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 putative two-component system, mtrA-mtrB, was isolated from M. tuberculosis H37Rv by using phoB from Pseudomonas aeruginosa as a hybridization probe. The predicted gene product of mtrA displayed high similarity with typical response regulators, including AfsQ1, PhoB, PhoP, and OmpR. The predicted gene product of mtrB displayed similarities with the histidine protein kinases AfsQ2, PhoR, and EnvZ and other members of this class of proteins. Expression analysis in the T7 system showed that mtrA encoded a polypeptide with an apparent molecular mass of 30 kDa. MtrA was overproduced, purified, and demonstrated to participate in typical phosphotransfer reactions using a heterologous histidine protein kinase, CheA, as a phosphoryl group donor. Mycobacterium bovis BCG, harboring an mtrA-gfp (green fluorescent protein cDNA) transcriptional fusion, was used to monitor mtrA expression in infected J774 monolayers. Flow cytometric and fluorescence microscopic analyses indicated that the mtrA promoter was activated upon entry and incubation in J774 macrophages. In contrast, the hsp60-gfp fusion displayed no change in expression under the growth conditions tested. These results suggest a potential role for mtrA in adaptation of the M. tuberculosis complex organisms to environmental changes which may include intracellular conditions. Mycobacterium tuberculosis is notorious for both its slow growth and global importance as a human pathogen (4). Although there is a relatively safe vaccine, Mycobacterium bovis bacille Calmette-Guérin (BCG), against tuberculosis, its effectiveness appears to be variable (29, 32). Efforts to develop new vaccines and therapeutic approaches are hampered by the paucity of information regarding potential virulence factors of M. tuberculosis (31). Improved knowledge and identification of genes and functions critical for host-pathogen interactions that render this organism such a successful human pathogen are needed in order to refine the existing treatments or provide novel targets for intervention. In this context, and in the area of M. tuberculosis fundamental biology, the perception of this bacterium as an inert participant in pathogenic processes continues to be challenged by the latest advances in molecular genetic analyses. However, in some instances the views of M. tuberculosis as a static organism have received affirmation. For example, it has been recently reported that at least in one case, M. tuberculosis has a nonfunctional regulator of oxidative stress, oxyR (11, 35), whose homologs in other organisms are critical to their adjustments to endogenous and exogenous reactive oxygen intermediates (13). The oxyR gene is inactivated by multiple lesions in all strains of M. tuberculosis tested and in all members of the M. tuberculosis complex, representing a phenomenon that has been linked to the exquisite sensitivity of this organism to isonicotinic acid hydrazide (11). This finding appears to support the notion of preset expression levels for at least some subsets of genes in M. tuberculosis.In ...

Understanding promoter regulation and signal-transduction systems in pathogenic mycobacteria is critical for uncovering the processes that govern interactions of these bacteria with the human host. In order to develop additional genetic tools for analysis of mycobacterial promoters, the xyIE gene from Pseudomonas was tested as a transcriptional fusion reporter in fast- and slow-growing mycobacteria. Initially, its utility was demonstrated by expression behind the hsp60 promoter in Mycobacterium smegmatis and Mycobacterium bovis BCG. The presence of an active promoter in front of the promoterless xyIE cassette on a plasmid was scored by development of a bright yellow colour upon spraying of mycobacterial colonies on plates with a solution of catechol. The gene product of xyIE, catechol 2,3 dioxygenase, was measurable in sonic extracts and whole cells, permitting quantitative determination of promoter activity in both fast- and slow-growing mycobacteria. The xyIE-based mycobacterial transcriptional fusion plasmid pRCX3 was constructed and used to assess promoter activity within the sequences located upstream of the newly characterized Mycobacterium tuberculosis H37Rv response regulator mtrA, a member of the superfamily of bacterial signal-transduction systems.

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