Phosphoglucosamine Mutase Research Articles

Effect of Phosphoglucosamine Mutase on Biofilm Formation and Antimicrobial Susceptibilities in M. smegmatis glmM Gene Knockdown Strain

UDP-N-acetylglucosamine (UDP-GlcNAc) is a direct glycosyl donor of linker unit (L-Rhamnose-D-GlcNAc) and an essential precursor of peptidoglycan in mycobacteria. Phosphoglucosamine mutase (GlmM) is involved in the formation of glucosamine-1-phosphate from glucosamine-6-phosphate, the second step in UDP-GlcNAc biosynthetic pathway. We have demonstrated that GlmM protein is essential for the growth of M. smegmatis. To facilitate the analysis of the GlmM protein function in mycobacteria, a tetracycline inducible M. smegmatis glmM gene knockdown strain was constructed by using an antisense RNA technology. After induction with 20 ng/ml tetracycline, the expression of GlmM protein in glmM gene knockdown strain was significantly decreased, resulting in a decline of cell growth. The morphological changes of glmM gene knockdown strain induced with 20 ng/ml tetracycline have been observed by scanning electron microscope and transmission electron microscope. Furthermore, insufficient GlmM protein reduced the biofilm formation and increased the sensitivity to isoniazid and ethambutol in M. smegmatis, indicating that GlmM protein had effect on the biofilm formation and the senstivity to some anti-tuberculosis drugs targeting the cell wall. These results provide a new insight on GlmM functions in mycobacteria, suggesting that GlmM could be a potential target for development of new anti-tuberculosis drug.

Identification of M. tuberculosis Rv3441c and M. smegmatis MSMEG_1556 and Essentiality of M. smegmatis MSMEG_1556

The normal growth of mycobacteria attributes to the integrity of cell wall core which consists of peptidoglycan (PG), arabinogalactan (AG) and mycolic acids. N-acetyl glucosamine (GlcNAc) is an essential component in both PG and AG of mycobacterial cell wall. The biosynthetic pathway for UDP-N-acetylglucosamine (UDP-GlcNAc), as a sugar donor of GlcNAc, is different in prokaryotes and eukaryotes. The conversion of glucosamine-6-phosphate to glucosamine-1-phosphate, which is catalyzed by phosphoglucosamine mutase (GlmM), is unique to prokaryotes. Bioinformatic analysis showed that Msm MSMEG_1556 and Mtb Rv3441c are homologous to Ec GlmM. In this study, soluble Msm MSMEG_1556 protein and Mtb Rv3441c protein were expressed in E. coli BL21(DE3) and their phosphoglucosamine mutase activity were detected. In order to further investigate the essentiality of MSMEG_1556 for the growth of M. smegmatis, we generated a conditional MSMEG_1556 knockout mutant, which harbored thermo-sensitive rescue plasmid carrying Mtb Rv3441c. As the rescue plasmid was unable to complement MSMEG_1556 deficiency at 42°C, MSMEG_1556 knockout mutant did not grow. The dramatic morphological changes of MSMEG_1556 knockout mutant after temperature shift from 30°C to 42°C have been observed by scanning electron microscope. These results demonstrated that MSMEG_1556 is essential for growth of M. smegmatis. This study provided evidence that GlmM enzyme could be as a potential target for developing anti-tuberculosis drugs.

Metabolic engineering of Lactobacillus casei for production of UDP‐N‐acetylglucosamine

