The Helicobacter pylori ureC gene codes for a phosphoglucosamine mutase.

The function of UreC, the product of a 1,335-bp-long open reading frame upstream from the urease structural genes (ureAB) of Helicobacter pylori, was investigated. We present data showing that the ureC gene product is a phosphoglucosamine mutase. D. Mengin-Lecreulx and J. van Heijenoort (J. Biol. Chem. 271:32-39, 1996) observed that UreC is similar (43% identity) to the GlmM protein of Escherichia coli. Those authors showed that GlmM is a phosphoglucosamine mutase catalyzing interconversion of glucosamine-6-phosphate into glucosamine-1-phosphate, which is subsequently transformed into UDP-N-acetylglucosamine. The latter product is one of the main cytoplasmic precursors of cell wall peptidoglycan and outer membrane lipopolysaccharides. The present paper reports that, like its E. coli homolog glmM, the H. pylori ureC gene is essential for cell growth. It was known that growth of a lethal conditional glmM mutant of E. coli at a nonpermissive temperature can be restored in the presence of the ureC gene. We showed that complete complementation of the glmM mutant can be obtained with a plasmid overproducing UreC. The peptidoglycan content and the specific phosphoglucosamine mutase activity of such a complemented strain were measured; these results demonstrated that the ureC gene product functions as a phosphoglucosamine mutase. Homologs of the UreC and GlmM proteins were identified in Haemophilus influenzae, Mycobacterium leprae, Clostridium perfringens, Synechocystis sp. strain PCC6803, and Methanococcus jannaschii. Significant conservation of the amino acid sequence of these proteins in such diverse organisms suggests a very ancient common ancestor for the genes and defines a consensus motif for the phosphoglucosamine mutase active site. We propose renaming the H. pylori ureC gene the glmM gene.     

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 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. Leit?o, 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.     

耻垢分枝杆菌MSMEG_6281 过表达对菌株生长和形态的影响

【目的】耻垢分枝杆菌(Mycobacterium smegmatis mc2155,mc2155)MSMEG_6281为结核分枝杆菌自溶素Rv3717的同源蛋白,通过建立过表达MSMEG_6281的耻垢分枝杆菌菌株,推测该蛋白对耻垢分枝杆菌肽聚糖代谢的影响。【方法】利用RT-PCR方法检测乙胺丁醇(Ethambutol,EMB)作用后MSMEG_6281基因的表达变化;以耻垢分枝杆菌基因组DNA为模板,采用PCR技术克隆MSMEG_6281基因,构建分枝杆菌表达质粒p VV16-MSMEG_6281,进一步建立MSMEG_6281过表达的耻垢分枝杆菌菌株;利用生长曲线检测MSMEG_6281过表达对耻垢分枝杆菌生长的影响;利用扫描电子显微镜分析MSMEG_6281过表达引起的耻垢分枝杆菌形态变化。【结果】EMB处理引起MSMEG_6281基因表达上调;构建了过表达MSMGE_6281的耻垢分枝杆菌菌株(mc2155/p VV16-MSMEG_6281);过表达MSMGE_6281的耻垢分枝杆菌生长缓慢,菌体形态由短杆状转变为长杆状。【结论】MSMGE_6281的过表达可改变耻垢分枝杆菌形态。MSMGE_6281的功能与细胞壁肽聚糖水解相关,在mc2155细胞壁形态维持方面发挥重要作用。     

Transfer of the first arabinofuranose residue to galactan is essential for Mycobacterium smegmatis viability

