Lack of methoxy-mycolates characterizes the geographically restricted lineage 7 of Mycobacterium tuberculosis complex.

Lineage 7 (L7) emerged in the phylogeny of the Mycobacterium tuberculosis complex (MTBC) subsequent to the branching of 'ancient' lineage 1 and prior to the Eurasian dispersal of 'modern' lineages 2, 3 and 4. In contrast to the major MTBC lineages, the current epidemiology suggests that prevalence of L7 is highly confined to the Ethiopian population, or when identified outside of Ethiopia, it has mainly been in patients of Ethiopian origin. To search for microbiological factors that may contribute to its restricted distribution, we compared the genome of L7 to the genomes of globally dispersed MTBC lineages. The frequency of predicted functional mutations in L7 was similar to that documented in other lineages. These include mutations characteristic of modern lineages - such as constitutive expression of nitrate reductase - as well as mutations in the VirS locus that are commonly found in ancient lineages. We also identified and characterized multiple lineage-specific mutations in L7 in biosynthesis pathways of cell wall lipids, including confirmed deficiency of methoxy-mycolic acids due to a stop-gain mutation in the mmaA3 gene that encodes a methoxy-mycolic acid synthase. We show that the abolished biosynthesis of methoxy-mycolates of L7 alters the cell structure and colony morphology on selected growth media and impacts biofilm formation. The loss of these mycolic acid moieties may change the host-pathogen dynamic for L7 isolates, explaining the limited geographical distribution of L7 and contributing to further understanding the spread of MTBC lineages across the globe.

Inositol Monophosphatase: A Bifunctional Enzyme in Mycobacterium smegmatis.

Inositol monophosphatase (IMPase) is a crucial enzyme for the biosynthesis of phosphatidylinositol, an essential component in mycobacterial cell walls. IMPase A (ImpA) from Mycobacterium smegmatis is a bifunctional enzyme that also functions as a fructose-1,6-bisphosphatase (FBPase). To better understand the bifunctional nature of this enzyme, point mutagenesis was conducted on several key residues and their enzyme activity was tested. Our results along with active site models support the fact that ImpA is a bifunctional enzyme with residues Gly94, Thr95 hypothesized to be contributing to the FBPase activity and residues Trp220, Asp221 hypothesized to be contributing to the IMPase activity. Double mutants, W220A + D221A reduced both FBPase and IMPase activity drastically while the double mutant G94A + T95A surprisingly partially restored the IMPase activity compared to the single mutants. This study establishes the foundation toward obtaining a better understanding of the bifunctional nature of this enzyme.

The assessment of changes to the nontuberculous mycobacterial metabolome in response to anti-TB drugs.

Mycobacterium species can cause a range of nontuberculous infections of healthy and immunocompromised people as well as infected people during and after surgical procedures. The similarity of nontuberculous mycobacteria (NTM) to the tuberculosis bacilli (TB) could ultimately enable the use of anti-TB drugs for the genus. Hence, three NTM (Mycobacterium smegmatis, Mycobacterium phlei and Mycobacterium avium) were cultured under different lab conditions, causing two mycobacterial phenotypes (active and dormant), and treated with isoniazid (INH) and ethambutol (EMB) independently or in combination. Metabolite profiling was applied to facilitate the investigation and characterisation of intracellular targets affected by the antibiotics. Aliquots of the cell culture were taken over the treatment period and the metabolite profile of the cells analysed by gas chromatography mass spectrometry. Comparative analysis of the metabolite levels to untreated mycobacteria confirmed the successful action of the antibiotics on the metabolism of all three species. Furthermore, single metabolites and metabolite pathways affected by the antibiotics could be identified and included, besides the known target sites for INH and EMB on mycobacterial cells, changes in e.g. nucleotide and saccharide levels. The combined treatment highlighted the property of EMB to enhance the effects of INH even under hypoxic culture conditions.

Metabolite analysis of Mycobacterium species under aerobic and hypoxic conditions reveals common metabolic traits.