UDP-sugars are used as glycosyl donors in many enzymatic glycosylation processes. In bacteria UDP-N-acetylglucosamine (UDP-GlcNAc) is synthesized from fructose-6-phosphate by four successive reactions catalyzed by three enzymes: Glucosamine-6-phosphate synthase (GlmS), phosphoglucosamine mutase (GlmM), and the bi-functional enzyme glucosamine-1-phosphate acetyltransferase/N-acetylglucosamine-1-phosphate uridyltransferase (GlmU). In this work several metabolic engineering strategies, aimed to increment UDP-GlcNAc biosynthesis, were applied in the probiotic bacterium Lactobacillus casei strain BL23. This strain does not produce exopolysaccharides, therefore it could be a suitable host for the production of oligosaccharides. The genes glmS, glmM, and glmU coding for GlmS, GlmM, and GlmU activities in L. casei BL23, respectively, were identified, cloned and shown to be functional by homologous over-expression. The recombinant L. casei strain over-expressing simultaneously the genes glmM and glmS showed a 3.47 times increase in GlmS activity and 6.43 times increase in GlmM activity with respect to the control strain. Remarkably, these incremented activities resulted in about fourfold increase of the UDP-GlcNAc pool. In L. casei BL23 wild type strain transcriptional analyses showed that glmM and glmU are constitutively transcribed. By contrast, glmS transcription is down-regulated with a 21-fold decrease of glmS mRNA in cells cultured with N-acetylglucosamine as the sole carbon source compared to cells cultured with glucose. Our results revealed for the first time that GlmS, GlmM, and GlmU are responsible for UDP-GlcNAc biosynthesis in lactobacilli.

Quaternary structure, conformational variability and global motions of phosphoglucosamine mutase

Phosphoglucosamine mutase (PNGM) is a bacterial enzyme that participates in the peptidoglycan biosynthetic pathway. Recent crystal structures of PNGM from two bacterial pathogens, Bacillus anthracis and Francisella tularensis, have revealed key structural features of this enzyme for the first time. Here, we follow up on several novel findings from the crystallographic studies, including the observation of a structurally conserved interface between polypeptide chains and conformational variability of the C-terminal domain. Small-angle X-ray scattering of B. anthracis PNGM shows that this protein is a dimer in solution. Comparisons of the four independent polypeptide chains from the two structures reveals conserved residues and structural changes involved in the conformational variability, as well as a significant rotation of the C-terminal domain, of nearly 60°, between the most divergent conformers. Furthermore, the fluctuation dynamics of PNGM are examined via normal mode analyses. The most mobile region of the protein is its C-terminal domain, consistent with observations from the crystal structures. Large regions of correlated, collective motions are identified exclusively for the dimeric state of the protein, comprising both contiguous and noncontiguous structural domains. The motions observed in the lowest frequency normal mode of the dimer result in dynamically coupled opening and closing of the two active sites. The global motions identified in this study support the importance of the conformational change of PNGM in function, and suggest that the dimeric state of this protein may confer advantages consistent with its evolutionary conservation.

Contribution of phosphoglucosamine mutase to determination of bacterial cell morphology in Streptococcus gordonii

Phosphoglucosamine mutase (GlmM; EC 5.4.2.10) catalyzes the interconversion of glucosamine-6-phosphate to glucosamine-1-phosphate, an essential step in the biosynthetic pathway leading to the formation of the peptidoglycan precursor uridine 5'-diphospho-N-acetylglucosamine. We have recently identified the gene (glmM) encoding the enzyme of Streptococcus gordonii, an early colonizer on the human tooth and an important cause of infective endocarditis, and indicated that the glmM mutation in S. gordonii appears to influence bacterial cell growth, morphology, and sensitivity to penicillins. Moreover, the glmM mutation results in increased sensitivity to polymorphonuclear leukocyte (PMN)-dependent killing. In the present study, we observed similarities in the utilization of sugar between the wild-type strain and the glmM mutant of S. gordonii when cultivated with medium containing 0.2% glucose, fructose, lactose, or sucrose. Morphological analyses clearly indicated that the glmM mutation causes marked elongation of the streptococcal chains, enlargement of bacterial cells, increased distortion of the bacterial cell surface, and defects in cell separation. These results suggest that mutations in glmM appear to influence bacterial cell growth and morphology, independent of the carbon source.

