Ethambutol (EMB), a frontline antituberculous drug, targets the mycobacterial cell wall, a unique structure among prokaryotes which consists of an outer layer of mycolic acids covalently bound to peptidoglycan via the arabinogalactan. EMB inhibits the polymerization of cell wall arabinan, and results in the accumulation of the lipid carrier decaprenol phosphoarabinose, which suggests that the drug interferes with the transfer of arabinose to the cell wall acceptor. Unfortunately, resistance to EMB has been described in up to 4% of clinical isolates of Mycobacterium tuberculosis and is prevalent among isolates from patients with multidrug-resistant tuberculosis. We used resistance to EMB as a tool to identify genes participating in the biosynthesis of the mycobacterial cell wall. This approach led to the identification of the embCAB gene cluster, recently proposed to encode for mycobacterial arabinosyl transferases. Resistance to EMB results from an accumulation of genetic events determining overexpression of the Emb protein(s), structural mutation in EmbB, or both. Further characterization of these proteins might provide information on targets for new chemotherapeutic agents and might help development of diagnostic strategies for the detection of resistant M. tuberculosis.

The emergence of multidrug-resistant strains ofMycobacterium tuberculosis has resulted in increased interest in the fluoroquinolones (FQs) as antituberculosis agents. To investigate the frequency and mechanisms of FQ resistance in M. tuberculosis, we cloned and sequenced the wild-type gyrA and gyrB genes, which encode the A and B subunits of the DNA gyrase, respectively; DNA gyrase is the main target of the FQs. On the basis of the sequence information, we performed DNA amplification for sequencing and single-strand conformation polymorphism analysis to examine the presumed quinolone resistance regions ofgyrA and gyrB from reference strains (n = 4) and clinical isolates (n = 55). Mutations in codons ofgyrA analogous to those described in other FQ-resistant bacteria were identified in all isolates (n = 14) for which the ciprofloxacin MIC was >2 ,ug/ml. In addition, we selected ciprofloxacin-resistant mutants of Mycobacterium bovis BCG and M. tuberculosis Erdman and H37ra. Spontaneously resistant mutants developed at a frequency of 1 in 107 to 108 at ciprofloxacin concentrations of 2 ,ug/ml, but no primary resistant colonies were selected at higher ciprofloxacin concentrations. Replating of those first-step mutants selected for mutants with high levels of resistance which harbored gyrA mutations similar to those found among clinical FQ-resistant isolates. The gyrA and gyrB sequence information will facilitate analysis of the mechanisms of resistance to drugs which target the gyrase and the implementation of rapid strategies for the estimation of FQ susceptibility in clinical M. tuberculosis isolates.The resurgence of tuberculosis and its incidence in human immunodeficiency virus-positive populations in both developing countries and the industrialized world have been accompanied by the alarming emergence of virulent multidrugresistant tuberculosis (MDR-TB) strains in North American cities (7). Many of these strains have acquired resistance to almost all first-and second-line antituberculosis agents. For this reason, there is an increasing interest in the antimycobacterial actions of the fluoroquinolones (FQs). Against Mycobacterium tuberculosis, the FQs show moderate in vitro activity (4), with sparfloxacin (MIC, 0.25 to 0.5 ,ug/ml) perhaps being the most effective compound (17). The principal target of the quinolones is the DNA gyrase, a type II DNA topoisomerase that is composed of two A and two B subunits (30) encoded by gyrA and gyrB, respectively. Mutations in the putative FQbinding region of the A subunit have been found to confer high-level FQ resistance in several bacterial species (8,19,22,31,33). Other mutations that confer resistance to quinolones have been found in gyrB, in genes that lower the intracellular concentration of the drug (although these tend to confer lower-level resistance than do the gyrA mutations [32,34]), or

An integrated map of the genome of the tubercle bacillus, Mycobacterium tuberculosis, was constructed by using a twin-pronged approach. Pulsed

The systems participating in detoxification of reactive oxygen intermediates in Mycobacterium tuberculosis are believed to play a dual role in the biology of this highly adapted human pathogen: (i) they may contribute to the survival of this bacterium in the host; and (ii) alterations in the gene encoding catalase/peroxidase have been linked to this organism's resistance to the front-line antituberculosis drug isoniazid. These relationships prompted us to extend investigations of the oxidative-stress-response systems in M. tuberculosis by analysing the alkyl hydroperoxide reductase gene ahpC and its putative regulator oxyR. Surprisingly, the oxyR gene was found to be inactivated by multiple lesions in M. tuberculosis H37Rv. These alterations were observed in all M. tuberculosis strains tested, and in members of the M. tuberculosis complex: Mycobacterium bovis BCG, Mycobacterium africanum, and Mycobacterium microti. The corresponding region carrying these genes in Mycobacterium leprae, an organism not sensitive to isoniazid, has a complete oxyR gene divergently transcribed from ahpC. An increase in minimal inhibitory concentration for isoniazid was observed upon transformation of M. tuberculosis H37Rv with cosmids carrying the oxyR-ahpC region of M. leprae. In keeping with the observed inactivation of oxyR, transcriptional activity of the corresponding region in M. tuberculosis was an order of magnitude lower than that of the oxyR gene from M. leprae. While the loss of this putative regulator of oxidative-stress response in M. tuberculosis is paradoxical considering the fact that survival in host macrophages is regarded as a critical feature of this pathogen, it offers a partial explanation for the exquisite sensitivity of M. tuberculosis to isoniazid.