The mycobacterial arabinan is an elaborate component of the cell wall with multiple glycosyl linkages and no repeating units. In Mycobacterium spp., the Emb proteins (EmbA, EmbB, and EmbC) have been identified as putative mycobacterial arabinosyltransferases implicated in the biogenesis of the cell wall arabinan. Furthermore, it is now evident that the EmbA and EmbB proteins are involved in the assembly of the nonreducing terminal motif of arabinogalactan and EmbC is involved in transferring arabinose, perhaps in the early stage of arabinan synthesis in lipoarabinomannan. It has also been shown that the Emb proteins are a target of the antimycobacterial drug ethambutol (EMB). In the search for additional mycobacterial arabinosyltransferases in addition to the Emb proteins, we disrupted MSMEG_6386 (an orthologue of Rv3792 and a gene upstream of embC) in Mycobacterium smegmatis. Allelic exchange at the chromosomal MSMEG_6386 locus of M. smegmatis could only be achieved in the presence of a rescue plasmid carrying a functional copy of MSMEG_6386 or Rv3792, strongly suggesting that MSMEG_6386 is essential. An in vitro arabinosyltransferase assay using a membrane preparation from M. smegmatis expressing Rv3792 and synthetic beta-d-Galf-(1-->5)-beta-D-Galf-(1-->6)-beta-D-Galf-octyl and beta-D-Galf-(1-->6)-beta-D-Galf-(1-->5)-beta-D-Galf-octyl showed that Rv3792 gene product can transfer an arabinose residue to the C-5 position of the internal 6-linked galactose. The reactions were insensitive to EMB, and when alpha-d-Manp-(1-->6)-alpha-D-Manp-(1-->6)-alpha-D-Manp-octylthiomethyl was used as an acceptor, no product was formed. These observations indicate that transfer of the first arabinofuranose residue to galactan is essential for M. smegmatis viability.     

Initial step in the catabolism of cholesterol by Mycobacterium smegmatis mc2 155

The first step in the catabolism of cholesterol, i.e. the transformation of cholesterol into cholestenone, has been investigated in Mycobacterium smegmatis. In silico analysis identified the MSMEG_1604 gene encoding a putative protein similar to the ChoD cholesterol oxidase of M. tuberculosis H37Rv (Rv3409c) and the MSMEG_5228 gene coding for a protein similar to the NAD(P)-dependent cholesterol dehydrogenase/isomerase of Nocardia sp. The expression of the MSMEG_5228 gene was inducible by cholesterol whereas the expression of MSMEG_1604 gene was constitutive. When both genes were expressed in Escherichia coli only the MSMEG_5228 protein was active on cholesterol. The function of ChoD-like MSMEG_1604 protein remains to be elucidated, but it does not appear to play a critical role in the mineralization of cholesterol as a MSMEG_1604(-) mutant was not affected in the production of cholestenone. However, a MSMEG_5228(-) mutant showed a drastic reduction in the synthesis of cholestenone. The finding that this mutant was still able to grow in cholesterol, allowed us to demonstrate that the cholesterol-inducible MSMEG_5233 gene encodes an additional cholesterol dehydrogenase/isomerase similar to the AcmA dehydrogenase of Sterolibacterium denitrificans. The observation that the double MSMEG_5228-5233(-) mutant was able to grow in cholesterol suggests that in addition to these enzymes other dehydrogenase/isomerases can also catalyse the first reaction of the cholesterol degradation pathway in M. smegmatis, which is not the limiting step of the process.     

Reaction mechanism of phosphoglucosamine mutase from Escherichia coli.

The phosphoglucosamine mutase (GlmM) from Escherichia coli, specifically required for the interconversion of glucosamine-6-phosphate and glucosamine-1-phosphate (an essential step in the pathway for cell-wall peptidoglycan and lipopolysaccharide biosyntheses) was purified to homogeneity and its kinetic properties were investigated. The enzyme was active in a phosphorylated form and catalysed its reaction according to a classical ping-pong bi-bi mechanism. The dephosphorylated and phosphorylated forms of GlmM could be separated by HPLC and coupled MS showed that only one phosphate was covalently linked to the active site of the enzyme. The site of phosphorylation was clearly identified as Ser102 in the 445-amino acid polypeptide. GlmM was also capable of catalysing the interconversion of glucose-1-phosphate and glucose-6-phosphate isomers, although at a much lower (1400-fold) rate. Interestingly, the mutational change of the Ser100 to a threonine residue resulted in a 20-fold increase of the nonspecific phosphoglucomutase activity of GlmM, suggesting that the presence of either a serine or a threonine at this position in the consensus sequence of hexosephosphate mutases could be one of the factors that determines the specificity of these enzymes for either sugar-phosphate or amino sugar-phosphate substrates.     

Identification of the Streptococcus gordonii glmM gene 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.     

