Characterization of an N-acetylmuramic acid/N-acetylglucosamine kinase of Clostridium acetobutylicum

We report here the cloning and characterization of a cytoplasmic kinase of Clostridium acetobutylicum, named MurK (for murein sugar kinase). The enzyme has a unique specificity for both amino sugars of the bacterial cell wall, N-acetylmuramic acid (MurNAc) and N-acetylglucosamine (GlcNAc), which are phosphorylated at the 6-hydroxyl group. Kinetic analyses revealed Km values of 190 and 127 μM for MurNAc and GlcNAc, respectively, and a kcat value (65.0 s(-1)) that was 1.5-fold higher for the latter substrate. Neither the non-N-acetylated forms of the cell wall sugars, i.e., glucosamine and/or muramic acid, nor epimeric hexoses or 1,6-anhydro-MurNAc were substrates for the enzyme. MurK displays low overall amino acid sequence identity (24%) with human GlcNAc kinase and is the first characterized bacterial representative of the BcrAD/BadFG-like ATPase family. We propose a role of MurK in the recovery of muropeptides during cell wall rescue in C. acetobutylicum. The kinase was applied for high-sensitive detection of the amino sugars in cell wall preparations by radioactive phosphorylation.     

Purification and characterization of an extracellular muramidase of Clostridium acetobutylicum ATCC 824 that acts on non-N-acetylated peptidoglycan.

An extracellular enzyme showing lytic activity on non-N-acetylated peptidoglycan has been isolated from Clostridium acetobutylicum ATCC 824. The lytic enzyme was purified to homogeneity by anion-exchange chromatography and gel filtration, with a recovery of 24%. The enzyme was monomeric and had an estimated molecular weight of 41,000 and an isoelectric point of 3.8. It has been characterized as a muramidase whose 23-amino-acid N terminus displayed 39% homology with the N,O-diacetyl muramidase of the fungus Chalaropsis sp. The muramidase hydrolyzed purified cell walls at an optimum pH of 3, with a maximum velocity of 9.1 mumol of reducing sugars released min-1 mg of muramidase-1 and a concentration of cell walls giving a half-maximum rate of 0.01 mg ml-1. Its activity was inhibited by glucosamine, N-acetylglucosamine, Hg2+, Fe3+, and Ag+ but not by choline. The muramidase-peptidoglycan complex rapidly dissociated before total hydrolysis of the chain and randomly reassociated on another peptidoglycan chain. The affinity of the muramidase was affected by the protein content and the acetylation of the cell wall.     

Purification and characterization of an extracellular muramidase of Clostridium acetobutylicum ATCC 824 that acts on non-N-acetylated peptidoglycan.

An extracellular enzyme showing lytic activity on non-N-acetylated peptidoglycan has been isolated from Clostridium acetobutylicum ATCC 824. The lytic enzyme was purified to homogeneity by anion-exchange chromatography and gel filtration, with a recovery of 24%. The enzyme was monomeric and had an estimated molecular weight of 41,000 and an isoelectric point of 3.8. It has been characterized as a muramidase whose 23-amino-acid N terminus displayed 39% homology with the N,O-diacetyl muramidase of the fungus Chalaropsis sp. The muramidase hydrolyzed purified cell walls at an optimum pH of 3, with a maximum velocity of 9.1 mumol of reducing sugars released min-1 mg of muramidase-1 and a concentration of cell walls giving a half-maximum rate of 0.01 mg ml-1. Its activity was inhibited by glucosamine, N-acetylglucosamine, Hg2+, Fe3+, and Ag+ but not by choline. The muramidase-peptidoglycan complex rapidly dissociated before total hydrolysis of the chain and randomly reassociated on another peptidoglycan chain. The affinity of the muramidase was affected by the protein content and the acetylation of the cell wall.     

