[show abstract][hide abstract] ABSTRACT: The glpX gene (Rv1099c) of Mycobacterium tuberculosis (Mtb) encodes Fructose 1,6-bisphosphatase II (FBPase II; EC 126.96.36.199); a key gluconeogenic enzyme. Mtb possesses glpX homologue as the major known FBPase. This study explored the expression, purification and enzymatic characterization of functionally active FBPase II from Mtb. The glpX gene was cloned, expressed and purified using a two step purification strategy including affinity and size exclusion chromatography. The specific activity of Mtb FBPase II is 1.3 U/mg. The enzyme is oligomeric, followed Michaelis-Menten kinetics with an apparent km = 44 μM. Enzyme activity is dependent on bivalent metal ions and is inhibited by lithium and inorganic phosphate. The pH optimum and thermostability of the enzyme have been determined. The robust expression, purification and assay protocols ensure sufficient production of this protein for structural biology and screening of inhibitors against this enzyme.
Applied biochemistry and biotechnology 03/2011; 164(8):1376-89. · 1.94 Impact Factor
[show abstract][hide abstract] ABSTRACT: Mycobacteria use inositol in phosphatidylinositol, for anchoring lipoarabinomannan (LAM), lipomannan (LM) and phosphatidylinosotol mannosides (PIMs) in the cell envelope, and for the production of mycothiol, which maintains the redox balance of the cell. Inositol is synthesized by conversion of glucose-6-phosphate to inositol-1-phosphate, followed by dephosphorylation by inositol monophosphate phosphatases (IMPases) to form myo-inositol. To gain insight into how Mycobacterium tuberculosis synthesises inositol we carried out genetic analysis of the four IMPase homologues that are present in the Mycobacterium tuberculosis genome.
Mutants lacking either impA (Rv1604) or suhB (Rv2701c) were isolated in the absence of exogenous inositol, and no differences in levels of PIMs, LM, LAM or mycothiol were observed. Mutagenesis of cysQ (Rv2131c) was initially unsuccessful, but was possible when a porin-like gene of Mycobacterium smegmatis was expressed, and also by gene switching in the merodiploid strain. In contrast, we could only obtain mutations in impC (Rv3137) when a second functional copy was provided in trans, even when exogenous inositol was provided. Experiments to obtain a mutant in the presence of a second copy of impC containing an active-site mutation, in the presence of porin-like gene of M. smegmatis, or in the absence of inositol 1-phosphate synthase activity, were also unsuccessful. We showed that all four genes are expressed, although at different levels, and levels of inositol phosphatase activity did not fall significantly in any of the mutants obtained.
We have shown that neither impA, suhB nor cysQ is solely responsible for inositol synthesis. In contrast, we show that impC is essential for mycobacterial growth under the conditions we used, and suggest it may be required in the early stages of mycothiol synthesis.
[show abstract][hide abstract] ABSTRACT: To better understand the global effects of "natural" lesions in genes involved in the pyruvate metabolism in Mycobacterium bovis, null mutations were made in the Mycobacterium tuberculosis H37Rv ald and pykA genes to mimic the M. bovis situation. Like M. bovis, the M. tuberculosis DeltapykA mutant yielded dysgonic colonies on solid medium lacking pyruvate, whereas colony morphology was eugonic on pyruvate-containing medium. Global effects of the loss of the pykA gene, possibly underlying colony morphology, were investigated by using proteomics on cultures grown in the same conditions. The levels of Icd2 increased and those of Icl and PckA decreased in the DeltapykA knockout. Proteomics suggested that the synthesis of enzymes involved in fatty acid and lipid biosynthesis were decreased, whereas those involved in beta-oxidation were increased in the M. tuberculosis DeltapykA mutant, as confirmed by direct assays for these activities. Thus, the loss of pykA from M. tuberculosis results in fatty acids being used principally for energy production, in contrast to the situation in the host when carbon from fatty acids is conserved through the glyoxylate cycle and gluconeogenesis; when an active pykA gene was introduced into M. bovis, the opposite effects occurred. Proteins involved in oxidative stress-AhpC, KatG, and SodA-showed increased synthesis in the DeltapykA mutant, and iron-regulated proteins were also affected. Ald levels were decreased in the DeltapykA knockout, explaining why an M. tuberculosis DeltapykA Deltaald double mutant showed little additional phenotypic effect. Overall, these data show that the loss of the pykA gene has powerful, global effects on proteins associated with central metabolism.
