Mycobacterium tuberculosis possesses a large number of genes of unknown or predicted function, undermining fundamental understanding of pathogenicity and drug susceptibility. To address this challenge, we developed a high-throughput functional genomics approach combining inducible CRISPR-interference and image-based analyses of morphological features and sub-cellular chromosomal localizations in the related non-pathogen, M. smegmatis. Applying automated imaging and analysis to 263 essential gene knockdown mutants in an arrayed library, we derive robust, quantitative descriptions of bacillary morphologies consequent on gene silencing. Leveraging statistical-learning, we demonstrate that functionally related genes cluster by morphotypic similarity and that this information can be used to inform investigations of gene function. Exploiting this observation, we infer the existence of a mycobacterial restriction-modification system, and identify filamentation as a defining mycobacterial response to histidine starvation. Our results support the application of large-scale image-based analyses for mycobacterial functional genomics, simultaneously establishing the utility of this approach for drug mechanism-of-action studies.

High-throughput essentiality screens have enabled genome-wide assessments of the genetic requirements for growth and survival of a variety of bacteria in different experimental models. The reliance in many of these studies on transposon (Tn)-based gene inactivation has, however, limited the ability to probe essential gene function or design targeted screens. We interrogated the potential of targeted, large-scale, pooled CRISPR interference (CRISPRi)-based screens to extend conventional Tn approaches in mycobacteria through the capacity for positionally regulable gene repression. Here, we report the utility of the "CRISPRi-Seq" method for targeted, pooled essentiality screening, confirming strong overlap with TnSeq datasets. In addition, we exploit this high-throughput approach to provide insight into CRISPRi functionality. By interrogating polar effects and combining image-based phenotyping with CRISPRimediated depletion of selected essential genes, we demonstrate that CRISPRi-Seq can functionally validate Transcriptional Units within operons. Together, these observations suggest the utility of CRISPRiSeq to provide insights into (myco)bacterial gene regulation and expression on a genome-wide scale.

Metabolism underpins the physiology and pathogenesis of Mycobacterium tuberculosis. However, although experimental mycobacteriology has provided key insights into the metabolic pathways that are essential for survival and pathogenesis, determining the metabolic status of bacilli during different stages of infection and in different cellular compartments remains challenging. Recent advances-in particular, the development of systems biology tools such as metabolomics-have enabled key insights into the biochemical state of M. tuberculosis in experimental models of infection. In addition, their use to elucidate mechanisms of action of new and existing antituberculosis drugs is critical for the development of improved interventions to counter tuberculosis. This review provides a broad summary of mycobacterial metabolism, highlighting the adaptation of M. tuberculosis as specialist human pathogen, and discusses recent insights into the strategies used by the host and infecting bacillus to influence the outcomes of the host -pathogen interaction through modulation of metabolic functions.Ultimately, the process of pathogenicity itself will be describable in terms of the chemistry of the mycobacterial cell. Cite this article as Cold Spring Harb Perspect Med 2015;5:a021121 www.perspectivesinmedicine.org BOX 1. RECOMMENDED RESOURCES Physiology of Mycobacterium tuberculosis † Cook GM, Berney M, Gebhard S, Heinemann M, Cox RA, Danilchanka O, Niederweis M. 2009. Physiology of mycobacteria. Adv Microb Physiol 55: 81-319. † Russell DG. 2013. The evolutionary pressures that have molded Mycobacterium tuberculosis into an infectious adjuvant. Curr Opin Microbiol 16: 78-84. Central Carbon Metabolism † Ehrt S, Rhee K. 2013. Mycobacterium tuberculosis metabolism and host interaction: Mysteries and paradoxes.