Autophosphorylation of phosphoglucosamine mutase from Escherichia coli

Phosphoglucosamine mutase (GlmM) catalyzes the formation of glucosamine-1-phosphate from glucosamine-6-phosphate, an essential step in the pathway for UDP-N-acetylglucosamine biosynthesis in bacteria. This enzyme must be phosphorylated to be active and acts according to a ping-pong mechanism involving glucosamine-1, 6-diphosphate as an intermediate (L. Jolly, P. Ferrari, D. Blanot, J. van Heijenoort, F. Fassy, and D. Mengin-Lecreulx, Eur. J. Biochem. 262:202-210, 1999). However, the process by which the initial phosphorylation of the enzyme is achieved in vivo remains unknown. Here we show that the phosphoglucosamine mutase from Escherichia coli autophosphorylates in vitro in the presence of [(32)P]ATP. The same is observed with phosphoglucosamine mutases from other bacterial species, yeast N-acetylglucosamine-phosphate mutase, and rabbit muscle phosphoglucomutase. Labeling of the E. coli GlmM enzyme with [(32)P]ATP requires the presence of a divalent cation, and the label is subsequently lost when the enzyme is incubated with either of its substrates. Analysis of enzyme phosphorylation by high-pressure liquid chromatography and coupled mass spectrometry confirms that only one phosphate has been covalently linked to the enzyme. Only phosphoserine could be detected after acid hydrolysis of the labeled protein, and site-directed mutagenesis of serine residues located in or near the active site identifies the serine residue at position 102 as the site of autophosphorylation of E. coli GlmM.     

Cell wall core galactofuran synthesis is essential for growth of mycobacteria

The mycobacterial cell wall core consists of an outer lipid (mycolic acid) layer attached to peptidoglycan via a galactofuranosyl-containing polysaccharide, arabinogalactan. This structural arrangement strongly suggests that galactofuranosyl residues are essential for the growth and viability of mycobacteria. Galactofuranosyl residues are formed in nature by a ring contraction of UDP-galactopyranose to UDP-galactofuranose catalyzed by the enzyme UDP-galactopyranose mutase (Glf). In Mycobacterium tuberculosis the glf gene overlaps, by 1 nucleotide, a gene, Rv3808c, that has been shown to encode a galactofuranosyl transferase. We demonstrate here that glf can be knocked out in Mycobacterium smegmatis by allelic replacement only in the presence of two rescue plasmids carrying functional copies of glf and Rv3808c. The glf rescue plasmid was designed with a temperature-sensitive origin of replication and the M. smegmatis glf knockout mutant is unable to grow at the higher temperature at which the glf-containing rescue plasmid is lost. In a separate experiment, the Rv3808c rescue plasmid was designed with a temperature-sensitive origin of replication and the glf-bearing plasmid was designed with a normal original of replication; this strain was also unable to grow at the nonpermissive temperature. Thus, both glf and Rv3808c are essential for growth. These findings and the fact that galactofuranosyl residues are not found in humans supports the development of UDP-galactopyranose mutase and galactofuranosyl transferase as important targets for the development of new antituberculosis drugs.     

结核杆菌活动期高表达基因Rv1776c的克隆及其在耻垢分枝杆菌中的表达

目的构建表达结核分枝杆菌Rv1776c基因的重组耻垢分支杆菌,并鉴定该基因在重组耻垢分支杆菌中的活性。方法采用PCR技术克隆结核分枝杆菌Rv1776c基因,构建大肠埃希菌-分支杆菌穿梭表达质粒pMV-Rv1776c,通过酶切和测序鉴定其正确性,用电穿孔法将重组质粒转染到耻垢分支杆菌mc^2155中。以SDS-PAGE及Western blot检测证实Rv1776c蛋白在重组耻垢分支杆菌内的表达。结果重组耻垢分支杆菌构建成功,生长曲线说明重组质粒不会影响耻垢分支杆菌的体外生长;SDSPAGE及Western blot检测证实Rv1776c在耻垢分枝杆菌内表达出相对分子量约56kD的Rv1776c蛋白。结论成功构建了Rv1776c基因的穿梭质粒pMV-Rv1776c,且该质粒在耻垢分枝杆菌内具有生物活性,为进一步研究其表达产物的功能提供基础。     

