Browsing by Author "Mizrahi, Valerie"
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- ItemOpen AccessCleavage of the moaX-encoded fused molybdopterin synthase from Mycobacterium tuberculosis is necessary for activity(BioMed Central, 2015-02-06) Narrandes, Nicole C; Machowski, Edith E; Mizrahi, Valerie; Kana, Bavesh DBackground: Molybdopterin cofactor (MoCo) biosynthesis in Mycobacterium tuberculosis is associated with a multiplicity of genes encoding several enzymes in the pathway, including the molybdopterin (MPT) synthase, a hetero tetramer comprising two MoaD and two MoaE subunits. In addition to moaD1, moaD2, moaE1, moaE2, the M. tuberculosis genome also contains a moaX gene which encodes an MPT-synthase in which the MoaD and MoaE domains are located on a single polypeptide. In this study, we assessed the requirement for post-translational cleavage of MoaX for functionality of this novel, fused MPT synthase and attempted to establish a functional hierarchy for the various MPT-synthase encoding genes in M. tuberculosis. Results: Using a heterologous Mycobacterium smegmatis host and the activity of the MoCo-dependent nitrate reductase, we confirmed that moaD2 and moaE2 from M. tuberculosis together encode a functional MPT synthase. In contrast, moaD1 displayed no functionality in this system, even in the presence of the MoeBR sulphurtransferase, which contains the rhodansese-like domain, predicted to activate MoaD subunits. We demonstrated that cleavage of MoaX into its constituent MoaD and MoaE subunits was required for MPT synthase activity and confirmed that cleavage occurs between the Gly82 and Ser83 residues in MoaX. Further analysis of the Gly81-Gly82 motif confirmed that both of these residues are necessary for catalysis and that the Gly81 was required for recognition/cleavage of MoaX by an as yet unidentified protease. In addition, the MoaE component of MoaX was able to function in conjunction with M. smegmatis MoaD2 suggesting that cleavage of MoaX renders functionally interchangeable subunits. Expression of MoaX in E. coli revealed that incorrect post-translational processing is responsible for the lack of activity of MoaX in this heterologous host. Conclusions: There is a degree of functional interchangeability between the MPT synthase subunits of M. tuberculosis. In the case of MoaX, post-translational cleavage at the Gly82 residue is required for function.
- ItemOpen AccessDisabling the intrinsic resistome of Mycobacterium tuberculosis: elucidating hierarchies of DNA repair and mutagenesis that undermine current antibiotic efficacy(2021) Gobe, Irene; Warner, Digby; Mizrahi, Valerie; Iorger, Thomas RDNA damage repair mechanisms are critical to the adaptive evolution of Mycobacterium tuberculosis as obligate human pathogen, including the emergence of drug-resistance during anti-tuberculosis (TB) chemotherapy. In experimental models, DnaE2-dependent translesion synthesis (TLS) and UvrB-dependent nucleotide excision repair (NER) have been identified as major mediators of DNA damage tolerance and repair, respectively. Given the inferred dominance of these pathways, this thesis aimed to elucidate otherwise cryptic repair mechanisms which might buffer loss of DnaE2 and UvrB in bacilli exposed to genotoxic stress. Using dnaE2 and uvrB deletion mutants of the model mycobacterium, M. smegmatis (MSM) mc2 155, we applied genome-wide transposon (Tn) mutagenesis to identify conditionally essential repair pathways under treatment with genotoxins of different mechanistic classes. To this end, the DNA crosslinking agent, mitomycin C (MMC), and the gyrase inhibitor and clinically relevant TB drug, moxifloxacin (MOX), were used. The goal was to reveal potential targets for co-drugs that might shorten treatment duration and reduce the risk of drug resistance by severely limiting the intrinsic capacity of MTB to tolerate lethal drugs for extended periods. Among others, our analysis identified GlgB, which is involved in glycogen biosynthesis, and mycothiol biosynthesis proteins, MSMEG_0933 and MSMEG_5261, as compensating the absence of UvrB during MMC treatment. Under MOX treatment, the absence of UvrB was compensated by the RecC/Single-strand Annealing pathway. In contrast, DnaE2 deficiency revealed the conditional essentiality of the PadR family transcriptional regulator, MSMEG_2868, under MMC exposure. Importantly, in all cases, results from the Tn screen were validated using CRISPR interference targeting the identified genes. Of particular interest, we observed that UvrB was essential to compensate loss of DnaE2, whereas the reciprocal was less definitive: while DnaE2 appeared dispensable in MMC-treated uvrB, Tn analyses suggested that dnaE2 might be essential in the untreated ∆uvrB mutant. This result, which is consistent with very recent results suggesting the co-ordination of NER and DnaE2 functions in Caulobacter crescentus, is intriguing in potentially revealing a previously unappreciated role for DnaE2 in mycobacterial NER function. Taken together, these results support the utility of Tn-based whole-genome screens in revealing unexpected genegene interaction networks, and provide additional impetus to explore ancillary, non-essential metabolic functions as alternative targets for novel combination therapies designed to cripple intrinsic mechanisms of mycobacterial resistance.