A DNA damage-inducible mutagenic gene cassette has been implicated in the emergence of drug resistance in Mycobacterium tuberculosis during anti-tuberculosis (TB) chemotherapy. However, the molecular composition and operation of the encoded 'mycobacterial mutasome' - minimally comprising DnaE2 polymerase and ImuA′ and ImuB accessory proteins - remain elusive. Following exposure of mycobacteria to DNA damaging agents, we observe that DnaE2 and ImuB co-localize with the DNA polymerase III β subunit (β clamp) in distinct intracellular foci. Notably, genetic inactivation of the mutasome in an imuBAAAAGG mutant containing a disrupted β clamp-binding motif abolishes ImuB-β clamp focus formation, a phenotype recapitulated pharmacologically by treating bacilli with griselimycin and in biochemical assays in which this β clamp-binding antibiotic collapses pre-formed ImuB-β clamp complexes. These observations establish the essentiality of the ImuB-β clamp interaction for mutagenic DNA repair in mycobacteria, identifying the mutasome as target for adjunctive therapeutics designed to protect anti-TB drugs against emerging resistance.

A DNA damage-inducible mutagenic gene cassette has been implicated in the emergence of drug resistance in Mycobacterium tuberculosis during anti-tuberculosis (TB) chemotherapy. However, the molecular composition and operation of the encoded “mycobacterial mutasome” – minimally comprising DnaE2 polymerase and ImuA′ and ImuB accessory proteins – remain elusive. Following exposure of mycobacteria to DNA damaging agents, we observe that DnaE2 and ImuB co-localize with the DNA polymerase III β subunit (β clamp) in distinct intracellular foci. Notably, genetic inactivation of the mutasome in an imuBAAAAGG mutant containing a disrupted β clamp-binding motif abolishes ImuB-β clamp focus formation, a phenotype recapitulated pharmacologically by treating bacilli with griselimycin and in biochemical assays in which this β clamp-binding antibiotic collapses pre-formed ImuB-β clamp complexes. These observations establish the essentiality of the ImuB-β clamp interaction for mutagenic DNA repair in mycobacteria, identifying the mutasome as target for adjunctive therapeutics designed to protect anti-TB drugs against emerging resistance.

Cobalamin is an essential co-factor in all domains of life, yet its biosynthesis is restricted to some bacteria and archaea. Mycobacterium smegmatis, an environmental saprophyte frequently used as surrogate for the obligate human pathogen, M. tuberculosis, carries approximately 30 genes predicted to be involved in de novo cobalamin biosynthesis. M. smegmatis also encodes multiple cobalamin-dependent enzymes, including MetH, a methionine synthase which catalyzes the final reaction in methionine biosynthesis. In addition to metH, M. smegmatis possesses a cobalamin-independent methionine synthase, metE, suggesting that enzyme use – MetH or MetE – is regulated by cobalamin availability. Consistent with this notion, we previously described a cobalamin-sensing riboswitch controlling metE expression in M. tuberculosis. Here, we apply a targeted mass spectrometry-based approach to confirm de novo cobalamin biosynthesis in M. smegmatis during aerobic growth in vitro. We also demonstrate that M. smegmatis can transport and assimilate exogenous cyanocobalamin (CNCbl; a.k.a. vitamin B12) and its precursor, dicyanocobinamide ((CN)2Cbi). However, the uptake of CNCbl and (CN)2Cbi in this organism is restricted and seems dependent on the conditional essentiality of the cobalamin-dependent methionine synthase. Using gene and protein expression analyses combined with single-cell growth kinetics and live-cell time-lapse microscopy, we show that transcription and translation of metE are strongly attenuated by endogenous cobalamin. These results support the inference that metH essentiality in M. smegmatis results from riboswitch-mediated repression of MetE expression. Moreover, differences observed in cobalamin-dependent metabolism between M. smegmatis and M. tuberculosis provide some insight into the selective pressures which might have shaped mycobacterial metabolism for pathogenicity. IMPORTANCE Alterations in cobalamin-dependent metabolism have marked the evolution of Mycobacterium tuberculosis as human pathogen. However, the role(s) of cobalamin in mycobacterial physiology remain poorly understood. Using the non-pathogenic saprophyte, M. smegmatis, we investigated the production of cobalamin, transport and assimilation of cobalamin precursors, and the role of cobalamin in regulating methionine biosynthesis. We confirm constitutive de novo cobalamin biosynthesis in M. smegmatis, in contrast with M. tuberculosis, which appears to lack de novo cobalamin biosynthetic capacity. We also show that uptake of cyanocobalamin (vitamin B12) and its precursors is restricted in M. smegmatis, apparently depending on the co-factor requirements of the cobalamin-dependent methionine synthase. These observations establish M. smegmatis as informative foil to elucidate key metabolic adaptations enabling mycobacterial pathogenicity.