Journal of bacteriology 10/2009; 191(24):7545-53. · 3.94 Impact Factor
[show abstract][hide abstract] ABSTRACT: There is massive gene replication predicted for the activation of fatty acids and their entry into the beta-oxidation cycle for fatty acid oxidation. These two steps in fatty acid metabolism are catalyzed by FadD and FadE enzymes with 36 genes predicted for each of these respective activities in Mycobacterium tuberculosis. Here we present methods for the cell-free assay of types of enzymes in live bacteria, as well as for fatty acid oxidation overall.
Methods in molecular biology (Clifton, N.J.) 01/2009; 465:47-59.
[show abstract][hide abstract] ABSTRACT: A procedure for metabolic labeling of all cellular lipids starting with a culture of mycobacteria is described in this chapter using either a pulse-chase or a simple labeling experimental design. Three fractions are produced for subsequent lipid analysis: (1) the culture filtrate; (2) a readily released surface lipid fraction; and (3) the killed, labeled bacteria. A standardized, TLC-based method for general lipid analysis that can be used to quantify the labeling of all the mycobacterial lipids is given as well as a protocol for analyzing the fatty acyl moieties of the lipids.
Methods in molecular biology (Clifton, N.J.) 01/2009; 465:61-81.
[show abstract][hide abstract] ABSTRACT: Previous work on the population structure of Mycobacterium bovis strains in Great Britain has identified highly successful clones which are expanding across the country. One such clone, designated M. bovis type 17, differs from all other members of the Mycobacterium tuberculosis complex in having a region of deletion, termed RDbovis(d)_0173, of seven genes between Mb1963c and Mb1971. Three of these genes have functions annotated in lipid metabolism. To explore the molecular basis for the success of this clone, we examined the impact of this deletion on lipid metabolism. While type 17 isolates had similar lipid composition to other M. bovis strains, their ability to incorporate propanoate into mycolic acids was remarkably low. When expressed as a reciprocal (the ratio of incorporation of label from acetate : propanoate into mycolic acids) the ratio was higher for all three type 17 field strains tested (mean: 18.90) than the values of 7.30 to 7.61 for other field strains (P < 0.002) and values < 6.50 for all other strains in the M. tuberculosis complex tested. The label from propanoate was diverted to pyruvate, at significantly higher levels in M. bovis type 17 than all other strains (P < 0.021). Complementation of M. bovis type 17 with an integrating cosmid, IE471, carrying the M. tuberculosis orthologues of Mb1963c-Mb1971 resulted in the ability of the recombinant strain to incorporate label from propanoate into mycolic acids in a manner similar to other strains. M. bovis type 17 : : IE471 labelled pyruvate from propanoate about four times more slowly than the parent strain. Thus, RDbovis(d)_0173 results in a profound effect on carbon metabolism, providing the ability to compensate for the inactivation of the ald and pykA genes, involved in pyruvate metabolism, that is seen in M. bovis (but not in M. tuberculosis). This shift in carbon metabolism may be a factor in the extraordinary clonal expansion reported for M. bovis type 17.
