Recently, a novel type of secretory pathway, type VII secretion systems (T7SSs), has been characterized in mycobacteria. The chromosomes of Mycobacterium tuberculosis and Mycobacterium bovis encode five T7SSs (ESX-1 to ESX-5). The best characterized of them, ESX-1, is involved in host-pathogen interactions, and its deletion is one of the main causes of M. bovis BCG attenuation. Another T7SS, ESX-3, has been previously shown to be transcriptionally controlled by the zinc uptake repressor (Zur) and by the iron-dependent transcriptional repressor (IdeR), suggesting that it might be involved in zinc and iron homeostasis. In this study, we characterized an M. tuberculosis conditional mutant in which transcription of the ESX-3 gene cluster can be downregulated by anhydrotetracycline. We showed that this T7SS is essential for growth and that this phenotype can be complemented by zinc, iron, or supernatant from a wild-type parental strain culture, demonstrating that the ESX-3 secretion system is responsible for the secretion of some soluble factor(s) required for growth that is probably involved in optimal iron and zinc uptake.

Tightly regulated gene expression systems represent invaluable tools for studying gene function and for the validation of drug targets in bacteria. While several regulated bacterial promoters have been characterized, few of them have been successfully used in mycobacteria. In this article we describe the development of a novel repressible promoter system effective in both fast- and slow-growing mycobacteria based on two chromosomally encoded repressors, dependent on tetracycline (TetR) and pristinamycin (Pip), respectively. This uniqueness results in high versatility and stringency. Using this method we were able to obtain an ftsZ conditional mutant in Mycobacterium smegmatis and a fadD32 conditional mutant in Mycobacterium tuberculosis, confirming their essentiality for bacterial growth in vitro. This repressible promoter system could also be exploited to regulate gene expression during M. tuberculosis intracellular growth.

In order to gain additional understanding of the physiological mechanisms used by bacteria to maintain surface homeostasis and to identify potential targets for new antibacterial drugs, we analysed the variation of the Mycobacterium tuberculosis transcriptional profile in response to inhibitory and subinhibitory concentrations of vancomycin. Our analysis identified 153 genes differentially regulated after exposing bacteria to a concentration of the drug ten times higher than the MIC, and 141 genes differentially expressed when bacteria were growing in a concentration of the drug eightfold lower than the MIC. Hierarchical clustering analysis indicated that the response to these different conditions is different, although with some overlap. This approach allowed us to identify several genes whose products could be involved in the protection from antibiotic stress targeting the envelope and help to confer the basal level of M. tuberculosis resistance to antibacterial drugs, such as Rv2623 (UspA-like), Rv0116c, PE20-PPE31, PspA and proteins related to toxin-antitoxin systems. Moreover, we also demonstrated that the alternative sigma factor s E confers basal resistance to vancomycin, once again underlining its importance in the physiology of the mycobacterial surface stress response. INTRODUCTIONMycobacterium tuberculosis remains one of the world's most prevalent and serious pathogens. It is estimated that every year 2 million people die as a direct result of tuberculosis and that there is a reservoir of 2 billion cryptically infected people (Dye et al., 1999). Of these asymptomatic carriers, around 5 % will develop active disease at some stage in their lives and in doing so will contribute to the ongoing transmission of infection (Raviglione, 2003). The recent emergence of multidrugresistant (MDR) and extensively drug-resistant (XDR-TB) strains (Gandhi et al., 2006) has raised the importance of searching for alternative targets to develop new antimycobacterial drugs.Due to the importance of its physiological role and its difference from the eukaryotic cell surface, the bacterial cell wall is one of the best candidates in the search for new drug targets. A large number of antibacterial drugs currently in use are directed against surface components or metabolic pathways that are involved in their synthesis. For example, b-lactams, cephalosporins, glycopeptides, phosphomycin, bacitracin and cycloserine inhibit peptidoglycan biosynthesis, while polymyxin interferes with the cell-membrane structure. Moreover, compounds such as isoniazid, pyrazinamide, ethambutol and ethionamide target typical components of the mycobacterial cell surface. In spite of a good knowledge of the chemical composition of the mycobacterial surface, not much is known about its organization and physiology (Barry, 2001). The recent demonstration of the presence of an outer membrane will surely boost research in this field (Hoffmann et al., 2008;Zuber et al., 2008).One strategy to study bacterial surface physiology is to characterize the variation o...

MmpL3 is an inner membrane transporter of Mycobacterium tuberculosis responsible for the export of trehalose momomycolate, a precursor of the mycobacterial outer membrane component trehalose dimycolate (TDM), as well as mycolic acids bound to arabinogalactan. MmpL3 represents an emerging target for tuberculosis therapy. In this paper, we describe the construction and characterization of an mmpL3 knockdown strain of M. tuberculosis. Downregulation of mmpL3 led to a stop in bacterial division and rapid cell death, preceded by the accumulation of TDM precursors. MmpL3 was also shown to be essential for growth in monocyte-derived human macrophages. Using RNA-seq we also found that MmpL3 depletion caused up-regulation of 47 genes and down-regulation of 23 genes (at least 3-fold change and false discovery rate ≤1%). Several genes related to osmoprotection and metal homeostasis were induced, while several genes related to energy production and mycolic acids biosynthesis were repressed suggesting that inability to synthesize a correct outer membrane leads to changes in cellular permeability and a metabolic downshift.