A DraI restriction map of the -435 Mb circular chromosome of the vaccine strain Mycobacterium bovis BCG Pasteur was constructed by linking all 21 DraI fragments, ranging in size from 6 to 820 kb, using specific clones that spanned the DraI recognition sites as hybridization probes. The positions of 20 known genes were also established. Comparison of the resultant genome map with that of the virulent tubercle bacillus Mycobacterium tuberculosis H37Rv revealed extensive global conservation of the genomes of these two members of the M. tuberculosis complex. Possible sites of evolutionary rearrangements were localized on the chromosome of M. bovis 6CG Pasteur by comparing the Asnl restriction profile with that of M. tuberculosis H37Rv. When selected cosmids from the corresponding areas of the genome of M. tuberculosis H37Rv were used as hybridization probes to examine different BCG strains, wild-type M. bovis and M. tuberculosis H37Rv, a number of deletions up t o 10 kb in size, insertions and other polymorphisms were detected. In addition to the known deletions covering the genes for the protein antigens ESAT-6 and mpt64, other genetic loci exhibiting polymorphisms or rearrangements were detected in M. bovis BCG Pasteur.

Using the phoA gene fusion methodology adapted to mycobacteria, several Mycobacterium tuberculosis DNA fragments encoding exported proteins were recently identified. In this paper, the molecular cloning, genomic positioning, nucleotide sequence determination and transcriptional start site mapping of a new M. tuberculosis gene, identified by this methodology, are reported. This gene was called erp (for exported repetitive protein) and has a sequence similar t o that of the Mycobacterium leprae 28 kDa antigen irg gene M. tuberculosis erp gene contains a putative iron box close to the mapped transcriptional start site. The predicted Erp protein displays a typical N-terminal signal sequence, a hydrophobic domain at the C-terminus and harbours repeated amino acid motifs. These structural features are reminiscent of cell-wall-associated surface proteins from Gram-positive bacteria. We found that these repeats are conserved among M. tuberculosis isolates, and are absent from the published M. leprae irg gene sequence. In addition to being present in M. leprae, erp sequences were found in other members of the M. tuberculosis complex, but not in other mycobacteria tested. These results suggest that erp might encode a cell surface component shared by major pat hog en ic m ycobacter ia.Keywords : Mycobacterium tuberculosis, erp, exported repetitive protein INTRODUCTIONMycobacteritlm ttrberctllosis and Mycobacteritlm bovis, the etiologic agents of tuberculosis, are facultative intracellular bacteria. The ability to invade, survive and multiply within macrophages contributes to their pathogenicity. For other intracellular bacterial pathogens, proteins compartmentalized on the outer surface mediate the interaction with the host cells. Examples include the 103 kDa invasin of Yersinia psetldottlberctllosis (Isberg e t al., 1987) or the 80 kDa internalin from Listeria monogdogenes (Gaillard e t al., 1991). Similarly, proteins exported by pathogenic mycobacteria are likely to be involved in the infection of macrophages and intracellular survival.The adaptation to M. ttlberctllosis of a genetic methodology for the identification and phenotypic selection of exported Abbreviation: PhoA, alkaline phosphatase.The GSDB accession number for the sequence reported in this paper is L38851.proteins was recently described by Lim et al. (1995) and Timm e t al. (1994). This method uses the Escbericbia coli periplasmic alkaline phosphatase (PhoA) (Hoffman & Wright, 1985; Manoil e t al., 1990) as a heterologous reporter for exportation in mycobacteria. A plasmid vector allowing gene fusions between a truncated pboA gene and mycobacterial genomic DNA was constructed and used to select translational fusions with PhoA activity.Using this system, a M. tuberculosis DNA fragment showing sequence similarities with a Mycobacterium leprae gene (irg) encoding a 28 kDa exported protein antigen (Lim etal., 1995;Cherayil & Young, 1987), was identified. This M . leprae protein has been previously reported to be a major target of the humoral immune response...

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