[show abstract][hide abstract] ABSTRACT: Much work in the mycobacterial field has focused on the identification and characterization of antigenic proteins (1,2); many have now been identified and assigned a function; for example, the immunodommant 65kDa antigen of Mycobacterzum tuberculosis has been identified as a chaperonin (3) and the 28-kDa antigen of Mycobacterzum leprae was shown to be superoxide dismutase (4). In the beginnings of mycobactertal molecular biology, antigenic proteins were identified by screening M. leprae and M tuberculosis expression libraries in Escherichia coli (5–9) with mouse monoclonal antibodies (MAbs) (7,8) and polyclonal sera from rabbits (10,11) and patients (12,13). Although E. coli is still being used for the overexpression and purification of mycobacterial proteins (14), the use of nonpathogenic mycobacterta, such as Mycobacterium smegmatis as surrogate hosts may be preferable (15). For example, several mycobacterial proteins have been shown to undergo post-translational modification, such as glycosylation
(15,16). Because glycosylation does not occur in E. coli, the study of such proteins in their native state requires the use of mycobacterial hosts. The preparation of cell-free extracts
and protein purification from mycobacteria is a prerequisite for this kind of work.
[show abstract][hide abstract] ABSTRACT: Through examination of one of the fundamental in vitro characteristics of Mycobacterium bovis--its requirement for pyruvate in glycerol medium--we have revealed a lesion in central metabolism that has profound implications for in vivo growth and nutrition. Not only is M. bovis unable to use glycerol as a sole carbon source but the lack of a functioning pyruvate kinase (PK) means that carbohydrates cannot be used to generate energy. This disruption in sugar catabolism is caused by a single nucleotide polymorphism in pykA, the gene which encodes PK, that substitutes glutamic acid residue 220 with an aspartic acid residue. Substitution of this highly conserved amino acid residue renders PK inactive and thus blocks the ATP generating roles of glycolysis and the pentose phosphate pathway. This mutation was found to occur in other members of the M. tuberculosis complex, namely M. microti and M. africanum. With carbohydrates unable to act as carbon sources, the importance of lipids and gluconeogenesis for growth in vivo becomes apparent. Complementation of M. bovis with the pykA gene from M. tuberculosis H37Rv restored growth on glycerol. Additionally, the presence of a functioning PK caused the colony morphology of the complemented strain to change from the characteristic dysgonic growth of M. bovis to eugonic growth, an appearance normally associated with M. tuberculosis. We also suggest that the glycerol-soaked potato slices used for the derivation of the M. bovis bacillus Calmette and Guérin (BCG) vaccine strain selected for an M. bovis PK+ mutant, a finding that explains the alteration in colony morphology noted during the derivation of BCG. In summary, the disruption of a key step in glycolysis divides the M. tuberculosis complex into two groups with distinct carbon source utilization.
[show abstract][hide abstract] ABSTRACT: Methionine can be used as the sole sulfur source by the Mycobacterium tuberculosis complex although it is not obvious from examination of the genome annotation how these bacteria utilize methionine. Given that genome annotation is a largely predictive process, key challenges are to validate these predictions and to fill in gaps for known functions for which genes have not been annotated. We have addressed these issues by functional analysis of methionine metabolism. Transport, followed by metabolism of (35)S methionine into the cysteine adduct mycothiol, demonstrated the conversion of exogenous methionine to cysteine. Mutational analysis and cloning of the Rv1079 gene showed it to encode the key enzyme required for this conversion, cystathionine gamma-lyase (CGL). Rv1079, annotated metB, was predicted to encode cystathionine gamma-synthase (CGS), but demonstration of a gamma-elimination reaction with cystathionine as well as the gamma-replacement reaction yielding cystathionine showed it encodes a bifunctional CGL/CGS enzyme. Consistent with this, a Rv1079 mutant could not incorporate sulfur from methionine into cysteine, while a cysA mutant lacking sulfate transport and a methionine auxotroph was hypersensitive to the CGL inhibitor propargylglycine. Thus, reverse transsulfuration alone, without any sulfur recycling reactions, allows M. tuberculosis to use methionine as the sole sulfur source. Intracellular cysteine was undetectable so only the CGL reaction occurs in intact mycobacteria. Cysteine desulfhydrase, an activity we showed to be separable from CGL/CGS, may have a role in removing excess cysteine and could explain the ability of M. tuberculosis to recycle sulfur from cysteine, but not methionine.