Crystallization and initial crystallographic analysis of phosphoglucosamine mutase fromBacillus anthracis

The enzyme phosphoglucosamine mutase catalyzes the conversion of glucosamine 6-phosphate to glucosamine 1-phosphate, an early step in the formation of the nucleotide sugar UDP-N-acetylglucosamine, which is involved in peptidoglycan biosynthesis. These enzymes are part of the large alpha-D-phosphohexomutase enzyme superfamily, but no proteins from the phosphoglucosamine mutase subgroup have been structurally characterized to date. Here, the crystallization of phosphoglucosamine mutase from Bacillus anthracis in space group P3(2)21 by hanging-drop vapor diffusion is reported. The crystals diffracted to 2.7 A resolution under cryocooling conditions. Structure determination by molecular replacement was successful and refinement is under way. The crystal structure of B. anthracis phosphoglucosamine mutase should shed light on the substrate-specificity of these enzymes and will also serve as a template for inhibitor design.

Role of phosphoglucosamine mutase on virulence properties ofStreptococcus mutans

Streptococcus mutans has been strongly implicated as the principal etiological agent in dental caries. As a gram-positive bacterium, S. mutans has a thick and compact cell wall to maintain the cell shape and protect the cells against mechanical or osmotic damage. Previous studies have proved that peptidoglycan is the main component of the cell wall involved in the autolysis or biofilm formation processes. In this study, we investigated the gene SMU.1426c in the amino-sugar metabolism pathway of S. mutans UA159, which encodes phosphoglucosamine mutase (GlmM). The glmM gene that functions in the biosynthesis of peptidoglycan has been well investigated in Escherichia coli. Here a glmM mutant strain of S. mutans UA159 was constructed and several virulence properties were investigated. The mutant devoid of the glmM gene displayed long chains, reduced growth rate and increased autolysis. Biofilm formation by the mutant was found to be attenuated. These results proved that peptidoglycan biosynthesis plays an important part in a series of bacterial morphologies. The glmM gene may have a constructive role in the virulence properties of S. mutans.

The highly conserved serine threonine kinase StkP of Streptococcus pneumoniae contributes to penicillin susceptibility independently from genes encoding penicillin-binding proteins

BackgroundThe serine/threonine kinase StkP of Streptococcus pneumoniae is a major virulence factor in the mouse model of infection. StkP is a modular protein with a N-terminal kinase domain a C-terminal PASTA domain carrying the signature of penicillin-binding protein (PBP) and prokaryotic serine threonine kinase. In laboratory cultures, one target of StkP is the phosphoglucosamine mutase GlmM involved in the first steps of peptidoglycan biosynthesis. In order to further elucidate the importance of StkP in S. pneumoniae, its role in resistance to β-lactams has been assessed by mutational analysis in laboratory cultures and its genetic conservation has been investigated in isolates from infected sites (virulent), asymptomatic carriers, susceptible and non-susceptible to β-lactams.ResultsDeletion replacement mutation in stkP conferred hypersensitivity to penicillin G and was epistatic on mutations in PBP2X, PBP2B and PBP1A from the resistant 9V clinical isolate URA1258. Genetic analysis of 55 clinical isolates identified 11 StkP alleles differing from the reference R6 allele. None relevant mutation in the kinase or the PASTA domains were found to account for susceptibility of the isolates. Rather the minimal inhibitory concentration (MIC) values of the strains appeared to be determined by their PBP alleles.ConclusionThe results of genetic dissection analysis in lab strain Cp1015 reveal that StkP is involved in the bacterial response to penicillin and is epistatic on mutations PBP 2B, 2X and 1A. However analysis of the clinical isolates did not allow us to find the StkP alleles putatively involved in determining the virulence or the resistance level of a given strain, suggesting a strong conservation of StkP in clinical isolates.