- ItemOpen AccessElucidation of mechanisms of antibiotic subversion in mycobacteria(2015) Naran, Krupa; Warner, Digby F; Mizrahi, ValerieThe intrinsic resistance of Mycobacterium tuberculosis ( Mtb ) to antibiotics is generally attributed to multiple factors, most significantly the low permeability of the mycobacterial cell wall, the operation of various drug inactivating systems, and the activity of efflux pumps. This study aimed to investigate the role of various components of the "intrinsic resistome" that limit the efficacy of antitubercular agents. The DNA damage response: the SOS response was hypothesized to play a role in antibiotic- mediated cellular death, and that disabling the mycobacterial SOS response, by generating non-cleavable LexA mutants (lexA Ind-), could be used as a tool to validate antibiotic-mediated cell death. To this end, the M. smegmatis (Msm) cleavable LexA was shown to be essential for induced mutagenesis and damage tolerance and that an intact DNA damage repair system is required to respond to antibiotic-mediated DNA damage. In contrast, Mtb cleavable LexA was required for induced mutagenesis but not necessarily damage sensitivity. In addition, the Mtb SOS response does not contribute significantly to remediation of antibiotic-mediated DNA damage. Collectively, these data suggest that DNA repair mechanisms differ between the mycobacterial species and despite effectively inactivating the LexA-dependent DNA repair mechanism(s) in Msm and Mtb, these organisms are able to circumvent this pathway and successfully remediate damaged DNA sustained under various conditions. Furthermore, Mtb auto-bioluminescent reporter strains were generated by introducing the lux operon downstream of the recA or radA promoters. Analysis of a panel of antimicrobials against these strains allowed for the identification of true DNA-damaging agents and the evaluation of the kinetics of the DNA-damage response, in a concentration- and time-dependent manner. Efflux-mediated drug resistance: This study aimed to evaluate the interactions between pairwise combinations of selected antimicrobials and efflux pump inhibitors (EPIs), in vitro and ex vivo, and to identify a novel verapamil (VER)-analogue with improved efficacy against Mtb. Subsequently, a candidate EPI was identified, with equivalent in vitro synergistic effects to VER when used in combination with various antibiotics but with reduced cytotoxic effects, ex vivo, when compared to VER. Mycothiol-mediated protection : It was hypothesized that undetectable levels of mycothiol ( MSH ) in Mtb would potentiate the use of current antibiotics. To investigate the contribution of the cellular antioxidant, MSH, to the mitigation of antimicrobial efficacy, this study aimed to disrupt MSH production by conditionally knocking-down expression of the essential gene, mshC. The mshC knock-down mutants (in all configurations) were not anhydrotetracycline (ATC)-regulatable in liquid or on solid medium, which was subsequently validate d with quantitative gene expression analysis. These data suggest that a tetracycline (Tet)-based conditional expression system may not be applicable to mshC. In conclusion, Mtb has a multitude of inherent mechanisms to subvert the effects of antimicrobial treatment. This study has contributed to the understanding of certain aspects of the intrinsic resistome and in doing so, established tools that can be used in future drug discovery programmes.