Journal of Biological Chemistry 04/2005; 280(9):8069-78. · 4.65 Impact Factor
[show abstract][hide abstract] ABSTRACT: There are now abundant data indicating that Mycobacterium tuberculosis uses fatty acids as a carbon source in vivo. A key enzyme in gluconeogenesis, missing in the original annotation of the M. tuberculosis genome, is fructose 1,6-bisphosphatase (FBPase; EC 188.8.131.52). The authors have shown that M. tuberculosis Rv1099c, a glpX homologue, can complement Escherichia coli mutants lacking FBPase. The protein encoded by Rv1099c was shown to have FBPase activity. Rv1099c was expressed at significant levels in M. tuberculosis, and may encode the major FBPase of this pathogen.
[show abstract][hide abstract] ABSTRACT: Mycobacterium bovis is the causative agent of tuberculosis in a range of animal species and man, with worldwide annual losses to agriculture of $3 billion. The human burden of tuberculosis caused by the bovine tubercle bacillus is still largely unknown. M. bovis was also the progenitor for the M. bovis bacillus Calmette-Guérin vaccine strain, the most widely used human vaccine. Here we describe the 4,345,492-bp genome sequence of M. bovis AF2122/97 and its comparison with the genomes of Mycobacterium tuberculosis and Mycobacterium leprae. Strikingly, the genome sequence of M. bovis is >99.95% identical to that of M. tuberculosis, but deletion of genetic information has led to a reduced genome size. Comparison with M. leprae reveals a number of common gene losses, suggesting the removal of functional redundancy. Cell wall components and secreted proteins show the greatest variation, indicating their potential role in host-bacillus interactions or immune evasion. Furthermore, there are no genes unique to M. bovis, implying that differential gene expression may be the key to the host tropisms of human and bovine bacilli. The genome sequence therefore offers major insight on the evolution, host preference, and pathobiology of M. bovis.
Proceedings of the National Academy of Sciences 06/2003; 100(13):7877-82. · 9.74 Impact Factor
[show abstract][hide abstract] ABSTRACT: Mycobacterium tuberculosis andMycobacterium bovis, the causative agents of human and bovine tuberculosis, have been reported to express a range of surface and secreted glycoproteins,
although only one of these has been subjected to detailed structural analysis. We describe the use of a genetic system, in
conjunction with lectin binding, to characterize the points of attachment of carbohydrate moieties to the polypeptide backbone
of a second mycobacterial glycoprotein, antigen MPB83 fromM. bovis. Biochemical and structural analysis of the native MPB83 protein and derived peptides demonstrated the presence of 3 mannose
units attached to two threonine residues. Mannose residues were joined by a (1 → 3) linkage, in contrast to the (1 → 2) linkage
previously observed in antigen MPT32 from M. tuberculosisand the (1 → 2) and (1 → 6) linkages in other mycobacterial glycolipids and polysaccharides. The identification of glycosylated
antigens within the M. tuberculosis complex raises the possibility that the carbohydrate moiety of these glycoproteins might be involved in pathogenesis, either
by interaction with mannose receptors on host cells, or as targets or modulators of the cell-mediated immune response. Given
such a possibility characterization of mycobacterial glycoproteins is a step toward understanding their functional role and
elucidating the mechanisms of mycobacterial glycosylation.