Contribution of phosphoglucosamine mutase to the resistance ofStreptococcus gordoniiDL1 to polymorphonuclear leukocyte killing

Phosphoglucosamine mutase (GlmM; EC 5.4.2.10) catalyzes the interconversion of glucosamine-6-phosphate to glucosamine-1-phosphate, an essential step in the biosynthetic pathway leading to the formation of the peptidoglycan precursor uridine 5'-diphospho-N-acetylglucosamine. We have recently identified the gene (glmM) encoding the enzyme of Streptococcus gordonii, an early colonizer on the human tooth and an important cause of infective endocarditis, and indicated that the glmM mutation in S. gordonii appears to influence bacterial cell growth, morphology, and sensitivity to penicillins. In the present study, we assessed whether the glmM mutation also affects escape from polymorphonuclear leukocyte (PMN)-dependent killing. Although no differences in attachment to human PMNs were observed between the glmM mutant and the wild-type S. gordonii, the glmM mutation resulted in increased sensitivity to PMN-dependent killing. Compared with the wild type, the glmM mutant induced increased superoxide anion production and lysozyme release by PMNs. Moreover, the glmM mutant is more sensitive to lysozyme, indicating that the GlmM may be required for synthesis of firm peptidoglycans for resistance to bacterial cell lysis. These findings suggest that the GlmM contributes to the resistance of S. gordonii to PMN-dependent killing. Enzymes such as GlmM could be novel drug targets for this organism.

DETECTION OF Helicobacter pylori IN GASTRIC MUCOSA OF SHEEP: PRELIMINARY RESULTS

Helicobacter pylori is an organism widespread in humans and sometimes responsible for serious illnesses. It has been hypothesized the existence of animal reservoirs, and that the infection route by H. pylori involves multiple pathways including food-borne transmission as the microorganism has been detected from sheep, goat and cow milk. This work reports the preliminary results of a survey conducted in order to investigate the presence of H. pylori in gastric mucosa of sheep slaughtered in Apulia region (Italy) employing a Nested Polymerase Chain Reaction (Nested-PCR) assay for the detection of the phosphoglucosamine mutase gene (glmM), as screening method followed by conventional bacteriological isolation. Out of the 50 gastric mucosa samples examined, 3 (6%) resulted positive for the presence of glmMgene, but at this time no strains were isolated. The results deserve further investigations to asses the role of ruminants as possible reservoirs of H. pylori.

Identification of theStreptococcus gordonii glmMgene encoding phosphoglucosamine mutase and its role in bacterial cell morphology, biofilm formation, and sensitivity to antibiotics

Phosphoglucosamine mutase (EC 5.4.2.10) catalyzes the interconversion of glucosamine-6-phosphate into glucosamine-1-phosphate, an essential step in the biosynthetic pathway leading to the formation of peptidoglycan precursor uridine 5'-diphospho-N-acetylglucosamine. The gene (glmM) of Escherichia coli encoding the enzyme has been identified previously. We have now identified a glmM homolog in Streptococcus gordonii, an early colonizer on the human tooth and an important cause of infective endocarditis, and have confirmed that the gene encodes phosphoglucosamine mutase by assaying the enzymatic activity of the recombinant GlmM protein. Insertional glmM mutant of S. gordonii did not produce GlmM, and had a growth rate that was approximately half that of the wild type. Morphological analyses clearly indicated that the glmM mutation causes marked elongation of the streptococcal chains, enlargement of bacterial cells, and increased roughness of the bacterial cell surface. Furthermore, the glmM mutation reduces biofilm formation and increases sensitivity to penicillins relative to wild type. All of these phenotypic changes were also observed in a glmM deletion mutant, and were restored by the complementation with plasmid-borne glmM. These results suggest that, in S. gordonii, mutations in glmM appear to influence bacterial cell growth and morphology, biofilm formation, and sensitivity to penicillins.

Evaluation of a Nested-PCR assay based on the phosphoglucosamine mutase gene ( glmM) for the detection of Helicobacter pylori from raw milk

Helicobacter pylori is an organism widespread in the human population and sometimes responsible for serious illnesses. Since H. pylori has been detected in Italy from an high percentage of sheep milk samples, it has been hypothesized that contaminated milk, may act as a vehicle of transmission of the microorganism to humans. In this work, a Nested Polymerase Chain Reaction approach has been used to detect H. pylori phosphoglucosamine mutase gene ( glmM) from sheep, goat and cow milk artificially contaminated with wild H. pylori strains isolated from human gastric biopsies and the reference strain ( H. pylori ATCC 43504). The technique showed a high sensitivity of 3 CFU/ml and proved to be both specific and rapid, The authors suggest that it could be used as a sensitive method for a rapid screening of sheep, goat and cow milk samples during the microbiological control of these large consumed foods.