Viability,morphology, and proteome of Mycobacterium smegmatis MSMEG_0319 knockout strain

Mycobacterium tuberculosis Rv0228, a membrane protein, is predicted as a drug target through computational methods. MSMEG_0319 (MS0319) in Mycobacterium smegmatis mc²155 is the ortholog of Rv0228. To study the effect of MS0319 protein on M. smegmatis, an MS0319 gene knockout strain (ΔMS0319) was generated via a homologous recombination technique in this study. The results showed that the lack of MS0319 protein in mc²155 cells led to the loss of viability at nonpermissive temperature. Scanning electron microscopy and transmission electron microscopy observations showed drastic changes in cellular shape especially cell wall disruption in ΔMS0319 cells. Proteomic analysis of ΔMS0319 cells through LC‐MS/MS revealed that 462 proteins had changes in their expressions by lacking MS0319 protein. The M. tuberculosis orthologs of these 462 proteins were found through BLASTp search and functional clustering and metabolic pathway enrichment were performed on the orthologs. The results revealed that most of them were enzymes involved in metabolism of carbohydrates and amino acids, indicating that Rv0228 played an important role in cellular metabolism. All these results suggested Rv0228 as a potential target for development of antituberculosis drugs.     

VapC toxins from Mycobacterium tuberculosis are ribonucleases that differentially inhibit growth and are neutralized by cognate VapB antitoxins

The chromosome of Mycobacterium tuberculosis (Mtb) encodes forty seven toxin-antitoxin modules belonging to the VapBC family. The role of these modules in the physiology of Mtb and the function(s) served by their expansion are unknown. We investigated ten vapBC modules from Mtb and the single vapBC from M. smegmatis. Of the Mtb vapCs assessed, only Rv0549c, Rv0595c, Rv2549c and Rv2829c were toxic when expressed from a tetracycline-regulated promoter in M. smegmatis. The same genes displayed toxicity when conditionally expressed in Mtb. Toxicity of Rv2549c in M. smegmatis correlated with the level of protein expressed, suggesting that the VapC level must exceed a threshold for toxicity to be observed. In addition, the level of Rv2456 protein induced in M. smegmatis was markedly lower than Rv2549c, which may account for the lack of toxicity of this and other VapCs scored as 'non-toxic'. The growth inhibitory effects of toxic VapCs were neutralized by expression of the cognate VapB as part of a vapBC operon or from a different chromosomal locus, while that of non-cognate antitoxins did not. These results demonstrated a specificity of interaction between VapCs and their cognate VapBs, a finding corroborated by yeast two-hybrid analyses. Deletion of selected vapC or vapBC genes did not affect mycobacterial growth in vitro, but rendered the organisms more susceptible to growth inhibition following toxic VapC expression. However, toxicity of 'non-toxic' VapCs was not unveiled in deletion mutant strains, even when the mutation eliminated the corresponding cognate VapB, presumably due to insufficient levels of VapC protein. Together with the ribonuclease (RNase) activity demonstrated for Rv0065 and Rv0617--VapC proteins with similarity to Rv0549c and Rv3320c, respectively--these results suggest that the VapBC family potentially provides an abundant source of RNase activity in Mtb, which may profoundly impact the physiology of the organism.     

Inactivation of lsr2 results in a hypermotile phenotype in Mycobacterium smegmatis

Mycobacterial species are characterized by the presence of lipid-rich, hydrophobic cell envelopes. These cell envelopes contribute to properties such as roughness of colonies, aggregation of cells in liquid culture without detergent, and biofilm formation. We describe here a mutant strain of Mycobacterium smegmatis, called DL1215, which demonstrates marked deviations from the above-mentioned phenotypes. DL1215 arose spontaneously from a strain deficient for the stringent response (M. smegmatis Delta rel(Msm) strain) and is not a reversion to a wild-type phenotype. The nature of the spontaneous mutation was a single base-pair deletion in the lsr2 gene, leading to the formation of a truncated protein product. The DL1215 strain was complicated by having both inactivated rel(Msm) and lsr2 genes, and so a single lsr2 mutant was created to analyze the gene's function. The lsr2 gene was inactivated in the wild-type M. smegmatis mc(2)155 strain by allelic replacement to create strain DL2008. Strain DL2008 shows characteristics unique from those of both the wild-type and Delta rel(Msm) strains, some of which include a greatly enhanced ability to slide over agar surfaces (referred to here as "hypermotility"), greater resistance to phage infection and to the antibiotic kanamycin, and an inability to form biofilms. Complementation of the DL2008 mutant with a plasmid containing lsr2 (pLSR2) reverts the strain to the mc(2)155 phenotype. Although these phenotypic differences allude to changes in cell surface lipids, no difference is observed in glycopeptidolipids, polar lipids, apolar lipids, or mycolic acids of the cell wall.     