13Mycobacterium tuberculosis possesses a large number of genes of unknown or merely 14 predicted function, undermining fundamental understanding of pathogenicity and drug 15 susceptibility. To address this challenge, we developed a high-throughput functional 16 genomics approach combining inducible CRISPR-interference and image-based analyses of 17 morphological features and sub-cellular molecular localizations in the related non-pathogen, 18M. smegmatis. Applying automated imaging and analysis to an arrayed library of 272 19 essential gene knockdown mutants, we derive robust, quantitative descriptions of bacillary 20 morphologies consequent on gene silencing. Leveraging statistical-learning, we demonstrate 21 that functionally related genes cluster by morphotypic similarity and that this information 22can be used to infer gene function. Exploiting this observation, we reveal a previously 23 unknown restriction-modification system, and identify filamentation as a defining 24 mycobacterial response to histidine starvation. Our results support the application of large-25 scale image-based analyses for mycobacterial functional genomics, simultaneously 26 establishing the utility of this approach for drug mechanism-of-action studies.

Conspectus Tuberculosis (TB) is the leading cause of mortality globally resulting from an infectious disease, killing almost 1.6 million people annually and accounting for approximately 30% of deaths attributed to antimicrobial resistance (AMR). This despite the widespread administration of a neonatal vaccine, and the availability of an effective combination drug therapy against the causative agent, Mycobacterium tuberculosis (Mtb). Instead, TB prevalence worldwide is characterized by high-burden regions in which co-epidemics, such as HIV, and social and economic factors, undermine efforts to control TB. These elements additionally ensure conditions that favor the emergence of drug-resistant Mtb strains, which further threaten prospects for future TB control. To address this challenge, significant resources have been invested in developing a TB drug pipeline, an initiative given impetus by the recent regulatory approval of two new anti-TB drugs. However, both drugs have been reserved for drug-resistant disease, and the seeming inevitability of new resistance plus the recognized need to shorten the duration of chemotherapy demands continual replenishment of the pipeline with high-quality “hits” with novel mechanisms of action. This represents a massive challenge, which has been undermined by key gaps in our understanding of Mtb physiology and metabolism, especially during host infection. Whereas drug discovery for other bacterial infections can rely on predictive in vitro assays and animal models, for Mtb, inherent metabolic flexibility and uncertainties about the nutrients available to infecting bacilli in different host (micro)environments instead requires educated predictions or demonstrations of efficacy in animal models of arguable relevance to human disease. Even microbiological methods for enumeration of viable mycobacterial cells are fraught with complication. Our research has focused on elucidating those aspects of mycobacterial metabolism that contribute to the robustness of the bacillus to host immunological defenses and applied antibiotics and that, possibly, drive the emergence of drug resistance. This work has identified a handful of metabolic pathways that appear vulnerable to antibiotic targeting. Those highlighted, here, include the inter-related functions of pantothenate and coenzyme A biosynthesis and recycling and nucleotide metabolism—the last of which reinforces our view that DNA metabolism constitutes an under-explored area for new TB drug development. Although nonessential functions have traditionally been deprioritized for antibiotic development, a common theme emerging from this work is that these very functions might represent attractive targets because of the potential to cripple mechanisms critical to bacillary survival under stress (for example, the Rel Mtb -dependent stringent response) or to adaptability under unfavorable, potentially lethal, conditions including antibiotic therapy (for example, ...