Bacillus cereus autolytic endoglucosaminidase active on cell wall peptidoglycan with N-unsubstituted glucosamine residues

An autolytic glycosidase from a lysozyme-resistant strain of Bacillus cereus capable of cleaving the glycosidic linkages of N-unsubstituted glucosamine in the cell wall peptidoglycan was studied. This glycosidase activity, together with N-acetylmuramyl-L-alanine amidase activity, was found in an autolytic enzyme preparation obtained from the 20,000 x g precipitate fraction by means of autolysis followed by ammonium sulfate fractionation. The major saccharide fragments resulting from digestion of the untreated, non-N-acetylated, cell wall peptidoglycan of B. cereus with the autolytic enzyme preparation were identified as N-acetylmuramyl-glucosamine and its dimer. The peptidoglycan N-acetylated with acetic anhydride could also be digested with the same enzyme preparation, giving N-acetylmuramyl-N-acetylglucosamine and its dimer as the major saccharide fragments.     

Conformational flexibility of the glycosidase NagZ allows it to bind structurally diverse inhibitors to suppress β‐lactam antibiotic resistance

NagZ is an N‐acetyl‐β‐d ‐glucosaminidase that participates in the peptidoglycan (PG) recycling pathway of Gram‐negative bacteria by removing N‐acetyl‐glucosamine (GlcNAc) from PG fragments that have been excised from the cell wall during growth. The 1,6‐anhydromuramoyl‐peptide products generated by NagZ activate β‐lactam resistance in many Gram‐negative bacteria by inducing the expression of AmpC β‐lactamase. Blocking NagZ activity can thereby suppress β‐lactam antibiotic resistance in these bacteria. The NagZ active site is dynamic and it accommodates distortion of the glycan substrate during catalysis using a mobile catalytic loop that carries a histidine residue which serves as the active site general acid/base catalyst. Here, we show that flexibility of this catalytic loop also accommodates structural differences in small molecule inhibitors of NagZ, which could be exploited to improve inhibitor specificity. X‐ray structures of NagZ bound to the potent yet non‐selective N‐acetyl‐β‐glucosaminidase inhibitor PUGNAc (O‐(2‐acetamido‐2‐deoxy‐d ‐glucopyranosylidene) amino‐N‐phenylcarbamate), and two NagZ‐selective inhibitors – EtBuPUG, a PUGNAc derivative bearing a 2‐N‐ethylbutyryl group, and MM‐156, a 3‐N‐butyryl trihydroxyazepane, revealed that the phenylcarbamate moiety of PUGNAc and EtBuPUG completely displaces the catalytic loop from the NagZ active site to yield a catalytically incompetent form of the enzyme. In contrast, the catalytic loop was found positioned in the catalytically active conformation within the NagZ active site when bound to MM‐156, which lacks the phenylcarbamate extension. Displacement of the catalytic loop by PUGNAc and its N‐acyl derivative EtBuPUG alters the active site conformation of NagZ, which presents an additional strategy to improve the potency and specificity of NagZ inhibitors.     

Benzylpenicillin-induced filament formation of Clostridium perfringens

Growth of Clostridium perfringens with low concentrations of benzylpenicillin inhibited septum formation and division of the organisms. This resulted in continued growth of the organisms as aseptate filaments. The effect was reversed on removal of the antibiotic. The composition of walls isolated from organisms grown with the antibiotic was similar to that of walls from untreated bacteria. In addition, both contained non-N-acetylated glucosamine residues in their peptidoglycan. No differences were detected in the degree of cross-linkage of peptidoglycan. Clostridium perfringens contains six membrane-associated penicillin-binding proteins (PBPs) which have different affinities for [3H]benzylpenicillin. Concentrations of the antibiotic which were sufficient to cause filamentation of apparently all organisms in a culture caused almost complete saturation of PBPs 3, 4, 5 and 6. At these concentrations there was no measurable interaction with PBPs 1 and 2. Thus interaction of the antibiotic with the lower molecular weight PBPs is correlated with the inhibition of septum formation in C. perfringens.     

How Bacteria Consume Their Own Exoskeletons (Turnover and Recycling of Cell Wall Peptidoglycan)

Summary: The phenomenon of peptidoglycan recycling is reviewed. Gram-negative bacteria such as Escherichia coli break down and reuse over 60% of the peptidoglycan of their side wall each generation. Recycling of newly made peptidoglycan during septum synthesis occurs at an even faster rate. Nine enzymes, one permease, and one periplasmic binding protein in E. coli that appear to have as their sole function the recovery of degradation products from peptidoglycan, thereby making them available for the cell to resynthesize more peptidoglycan or to use as an energy source, have been identified. It is shown that all of the amino acids and amino sugars of peptidoglycan are recycled. The discovery and properties of the individual proteins and the pathways involved are presented. In addition, the possible role of various peptidoglycan degradation products in the induction of β-lactamase is discussed.     