A metabolite profiling approach has been implemented to elucidate metabolic adaptation at set culture conditions in five Mycobacterium species (two fast- and three slow-growing) with the potential to act as model organisms for Mycobacterium tuberculosis (Mtb). Analysis has been performed over designated growth phases and under representative environments (nutrient and oxygen depletion) experienced by Mtb during infection. The procedure was useful in determining a range of metabolites (60-120 compounds) covering nucleotides, amino acids, organic acids, saccharides, fatty acids, glycerols, -esters, -phosphates and isoprenoids. Among these classes of compounds, key biomarker metabolites, which can act as indicators of pathway/process activity, were identified. In numerous cases, common metabolite traits were observed for all five species across the experimental conditions (e.g. uracil indicating DNA repair). Amino acid content, especially glutamic acid, highlighted the different properties between the fast- and slow-growing mycobacteria studied (e.g. nitrogen assimilation). The greatest similarities in metabolite composition between fast- and slow-growing mycobacteria were apparent under hypoxic conditions. A comparison to previously reported transcriptomic data revealed a strong correlation between changes in transcription and metabolite content. Collectively, these data validate the changes in the transcription at the metabolite level, suggesting transcription exists as one of the predominant modes of cellular regulation in Mycobacterium. Sectors with restricted correlation between metabolites and transcription (e.g. hypoxic cultivation) warrant further study to elucidate and exploit post-transcriptional modes of regulation. The strong correlation between the laboratory conditions used and data derived from in vivo conditions, indicate that the approach applied is a valuable addition to our understanding of cell regulation in these Mycobacterium species.

The application of metabolite profiling to Mycobacterium spp.: determination of metabolite changes associated with growth.

In order to decipher the complex biological networks underlying biochemical and physiological processes, cellular regulation at all levels must be studied. The metabolites determined by metabolomics represent the end-point of cellular regulation and thus vital components of any integrative network. In the case of pathogenic agents such as Mycobacterium tuberculosis metabolomics offers an ideal opportunity to gain a better understanding of how this species adapts to environmental conditions and antimicrobial treatments. In the present study a metabolite profiling protocol for Mycobacterium including optimised quenching, extraction and analysis has been devised. These methods have been applied to three different Mycobacterium spp. demonstrating potential translation across the genus. Steady-state levels of metabolites during growth have been determined for Mycobacterium smegmatis, Mycobacterium phlei and Mycobacterium bovis BCG (Bacillus Calmette-Guérin). The changes of designated biomarkers emphasised phenotypical differences (e.g. nitrogen metabolism) and similarities (e.g. cysteine biosynthesis) between the bacteria. Each time point showed distinguishable metabolic characteristics from early lag to late stationary phase/beginning of non-replicating phase. The combination of the metabolic results with published "omics" data indicated that transcription appeared to be the most predominant mode of cellular regulation utilised by these bacteria studied.

Iron-regulated protein HupB of Mycobacterium tuberculosis positively regulates siderophore biosynthesis and is essential for growth in macrophages.

Mycobacterium tuberculosis expresses the 28-kDa protein HupB (Rv2986c) and the Fe(3+)-specific high-affinity siderophores mycobactin and carboxymycobactin upon iron limitation. The objective of this study was to understand the functional role of HupB in iron acquisition. A hupB mutant strain of M. tuberculosis, subjected to growth in low-iron medium (0.02 µg Fe ml(-1)), showed a marked reduction of both siderophores with low transcript levels of the mbt genes encoding the MB biosynthetic machinery. Complementation of the mutant strain with hupB restored siderophore production to levels comparable to that of the wild type. We demonstrated the binding of HupB to the mbtB promoter by both electrophoretic mobility shift assays and DNA footprinting. The latter revealed the HupB binding site to be a 10-bp AT-rich region. While negative regulation of the mbt machinery by IdeR is known, this is the first report of positive regulation of the mbt operon by HupB. Interestingly, the mutant strain failed to survive inside macrophages, suggesting that HupB plays an important role in vivo.

Systems-based approaches to probing metabolic variation within the Mycobacterium tuberculosis complex.

