Browsing by Author "Warner, Digby"
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- ItemOpen AccessBarriers to tuberculosis drug discovery: the mycobacterial cell wall(2023) Whittaker, Caitlin; Warner, Digby; Egan Timothy JohnThe mycobacterial cell wall is a highly complex macromolecular structure that provides intrinsic resistance to several anti-tuberculosis drugs, making it critical in the success of Mycobacterium tuberculosis infection. Multiple layers encapsulate the cell membrane, including arabinogalactan chains and mycolic acids that are covalently bonded to the peptidoglycan layer, resulting in a highly selectively permeable barrier that is unique to this genus. The current treatment for tuberculosis (TB) utilizes antibiotics that weaken the structural integrity of the cell wall to allow easier access for drugs that have intracellular targets. Although this approach is theoretically effective, patient adherence is often poor owing to the lengthy treatment times and negative side effects associated with the multidrug combination regimen. As such, rational drug design to develop more potent, faster-acting anti-TB compounds requires a comprehensive understanding of the composition and functioning of the mycobacterial cell envelope to ensure effective penetration through this barrier. Bioinformatic approaches to compound validation provide a crucial foundation for drug development, but empirical validation of these molecules can present a serious bottleneck in the drug discovery pipeline. Here, we investigated fluorescent click chemistry as a rapid and inexpensive means of ascertaining molecular properties that impact compound permeation of the mycobacterial cell envelope. The variability in permeation of different click-reactive moieties could be rapidly determined using fluorescent read-outs; this, in combination with the availability of a wide array of click-reactive side chains, presents a potentially powerful platform for establishing the properties required by a compound to effectively cross the mycomembrane. Enzymatic degradation of cell wall components further revealed the resilience of mycobacteria as the resulting organisms, spheroplasts, were capable of surviving in the absence of this seemingly essential protective layer. This presents a potentially novel form of intrinsic resistance whereby stripping of the cell wall could allow for tolerance to cell wall active antibiotics, a previously under-appreciated strategy that has been reported in other pathogenic bacteria. Together, these findings highlight the highly dynamic nature of the mycobacterial cell envelope and the need for further investigation into the properties of this structure that allow for such efficient antibiotic evasion.
- ItemOpen AccessCatching a glimpse: the visualization of Mycobacterium tuberculosis from TB patient bioaerosols(2023) Dinkele, Ryan; Warner, Digby; Gessner, SophiaTransmission between hosts is crucial for the success and survival of the obligate human pathogen and aetiological agent of tuberculosis (TB), Mycobacterium tuberculosis (Mtb). Despite this, little is known about how and when Mtb is aerosolized nor the key metabolic and morphological determinants driving successful transmission. To address these knowledge gaps, my doctoral research sought to develop a microscopic method for the detection of aerosolized Mtb following liquidcapture within the respiratory aerosol sampling chamber (RASC). This was achieved through the combination of the mycobacterial cell wall probe, 4-N,Ndimethylamino-1,8-naphthalimide-trehalose (DMN-tre), with the arraying of bioaerosol samples on bespoke nanowell devices amenable to fluorescence microscopy. With this method, a median of 14 live Mtb bacilli (range 0-36) were detected in 90% of confirmed TB patients following 60 minutes of bioaerosol sampling. Three distinct DMN-tre staining patterns were identified among aerosolized Mtb, strongly suggestive of metabolic heterogeneity. Moreover, a low proportion of patients produced Mtb in small clumps. These observations highlight the advantages of using microscopy over conventional culture- or molecular-based techniques for probing the metabolic and morphological characteristics of aerosolized Mtb. Applying this method in a second study, we sought to understand how and when Mtb is aerosolized. To this end, we aimed to compare the aerosolization of Mtb and total particulate matter from patients with TB during three respiratory manoeuvres: tidal breathing (TiBr), forced vital capacity (FVC), and cough. Although total particle counts were 4.8-fold greater in cough samples than either TiBr or FVC, all three manoeuvres returned similar rates of positivity for Mtb. No correlation was observed between total particle production and Mtb count. Instead, for total Mtb counts, the variability between individuals was greater than the variability between sampling manoeuvres. Finally, when modelled using 24-hour breath and cough frequencies, our data indicate that TiBr might contribute more than 90% of the daily aerosolized Mtb among symptomatic TB patients. Assuming the number of viable Mtb organisms detected provides a proxy measure of patient infectiousness, this method suggests that TiBr is a significant contributor to TB transmission. In developing a novel platform for the detection of aerosolized Mtb, this work has suggested the need to re-examine old assumptions about Mtb transmission.