- ItemOpen AccessA high-throughput screen against pantothenate synthetase (PanC) identifies 3-biphenyl-4-cyanopyrrole-2-carboxylic acids as a new class of inhibitor with activity against Mycobacterium tuberculosis(Public Library of Science, 2013) Kumar, Anuradha; Casey, Allen; Odingo, Joshua; Kesicki, Edward A; Abrahams, Garth; Vieth, Michal; Masquelin, Thierry; Mizrahi, Valerie; Hipskind, Philip A; Sherman, David RThe enzyme pantothenate synthetase, PanC, is an attractive drug target in Mycobacterium tuberculosis . It is essential for the in vitro growth of M. tuberculosis and for survival of the bacteria in the mouse model of infection. PanC is absent from mammals. We developed an enzyme-based assay to identify inhibitors of PanC, optimized it for high-throughput screening, and tested a large and diverse library of compounds for activity. Two compounds belonging to the same chemical class of 3-biphenyl-4- cyanopyrrole-2-carboxylic acids had activity against the purified recombinant protein, and also inhibited growth of live M. tuberculosis in manner consistent with PanC inhibition. Thus we have identified a new class of PanC inhibitors with whole cell activity that can be further developed.
- ItemOpen AccessIdentification of new drug targets and resistance mechanisms in Mycobacterium tuberculosis(Public Library of Science, 2013) Ioerger, Thomas R; O'Malley, Theresa; Liao, Reiling; Guinn, Kristine M; Hickey, Mark J; Mohaideen, Nilofar; Murphy, Kenan C; Boshoff, Helena I M; Mizrahi, Valerie; Rubin, Eric JIdentification of new drug targets is vital for the advancement of drug discovery against Mycobacterium tuberculosis , especially given the increase of resistance worldwide to first- and second-line drugs. Because traditional target-based screening has largely proven unsuccessful for antibiotic discovery, we have developed a scalable platform for target identification in M. tuberculosis that is based on whole-cell screening, coupled with whole-genome sequencing of resistant mutants and recombineering to confirm. The method yields targets paired with whole-cell active compounds, which can serve as novel scaffolds for drug development, molecular tools for validation, and/or as ligands for co-crystallization. It may also reveal other information about mechanisms of action, such as activation or efflux. Using this method, we identified resistance-linked genes for eight compounds with anti-tubercular activity. Four of the genes have previously been shown to be essential: AspS, aspartyl-tRNA synthetase, Pks13, a polyketide synthase involved in mycolic acid biosynthesis, MmpL3, a membrane transporter, and EccB3, a component of the ESX-3 type VII secretion system. AspS and Pks13 represent novel targets in protein translation and cell-wall biosynthesis. Both MmpL3 and EccB3 are involved in membrane transport. Pks13, AspS, and EccB3 represent novel candidates not targeted by existing TB drugs, and the availability of whole-cell active inhibitors greatly increases their potential for drug discovery.
