation of lipid II. In a second step, vancomycin inhibition was relieved by addition of a tripeptide (Lys-d-ala-d-ala) or UDP-MurNAc(pp), resulting in conversion of lipid II to cross-linked peptidoglycan. Inhibitors of the transglycosylase or transpeptidase were added at step 2. Moenomycin, a transglycosylase inhibitor, had an IC₅₀ of 8 nM. Vancomycin and nisin also inhibited the assay. Surprisingly, the transpeptidase inhibitors penicillin and ampicillin showed no inhibition. In a pathway assay of peptidoglycan synthesis, starting from the UDP linked sugar precursors, inhibition by penicillin was reversed by a ‘neutral’ combination of vancomycin plus tripeptide, suggesting an interaction thus far unreported.     

Structure and function of the first full-length murein peptide ligase (Mpl) cell wall recycling protein

Bacterial cell walls contain peptidoglycan, an essential polymer made by enzymes in the Mur pathway. These proteins are specific to bacteria, which make them targets for drug discovery. MurC, MurD, MurE and MurF catalyze the synthesis of the peptidoglycan precursor UDP-N-acetylmuramoyl-L-alanyl-γ-D-glutamyl-meso-diaminopimelyl-D-alanyl-D-alanine by the sequential addition of amino acids onto UDP-N-acetylmuramic acid (UDP-MurNAc). MurC-F enzymes have been extensively studied by biochemistry and X-ray crystallography. In Gram-negative bacteria, ∼30–60% of the bacterial cell wall is recycled during each generation. Part of this recycling process involves the murein peptide ligase (Mpl), which attaches the breakdown product, the tripeptide L-alanyl-γ-D-glutamyl-meso-diaminopimelate, to UDP-MurNAc. We present the crystal structure at 1.65 Å resolution of a full-length Mpl from the permafrost bacterium Psychrobacter arcticus 273-4 (PaMpl). Although the Mpl structure has similarities to Mur enzymes, it has unique sequence and structure features that are likely related to its role in cell wall recycling, a function that differentiates it from the MurC-F enzymes. We have analyzed the sequence-structure relationships that are unique to Mpl proteins and compared them to MurC-F ligases. We have also characterized the biochemical properties of this enzyme (optimal temperature, pH and magnesium binding profiles and kinetic parameters). Although the structure does not contain any bound substrates, we have identified ∼30 residues that are likely to be important for recognition of the tripeptide and UDP-MurNAc substrates, as well as features that are unique to Psychrobacter Mpl proteins. These results provide the basis for future mutational studies for more extensive function characterization of the Mpl sequence-structure relationships.     

Studies on bacterial cell wall inhibitors: II. Inhibition of peptidoglycan synthesis in vivo and in vitro by amphomycin

Amphomycin has been reported by the present authors to be a selective inhibitor of cell wall peptidoglycan synthesis in Bacillus cereus T (ōmura, S., Tanaka, H., Shinohara, M., ōiwa, R. and Hata, T. (1975) Chemotherapy 5, 365–369). Investigations were carried out to clarify the target of amphomycin.Amphomycin (10 μg/ml) lysed growing cells of B. cereus T, and inhibited peptidoglycan synthesis, accompanied by accumulation of uridine diphosphate-N-acetylmuramyl (UDP-MurNAc) peptides. The nucleotide precursors that accumulated in cells of Staphylococcus aureus FDA 209P in the presence of amphomycin were identified as UDP-MurNAc-L-Ala-D-Glu-L-Lys-D-Ala-D-Ala, UDP-MurNAc-L-Ala and UDP-MurNAc. In the experiments using a particulate enzyme system of Bacillus megaterium KM, amphomycin inhibited the polymerization of UDP-MurNAc-L-Ala-D-Glu-meso-diaminopimelic acid-D-Ala-D-Ala (UDP-MurNAc-pentapeptide) and UDP-N-acetylglucosamine, and also inhibited the formation of lipid intermediates, but did not inhibit the cross-linking, the last step of peptidoglycan synthesis. Unlike bacitracin, amphomycin did not lyse protoplasts of B. megaterium KM.We conclude that the site of action of amphomycin is the formation of MurNAc-(pentapeptide)-P-P-lipid from MurNAc-pentapeptide and undecaprenol (lipid) phosphate.     