High occurrence of Helicobacter pylori in raw goat, sheep and cow milk inferred by glmM gene: A risk of food-borne infection?

Helicobacter pylori is an organism widespread in humans and sometimes responsible for serious illnesses, such as gastric and duodenal ulcers, MALToma and even gastric cancer. It has been hypothesized that the infection route by H. pylori involves multiple pathways including food-borne transmission, as the microorganism has been detected from foods such as sheep and cow milk. This work reports the results of a survey conducted in order to investigate the presence of H. pylori in raw goat, sheep and cow milk produced in Southern Italy, employing a Nested Polymerase Chain Reaction (Nested-PCR) assay for the detection of the phosphoglucosamine mutase gene ( glmM), as screening method followed by conventional bacteriological isolation. Out of the 400 raw milk samples examined, 139 (34.7%) resulted positive for the presence of glmM gene, but no strains were isolated. In this work H. pylori DNA has been firstly detected from 41 (25.6%) raw goat milk samples. The results deserve further investigations on the contamination source/s of the milk samples and on the major impact that it may have on consumers.

Characterization of a eukaryotic type serine/threonine protein kinase and protein phosphatase of Streptococcus pneumoniae and identification of kinase substrates

Searching the genome sequence of Streptococcus pneumoniae revealed the presence of a single Ser/Thr protein kinase gene stkP linked to protein phosphatase phpP. Biochemical studies performed with recombinant StkP suggest that this protein is a functional eukaryotic-type Ser/Thr protein kinase. In vitro kinase assays and Western blots of S. pneumoniae subcellular fractions revealed that StkP is a membrane protein. PhpP is a soluble protein with manganese-dependent phosphatase activity in vitro against a synthetic substrate RRA(pT)VA. Mutations in the invariant aspartate residues implicated in the metal binding completely abolished PhpP activity. Autophosphorylated form of StkP was shown to be a substrate for PhpP. These results suggest that StkP and PhpP could operate as a functional pair in vivo. Analysis of phosphoproteome maps of both wild-type and stkP null mutant strains labeled in vivo and subsequent phosphoprotein identification by peptide mass fingerprinting revealed two possible substrates for StkP. The evidence is presented that StkP can phosphorylate in vitro phosphoglucosamine mutase GlmM which catalyzes the first step in the biosynthetic pathway leading to the formation of UDP-N-acetylglucosamine, an essential common precursor to cell envelope components.

Chromosomal organization and transcription analysis of genes in the vicinity of Pseudomonas aeruginosa glmM gene encoding phosphoglucosamine mutase

A computer-aided analysis of the Pseudomonas aeruginosa PAO1 genome surrounding the glmM gene was carried out and the organization of this chromosomal region was compared with the equivalent regions in other γ-proteobacteria species with the genome sequence available. glmM encodes the enzyme phosphoglucosamine mutase which catalyses the interconversion of glucosamine-6-phosphate into glucosamine-1-phosphate in the biosynthetic pathway leading to the synthesis of UDP- N-acetylglucosamine which is simultaneously a precursor for the biosynthesis of cell-wall peptidoglycan and outer membrane lipopolysaccharide. Northern blot analysis suggests that glmM may be a part of the five-cistron operonic structure composed by the Escherichia coli homologues ftsJ, ftsH, folP, glmM, and tpiA. The secG gene, downstream tpiA, does not make part of this polygenic organization, being actively transcribed as a monocistronic mRNA during transition to the stationary phase of growth. Differently, transcription of genes in the glmM operon is more active in the early exponential phase, decreasing with the increase of cell density during exponential growth and reaching negligible values in stationary phase cells.

Donor substrate regeneration for efficient synthesis of globotetraose and isoglobotetraose.

