A new evolutionary scenario for the Mycobacterium tuberculosis complex.

The distribution of 20 variable regions resulting from insertion-deletion events in the genomes of the tubercle bacilli has been evaluated in a total of 100 strains of Mycobacterium tuberculosis, Mycobacterium africanum, Mycobacterium canettii, Mycobacterium microti, and Mycobacterium bovis. This approach showed that the majority of these polymorphisms did not occur independently in the different strains of the M. tuberculosis complex but, rather, resulted from ancient, irreversible genetic events in common progenitor strains. Based on the presence or absence of an M. tuberculosis specific deletion (TbD1), M. tuberculosis strains can be divided into ancestral and "modern" strains, the latter comprising representatives of major epidemics like the Beijing, Haarlem, and African M. tuberculosis clusters. Furthermore, successive loss of DNA, reflected by region of difference 9 and other subsequent deletions, was identified for an evolutionary lineage represented by M. africanum, M. microti, and M. bovis that diverged from the progenitor of the present M. tuberculosis strains before TbD1 occurred. These findings contradict the often-presented hypothesis that M. tuberculosis, the etiological agent of human tuberculosis evolved from M. bovis, the agent of bovine disease. M. canettii and ancestral M. tuberculosis strains lack none of these deleted regions, and, therefore, seem to be direct descendants of tubercle bacilli that existed before the M. africanum-->M. bovis lineage separated from the M. tuberculosis lineage. This observation suggests that the common ancestor of the tubercle bacilli resembled M. tuberculosis or M. canettii and could well have been a human pathogen already.

Genomics of Mycobacterium bovis.

The imminent completion of the genome sequence of Mycobacterium bovis will reveal the genetic blueprint for this most successful pathogen. Comparative analysis with the genome sequences of M. tuberculosis and M. bovis BCG promises to expose the genetic basis for the phenotypic differences between the tubercle bacilli, offering unparalleled insight into the virulence factors of the M. tuberculosis complex. Initial analysis of the sequence data has already revealed a novel deletion from M. bovis, as well as identifying variation in members of the PPE family of proteins. As the study of bacterial pathogenicity enters the postgenomic phase, the genome sequence of M. bovis promises to serve as a cornerstone of mycobacterial genetics.

Massive gene decay in the leprosy bacillus.

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.

The decaying genome of Mycobacterium leprae.

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.

The integrated genome map of Mycobacterium leprae.

The integrated map of the Mycobacterium leprae genome unveiled for the first time the genomic organization of this obligate intracellular parasite. Selected cosmid clones, isolated from a genomic library created in the cosmid vector Lorist6, were identified as representing nearly the complete genome and were subsequently used in the M. leprae genome sequencing project. Now a new version of the integrated map of M. leprae can be presented, combining the mapping results from the Lorist6 cosmids with data obtained from a second genomic library constructed in an Escherichia coli-mycobacterium shuttle cosmid, pYUB18. More than 98% of the M. leprae genome is now covered by overlapping large insert genomic clones representing a renewable source of well defined DNA segments and a powerful tool for functional genomics.

Comparative genomics of the mycobacteria.

The genus mycobacteria includes two important human pathogens Mycobacterium tuberculosis and Mycobacterium lepra. The former is reputed to have the highest annual global mortality of all pathogens. Their slow growth, virulence for humans and particular physiology makes these organisms extremely difficult to work with. However the rapid development of mycobacterial genomics following the completion of the Mycobacterium tuberculosis genome sequence provides the basis for a powerful new approach for the understanding of these organisms. Five further genome sequencing projects of closely related mycobacterial species with differing host range, virulence for humans and physiology are underway. A comparative genomic analysis of these species has the potential to define the genetic basis of these phenotypes which will be invaluable for the development of urgently needed new vaccines and drugs. This minireview summarises the different techniques that have been employed to compare these genomes and gives an overview of the wealth of data that has already been generated by mycobacterial comparative genomics.

Comparative genomics of the leprosy and tubercle bacilli.

To achieve the quantum leap in understanding required to overcome two major human diseases, leprosy and tuberculosis, systematic and comparative genome analysis has been undertaken. New insight into the biology of their causative agents has been obtained and the principle findings are reported here.

Identification of variable regions in the genomes of tubercle bacilli using bacterial artificial chromosome arrays.