Crystal structure of UDP-N-acetylglucosamine-enolpyruvate reductase (MurB) from Mycobacterium tuberculosis

The biosynthesis of UDP-N-acetylmuramic acid (UDP-MurNAc) by reduction of UDP-N-acetylglucosamine-enolpyruvate (UDP-GlcNAc-EP) in an NADPH and FAD-dependent reaction in bacteria is one of the key steps in peptidoglycan biosynthesis catalyzed by UDP-N-acetylglucosamine-enolpyruvate reductase (MurB). Here, we present the crystal structure of Mycobacterium tuberculosis MurB (MtbMurB) with FAD as the prosthetic group at 2.0 Å resolution. There are six molecules in asymmetric unit in the form of dimers. Each protomer can be subdivided into three domains and the prosthetic group, FAD is bound in the active site between domain I and domain II. Comparison of MtbMurB structure with the structures of the Escherichia coli MurB (in complex with UDP-GlcNAc-EP) and Pseudomonas aeruginosa MurB (in complex with NADPH) showed all three structures share similar domain architecture and residues in the active site. The nicotinamide and the enol pyruvyl moieties are well aligned upon superimposition, both positioned in suitable position for hydride transfer to and from FAD. The comparison studies and MD simulations demonstrate that the two lobes of domain-III become more flexible. The substrates (NADPH and UDP-GlcNAc-EP) binding responsible for open conformation of MurB, suggesting that NADPH and UDP-GlcNAc-EP interactions are conformationally stable. Our findings provide a detail mechanism about the closed to open state by binding of NADPH and UDP-GlcNAc-EP induces the conformational changes of MurB structure that may trigger the MurB catalytic reaction.     

Kinetic and mechanistic characterization of recombinant Lactobacillus viridescens FemX (UDP-N-acetylmuramoyl pentapeptide-lysine N6-alanyltransferase)

The FemABX family encodes enzymes that incorporate l-amino acids into the interchain peptide bridge of Gram-positive cell wall peptidoglycan and are novel nonribosomal peptidyl transferases that use aminoacyl-tRNA as the amino acid donor. We previously reported the identification of the femX gene from Lactobacillus viridescens and recombinant expression of active FemX (LvFemX) that catalyzes the transfer of l-Ala from Ala-tRNAAla to the epsilon-amino group of l-lysine of UDP-MurNAc pentapeptide (Hegde, S. S., and Shrader, T. E. (2001) J. Biol. Chem. 276, 6998-7003). Recombinant LvFemX exhibits Km values of 42 and 15 microm for UDP-MurNAc pentapeptide and Escherichia coli Ala-tRNAAla, respectively, and exhibited a kcat value of 660 min-1. Initial velocity and inhibition kinetic studies support an ordered sequential mechanism for the enzyme, and we propose that catalysis proceeds via a ternary complex. The pH dependence of the activity was bell-shaped, depending on the ionization state of two groups exhibiting apparent pKa values of 5.5 and 9.3. Chemical modification of the enzyme and the kinetics of inactivation, and protection by substrate, indicated the involvement of carboxyl groups in the catalytic function of the enzyme. Site-directed mutagenesis identified Asp109 as a candidate for the catalytic base and Glu320 plays an additional important role in the catalytic function of the enzyme.     

Biosynthesis of peptidoglycan in Pseudomonas aeruginosa. 1. The incorporation of peptidoglycan into the cell wall.

