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.

ESX-3 is one of the five type VII secretion systems encoded by the Mycobacterium tuberculosis genome. We recently showed the essentiality of ESX-3 for M. tuberculosis viability and proposed its involvement in iron and zinc metabolism. In this study we confirmed the role of ESX-3 in iron uptake and its involvement in the adaptation to low zinc environment in M. tuberculosis. Moreover, we unveiled functional differences between the ESX-3 roles in M. tuberculosis and M. smegmatis showing that in the latter ESX-3 is only involved in the adaptation to iron and not to zinc restriction. Finally, we also showed that in M. tuberculosis this secretion system is essential for iron and zinc homeostasis not only in conditions in which the concentrations of these metals are limiting but also in metal sufficient conditions.

SummaryIn Mycobacterium tuberculosis the decaprenylphospho-D-arabinofuranose (DPA) pathway is a validated target for the drugs ethambutol and benzothiazinones. To identify other potential drug targets in the pathway, we generated conditional knock-down mutants of each gene involved using the TET-PIP OFF system. dprE1, dprE2, ubiA, prsA, rv2361c, tkt and rpiB were confirmed to be essential under non-permissive conditions, whereas rv3807c was not required for survival. In the most vulnerable group, DprE1-depleted cells died faster in vitro and intracellularly than those lacking UbiA and PrsA. Downregulation of DprE1 and UbiA resulted in similar phenotypes, namely swelling of the bacteria, cell wall damage and lysis as observed at the single cell level, by real time microscopy and electron microscopy. By contrast, depletion of PrsA led to cell elongation and implosion, which was suggestive of a more pleiotropic effect. Drug sensitivity assays with known DPA-inhibitors supported the use of conditional knock-down strains for target-based whole-cell screens. Together, our work provides strong evidence for the vulnerability of all but one of the enzymes in the DPA pathway and generates valuable tools for the identification of lead compounds targeting the different biosynthetic steps. PrsA, phosphoribosyl-pyrophosphate synthetase, appears to be a particularly attractive new target for drug discovery.

SummaryBacterial nutrition is an essential aspect of host–pathogen interaction. For the intracellular pathogen Mycobacterium tuberculosis (Mtb), the causative agent of tuberculosis in humans, fatty acids derived from lipid droplets are considered the major carbon source. However, many other soluble nutrients are available inside host cells and may be used as alternative carbon sources. Lactate and pyruvate are abundant in human cells and fluids, particularly during inflammation. In this work, we study Mtb metabolism of lactate and pyruvate combining classic microbial physiology with a ‘multi‐omics’ approach consisting of transposon‐directed insertion site sequencing (TraDIS), RNA‐seq transcriptomics, proteomics and stable isotopic labelling coupled with mass spectrometry‐based metabolomics. We discovered that Mtb is well adapted to use both lactate and pyruvate and that their metabolism requires gluconeogenesis, valine metabolism, the Krebs cycle, the GABA shunt, the glyoxylate shunt and the methylcitrate cycle. The last two pathways are traditionally associated with fatty acid metabolism and, unexpectedly, we found that in Mtb the methylcitrate cycle operates in reverse, to allow optimal metabolism of lactate and pyruvate. Our findings reveal a novel function for the methylcitrate cycle as a direct route for the biosynthesis of propionyl‐CoA, the essential precursor for the biosynthesis of the odd‐chain fatty acids.

The LSM4 gene of Saccharomyces cerevisiae codes for an essential protein involved in pre-mRNA splicing and also in mRNA decapping, a crucial step for mRNA degradation. We previously demonstrated that the first 72 amino acids of the Kluyveromyces lactis Lsm4p (KlLsm4p), which contain the Sm-like domains, can restore cell viability in both K. lactis and S. cerevisiae cells not expressing the endogenous protein. However, the absence of the carboxy-terminal region resulted in a remarkable loss of viability in stationary phase cells (Mazzoni and Falcone, 2001). Herein, we demonstrate that S. cerevisiae cells expressing the truncated LSM4 protein of K. lactis showed the phenotypic markers of yeast apoptosis such as chromatin condensation, DNA fragmentation, and accumulation of reactive oxygen species. The study of deletion mutants revealed that apoptotic markers were clearly evident also in strains lacking genes involved in mRNA decapping, such as LSM1, DCP1, and DCP2, whereas a slight effect was observed in strains lacking the genes DHH1 and PAT1. This is the first time that a connection between mRNA stability and apoptosis is reported in yeast, pointing to mRNA decapping as the crucial step responsible of the observed apoptotic phenotypes. INTRODUCTIONApoptosis is a kind of programmed cell suicide crucial for health, homeostasis, and embryonic development. Its important role in different diseases such as cancer, neurodegenerative disorders, or stroke, and its very complex regulatory network were discovered in model organisms such as Drosophila melanogaster or Caenorhabditis elegans. Recent studies support the notion that apoptosis also occurs in Saccharomyces cerevisiae (Frohlich and Madeo, 2000;Madeo et al., 2002a). Apoptosis in yeast has been demonstrated in very different cases, e.g., in cdc48 mutants (Madeo et al., 1997); in cells expressing Bax, the mammalian apoptosis inducer gene (Ligr et al., 1998); during perturbation of the vesicular trafficking (Levine et al., 2001) and salt stress (Huh et al., 2002); and after cell treatment with osmotin, an antifungal protein from tobacco implicated in hostplant defense (Narasimhan et al., 2001). Very recently, evidence for the existence of a caspase-related protease regulating apoptosis has been reported in yeast cells (Madeo et al., 2002b). Most of these scenarios were related to oxygen stress, suggesting that reactive oxygen species (ROS) are key regulators of yeast apoptosis (Madeo et al., 1999).Interestingly, it has also been reported that altered premRNA splicing or mRNA stability are involved in mammalian apoptotic-linked diseases (Guhaniyogi and Brewer, 2001;Nissim-Rafinia and Kerem, 2002).In previous work (Mazzoni and Falcone, 2001), we demonstrated that the Kluyveromyces lactis LSM4 gene (KlLSM4), and a truncated form (Kllsm4⌬1) still containing the Sm-like domains, could restore viability in a S. cerevisiae strain not expressing the endogenous Lsm4p, a subunit of the Lsm complex that is involved in mRNA decapping and splicing (Cooper et al., 1995;Tharun et al...