- ItemOpen AccessCRISPRI-based high-throughput functional genomic approaches for use in mycobacteria(2021) De Wet, Timothy; Warner, DigbyIn the 20 years since the pioneering publication of the genome of Mycobacterium tuberculosis, significant efforts have been made to complete functional annotation of the genome. However, these efforts have generally been performed on a single-gene basis, ensuring slow progress and leaving large portions of the genome unannotated. High-throughput approaches to understanding the functional genome, such as transposon-insertion sequencing, have been developed and applied to mycobacteria in a variety of conditions; however, they have several limitations, particularly in their ability to study genes essential for viability. The recent optimisation of inducible CRISPR-interference for mycobacteria offers the potential to expand the high-throughput functional genomic toolkit. This thesis utilises CRISPR-interference for the development and validation of two high-throughput functional genomic approaches in the model mycobacterium M. smegmatis. The first approach combines large-scale pooled oligonucleotide synthesis and nextgeneration sequencing, and is termed CRISPRi-Seq. A pooled library of 11 367 mutants, targeting 2 385 M. smegmatis genes with M. tuberculosis homologues, was constructed and used to infer gene essentialities which were compared with corresponding predictions from transposon-insertion sequencing data. This process validated the CRISPRi-Seq technique and identified practical considerations for its future use. The second approach utilises data derived from CRISPRi-Seq to create an arrayed library of 263 individual M. smegmatis inducible CRISPRi mutants targeting essential genes. This library is applied to a quantitative imaging pipeline to produce detailed data-driven profiles of the morphological impact of essential gene suppression. These morphological profiles are used to statistically predict genetic function, as well as antimicrobial mechanism-of-action. The two novel approaches developed in this work represent valuable technical advances and produce large datasets of functional genomic data which are available interactively online. Taken individually, or in combination, these methodologies can be utilised to increase fundamental understanding of mycobacteria, including the pathogenic M. tuberculosis
- 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 AccessDrug discovery at the site of pulmonary tuberculosis(2018) Tanner, Lloyd; Wiesner, Lubbe; Warner, Digby; Haynes, RichardThe protracted duration of standard tuberculosis (TB) therapy suggests the inadequacy of current first line TB drugs to eliminate the causative agent Mycobacterium tuberculosis (Mtb). Among multiple potential causes, this may be due to poor distribution of TB drugs into the pulmonary lesions in which the bacilli reside. In attempting to explore this possibility, the study described here aimed to assess selected novel compounds (phenoxazine, artemisinin, and decoquinate derivatives) proposed to induce redox-cycling in efficacious combinations, as well as their distribution to TB-relevant lesions, for potential clinical use against TB. To this end, specific in vitro absorption, distribution, metabolism, and excretion (ADME) assays were performed to predict the abilities of the compounds to penetrate different TB microenvironments. Penetration into murine blood and organs was assessed via pharmacokinetic (PK) profiling. Complementary analyses involving murine epithelial lining fluid allowed for more detailed analyses of the potential of these novel compounds to penetrate the deeper recesses of the lung. In order to gain a greater understanding of the potential efficacy of the compounds in an intracellular environment, THP-1 macrophage-like cells were infected with Mtb, treated with anti-TB agents, and sampled at different time-points. Samples were analyzed via liquid chromatography-tandem mass spectrometry (LC-MS/MS) assays and drug concentrations were determined and related to efficacy measurements using colony forming unit counts. Promising combinations of novel drugs were identified in a two dimensional synergy assay; these combinations showed synergistic activity in the infected macrophage model. The compounds showed marked differences in their abilities to accumulate within infected macrophages. Differential uptake was also indicated by the results of the PK studies involving murine blood and organ uptake, and the in vitro ADME assays. These results enable PK modelling on the putative drug target to be carried out, allowing for the determination of more accurate dosing. In addition, results indicate the need for further studies, including investigations of the impact of macrophage structural organisation (three-dimensional model) on compound efficacy and in vivo studies in a relevant mouse model of TB disease. The identification of a potentially efficacious two drug combination which might penetrate to the site of pulmonary TB supports the utility of this approach in the preclinical drug discovery and development pipeline.
