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The ability to construct defined deletions of Mycobacterium tuberculosis has allowed many genes involved in virulence to be identified. Deletion of nutritional genes leads to varying levels of attenuation, presumably reflecting the need for a particular molecule, and the availability (or lack) of that molecule in vivo. We have previously shown that M. tuberculosis mutants lacking either the trpD or ino1 gene are highly attenuated in mouse models of infection, but can grow when supplemented with tryptophan or inositol, respectively. In this paper we have constructed a double Delta trpDDelta ino1 mutant, and show that this is severely attenuated in SCID mouse and guinea pig models. As the strain will grow in the presence of supplements, we propose that this strain could be used for research and antigen preparative purposes, with reduced risks to laboratory workers.

Induction of the Mycobacterium tuberculosis dosR gene, which is known to respond to hypoxia, was measured using RTq-PCR following exposure to different stresses. Increased expression was seen after exposure to S-nitrosoglutathione (GSNO), ethanol and (to a lesser extent) H2O2, but not heat- or cold-shock. We also demonstrated that hspX, which is dependent on dosR for expression, is induced when cultures are left standing for 30 min, while significant but minor induction was seen following a 10 min centrifugation. Microarray analysis was used to compare gene expression in wild-type and deltadosR strains following 30 min standing. Fifty-two genes were significantly up-regulated, and 19 genes were down-regulated. These included genes that had previously been reported as being part of the dosR regulon, and also some novel ones.

Unlabelled: SWTTING: Demonstrating that a gene is essential is always difficult, but this is particularly true for a slow-growing organism such as Mycobacterium tuberculosis. One method currently used is to show that homologous recombination leading to gene inactivation only occurs in the presence of a second copy of the gene, but obtaining statistically significant data can be prohibitively difficult. L5-based integrating plasmids have been widely used in the genetic analysis of mycobacteria. The L5 excisionase has been used in Mycobacterium smegmatis to excise and recover these plasmids from chromosome., Objective: Our aims were to establish whether the L5 excisionase could function in M. tuberculosis to remove an L5-based integrated plasmid and, if so, to use this technology as the basis for an improved method for determining whether a gene is essential., Design: We took two strains of M. tuberculosis carrying the essential gene glnE integrated into the chromosome on an L5-based plasmid, one of which lacked the functional chromosomal copy of the gene. We transformed these with vectors expressing the L5 excisionase and looked for loss of the integrated plasmid., Results: We obtained efficient excision of an integrated vector from the wild-type strain. However, when the integrated vector carried the only functional copy of the essential gene glnE, the numbers of colonies recovered were reduced to background levels., Conclusion: The L5 excisionase does function in M. tuberculosis and can be used to confirm the essentiality of a gene. This technology also allows further analysis of essential genes that is difficult or impossible using current methods., (Copyright 2001 Harcourt Publishers Ltd.)

Drug-resistant tuberculosis has become a major public health problem. Resistance to rifampicin probably arises through mutations in the mycobacterial RNA polymerase. Patients may acquire rifampicin resistant tuberculosis by three mechanisms: (1) infection with a resistant organism, (2) selection of a sub-population of resistant organisms that remain contained whilst the more virulent wild type is present, (3) mutations within the population of bacilli causing the original infection. Sequential isolates of Mycobacterium tuberculosis were collected from 2 patients who developed rifampicin resistance whilst on treatment. One patient was immunosuppressed with HIV-infection; in the other patient the original isolate was also resistant to isoniazid. DNA fingerprinting techniques were used to type the isolates. No differences were found between the fingerprints of isolates from before and after the development of resistance. These data suggest that the third of the mechanisms listed above was responsible for the acquisition of rifampicin resistance in these 2 patients.