Ether-treated cells of Pseudomonas aeruginosa catalyze the formation of crosslinked peptidoglycan from the two nucleotide precursors uridinediphospho-N-acetylglucosamine and uridinediphospho-N-acetylmuramyl-L-alanyl-D-gamma-glutamyl-meso-diaminopimelyl-D-alanyl-D-alanine. The main enzymatic reactions of biosynthesis were similar to those found in Escherichia coli. Part of the reaction products were soluble in 4% sodium dodecylsulfate whereas the other part was covalently bound to the preexisting cell wall peptidoglycan sacculus. The incorporation into cell wall is carried out by a transpeptidation reaction in which the nascent peptidoglycan functions mainly as the donor and the preexisting one as acceptor. The detergent-soluble peptidoglycan is composed of partially crosslinked peptidoglycan strands as well as low-molecular-weight peptidoglycan fragments. Pulse-chase biosynthesis experiments show that the detergent-soluble peptidoglycan is an intermediate that eventually becomes covalently bound to the wall. The DD-carboxypeptidase activity of P. aeruginosa is membrane-bound and does not hydrolyse C-terminal D-alanine residues from the L-lysine-containing nucleotide-precursor analogue. An LD-carboxypeptidase was also detected in P. aeruginosa.     

In vivo and in vitro action of new antibiotics interfering with the utilization of N-acetyl-glucosamine-N-acetyl-muramyl-pentapeptide

Recent literature on the antibiotics enduracidin, moenomycin, prasinomycin, and 11.837 RP suggested an interaction with murein synthesis. Incubation of sensitive strains from Bacillus cereus and Staphylococcus aureus in a "wall medium" containing labeled l-alanine showed that all four antibiotics inhibited the incorporation of alanine into murein and gave rise to accumulation of radioactive uridine diphosphate-N-acetyl-muramyl (UDP-MurNAc)-pentapeptide. Peptidoglycan was synthesized when the particulate enzyme of B. stearothermophilus was incubated with the murein precursors UDP-N-acetyl-glucosamine (UDP-GlcNAc) and UDP-MurNAc-pentapeptide. The newly formed polymer was less accessible for lysozyme and more strongly bound to the acceptor than the same product from the Escherichia coli particulate enzyme. After incubation in the presence of penicillin, a greater part of the peptidoglycan was lysozyme sensitive and more loosely bound to the acceptor. The antibiotics enduracidin, moenomycin, prasinomycin, and 11.837 RP inhibited peptidoglycan synthesis by the B. stearothermophilus particulate enzyme. The rate of synthesis of GlcNAc-MurNAc(-pentapeptide)-P-P-phospholipid was independent from the addition of these antibiotics, but its utilization was strongly inhibited. With the present results, it is not possible to distinguish the mechanisms of action of enduracidin, moenomycin, prasinomycin, and 11.837 RP from the mechanisms of action of vancomycin and ristocetin.     

Inhibition of peptidoglycan biosynthesis at a postcytoplasmic reaction in a stable L-phase variant of Streptococcus faecium.

Cultures of a stable L-phase variant of Streptococcus faecium F24 produced and retained peptidoglycan precursors intracellularly over the entire growth cycle in a chemically defined medium. The identity of the most abundant precursor, UDP N-acetylmuramyl-L-alanyl-D-glutamyl-L-lysyl-D-alanyl-D-alanine (UDP-MurNAc-pentapeptide), was confirmed by demonstrating in vitro the presence of enzymes required for the cytoplasmic stage of peptidoglycan biosynthesis. The initial membrane-bound reaction in peptidoglycan biosynthesis involving phospho-MurNAc-pentapeptide translocase and undecaprenyl-phosphate membrane carrier was catalyzed by protoplast membrane preparations but not by L-phase membrane preparations. However, both protoplast and L-phase membranes incorporated radioactivity from dTDP-L-[14C]rhamnose, the presumed precursor to a non-peptidoglycan cell surface component, into high-molecular-weight material. dTDP-L-rhamnose did not accumulate in growing cultures but was synthesized from D-glucose-1-phosphate and dTTP by cell-free extracts of the streptococcus and L-phase variant. Neither rhamnose- nor muramic acid-containing compounds were detected in culture fluids. It is suggested that continued inhibition of cell wall biosynthesis in this stable L-phase variant is the result of a defect expressed at the membrane stage of peptidoglycan biosynthesis specifically involving the translocation step.     