Acetyl-coenzyme A:alpha-glucosaminide N-acetyltransferase. Evidence for an active site histidine residue

The lysosomal membrane enzyme acetyl-CoA:alpha-glucosaminide N-acetyltransferase catalyzes the transfer of the acetyl group from acetyl-CoA to terminal alpha-linked glucosamine residues of heparan sulfate. The reaction appears to be a transmembrane process: the enzyme is acetylated on the outside of the lysosome, and the acetyl group is transferred across the membrane to the inside of the lysosome where it is used to acetylate glucosamine. To determine the reactive site residues involved in the acetylation reaction, lysosomal membranes were treated with various amino acid modification reagents and assayed for enzyme activity. Although four thiol modification reagents were examined, only one, p-chloromercuribenzoate inactivated the N-acetyltransferase. Thiol modification by p-chloromercuribenzoate did not appear to occur at the active site since inactivation was still observed in the presence of the substrate acetyl-CoA. N-Acetyltransferase could be inactivated by N-bromosuccinimide, even after pretreatment with reagents specific for tyrosine and tryptophan, suggesting that the modified residue is a histidine. Diethyl pyrocarbonate, another histidine modification reagent, could also inactivate the enzyme; this inactivation could be reversed by incubation with hydroxylamine. N-Bromosuccinimide and diethyl pyrocarbonate modifications appear to be at the active site of the enzyme since co-incubation with acetyl-CoA protects the N-acetyltransferase from inactivation. This protection is lost if glucosamine is also present. Pre-acetylated lysosomal membranes are also able to provide protection from N-bromosuccinimide inactivation, providing further evidence for a histidine moiety at the active site and for the existence of an acetyl-enzyme intermediate.     

Cadaverine is covalently linked to peptidoglycan in Selenomonas ruminantium.

Cadaverine was found to exist as a component of cell wall peptidoglycan of Selenomonas ruminantium, a strictly anaerobic bacterium. [14C]cadaverine added to the growth medium was incorporated into the cells, and about 70% of the total radioactivity incorporated was found in the peptidoglycan fraction. When the [14C]cadaverine-labeled peptidoglycan preparation was acid hydrolyzed, all of the 14C counts were recovered as cadaverine. The [14C]cadaverine-labeled peptidoglycan preparation was digested with lysozyme into three small fragments which were radioactive and were positive in ninhydrin reaction. One major spot, a compound of the fragments, was composed of alanine, glutamic acid, diaminopimelic acid, cadaverine, muramic acid, and glucosamine. One of the two amino groups of cadaverine was covalently linked to the peptidoglycan, and the other was free. The chemical composition of the peptidoglycan preparation of this strain was determined to be as follows: L-alanine-D-alanine-D-glutamic acid-meso-diaminopimelic acid-cadaverine-muramic acid-glucosamine (1.0:1.0:1.0:1.0:1.1:0.9:1.0).     

Enzymatic deacetylation of N-acetylglucosamine residues in cell wall peptidoglycan

An enzyme which catalyzes the hydrolysis of acetamido groups of N-acetylglucosamine residues in cell wall peptidoglycan was found in the supernatant and 20,000 X g pellet fractions of Bacillus cereus. Autolysis of the latter fraction resulted in solubilization and activation of the deacetylase. Among various bacteria, strains of B. cereus which contain high proportions of N-unsubstituted glucosamine residues in their cell wall peptidoglycan components are particularly rich in the deacetylase. The peptidoglycan deacetylase is distinguishable from N-acetylglucosamine-6-phosphate deacetylase [EC 3.5.1.25] on the basis of their cellular distribution and chromatographic behavior. The rate of reaction of the deacetylase with (N-acetylglucosaminyl-N-acetylmuramic acid)3 [abbreviated as (GlcNAc-MurNAc)3] is less than 1/100 of that with peptidoglycan, while the enzyme is inactive towards (GlcNAc-MurNAc)2, GlcNAc-MurNAc, and monomeric N-acetylglucosamine derivatives. The enzyme also deacetylates partially O-hydroxyethylated chitin. The concentrations of peptidoglycan and partially O-hydroxyethylated chitin required for half-maximum activities were found to be 0.29 and 6.9 mg per ml (or 0.17 and 20 mM with respect to N-acetylglucosamine residues), respectively. The occurrence of this enzyme accounts for the formation of cell wall peptidoglycan N-unsubstituted at the glucosamine residues.     

