Gene_locus Report for: myctu-MBTB

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-Guerin 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.

Virulence and immunity are poorly understood in Mycobacterium tuberculosis. We sequenced the complete genome of the M. tuberculosis clinical strain CDC1551 and performed a whole-genome comparison with the laboratory strain H37Rv in order to identify polymorphic sequences with potential relevance to disease pathogenesis, immunity, and evolution. We found large-sequence and single-nucleotide polymorphisms in numerous genes. Polymorphic loci included a phospholipase C, a membrane lipoprotein, members of an adenylate cyclase gene family, and members of the PE/PPE gene family, some of which have been implicated in virulence or the host immune response. Several gene families, including the PE/PPE gene family, also had significantly higher synonymous and nonsynonymous substitution frequencies compared to the genome as a whole. We tested a large sample of M. tuberculosis clinical isolates for a subset of the large-sequence and single-nucleotide polymorphisms and found widespread genetic variability at many of these loci. We performed phylogenetic and epidemiological analysis to investigate the evolutionary relationships among isolates and the origins of specific polymorphic loci. A number of these polymorphisms appear to have occurred multiple times as independent events, suggesting that these changes may be under selective pressure. Together, these results demonstrate that polymorphisms among M. tuberculosis strains are more extensive than initially anticipated, and genetic variation may have an important role in disease pathogenesis and immunity.

Countless millions of people have died from tuberculosis, a chronic infectious disease caused by the tubercle bacillus. The complete genome sequence of the best-characterized strain of Mycobacterium tuberculosis, H37Rv, has been determined and analysed in order to improve our understanding of the biology of this slow-growing pathogen and to help the conception of new prophylactic and therapeutic interventions. The genome comprises 4,411,529 base pairs, contains around 4,000 genes, and has a very high guanine + cytosine content that is reflected in the biased amino-acid content of the proteins. M. tuberculosis differs radically from other bacteria in that a very large portion of its coding capacity is devoted to the production of enzymes involved in lipogenesis and lipolysis, and to two new families of glycine-rich proteins with a repetitive structure that may represent a source of antigenic variation.

The alpha/beta hydrolase fold superfamily is an ancient and widely diversified group of primarily hydrolytic enzymes. In this review, the adaptations of these proteins to the pathogenic lifestyle of Mycobacterium tuberculosis (Mtb), the causative agent of tuberculosis, are examined. Of the 105 alpha/beta hydrolases identified in Mtb, many are associated with lipid metabolism, particularly in the biosynthesis and maintenance of the Mtb's unique cell envelope, as well in the large number of extracellular lipases that are likely responsible for degradation of host lipid material. alpha/beta hydrolase fold proteins are also involved in the evasion and modulation of the immune response, detoxification and metabolic adaptations, including growth, response to acidification of the intracellular environment and dormancy. A striking feature of Mtb's alpha/beta hydrolases is their diversification into virulence-associated niches. It is clear that the alpha/beta hydrolase fold family has made a significant contribution to Mtb's remarkable success as a pathogen.

The genome sequencing of H37Rv strain of Mycobacterium tuberculosis was completed in 1998 followed by the whole genome sequencing of a clinical isolate, CDC1551 in 2002. Since then, the genomic sequences of a number of other strains have become available making it one of the better studied pathogenic bacterial species at the genomic level. However, annotation of its genome remains challenging because of high GC content and dissimilarity to other model prokaryotes. To this end, we carried out an in-depth proteogenomic analysis of the M. tuberculosis H37Rv strain using Fourier transform mass spectrometry with high resolution at both MS and tandem MS levels. In all, we identified 3176 proteins from Mycobacterium tuberculosis representing ~80% of its total predicted gene count. In addition to protein database search, we carried out a genome database search, which led to identification of ~250 novel peptides. Based on these novel genome search-specific peptides, we discovered 41 novel protein coding genes in the H37Rv genome. Using peptide evidence and alternative gene prediction tools, we also corrected 79 gene models. Finally, mass spectrometric data from N terminus-derived peptides confirmed 727 existing annotations for translational start sites while correcting those for 33 proteins. We report creation of a high confidence set of protein coding regions in Mycobacterium tuberculosis genome obtained by high resolution tandem mass-spectrometry at both precursor and fragment detection steps for the first time. This proteogenomic approach should be generally applicable to other organisms whose genomes have already been sequenced for obtaining a more accurate catalogue of protein-coding genes.