Journal of Biological Chemistry 05/2003; 278(18):16423-16432. · 4.65 Impact Factor
[show abstract][hide abstract] ABSTRACT: Deletions and the appearance of pseudogenes in pathways of carbon source utilisation and energy metabolism best explain the host-dependency and failure to culture Mycobacterium leprae axenically. From the genome sequence it is possible to predict that acetate and galactose cannot be used as carbon sources, while pyruvate can only be catabolised. Glycerol, glucose, and fatty acids could be used for glycolysis, the pentose cycle and -oxidation which are complete. Retrospective functional genomics – interpreting work before the completion of the genome project – supports the failure of M. leprae to use acetate as well as another prediction that metabolic flux from pyruvate to acetyl-CoA would be very low. However, the loss of a second icd gene (compared with M. tuberculosis), predicted to encode isocitrate dehydrogenase, did not diminish the specific activity of the enzyme. The genes for respiratory pathways are extremely limited, being present for oxidative phosphorylation as a result of electron transport only using FADH as an electron donor. In contrast, all the major biosynthetic pathways are complete except that M. leprae is a natural methionine auxotroph: this is predicted not to be attenuating, or explain host-dependency since methionine would be present in rich culture media.
World Journal of Microbiology and Biotechnology 01/2003; 19(1):1-16. · 1.26 Impact Factor
[show abstract][hide abstract] ABSTRACT: Sulphur is essential for some of the most vital biological activities such as translation initiation and redox maintenance, and genes involved in sulphur metabolism have been implicated in virulence. Mycobacterium tuberculosis has three predicted genes for the prototrophic acquisition of sulphur as sulphate: cysA, part of an ABC transporter, and cysA2 and A3, SseC sulphotransferases. Screening for amino acid auxotrophs of Mycobacterium bovis BCG, obtained by transposon mutagenesis, was used to select methionine auxotrophs requiring a sulphur-containing amino acid for growth. We have characterized one of these auxotrophs as being disrupted in cysA. Both the cysA mutant and a previously identified mutant in an upstream gene, subI, were functionally characterized as being completely unable to take up sulphate. Complementation of the cysA mutant with the wild-type gene from M. tuberculosis restored prototrophy and the ability to take up sulphate with the functional characteristics of an ABC transporter. Hence, it appears that this is the sole locus encoding inorganic sulphur transport in the M. tuberculosis complex.
[show abstract][hide abstract] ABSTRACT: Everything that we need to know about Mycobacterium leprae, a close relative of the tubercle bacillus, is encrypted in its genome. Inspection of the 3.27 Mb genome sequence of an armadillo-derived Indian isolate of the leprosy bacillus identified 1,605 genes encoding proteins and 50 genes for stable RNA species. Comparison with the genome sequence of Mycobacterium tuberculosis revealed an extreme case of reductive evolution, since less than half of the genome contains functional genes while inactivated or pseudogenes are highly abundant. The level of gene duplication was approximately 34% and, on classification of the proteins into families, the largest functional groups were found to be involved in the metabolism and modification of fatty acids and polyketides, transport of metabolites, cell envelope synthesis and gene regulation. Reductive evolution, gene decay and genome downsizing have eliminated entire metabolic pathways, together with their regulatory circuits and accessory functions, particularly those involved in catabolism. This may explain the unusually long generation time and account for our inability to culture the leprosy bacillus.
[show abstract][hide abstract] ABSTRACT: Leprosy, a chronic human neurological disease, results from infection with the obligate intracellular pathogen Mycobacterium leprae, a close relative of the tubercle bacillus. Mycobacterium leprae has the longest doubling time of all known bacteria and has thwarted every effort at culture in the laboratory. Comparing the 3.27-megabase (Mb) genome sequence of an armadillo-derived Indian isolate of the leprosy bacillus with that of Mycobacterium tuberculosis (4.41 Mb) provides clear explanations for these properties and reveals an extreme case of reductive evolution. Less than half of the genome contains functional genes but pseudogenes, with intact counterparts in M. tuberculosis, abound. Genome downsizing and the current mosaic arrangement appear to have resulted from extensive recombination events between dispersed repetitive sequences. Gene deletion and decay have eliminated many important metabolic activities including siderophore production, part of the oxidative and most of the microaerophilic and anaerobic respiratory chains, and numerous catabolic systems and their regulatory circuits.