Peptidoglycan of Rhodopseudomonas viridis: partial lack of N-acetyl substitution of glucosamine

A lack of at least 70% of N-acetyl substitution of glucosamine in the glycan strands of the peptidoglycan from the gram-negative bacterium Rhodopseudomonas viridis is reported. A disaccharide, very likely GlcN beta(1 leads to 4) Mur, was observed in hydrolysates of the isolated peptidoglycan. The disaccharide was not observed when peptidoglycan was N-acetylated before hydrolysis. The peptidoglycan of R. viridis was resistant to lysozyme but became sensitive after N-acetylation with acetic anhydride. The disaccharide was found with peptidoglycan from all R. viridis strains investigated, as well as with R. sulfoviridis P1 and R. palustris strains, but not with peptidoglycan from R. gelatinosa, Rhodospirillum tenue, and Pseudomonas diminuta NCTC 8545.     

Heterodisaccharide 4-O-(N-acetyl-β-D-glucosaminyl)-D-glucosamine is an effective chemotactic attractant for Vibrio bacteria that produce chitin oligosaccharide deacetylase

Aims: To investigate the attractant effect of 4‐O‐(N‐acetyl‐β‐d ‐glucosaminyl)‐d ‐glucosamine (GlcNAc‐GlcN) in the chemotaxis of Vibrio bacteria that produce carbohydrate esterase (CE) family 4 chitin oligosaccharide deacetylase (COD), an enzyme that catalyzes the production of GlcNAc‐GlcN from N,N′‐diacetylchitobiose (GlcNAc)₂. Methods and Results: The chemotactic effect of disaccharides from chitin on several strains of Vibrio bacteria was investigated using an agar gel lane‐migration method. The results demonstrated that GlcNAc‐GlcN functions as an effective chemoattractant in the CE family 4 COD‐producing vibrios, Vibrio parahaemolyticus and Vibrio alginolyticus. In contrast, this phenomenon was not observed in Vibrio nereis or Vibrio furnissii, which lack genes encoding this enzyme. From transmission electron microscope observation of V. parahaemolyticus cells following the chemotaxis assay, GlcNAc‐GlcN appears to stimulate polar flagellum rotation. Conclusions: GlcNAc‐GlcN is a specific chemoattractant for the CE family 4 COD‐producing vibrios, V. parahaemolyticus and V. alginolyticus. Significance and Impact of the Study: It was clarified for the first time that GlcNAc‐GlcN functions as a signalling molecule in the chemotaxis of Vibrio bacteria that have an ability to produce CE family 4 COD, which generate GlcNAc‐GlcN from (GlcNAc)₂.     

Cellular location of N-acetyltransfer activities toward glucosamine and glucosamine-6-phosphate in cultured human skin fibroblasts

The intracellular location in normal human cultured skin fibroblasts of the N-acetyltransferase activities that transfer the acetyl group from acetyl-CoA to the 2-amino group of glucosamine and glucosamine-6-phosphate have been investigated. Organelles have been separated using a combination of differential centrifugation and free flow electrophoresis. The intracellular distribution of the enzyme involved in the N-acetyltransfer to glucosamine and an alpha-glucosaminide disaccharide indicated that this enzyme activity concentrates mainly with lysosomal organelles whereas the activity associated with N-acetyltransferase to glucosamine-6-phosphate is non-lysosomal. It is proposed that acetyl-CoA: alpha-glucosaminide N-acetyltransferase may be used as a convenient enzyme marker of lysosomal organelle membranes.     