Aminoglycoside multiacetylating activity of the enhanced intracellular survival protein from Mycobacterium smegmatis and its inhibition

The enhanced intracellular survival (Eis) protein improves the survival of Mycobacterium smegmatis (Msm) in macrophages and functions as the acetyltransferase responsible for kanamycin A resistance, a hallmark of extensively drug-resistant (XDR) tuberculosis, in a large number of Mycobacterium tuberculosis (Mtb) clinical isolates. We recently demonstrated that Eis from Mtb (Eis_Mtb) efficiently multiacetylates a variety of aminoglycoside (AG) antibiotics. Here, to gain insight into the origin of substrate selectivity of AG multiacetylation by Eis, we analyzed AG acetylation by Eis_Msm, investigated its inhibition, and compared these functions to those of Eis_Mtb. Even though for several AGs the multiacetylation properties of Eis_Msm and Eis_Mtb are similar, there are three major differences. (i) Eis_Msm diacetylates apramycin, a conformationally constrained AG, which Eis_Mtb cannot modify. (ii) Eis_Msm triacetylates paromomycin, which can be only diacetylated by Eis_Mtb. (iii) Eis_Msm only monoacetylates hygromycin, a structurally unique AG that is diacetylated by Eis_Mtb. Several nonconserved amino acid residues lining the AG-binding pocket of Eis are likely responsible for these differences between the two Eis homologues. Specifically, we propose that because the AG-binding pocket of Eis_Msm is more open than that of Eis_Mtb, it accommodates apramycin for acetylation in Eis_Msm, but not in Eis_Mtb. We also demonstrate that inhibitors of Eis_Mtb that we recently discovered can inhibit Eis_Msm activity. These observations help define the structural origins of substrate preference among Eis homologues and suggest that Eis_Mtb inhibitors may be applied against all pathogenic mycobacteria to overcome AG resistance caused by Eis upregulation.     

Design and synthesis of novel cell wall inhibitors of Mycobacterium tuberculosis GlmM and GlmU

GlmM and GlmU are key enzymes in the biosynthesis of UDP-N-acetyl-d-glucosamine (UDP-GlcNAc), an essential precursor of peptidoglycan and the rhamnose–GlcNAc linker region in the mycobacterial cell wall. These enzymes are involved in the conversion of two important precursors of UDP-GlcNAc, glucosamine-6-phosphate (GlcN-6-P) and glucosamine-1-phosphate (GlcN-1-P). GlmM converts GlcN-6-P to GlcN-1-P, GlmU is a bifunctional enzyme, whereby GlmU converts GlcN-1-P to GlcNAc-1-P and then catalyzes the formation of UDP-GlcNAc from GlcNAc-1-P and uridine triphosphate. In the present study, methyl 2-amino-2-deoxyl-α-d-glucopyranoside 6-phosphate (1α), methyl 2-amino-2-deoxyl-β-d-glucopyranoside 6-phosphate (1β), two analogs of GlcN-6-P, were synthesized as GlmM inhibitors; 2-azido-2-deoxy-α-d-glucopyranosyl phosphate (2) and 2-amino-2,3-dideoxy-3-fluoro-α-d-glucopyranosyl phosphate (3), analogs of GlcN-1-P, were synthesized firstly as GlmU inhibitors. Compounds 1α, 1β, 2, and 3 as possible inhibitors of mycobacterial GlmM and GlmU are reported herein. Compound 3 showed promising inhibitory activities against GlmU, whereas 1α, 1β and 2 were inactive against GlmM and GlmU even at high concentrations.