- ItemOpen AccessMolecular mechanisms of DNA repair in Mycobacterium tuberculosis(2017) Gessner, Sophia Johanna; Warner, Digby F; Mizrahi, ValerieThe mycobacterial DNA damage and repair pathways involved in the emergence of drug-resistance during host infection remain poorly understood, yet are critical to any efforts to develop novel "anti-evolution" drugs aimed at reducing the capacity of Mycobacterium tuberculosis to adapt genetically during tuberculosis (TB) treatment. The thesis presented here aimed to investigate the contribution of the DNA damage (SOS) response in adaptive mutagenesis, and focused on two specific components: the role of the specialist translesion synthesis DNA polymerase, DnaE2, in mutagenesis under stress and, secondly, the function of the mycobacterial homologue of a putative SOS response associated peptidase (SRAP) protein which has been identified in comparative genomics analyses of organisms possessing a DnaE2-type C family DNA polymerase. This work focused on the putative SRAP protein which was predicted to form part of the mycobacterial DNA damage response as a functional switch by binding DNA in an autoproteolytic dependent manner. To this end, SRAP deletion mutants were generated for both M. smegmatis (MSMEG_1891) and M. tuberculosis (Rv3226c). Despite the fact that SRAP was upregulated in both M. smegmatis and M. tuberculosis following genotoxic stress, no DNA damage phenotype was detected in any SRAP deletion mutant using a variety of DNA damaging agents. In parallel, an eGFP-tagged M. smegmatis SRAP allele was constructed to enable visualisation of SRAP upregulation and sub-cellular recruitment using fluorescent microscopy; however no eGFP expression could be visualised after MMC treatment. It was not clear whether this was due to faulty eGFP expression in the fusion protein, or to low-level induction of SRAP. In a biochemical approach to elucidate SRAP function, soluble M. smegmatis SRAP protein was expressed and purified using a N-terminal hexa-histidine tag. No proteolytic activity was detected in gelatine or casein zymography, perhaps indicating that SRAP has a very specific substrate. Moreover, while it was predicted that autocatalytic cleavage of the C-terminus was required for activation of SRAP, no such cleavage was detected using hexa-histidine tag staining, possibly pointing to a set of very specific conditions for activation. In combination, therefore, neither microbiological nor biochemical assays could elucidate a definitive role for SRAP in the mycobacterial DNA damage response. DnaE2 has been directly implicated in induced mutagenesis to rifampicin (Rif) resistance in Mycobacterium tuberculosis following exposure of bacilli to genotoxic stress. In previous work in our group, a vitamin B₁₂-sensitive ΔmetH strain was found to form "B₁₂-resistant" suppressor mutants at a frequency higher than could be explained by spontaneous mutagenesis alone. The first part of this thesis investigated the potential role of DnaE2 in the high-frequency emergence of B₁₂-resistance by mutating DnaE2 in the ΔmetH background. Whereas elimination of polymerase function in a DnaE2ᴬᴵᴬ mutant abrogated DNA damage-induced mutagenesis to Rif resistance, no change in B₁₂ sensitivity was detected in a ΔmetH dnaE2ᴬᴵᴬ double mutant. PCR sequencing of spontaneous B₁₂-resistant mutants revealed mutations in genes previously associated with the suppressor phenotype; moreover, there was no apparent difference in the nature of mutations observed in both parental and dnaE2ᴬᴵᴬ mutant strains. Instead, these results suggest that an alternative mechanism must exist to enable adaptive mutagenesis in methionine-starved mycobacteria.
- ItemOpen AccessReal-time investigation of tuberculosis transmission: developing the Respiratory Aerosol Sampling Chamber (RASC)(Public Library of Science, 2016) Wood, Robin; Morrow, Carl; III, Clifton E Barry; Bryden, Wayne A; Call, Charles J; Hickey, Anthony J; Rodes, Charles E; Scriba, Thomas J; Blackburn, Jonathan; Issarow, Chacha; Mulder, Nicola; Woodward, Jeremy; Moosa, Atica; Singh, Vinayak; Mizrahi, Valerie; Warner, Digby FKnowledge of the airborne nature of respiratory disease transmission owes much to the pioneering experiments of Wells and Riley over half a century ago. However, the mechanical, physiological, and immunopathological processes which drive the production of infectious aerosols by a diseased host remain poorly understood. Similarly, very little is known about the specific physiological, metabolic and morphological adaptations which enable pathogens such as Mycobacterium tuberculosis ( Mtb ) to exit the infected host, survive exposure to the external environment during airborne carriage, and adopt a form that is able to enter the respiratory tract of a new host, avoiding innate immune and physical defenses to establish a nascent infection. As a first step towards addressing these fundamental knowledge gaps which are central to any efforts to interrupt disease transmission, we developed and characterized a small personal clean room comprising an array of sampling devices which enable isolation and representative sampling of airborne particles and organic matter from tuberculosis (TB) patients. The complete unit, termed the Respiratory Aerosol Sampling Chamber (RASC), is instrumented to provide real-time information about the particulate output of a single patient, and to capture samples via a suite of particulate impingers, impactors and filters. Applying the RASC in a clinical setting, we demonstrate that a combination of molecular and microbiological assays, as well as imaging by fluorescence and scanning electron microscopy, can be applied to investigate the identity, viability, and morphology of isolated aerosolized particles. Importantly, from a preliminary panel of active TB patients, we observed the real-time production of large numbers of airborne particles including Mtb , as confirmed by microbiological culture and polymerase chain reaction (PCR) genotyping. Moreover, direct imaging of captured samples revealed the presence of multiple rod-like Mtb organisms whose physical dimensions suggested the capacity for travel deep into the alveolar spaces of the human lung.