A unique cell wall synthetic response evoked by glucosamine determines pathogenicity-associated fungal cellular differentiation

The yeast-to-hypha transition is tightly associated with pathogenicity in many human pathogenic fungi, such as the model fungal pathogen Cryptococcus neoformans, which is responsible for approximately 180,000 deaths annually. In this pathogen, the yeast-to-hypha transition can be initiated by distinct stimuli: mating stimulation or glucosamine (GlcN), the monomer of cell wall chitosan. However, it remains poorly understood how the signal specificity for Cryptococcus morphological transition by disparate stimuli is ensured. Here, by integrating temporal expression signature analysis and phenome-based clustering evaluation, we demonstrate that GlcN specifically triggers a unique cellular response, which acts as a critical determinant underlying the activation of GlcN-induced filamentation (GIF). This cellular response is defined by an unusually hyperactive cell wall synthesis that is highly ATP-consuming. A novel cell surface protein Gis1 was identified as the indicator molecule for the GlcN-induced cell wall response. The Mpk1-directed cell wall pathway critically bridges global cell wall gene induction and intracellular ATP supply, ensuring the Gis1-dependent cell wall response and the stimulus specificity of GIF. We further reveal that the ability of Mpk1 to coordinate the cell wall response and GIF activation is conserved in different Cryptococcus pathogens. Phosphoproteomics-based profiling together with genetic and phenotypic analysis revealed that the Mpk1 kinase mediates the regulatory specificity of GIF through a coordinated downstream regulatory network centered on Skn7 and Crz1. Overall, our findings discover an unprecedented and conserved cell wall biosynthesis-dependent fungal differentiation commitment mechanism, which enables the signal specificity of pathogenicity-related dimorphism induced by GlcN in Cryptococcus pathogens.     

Structural studies on the lipid A component of enterobacterial lipopolysaccharides by laser desorption mass spectrometry. Location of acyl groups at the lipid A backbone

In the present paper laser desorption mass spectrometry (LDMS) was applied to dephosphorylated free lipid A preparations obtained from lipopolysaccharides of Re mutants of Salmonella minnesota, Escherichia coli and Proteus mirabilis. The purpose of this study was to elucidate the location of (R)-3-hydroxytetradecanoic acid and 3-O-acylated (R)-3-hydroxytetradecanoic acid residues which are bound to amino and hydroxyl groups of the glucosamine disaccharide backbone of lipid A. Based on the previous finding from biochemical analyses that the amino group of the nonreducing glucosamine residue (GlcN II) of the backbone carries, in S. minnesota and E. coli, 3-dodecanoyloxytetradecanoic acid and, in P. mirabilis, 3-tetradecanoyloxytetradecanoic acid, a self-consistent interpretation of the LDMS was possible. It was found that: (a) in all three lipids A GlcN II is, besides the amide-linked 3-acyloxyacyl residue, substituted by ester-linked 3-tetradecanoyloxytetradecanoic acid; (b) the reducing glucosamine (GlcN I) is substituted by ester-linked 3-hydroxytetradecanoic acid; (c) the amino group of GlcN I carries a 3-hydroxytetradecanoic acid which is non-acylated in E. coli and which is partially acylated by hexadecanoic acid in S. minnesota and P. mirabilis. In lipids A which were obtained from the P. mirabilis Re mutant grown at low temperature (12 degrees C) LDMS analysis revealed that specifically the one fatty acid bound to the 3-hydroxyl group of amide-linked 3-hydroxytetra-decanoic acid at GlcN II is positionally replaced by delta 9-hexadecenoic acid (palmitoleic acid). It appears, therefore, that enterobacterial lipids A resemble each other in that the 3-hydroxyl groups of the two 3-hydroxytetradecanoic acid residues linked to GlcN II are fully acylated, while those of the two 3-hydroxytetradecanoic acid groups attached to GlcN I are free or only partially substituted.     

Evidence for N----O acetyl migration as the mechanism for O acetylation of peptidoglycan in Proteus mirabilis.