Here we describe the efficient synthesis of two oligosaccharide moieties of human glycosphingolipids, globotetraose (GalNAcbeta1-->3Galalpha1-->4Galbeta1-->4Glc) and isoglobotetraose (GalNAcbeta1-->3Galalpha1-->3Galbeta1-->4Glc), with in situ enzymatic regeneration of UDP-N-acetylgalactosamine (UDP-GalNAc). We demonstrate that the recombinant beta-1,3-N-acetylgalactosaminyltransferase from Haemophilus influenzae strain Rd can transfer N-acetylgalactosamine to a wide range of acceptor substrates with a terminal galactose residue. The donor substrate UDP-GalNAc can be regenerated by a six-enzyme reaction cycle consisting of phosphoglucosamine mutase, UDP-N-acetylglucosamine pyrophosphorylase, phosphate acetyltransferase, pyruvate kinase, and inorganic pyrophosphatase from Escherichia coli, as well as UDP-N-acetylglucosamine C4 epimerase from Plesiomonas shigelloides. All these enzymes were overexpressed in E. coli with six-histidine tags and were purified by one-step nickel-nitrilotriacetic acid affinity chromatography. Multiple-enzyme synthesis of globotetraose or isoglobotetraose with the purified enzymes was achieved with relatively high yields.

Comparative proteomics of Staphylococcus aureus and the response of methicillin-resistant and methicillin-sensitive strains to Triton X-100.

Proteomics is a powerful tool for analysing differences in gene expression between bacterial strains with alternate phenotypes. Staphylococcus aureus strains are grouped on the basis of their sensitivity to methicillin. Two-dimensional gel electrophoresis was combined with MS to compare the protein profiles of S. aureus strains COL (methicillin-resistant) and 8325 (methicillin-sensitive). Reference mapping via this approach identified 377 proteins that corresponded to 266 distinct ORFs. Amongst these identified proteins were 14 potential virulence factors. The production of 41 'hypothetical' proteins was confirmed, and eight of these appeared to be unique to S. aureus. Strain COL displayed 12 protein spots, which included alkaline-shock protein 23 (Asp23) and cold-shock proteins CspABC, which either were not present in strain 8325 or were present at a significantly lower intensity in this strain. Comparative maps were used to characterize the S. aureus response to treatment with Triton X-100 (TX-100), a detergent that has been shown to reduce methicillin resistance independently of an interaction with the mecA-encoded penicillin-binding protein 2a. In response to growth of the bacteria in the presence of TX-100, 44 protein spots showed altered levels of abundance, and 11 of these spots were found only in COL. The products of genes regulated by sigma(B) (the alternative sigma factor), including Asp23 and three proteins of unknown function, and SarA (a regulator of virulence genes) were shown to be present at significantly altered levels. SarA production was induced in TX-100-treated cultures. A protein of the sigma(B) operon, RsbV, was only detected in COL and its production was down-regulated in COL when the strain was treated with TX-100, whereas RsbW was present at reduced levels in both strains. Upon growth of both strains in the presence of TX-100, no effects on the production of the essential methicillin-resistance factor FemA were detected, whereas phosphoglucosamine mutase (GlmM) production was reduced in COL alone. This study suggests that proteins of the sigma(B) and sarA regulons, as well as other factors, are involved in methicillin resistance in S. aureus.

Identification of the Pseudomonas aeruginosa glmM gene, encoding phosphoglucosamine mutase.

A search for a potential algC homologue within the Pseudomonas aeruginosa PAO1 genome database has revealed an open reading frame (ORF) of unknown function, ORF540 in contig 54 (July 1999 Pseudomonas genome release), that theoretically coded for a 445-amino-acid-residue polypeptide (I. M. Tavares, J. H. Leitão, A. M. Fialho, and I. Sá-Correia, Res. Microbiol. 150:105-116, 1999). The product of this gene is here identified as the phosphoglucosamine mutase (GlmM) which catalyzes the conversion of glucosamine-6-phosphate to glucosamine-1-phosphate, an essential step in the formation of the cell wall precursor UDP-N-acetylglucosamine. The P. aeruginosa gene has been cloned into expression vectors and shown to restore normal peptidoglycan biosynthesis and cell growth of a glmM Escherichia coli mutant strain. The GlmM enzyme from P. aeruginosa has been overproduced to high levels and purified to homogeneity in a six-histidine-tagged form. Beside its phosphoglucosamine mutase activity, the P. aeruginosa enzyme is shown to exhibit phosphomannomutase and phosphoglucomutase activities, which represent about 20 and 2% of its GlmM activity, respectively.