- ItemOpen AccessReplication fidelity in the mircroevolution of mycobacteria(2015) Ditse, Zanele; Warner, Digby F; Mizrahi, ValerieThis thesis aimed to elucidate the structure-function relationships determining the differential fidelities of the dnaE1- and dnaE2-encoded mycobacterial PolIIIα subunits under conditions of genotoxic stress. To this end, the role in DnaE1 intrinsic fidelity of highly conserved PHP domain residues was explored by site-directed replacement of targeted amino acids, resulting in a panel of Mycobacterium smegmatis mutants carrying selected dnaE1 alleles. A complementary approach investigated the contribution of the mycobacterial proofreading DnaQ subunit homolog to the maintenance of DnaE1-dependent replicative fidelity by generating a targeted dnaQ knockout mutant. The third component of this study focused on the inferred role of a highly-conserved N-terminal extension and C-terminal pentapeptide motif in the function of the alternative, error-prone DNA PolIIIα subunit, DnaE2.
- ItemOpen AccessRiboswitch regulation of methionine metabolism and vitamin B12 uptake in mycobacteria – implications for drug susceptibility and pathogenesis(2019) Kipkorir, Terry; Warner, Digby; Mizrahi, ValerieAlterations in the genetic capacity for cobamide biosynthesis have been identified as potentially critical in the evolution of Mycobacterium tuberculosis from a putative environmental ancestor. Moreover, recent studies have implicated cobamide biosynthesis pathway genes in the adaptation of the bacillus to intracellular pathogenesis. Although mycobacteria retain essential biochemical reactions that require cobamides, the specific role of these co-factors during tuberculosis (TB) disease remains unresolved. This thesis aimed to examine the production, uptake, and utilization of cobamides in mycobacteria using M. smegmatis as a model. To this end, the genetic capacity for de novo production and uptake of cobamide in host-associated and environmental mycobacteria was assessed, followed by direct validation in M. smegmatis. A combination of genetics, gene expression analysis, live-cell time-lapse microscopy and targeted metabolite and protein analysis via mass spectrometry (MS) was then employed to investigate cobamide riboswitch-dependent regulation of methionine biosynthesis in M. smegmatis. Results indicated that, in wild-type M. smegmatis, de novo cobamide biosynthesis ensured constitutive repression of metE, the gene encoding the mycobacterial cobalamin-independent methionine synthase. Owing to this repression, metH, a gene encoding the cobalamin-dependent methionine synthase, was found to be conditionally essential for bacillary replication in vitro. Drug susceptibility testing to investigate the link between cobamides and the intrinsic resistance to anti-folate antibiotics confirmed novel mycobacterial vulnerabilities in cobamide-related methionine metabolism, indicating that the outcomes of cobamidedependent regulation may have relevance to mycobacterial pathogenesis and drug discovery. In contrast to M. tuberculosis, which was previously shown to transport exogenous CNCbl readily, M. smegmatis poorly assimilated exogenous co-factor despite the presence of multiple putative cobamide transporters. However, uptake was enhanced in a mutant requiring CNCbl for growth. Elucidating the factors which regulate cobamide biosynthesis and co-factor utilization in M. smegmatis, an environmental mycobacterium, might provide a lens through which to consider the differential regulation and utilization of cobamides in M. tuberculosis, an obligate pathogen with a limited host range.