O-acetylated peptidoglycan was purified from Proteus mirabilis grown in the presence of specifically radiolabelled glucosamine derivatives, and the migration of the radiolabel was monitored. Mild-base hydrolysis of the isolated peptidoglycan (to release ester-linked acetate) from cells grown in the presence of 40 microM [acetyl-3H]N-acetyl-D-glucosamine resulted in the release of [3H]acetate, as detected by high-pressure liquid chromatography. The inclusion of either acetate, pyruvate, or acetyl phosphate, each at 1 mM final concentration, did not result in a diminution of mild-base-released [3H]acetate levels. No such release of [3H]acetate was observed with peptidoglycan isolated from either Escherichia coli incubated with the same radiolabel or P. mirabilis grown with [1,6-3H]N-acetyl-D-glucosamine or D-[1-14C]glucosamine. These observations support a hypothesis that O acetylation occurs by N----O acetyl transfer within the sacculus. A decrease in [3H]acetate release by mild-base hydrolysis was observed with the peptidoglycan of P. mirabilis cultures incubated in the presence of antagonists of peptidoglycan biosynthesis, penicillin G and D-cycloserine. The absence of free-amino sugars in the peptidoglycan of P. mirabilis but the detection of glucosamine in spent culture broths implies that N----O transacetylation is intimately associated with peptidoglycan turnover.     

Halococcus morrhuae: a sulfated heteropolysaccharide as the structural component of the bacterial cell wall.

The qualitative and quantitative composition of purifed cell wall of Halococcus morrhuae CCM 859 was determined. Glucose, mannose, galactose; glucuronic and galacturonic acids; glucosamine, galactosamine, gulosaminuronic acid; acetate, glycine and sulfate are found as major constituents. The amino sugars are N-acetylated. It was not possible to fractionate the cell wall in chemically different polymers. Evidence is presented that the major cell wall polymer of this strain is a complex heterolgycan which seems, like the peptidoglycan of most bacteria, to be responsible for the rigidity and stability of the cell wall. In addition it could be proved that this heteroglycan is sulfated and therefore differs considerably from previously described bacterial cell wall polymers.     

Backbone and nucleobase contacts to glucosamine-6-phosphate in the glmS ribozyme

The glmS ribozyme resides in the 5' untranslated region of glmS mRNA and functions as a catalytic riboswitch that regulates amino sugar metabolism in certain Gram-positive bacteria. The ribozyme catalyzes self-cleavage of the mRNA and ultimately inhibits gene expression in response to binding of glucosamine-6-phosphate (GlcN6P), the metabolic product of the GlmS protein. We have used nucleotide analog interference mapping (NAIM) and suppression (NAIS) to investigate backbone and nucleobase functional groups essential for ligand-dependent ribozyme function. NAIM using GlcN6P as ligand identified requisite structural features and potential sites of ligand and/or metal ion interaction, whereas NAIS using glucosamine as ligand analog revealed those sites that orchestrate recognition of ligand phosphate. These studies demonstrate that the ligand-binding site lies in close proximity to the cleavage site in an emerging model of ribozyme structure that supports a role for ligand within the catalytic core.     

Peptidoglycan biosynthesis by preparations from Bacillus licheniformis: cross-linking of newly synthesized chains to preformed cell wall (Short Communication)

A wall-plus-membrane preparation from a Bacillus licheniformis mutant incorporated radioactivity from a peptidoglycan precursor in which the free amino group of diaminopimelic acid was blocked by (14)C-labelled acetyl group. This incorporation was penicillin-sensitive. The enzymically degraded product contained cross-linked dimers, showing that newly synthesized peptidoglycan chains had been cross-linked to the pre-existing cell wall.     

Subunit Cell Wall of Sulfolobus acidocaldarius

The cell wall of Sulfolobus acidocaldarius has been isolated. Cells were mechanically disrupted with a French press, and the cytoplasmic membrane was removed by extracting cell-envelope fragments with Triton X-100. The Triton-insoluble cell wall material retained the characteristic subunit structure when examined in the electron microscope. Isolated cell wall fragments formed in open sheets that were easily separated from cytoplasmic contamination. Chemical studies showed that the Triton-insoluble cell wall fragments consisted of lipoprotein with small amounts of carbohydrate and hexosamine. The amino acid composition indicated a highly charged hydrophobic cell surface. The presence of diaminopimelic acid with only traces of muramic acid indicates that the cell envelope does not have a rigid peptidoglycan layer. The results of chemical analyses and electron microscopy suggest a wall-membrane interaction stabilizing the cell envelope. The chemical and physical properties of this type of cell envelope would appear to form the basis for a new major division of bacteria with the definitive characteristics of a morphologically distinct subunit cell wall devoid of peptidoglycan.