The Mycobacterium tuberculosis complex includes bovine and human strains of the tuberculosis bacillus, including Mycobacterium tuberculosis, Mycobacterium bovis and the Mycobacterium bovis BCG vaccine strain. M. bovis has evolved from a M. tuberculosis-like ancestor and is the ancestor of the BCG vaccine. The pathogens demonstrate distinct differences in virulence, host range and metabolism, but the role of metabolic differences in pathogenicity is poorly understood. Systems biology approaches have been used to investigate the metabolism of M. tuberculosis, but not to probe differences between tuberculosis strains. In this study genome scale metabolic networks of M. bovis and M. bovis BCG were constructed and interrogated, along with a M. tuberculosis network, to predict substrate utilisation, gene essentiality and growth rates. The models correctly predicted 87-88% of high-throughput phenotype data, 75-76% of gene essentiality data and in silico-predicted growth rates matched measured rates. However, analysis of the metabolic networks identified discrepancies between in silico predictions and in vitro data, highlighting areas of incomplete metabolic knowledge. Additional experimental studies carried out to probe these inconsistencies revealed novel insights into the metabolism of these strains. For instance, that the reduction in metabolic capability observed in bovine tuberculosis strains, as compared to M. tuberculosis, is not reflected by current genetic or enzymatic knowledge. Hence, the in silico networks not only successfully simulate many aspects of the growth and physiology of these mycobacteria, but also provide an invaluable tool for future metabolic studies.

High throughput phenotypic analysis of Mycobacterium tuberculosis and Mycobacterium bovis strains' metabolism using biolog phenotype microarrays.

Tuberculosis is a major human and animal disease of major importance worldwide. Genetically, the closely related strains within the Mycobacterium tuberculosis complex which cause disease are well-characterized but there is an urgent need better to understand their phenotypes. To search rapidly for metabolic differences, a working method using Biolog Phenotype MicroArray analysis was developed. Of 380 substrates surveyed, 71 permitted tetrazolium dye reduction, the readout over 7 days in the method. By looking for ≥5-fold differences in dye reduction, 12 substrates differentiated M. tuberculosis H37Rv and Mycobacterium bovis AF2122/97. H37Rv and a Beijing strain of M. tuberculosis could also be distinguished in this way, as could field strains of M. bovis; even pairs of strains within one spoligotype could be distinguished by 2 to 3 substrates. Cluster analysis gave three clear groups: H37Rv, Beijing, and all the M. bovis strains. The substrates used agreed well with prior knowledge, though an unexpected finding that AF2122/97 gave greater dye reduction than H37Rv with hexoses was investigated further, in culture flasks, revealing that hexoses and Tween 80 were synergistic for growth and used simultaneously rather than in a diauxic fashion. Potential new substrates for growth media were revealed, too, most promisingly N-acetyl glucosamine. Osmotic and pH arrays divided the mycobacteria into two groups with different salt tolerance, though in contrast to the substrate arrays the groups did not entirely correlate with taxonomic differences. More interestingly, these arrays suggested differences between the amines used by the M. tuberculosis complex and enteric bacteria in acid tolerance, with some hydrophobic amino acids being highly effective. In contrast, γ-aminobutyrate, used in the enteric bacteria, had no effect in the mycobacteria. This study proved principle that Phenotype MicroArrays can be used with slow-growing pathogenic mycobacteria and already has generated interesting data worthy of further investigation.

glpX gene of Mycobacterium tuberculosis: heterologous expression, purification, and enzymatic characterization of the encoded fructose 1,6-bisphosphatase II.

The glpX gene (Rv1099c) of Mycobacterium tuberculosis (Mtb) encodes Fructose 1,6-bisphosphatase II (FBPase II; EC 3.1.3.11); 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.

Inositol monophosphate phosphatase genes of Mycobacterium tuberculosis.

BACKGROUND: 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. RESULTS: 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. CONCLUSIONS: 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.

Global effects of inactivation of the pyruvate kinase gene in the Mycobacterium tuberculosis complex.

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.

Functional analysis of a clonal deletion in an epidemic strain of Mycobacterium bovis reveals a role in lipid metabolism.

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.

The pyruvate requirement of some members of the Mycobacterium tuberculosis complex is due to an inactive pyruvate kinase: implications for in vivo growth.

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.

Transposon mutagenesis of Mb0100 at the ppe1-nrp locus in Mycobacterium bovis disrupts phthiocerol dimycocerosate (PDIM) and glycosylphenol-PDIM biosynthesis, producing an avirulent strain with vaccine properties at least equal to those of M. bovis BCG.