- ItemOpen AccessThe structure and reactivity of N-Acyl phosphoric amides and related systems(1983) Mizrahi, Valerie; Modro, Tomasz ATwo synthetic approaches towards the N-acyl phosphylamide system Z₂P(O)-NR-C(O)R' (1; Z =alkyl, O-alkyl; R = H, Me; R' =M e, Ph), from phosphylamide and carboxamide precursors are discussed. The infrared, ¹H and ¹³C NMR spectral features of system (1), indicate predominant resonance interaction of the nitrogen non-bonding electrons with the adjacent carboxyacyl, rather than phosphacyl centre. The electron-with- drawing effect of the phosphyl substituent Z₂P(O), is nonetheless sub- stantial, thus weakening the basicity and nucleophilicity of the nitrogen atom and enhancing the electrophilicity of the carbonyl centre. The influence of the electronic distribution within the OPNCO moiety upon the structure and reactivity of (1), has been investigated.
- ItemOpen AccessSubcellular localization and visualization of RecA and ImuAʹ in mycobacteria(2021) Ramudzuli, Atondaho Angelah; Warner, Digby; Gessner, Sophia; Mizrahi, ValerieAntibiotic-resistant strains of Mycobacterium tuberculosis (Mtb) are threatening global efforts to eradicate tuberculosis (TB). One attractive approach for target-based drug design proposes to curb the evolution of Mtb during both immune and drug assault. The potential target: mycobacterial DNA metabolism. For this, an in-depth understanding of the mechanisms of DNA repair and mutagenesis in mycobacteria is required. RecA and ImuAʹ are DNA damageinducible proteins implicated in DNA damage repair and tolerance in Mtb. RecA is a key regulatory protein of the SOS response and ImuAʹ is a component of the mycobacterial mutasome, effecting DNA damage tolerance and mutagenesis. In this study, a comprehensive panel of M. smegmatis (Msm) RecA and ImuAʹ reporter strains was generated to explore the dynamics of their expression and subcellular localization within live Msm cells. To this end, fluorescently tagged versions of ImuAʹ and RecA were constructed and shown to retain functional activity in UV-induced mutagenesis but not survival of mitomycin C (MMC) treatment. The discrepant complementation phenotypes observed in UV and MMC assays was unexpected and suggested disruption of a critical protein-protein interaction(s) owing to the presence of the fluorophore. Using fluorescence microscopy, RecA and ImuAʹ expression were monitored in Msm exposed to different types of genotoxic stresses conditions. When mScarletImuAʹ was introduced into wild-type (WT) and ∆imuAʹ backgrounds, diffuse bright red fluorescence was observed in cells treated with MMC and UV; in contrast, no fluorescence expression was observed in untreated cells, confirming the DNA damage-dependent induction of imuAʹ. Following the introduction of RecA-msfGFP into WT and ∆recA backgrounds, discrete green, fluorescent foci were observed in treated and untreated cells in both backgrounds, consistent with the role of RecA in DNA replication in the absence of external DNA damage, and elevated expression under genotoxic conditions. Taken together, these observations support the utility of the fluorescently tagged translational fusions as bioreporters to elucidate the function and regulation of ImuA' and RecA in mycobacteria.
- ItemOpen AccessTranslating genomics research into control of tuberculosis: lessons learned and future prospects(BioMed Central, 2014-11-07) Warner, Digby F; Mizrahi, ValerieGenomics research has enabled crucial insights into the adaptive evolution of Mycobacterium tuberculosis as an obligate human pathogen. Here, we highlight major recent advances and evaluate the potential for genomics approaches to inform tuberculosis control efforts in high-burden settings.
- ItemOpen AccessTranslating genomics research into control of tuberculosis: lessons learned and future prospects(2014-11-07) Warner, Digby F; Mizrahi, ValerieAbstract Genomics research has enabled crucial insights into the adaptive evolution of Mycobacterium tuberculosis as an obligate human pathogen. Here, we highlight major recent advances and evaluate the potential for genomics approaches to inform tuberculosis control efforts in high-burden settings.