Characterization of the peptidoglycan of vancomycin-susceptible Enterococcus faecium

Vancomycin and other antibacterial glycopeptide analogues target the cell wall and affect the enzymatic processes involved with cell-wall biosynthesis. Understanding the structure and organization of the peptidoglycan is the first step in establishing the mode of action of these glycopeptides. We have used solid-state NMR to determine the relative concentrations of stem-links (64%), bridge-links (61%), and cross-links (49%) in the cell walls of vancomycin-susceptible Enterococcus faecium (ATTC 49624). Furthermore, we have determined that in vivo only 7% of the peptidoglycan stems terminate in d-Ala- d-Ala, the well-known vancomycin-binding site. Presumably, d-Ala- d-Ala is cleaved from uncross-linked stems in mature peptidoglycan by an active carboxypeptidase. We believe that most of the few pentapeptide stems ending in d-Ala- d-Ala occur in the template and nascent peptidoglycan strands that are crucial for cell-wall biosynthesis.     

N-acetylmuramoyl-L-alanine amidase of Escherichia coli K12. Possible physiological functions

Various experiments were carried out in an attempt to determine the possible physiological function of the N-acetylmuramoyl-L-alanine amidase purified from Escherichia coli K12 on the basis of its activity on N-acetylmuramoyl-L-alanyl-D-gamma-glutamyl-meso-diaminopimelic acid [MurNAc-LAla-DGlu(msA2pm)]. A Km value of 0.04 mM was determined with this substrate. Specificity studies revealed that compounds with a MurNAc-LAla linkage are the most probable substrates of this enzyme in vivo. Purified amidase had no effect on purified peptidoglycan and only low levels (1-2.5%) of cleaved MurNAc-LAla linkages were detected in peptidoglycan isolated from normally growing cells. However, the action of the amidase in vivo on peptidoglycan was clearly detectable during autolysis. The amidase activity of cells treated by osmotic shock, ether or toluene, as well as that of mutants with altered outer membrane composition was investigated. Attempts to reveal a transfer reaction catalysed by amidase were unsuccessful. Furthermore, by its location and specificity, amidase was clearly not involved in the formation of UDP-MurNAc. The possibility that it might be functioning in vivo as a hydrolase degrading exogeneous peptidoglycan fragments in the periplasma was substantiated by the fact that MurNAc itself and MurNAc-peptides could sustain growth of E. coli as sole carbon and nitrogen sources. Finally, out of 200 thermosensitive mutants examined for altered amidase activity, only two strains had less than 50% of the normal level of activity, whereas ten strains were found to possess more than 50%. In fact, two of the overproducers encountered presented a 4-5-fold increase in activity.     

Inhibition of lipopolysaccharide O-antigen synthesis by colicin M

Colicin M inhibits peptidoglycan biosynthesis at the level of the bactoprenyl carrier lipid. Since the synthesis of O-antigen also requires bactoprenyl carrier lipid, the effect of colicin M on O-antigen biosynthesis was studied using a colicin-sensitive strain of Salmonella typhimurium. Determination of O-antigen intermediates by two different methods showed that bactoprenyl-dependent O-antigen biosynthesis was inhibited by colicin M. Synthesis of both O-antigen and peptidoglycan was almost immediately inhibited following colicin addition. This was followed some 20 min later by cell lysis. The only known common step between O-antigen and peptidoglycan synthesis is formation of bactoprenyl phosphate by dephosphorylation of bactoprenyl pyrophosphate. Determination of bactoprenyl phosphates showed an accumulation of bactoprenyl pyrophosphate in colicin-treated cultures. It was concluded that dephosphorylation of the bactoprenyl lipid carrier was inhibited by colicin M, and this in turn prevented both O-antigen and peptidoglycan synthesis.