Whole-genome comparisons of the tubercle bacilli were undertaken using ordered bacterial artificial chromosome (BAC) libraries of Mycobacterium tuberculosis and the vaccine strain, Mycobacterium bovis BCG-Pasteur, together with the complete genome sequence of M. tuberculosis H37Rv. Restriction-digested BAC arrays of M. tuberculosis H37Rv were used in hybridization experiments with radiolabelled M. bovis BCG genomic DNA to reveal the presence of 10 deletions (RD1-RD10) relative to M. tuberculosis. Seven of these regions, RD4-RD10, were also found to be deleted from M. bovis, with the three M. bovis BCG-specific deletions being identical to the RD1-RD3 loci described previously. The distribution of RD4-RD10 in Mycobacterium africanum resembles that of M. tuberculosis more closely than that of M. bovis, whereas an intermediate arrangement was found in Mycobacterium microti, suggesting that the corresponding genes may affect host range and virulence of the various tubercle bacilli. Among the known products encoded by these loci are a copy of the proposed mycobacterial invasin Mce, three phospholipases, several PE, PPE and ESAT-6 proteins, epoxide hydrolase and an insertion sequence. In a complementary approach, direct comparison of BACs uncovered a third class of deletions consisting of two M. tuberculosis H37Rv loci, RvD1 and RvD2, deleted from the genome relative to M. bovis BCG and M. bovis. These deletions affect a further seven genes, including a fourth phospholipase, plcD. In summary, the insertions and deletions described here have important implications for our understanding of the evolution of the tubercle complex.

Deciphering the biology of Mycobacterium tuberculosis from the complete genome sequence.

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.

Use of a Mycobacterium tuberculosis H37Rv bacterial artificial chromosome library for genome mapping, sequencing, and comparative genomics.

The bacterial artificial chromosome (BAC) cloning system is capable of stably propagating large, complex DNA inserts in Escherichia coli. As part of the Mycobacterium tuberculosis H37Rv genome sequencing project, a BAC library was constructed in the pBeloBAC11 vector and used for genome mapping, confirmation of sequence assembly, and sequencing. The library contains about 5,000 BAC clones, with inserts ranging in size from 25 to 104 kb, representing theoretically a 70-fold coverage of the M. tuberculosis genome (4.4 Mb). A total of 840 sequences from the T7 and SP6 termini of 420 BACs were determined and compared to those of a partial genomic database. These sequences showed excellent correlation between the estimated sizes and positions of the BAC clones and the sizes and positions of previously sequenced cosmids and the resulting contigs. Many BAC clones represent linking clones between sequenced cosmids, allowing full coverage of the H37Rv chromosome, and they are now being shotgun sequenced in the framework of the H37Rv sequencing project. Also, no chimeric, deleted, or rearranged BAC clones were detected, which was of major importance for the correct mapping and assembly of the H37Rv sequence. The minimal overlapping set contains 68 unique BAC clones and spans the whole H37Rv chromosome with the exception of a single gap of approximately 150 kb. As a postgenomic application, the canonical BAC set was used in a comparative study to reveal chromosomal polymorphisms between M. tuberculosis, M. bovis, and M. bovis BCG Pasteur, and a novel 12.7-kb segment present in M. tuberculosis but absent from M. bovis and M. bovis BCG was characterized. This region contains a set of genes whose products show low similarity to proteins involved in polysaccharide biosynthesis. The H37Rv BAC library therefore provides us with a powerful tool both for the generation and confirmation of sequence data as well as for comparative genomics and other postgenomic applications. It represents a major resource for present and future M. tuberculosis research projects.

Multiplex sequencing of 1.5 Mb of the Mycobacterium leprae genome.

The nucleotide sequence of 1.5 Mb of genomic DNA from Mycobacterium leprae was determined using computer-assisted multiplex sequencing technology. This brings the 2.8-Mb M. leprae genome sequence to approximately 66% completion. The sequences, derived from 43 recombinant cosmids, contain 1046 putative protein-coding genes, 44 repetitive regions, 3 tRNAs, and 15 tRNAs. The gene density of one per 1.4 kb is slightly lower than that of Mycoplasma (1.2 kb). Of the protein coding genes, 44% have significant matches to genes with well-defined functions. Comparison of 1157 M. leprae and 1564 Mycobacterium tuberculosis proteins shows a complex mosaic of homologous genomic blocks with up to 22 adjacent proteins in conserved map order. Matches to known enzymatic, antigenic, membrane, cell wall, cell division, multidrug resistance, and virulence proteins suggest therapeutic and vaccine targets. Unusual features of the M. leprae genome include large polyketide synthase (pks) operons, inteins, and highly fragmented pseudogenes.