To investigate the molecular characteristics of bacillus Calmette-Guerin (BCG) vaccines, the complete genomic sequence of Mycobacterium bovis BCG Tokyo 172 was determined, and the results were compared with those for BCG Pasteur and other M. tuberculosis complex. The genome of BCG Tokyo had a length of 4,371,711bp and contained 4033 genes, including 3950 genes coding for proteins (CDS). There were 18 regions of difference (showing differences of more than 20bp), 20 insertion or deletion (ins/del) mutations of less than 20bp, and 68 SNPs between the two BCG substrains. These findings are useful for better understanding of the genetic differences in BCG substrains due to in vitro evolution of BCG.

To understand the evolution, attenuation, and variable protective efficacy of bacillus Calmette-Guerin (BCG) vaccines, Mycobacterium bovis BCG Pasteur 1173P2 has been subjected to comparative genome and transcriptome analysis. The 4,374,522-bp genome contains 3,954 protein-coding genes, 58 of which are present in two copies as a result of two independent tandem duplications, DU1 and DU2. DU1 is restricted to BCG Pasteur, although four forms of DU2 exist; DU2-I is confined to early BCG vaccines, like BCG Japan, whereas DU2-III and DU2-IV occur in the late vaccines. The glycerol-3-phosphate dehydrogenase gene, glpD2, is one of only three genes common to all four DU2 variants, implying that BCG requires higher levels of this enzyme to grow on glycerol. Further amplification of the DU2 region is ongoing, even within vaccine preparations used to immunize humans. An evolutionary scheme for BCG vaccines was established by analyzing DU2 and other markers. Lesions in genes encoding sigma-factors and pleiotropic transcriptional regulators, like PhoR and Crp, were also uncovered in various BCG strains; together with gene amplification, these affect gene expression levels, immunogenicity, and, possibly, protection against tuberculosis. Furthermore, the combined findings suggest that early BCG vaccines may even be superior to the later ones that are more widely used.

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-Guerin 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.

Virulence and immunity are poorly understood in Mycobacterium tuberculosis. We sequenced the complete genome of the M. tuberculosis clinical strain CDC1551 and performed a whole-genome comparison with the laboratory strain H37Rv in order to identify polymorphic sequences with potential relevance to disease pathogenesis, immunity, and evolution. We found large-sequence and single-nucleotide polymorphisms in numerous genes. Polymorphic loci included a phospholipase C, a membrane lipoprotein, members of an adenylate cyclase gene family, and members of the PE/PPE gene family, some of which have been implicated in virulence or the host immune response. Several gene families, including the PE/PPE gene family, also had significantly higher synonymous and nonsynonymous substitution frequencies compared to the genome as a whole. We tested a large sample of M. tuberculosis clinical isolates for a subset of the large-sequence and single-nucleotide polymorphisms and found widespread genetic variability at many of these loci. We performed phylogenetic and epidemiological analysis to investigate the evolutionary relationships among isolates and the origins of specific polymorphic loci. A number of these polymorphisms appear to have occurred multiple times as independent events, suggesting that these changes may be under selective pressure. Together, these results demonstrate that polymorphisms among M. tuberculosis strains are more extensive than initially anticipated, and genetic variation may have an important role in disease pathogenesis and immunity.

In this work, we characterize genes in Mycobacterium tuberculosis that are regulated by IdeR (iron-dependent regulator), an iron-responsive DNA-binding protein of the DtxR family that has been shown to regulate iron acquisition in Mycobacterium smegmatis. To identify some of the genes that constitute the IdeR regulon, we searched the M. tuberculosis genome for promoter regions containing the consensus IdeR/DxR binding sequence. Genes preceded by IdeR boxes included a set encoding proteins necessary for iron acquisition, such as the biosynthesis of siderophores (mbtA, mbtB, mbtI), aromatic amino acids (pheA, hisE, hisB-like) and others annotated to be involved in the synthesis of iron-storage proteins (bfrA, bfrB). Some putative IdeR-regulated genes identified in this search encoded proteins predicted to be engaged in the biosynthesis of lipopolysaccharide (LPS)-like molecules (rv3402c), lipids (acpP) and peptidoglycan (murB). We analysed four promoter regions containing putative IdeR boxes, mbtA-mbtB, mbI, rv3402c and bfrA-bfd, for interaction with IdeR and for iron-dependent expression. Gel retardation experiments and DNase footprinting analyses with purified IdeR showed that IdeR binds to these IdeR boxes in vitro. Analysis of the promoters by primer extension indicated that the IdeR boxes are located near the -10 position of each promoter, suggesting that IdeR acts as a transcriptional repressor by blocking RNA polymerase binding. Using quantitative reverse transcriptase-polymerase chain reaction (RT-PCR) coupled to molecular beacons, we showed that mRNA levels of mbtA, mbtB, mbtI, rv3402c and bfd are induced 14- to 49-fold in cultures of M. tuberculosis starved for iron, whereas mRNA levels of bfrA decreased about threefold. We present evidence that IdeR not only acts as a transcriptional repressor but also functions as an activator of bfrA. Three of the IdeR- and iron-repressed genes, mbtB, mbtI and rv3402c, were induced during M. tuberculosis infection of human THP-1 macrophages.