- ItemOpen AccessTranslational research for tuberculosis elimination: priorities, challenges, and actions(Public Library of Science, 2016) Lienhardt, Christian; Lönnroth, Knut; Menzies, Dick; Balasegaram, Manica; Chakaya, Jeremiah; Cobelens, Frank; Cohn, Jennifer; Denkinger, Claudia M; Evans, Thomas G; Källenius, Gunilla; Kaplan, Gilla; Kumar, Ajay M V; Matthiessen, Line; Mgone, Charles S; Mizrahi, Valerie; Mukadi, Ya-diul; Nguyen, Viet Nhung; Nordström, Anders; Sizemore, Christine F; Spigelman, Melvin; Squire, S Bertel; Swaminathan, Soumya; Helden, Paul D Van; Zumla, Alimuddin; Weyer, Karin; Weil, Diana; Raviglione, MarioChristian Lienhardt and colleagues describe the research efforts needed to end the global tuberculosis epidemic by 2035.
- ItemOpen AccessWhole-genome transposon mutagenesis to elucidate the genetic requirements for vitamin B12 biosynthesis and assimilation in mycobacteria(2022) Mbau, Rendani Donald; Warner, Digby; Mizrahi, ValerieComparative genomic analyses have identified an altered capacity for cobalamin biosynthesis as a critical step in the evolution of the pathogenic Mycobacterium tuberculosis Complex strains from a common environmental ancestor. However, resolving the full gene complement involved in the complex, multi-step pathway for de novo cobalamin biosynthesis, assimilation, and salvage in different mycobacterial species is challenging. A genome-scale approach was adopted to yield detailed genetic maps of de novo cobalamin biosynthesis in M. smegmatis, a non-pathogenic saprophyte. To this end, a combination of whole-genome transposon (Tn) mutagenesis and next generation sequencing (TnSeq) was applied in M. smegmatis ΔmetE, a gene-deletion mutant in which the cobalamin-independent methionine synthase is inactivated, rendering the cobalamin-dependent isoform, MetH, essential for viability. Following growth of the metE mutant in rich medium, genomic DNA was extracted, amplified by PCR, and subjected to high-throughput sequencing to quantify all Tn junctions. Thereafter, the library was cultivated in defined minimal medium to enable identification of all conditionally essential genes – including those required for de novo cobalamin biosynthesis. A ∆metE library comprising 400,000 individual Tn insertion mutants (cfu/ml) was generated. Of the predicted 6,716 genes in the M. smegmatis genome, 213 genes were identified as essential for growth on rich agar while 356, 301, and 337 genes were identified as essential in unsupplemented, cyanocobalamin (CNCbl; vitamin B12)-supplemented and cobalt-supplemented Sauton's minimal medium, respectively. A total of 424 genes were identified as essential across all conditions tested with only 10, 13 and 24 genes (ES plus GD) uniquely required for growth in unsupplemented, CNCbl-supplemented and cobalt supplemented Sauton's minimal medium, respectively. On average, predicted cobalamin pathway genes were underrepresented in number of Tn insertions and read counts, indicating the likely essentiality of these genes during growth of the metE mutant in minimal medium. Notably, elucidation of cobalamin biosynthetic and assimilatory genes required the analysis of libraries exposed to CNCbl-unsupplemented minimal media for extended durations, probably reflecting the need to exhaust the organism's capacity for co-factor storage and recycling. Utilizing targeted silencing of individual genes by CRISPR interference, candidate cobalamin biosynthesis genes were validated, providing functional evidence of their essentiality for metE survival in minimal medium, in turn supporting the validity of the cobalamin biosynthetic pathway constructed from the TnSeq results. In addition, the results add further evidence in support of the functionality of the cobalamin riboswitch upstream of metE. This is an important observation as it suggests the potential to apply an analogous approach in M. tuberculosis, a major human pathogen whose ability to synthesize cobalamins remains unresolved. Moreover, elucidating the genetic requirements for optimal growth under specific conditions can inform our basic understanding of mycobacterial physiology and pathogenicity, identifying potential vulnerabilities for novel anti-tuberculosis therapeutics.