On the catalase-peroxidase gene, katG, of Mycobacterium leprae and the implications for treatment of leprosy with isoniazid.

The toxicity of the potent tuberculocidal agent, isoniazid, is mediated by the heme-containing enzyme, catalase-peroxidase, encoded by the katG gene. Although isoniazid has been used for the treatment of leprosy, it is shown here that the katG gene of Mycobacterium leprae is a pseudogene, which has probably been inactivated by multiple mutations. Inactive genes were detected by the polymerase chain reaction in several isolates of M. leprae, of different geographical origins, and attempts to complement an isoniazid-resistant strain of Mycobacterium smegmatis with the katG pseudogene were unsuccessful. Isoniazid is thus likely to be of no therapeutic benefit to leprosy patients.

Gene arrangement and organization in a approximately 76 kb fragment encompassing the oriC region of the chromosome of Mycobacterium leprae.

A continuous 75627 bp segment of the Mycobacterium leprae chromosome spanning the oriC region was sequenced. The gene order at this locus was similar to that found in the replication origin region of many other prokaryotes, particularly Mycobacterium tuberculosis and Streptomyces coelicolor. As in the case of several Gram-positive bacteria, essential genes involved in basic cellular functions, such as DNA or RNA metabolism (dnaA, dnaB, dnaN, gyrB, gyrA, pcnB, recF, rnpA, ssb), cell wall synthesis (ponA, pbpA) and probably cell division (gidB, rodA) were found. Strikingly, the gidA gene was absent from this part of the genome and there was no rRNA operon near oriC. The gyrA gene harbours an intein coding sequence indicating that protein splicing is required to produce the mature A subunit of DNA gyrase. Among the many other noteworthy features were ORFs encoding putative serine/threonine protein kinases and a protein phosphatase, three tRNA genes, one M. leprae-specific repetitive element and a glnQ pseudogene.

An integrated map of the genome of the tubercle bacillus, Mycobacterium tuberculosis H37Rv, and comparison with Mycobacterium leprae.

An integrated map of the genome of the tubercle bacillus, Mycobacterium tuberculosis, was constructed by using a twin-pronged approach. Pulsed-field gel electrophoretic analysis enabled cleavage sites for Asn I and Dra I to be positioned on the 4.4-Mb circular chromosome, while, in parallel, clones from two cosmid libraries were ordered into contigs by means of fingerprinting and hybridization mapping. The resultant contig map was readily correlated with the physical map of the genome via the landmarked restriction sites. Over 165 genes and markers were localized on the integrated map, thus enabling comparisons with the leprosy bacillus, Mycobacterium leprae, to be undertaken. Mycobacterial genomes appear to have evolved as mosaic structures since extended segments with conserved gene order and organization are interspersed with different flanking regions. Repetitive sequences and insertion elements are highly abundant in M. tuberculosis, but the distribution of IS6110 is apparently nonrandom.

Cloning, sequencing, and expression of the apa gene coding for the Mycobacterium tuberculosis 45/47-kilodalton secreted antigen complex.