Functional cloning and mutational analysis of the human cDNA for phosphoacetylglucosamine mutase: identification of the amino acid residues essential for the catalysis

In Saccharomyces cerevisiae, phosphoacetylglucosamine mutase is encoded by an essential gene called AGM1. The human AGM1 cDNA ( HsAGM1) and the Candida albicans AGM1 gene ( CaAGM1) were functionally cloned and characterized by using an S. cerevisiae strain in which the endogenous phosphoacetylglucosamine mutase was depleted. When expressed in Escherichia coli as fusion proteins with glutathione S-transferase, both HsAgm1 and CaAgm1 proteins displayed phosphoacetylglucosamine mutase activities, demonstrating that they indeed specify phosphoacetylglucosamine mutase. Sequence comparison of HsAgm1p with several hexose-phosphate mutases yielded three domains that are highly conserved among phosphoacetylglucosamine mutases and phosphoglucomutases of divergent organisms. Mutations of the conserved amino acids found in these domains, which were designated region I, II, and III, respectively, demonstrated that alanine substitutions for Ser 64 and His 65 in region I, and for Asp 276, Asp 278, and Arg 281 in region II of HsAgm1p severely diminished the enzyme activity and the ability to rescue the S. cerevisiae agm1Δ null mutant. Conservative mutations of His 65 and Asp 276 restored detectable activities, whereas those of Ser 64, Asp 278, and Arg 281 did not. These results indicate that Ser 64, Asp 278, and Arg 281 of HsAgm1p are residues essential for the catalysis. Because Ser 64 corresponds to the phosphorylating serine in the E. coli phosphoglucosamine mutase, it is likely that the activation of HsAgm1p also requires phosphorylation on Ser 64. Furthermore, alanine substitution for Arg 496 in region III significantly increased the K m value for N-acetylglucosamine-6-phosphate, demonstrating that Arg 496 serves as a binding site for N-acetylglucosamine-6-phosphate.

Tn551-mediated insertional inactivation of the fmtB gene encoding a cell wall-associated protein abolishes methicillin resistance in Staphylococcus aureus.

A Tn551 insert in a gene termed fmtB was shown to reduce oxacillin as well as Triton X-100 resistance in highly methicillin-resistant Staphylococcus aureus (MRSA) COL. Backcrosses of fmtB::Tn551 into S. aureus COL and into two genetically distinct MRSA strains, KSA8 and NCTC10443, confirmed the linkage of fmtB::Tn551 with loss of oxacillin resistance. The fmtB gene codes for a protein of a deduced molecular mass of 263 kDa that contains 17 tandem repeats of 75 amino acids and a C-terminal LPXTG cell wall-sorting motif. Immunoblots with anti-FmtB antibodies confirmed its localization in the cell wall fraction. The fmtB gene was mapped downstream of the phosphoglucosamine mutase operon glmM which catalyses formation of glucosamine-1-phosphate. Oxacillin resistance was not restored in fmtB mutants by trans-complementation with fmtB. However, although GlmM production was not affected by fmtB inactivation, oxacillin resistance was increased in fmtB mutants by introducing a plasmid-borne glmM gene, presumably by GlmM overexpression. Interestingly, a similar phenotypic complementation was obtained in fmtB mutants by including substrate level concentrations of N-acetylglucosamine or glucosamine in the growth medium. Inactivation of the fmtB gene seems therefore to have an indirect effect on methicillin resistance which can be relieved by increasing the production of the cell wall precursor glucosamine-1-phosphate.