The unusual and complex cell wall of pathogenic mycobacteria plays a major role in pathogenesis, with specific complex lipids acting as defensive, offensive, or adaptive effectors of virulence. The phthiocerol and phthiodiolone dimycocerosate esters (PDIMs) comprise one such category of virulence-enhancing lipids. Recent work in several laboratories has established that the Mycobacterium tuberculosis fadD26-mmpL7 (Rv2930-Rv2942) locus plays a major role in PDIM biosynthesis and secretion and that PDIM is required for virulence. Here we describe two independent transposon mutants (WAg533 and WAg537) of Mycobacterium bovis, both of which carry an insertion in Mb0100 (= M. tuberculosis Rv0097) to reveal a new locus involved in PDIM biosynthesis. The mutations have a polar effect on expression of the downstream genes Mb0101, Mb0102 (fadD10), Mb0103, and Mb0104 (nrp), and Mb0100 is shown to be in an operon comprising these genes and Mb0099. Reverse transcription-PCR analysis shows elevated transcription of genes in the operon upstream from the transposon insertion sites in both mutants. Both mutants have altered colony morphology and do not synthesize PDIMs or glycosylphenol-PDIM. Both mutants are avirulent in a guinea pig model of tuberculosis, and when tested as a vaccine, WAg533 conferred protective immunity against M. bovis infection at least equal to that afforded by M. bovis bacillus Calmette-Guerin.

Functional demonstration of reverse transsulfuration in the Mycobacterium tuberculosis complex reveals that methionine is the preferred sulfur source for pathogenic Mycobacteria.

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.

Tubercle bacilli generate a novel cell wall-associated pigment after long-term anaerobic culture.

Many cases of tuberculosis result from reactivation of previously acquired latent infections. Models to study such persister forms often involve gradual depletion of oxygen during culture as poor aeration is a characteristic of non-progressive TB granulomas. Anaerobically cultured bacilli develop a thickened outer-most cell wall layer. Here, we analyzed this layer from anaerobically cultured Mycobacterium tuberculosis and Mycobacterium bovis BCG. By six weeks of anaerobiosis a pigment was detected at levels > 60-fold higher in anaerobic than aerobic bacilli. This pigment was responsible for the electron-dense appearance of the thickened cell wall layer and gave an electrospray mass spectrometry peak at 409 Da (M+Na)+ or (M+H)+. We termed this pigment APP1, anaerobically produced pigment 1, the first pigment identified in M. tuberculosis.

Biological implications of Mycobacterium leprae gene expression during infection.

The genome of Mycobacterium leprae, the etiologic agent of leprosy, has been sequenced and annotated revealing a genome in apparent disarray and in stark contrast to the genome of the related human pathogen, M. tuberculosis. With less than 50% coding capacity of a 3.3-Mb genome and 1,116 pseudogenes, the remaining genes help define the minimal gene set necessary for in vivo survival of this mycobacterial pathogen as well as genes potentially required for infection and pathogenesis seen in leprosy. To identify genes transcribed during infection, we surveyed gene transcripts from M. leprae growing in athymic nude mice using reverse transcriptase-polymerase chain reaction (RT-PCR) and cross-species DNA microarray technologies. Transcripts were detected for 221 open reading frames, which included genes involved in DNA replication, cell division, SecA-dependent protein secretion, energy production, intermediary metabolism, iron transport and storage and genes associated with virulence. These results suggest that M. leprae actively catabolizes fatty acids for energy, produces a large number of secretory proteins, utilizes the full array of sigma factors available, produces several proteins involved in iron transport, storage and regulation in the absence of recognizable genes encoding iron scavengers and transcribes several genes associated with virulence in M. tuberculosis. When transcript levels of 9 of these genes were compared from M. leprae derived from lesions of multibacillary leprosy patients and infected nude mouse foot pad tissue using quantitative real-time RT-PCR, gene transcript levels were comparable for all but one of these genes, supporting the continued use of the foot pad infection model for M. leprae gene expression profiling. Identifying genes associated with growth and survival during infection should lead to a more comprehensive understanding of the ability of M. leprae to cause disease.

The complete genome sequence of Mycobacterium bovis.

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.

The MPB83 antigen from Mycobacterium bovis contains O-linked mannose and (1-->3)-mannobiose moieties.

Mycobacterium tuberculosis and Mycobacterium 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 from M. 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. tuberculosis and 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.