Effective protection against a virulent challenge with Mycobacterium tuberculosis is induced mainly by previous immunization with living attenuated mycobacteria, and it has been hypothesized that secreted proteins serve as major targets in the specific immune response. To identify and purify molecules present in culture medium filtrate which are dominant antigens during effective vaccination, a two-step selection procedure was used to select antigens able to interact with T lymphocytes and/or antibodies induced by immunization with living bacteria and to counterselect antigens interacting with the immune effectors induced by immunization with dead bacteria. A Mycobacterium bovis BCG 45/47-kDa antigen complex, present in BCG culture filtrate, has been previously identified and isolated (F. Romain, A. Laqueyrerie, P. Militzer, P. Pescher, P. Chavarot, M. Lagranderie, G. Auregan, M. Gheorghiu, and G. Marchal, Infect. Immun. 61:742-750, 1993). Since the cognate antibodies recognize the very same antigens present in M. tuberculosis culture medium filtrates, a project was undertaken to clone, express, and sequence the corresponding gene of M. tuberculosis. An M. tuberculosis shuttle cosmid library was transferred in Mycobacterium smegmatis and screened with a competitive enzyme-linked immunosorbent assay to detect the clones expressing the proteins. A clone containing a 40-kb DNA insert was selected, and by means of subcloning in Escherichia coli, a 2-kb fragment that coded for the molecules was identified. An open reading frame in the 2,061-nucleotide sequence codes for a secreted protein with a consensus signal peptide of 39 amino acids and a predicted molecular mass of 28,779 Da. The gene was referred to as apa because of the high percentages of proline (21.7%) and alanine (19%) in the purified protein. Southern hybridization analysis of digested total genomic DNA from M. tuberculosis (reference strains H37Rv and H37Ra) indicated that the apa gene was present as a single copy on the genome. The N-terminal identity or homology of the M. tuberculosis and M. bovis BCG purified molecules and their similar global and deduced amino acid compositions demonstrated the perfect correspondence between the molecular and chemical analyses. The presence of a high percentage of proline (21.7%) was confirmed and explained the apparent higher molecular mass (45/47 kDa) determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis resulting from the increased rigidity of molecules due to proline residues.(ABSTRACT TRUNCATED AT 400 WORDS)

Characterization of the gene encoding the immunodominant 35 kDa protein of Mycobacterium leprae.

Analysis of the interaction between the host immune system and the intracellular parasite Mycobacterium leprae has identified a 35 kDa protein as a dominant antigen. The native 35 kDa protein was purified from the membrane fraction of M. leprae and termed MMPI (major membrane protein I). As the purified protein was not amenable to N-terminal sequencing, partial proteolysis was used to establish the sequences of 21 peptides. A fragment of the 35 kDa protein-encoding gene was amplified by the polymerase chain reaction from M. leprae chromosomal DNA with oligonucleotide primers derived from internal peptide sequences and the whole gene was subsequently isolated from a M. leprae cosmid library. The nucleotide sequence of the gene revealed an open reading frame of 307 amino acids containing most of the peptide sequences derived from the native 35 kDa protein. The calculated subunit mass was 33.7 kDa, but the native protein exists as a multimer of 950 kDa. Database searches revealed no identity between the 35 kDa antigen and known protein sequences. The gene was expressed in Mycobacterium smegmatis under the control of its own promoter or at a higher level using an 'up-regulated' promoter derived from Mycobacterium fortuitum. The gene product reacted with monoclonal antibodies raised to the native protein. Using the bacterial alkaline phosphatase reporter system, we observed that the 35 kDa protein was unable to be exported across the membrane of recombinant M. smegmatis. The 35 kDa protein-encoding gene is absent from members of the Mycobacterium tuberculosis complex, but homologous sequences were detected in Mycobacterium avium, Mycobacterium haemophilum and M. smegmatis. The availability of the recombinant 35 kDa protein will permit dissection of both antibody- and T-cell-mediated immune responses in leprosy patients.

Use of an ordered cosmid library to deduce the genomic organization of Mycobacterium leprae.

In an attempt to unify the genetic and biological research on Mycobacterium leprae, the aetiological agent of leprosy, a cosmid library was constructed and then ordered by a combination of fingerprinting and hybridization techniques. The genome of M. leprae is represented by four contigs of overlapping clones which, together, account for nearly 2.8Mb of DNA. Several arguments suggest that the gaps between the contigs are small in size and that virtually complete coverage of the chromosome has been obtained. All of the cloned M. leprae genes have been positioned on the contig maps together with the 29 copies of the dispersed repetitive element, RLEP. These have been classified into four groups on the basis of differences in their organization. Several key housekeeping genes were identified and mapped by hybridization with heterologous probes, and the current genome map of this uncultivable pathogen comprises 72 loci.

Nucleotide sequence of the first cosmid from the Mycobacterium leprae genome project: structure and function of the Rif-Str regions.

The nucleotide sequence of cosmid B1790, carrying the Rif-Str regions of the Mycobacterium leprae chromosome, has been determined. Twelve open reading frames were identified in the 36716bp sequence, representing 40% of the coding capacity. Five ribosomal proteins, two elongation factors and the beta and beta' subunits of RNA polymerase have been characterized and two novel genes were found. One of these encodes a member of the so-called ABC family of ATP-binding proteins while the other appears to encode an enzyme involved in repairing genomic lesions caused by free radicals. This finding may well be significant as M. leprae, an intracellular pathogen, lives within macrophages.