Mycobacterium tuberculosis is an important pathogen of mammals that relies on 2-hydroxyphenyloxazoline-containing siderophore molecules called mycobactins for the acquisition of iron in the restrictive environment of the mammalian macrophage. These compounds have been proposed to be biosynthesized through the action of a cluster of genes that include both nonribosomal peptide synthase and polyketide synthase components. One of these genes encodes a protein, MbtB, that putatively couples activated salicylic acid with serine or threonine and then cyclizes this precursor to the phenyloxazoline ring system. We have used gene replacement through homologous recombination to delete the mbtB gene and replace this with a hygromycin-resistance cassette in the virulent strain of M. tuberculosis H37Rv. The resulting mutant is restricted for growth in iron-limited media but grows normally in iron-replete media. Analysis of siderophore production by this organism revealed that the biosynthesis of all salicylate-derived siderophores was interrupted. The mutant was found to be impaired for growth in macrophage-like THP-1 cells, suggesting that siderophore production is required for virulence of M. tuberculosis. These results provide conclusive evidence linking this genetic locus to siderophore production.

Countless millions of people have died from tuberculosis, a chronic infectious disease caused by the tubercle bacillus. The complete genome sequence of the best-characterized strain of Mycobacterium tuberculosis, H37Rv, has been determined and analysed in order to improve our understanding of the biology of this slow-growing pathogen and to help the conception of new prophylactic and therapeutic interventions. The genome comprises 4,411,529 base pairs, contains around 4,000 genes, and has a very high guanine + cytosine content that is reflected in the biased amino-acid content of the proteins. M. tuberculosis differs radically from other bacteria in that a very large portion of its coding capacity is devoted to the production of enzymes involved in lipogenesis and lipolysis, and to two new families of glycine-rich proteins with a repetitive structure that may represent a source of antigenic variation.

BACKGROUND: Many pathogenic bacteria secrete iron-chelating siderophores as virulence factors in the iron-limiting environments of their vertebrate hosts to compete for ferric iron. Mycobacterium tuberculosis mycobactins are mixed polyketide/nonribosomal peptides that contain a hydroxyaryloxazoline cap and two N-hydroxyamides that together create a high-affinity site for ferric ion. The mycobactin structure is analogous to that of the yersiniabactin and vibriobactin siderophores from the bacteria that cause plague and cholera, respectively.
RESULTS: A ten-gene cluster spanning 24 kilobases of the M. tuberculosis genome, designated mbtA-J, contains the core components necessary for mycobactin biogenesis. The gene products MbtB, MbtE and MbtF are proposed to be peptide synthetases, MbtC and MbtD polyketide synthases, MbtI an isochorismate synthase that provides a salicylate activated by MbtA, and MbtG a required hydroxylase. An aryl carrier protein (ArCP) domain is encoded in mbtB, and is probably the site of siderophore chain initiation. Overproduction and purification of the mbtB ArCP domain and MbtA in Escherichia coli allowed validation of the mycobactin initiation hypothesis, as sequential action of PptT (a phosphopantetheinyl transferase) and MbtA (a salicyl-AMP ligase) resulted in the mbtB ArCP domain being activated as salicyl-S-ArCP.
CONCLUSIONS: Mycobactins are produced in M. tuberculosis using a polyketide synthase/nonribosomal peptide synthetase strategy. The mycobactin gene cluster has organizational homologies to the yersiniabactin and enterobactin synthetase genes. Enzymatic targets for inhibitor design and therapeutic intervention are suggested by the similar ferric-ion ligation strategies used in the siderophores from Mycobacteria, Yersinia and E. coli pathogens.