- ItemOpen AccessExploring the molecular diversity and biomedicinal potential of marine invertebrates and South African actinomycetes for tuberculosis drug discovery(2021) Acquah, Kojo Sekyi; Gammon, David; Beukes, Denzil; Meyers, Paul; Warner, DigbyTuberculosis (TB) which is caused by Mycobacterium tuberculosis (Mtb), is the leading cause of death from a single infectious disease and remains a global health threat. Although there is medicine for treating TB, it still kills millions each year. This is due to a lengthy and demanding TB treatment regimen with associated problems of adverse drug-drug interactions and toxicities. Several resistant strains of Mtb, which are difficult and more expensive to treat, have also emerged. Therefore, there is a need to discover new potent and safe TB drugs to effectively treat Mtb with its resistant strains. Less explored and biodiversity-rich ecosystems such as the marine environment and South Africa (SA)'s fauna and flora have been a source of new bioactive natural products (NPs). A range of marine invertebrates and SA actinomycetes were therefore studied for the discovery of new antimycobacterial NPs. Several organic and aqueous marine invertebrate extracts were screened for their in vitro inhibitory activity against the Mtb strain H37Rv. The chemical components of two out of 54 active extracts were dereplicated using 1H NMR, HR-LCMS with GNPS molecular networking. The extracts were subsequently subjected to an activity-guided isolation process to yield heteronemin from Hyrtios reticulatus extract, and bengamides P and Q from Jaspis splendens extract. A new bengamide derivative was putatively identified in the molecular network of Jaspis splendens extract, and its structure predicted based on the similarity of its MS/MS fragmentation pattern to that of other bengamides. The isolated bioactive metabolites and semi-pure fractions exhibited antimycobacterial activity with MIC90 in the range of < 0.24 to 62.50 µg/mL. This is first report of antitubercular activity of bengamides P and Q. In studies on actinomycetes, the organic extract of a liquid culture of the South African Streptomyces strain Muiz4Y exhibited antimycobacterial activity against Mycobacterium aurum strain A+ and Mtb strain H37Rv. HR-LCMS analysis of the crude extract for dereplication suggested the presence of new compounds. A bioactivity, spectroscopy and spectrometry guided isolation procedure led to the isolation of three new natural products, a β-carboline alkaloid (1- (1,2-dihydroxyethyl)-1,2,3,4-tetrahydro-β-carboline-3-carboxylic acid), a peptide (N-(2- phenylacetyl)-serine), and a glycosylated lactone (4-O-(β-glucopyranosyl)-5-(hydroxymethyl)-3- (4-methylpentyl)-5,6-dihydro-2H-pyran-2-one), together with known compounds 3,6- bis(phenylmethyl)-2,5-piperazinedione and 2,4,6-triphenylhex-1-ene. The structures of the isolated compounds were elucidated based on spectroscopic methods including 1D and 2D NMR, MS, as well as by comparison with the relevant literature. Only 2,4,6-triphenylhex-1-ene exhibited antimycobacterial activity against Mtb strain H37Rv with an MIC90 of 5.8 µg/mL. The novel rare South African Actinomycete Kribbella speibonae strain SK5 exhibited antimycobacterial activity against Mycobacterium aurum A+. Dereplicating the crude extract of a large-scale culture of strain SK5 using 1H NMR, genome mining and HR-LCMS with GNPS molecular networking showed that it is a prolific producer of hydroxamate siderophores including new congeners. Two new analogues, dehydroxylated desferrioxamines, speibonoxamine and desoxy-desferrioxamine D1, were isolated, together with four known hydroxamates, desferrioxamine D1, desferrioxamine B, desoxy-nocardamine and nocardamine, and a diketopiperazine (DKP). The isolated compounds were characterized by the analysis of HRESIMS and 1D and 2D NMR data, as well as by comparison with the relevant literature. Three new dehydroxy desferrioxamine derivatives were tentatively identified in the molecular network of K. speibonae strain SK5 extracts, and structures were proposed based on their MS/MS fragmentation patterns. A plausible spb biosynthetic pathway was proposed. To the best of our knowledge, this is the first report of the isolation of desferrioxamines from the actinobacterial genus Kribbella. The isolated compounds were inactive against Mtb strain H37Rv and Mycobacterium aurum A+. This study confirmed the marine environment as a source of new antimycobacterial NPs and established South African actinomycete as a source of new bioactive NPs.
- ItemOpen AccessInvestigating the chemical diversity and biomedicinal potential of South African actinomycetes for tuberculosis drug discovery(2018) De Cerf, Christopher; Sunassee, Suthananda N; Meyers, Paul R; Warner, DigbyThe chemical diversity and biomedicinal potential of three South African actinomycetes, Streptomyces speibonae PK-BlueT , Streptomyces africanus CPJVR-H T and Streptomyces pharetrae CZA14T , was investigated. The primary aim was the isolation and structure elucidation of anti-tubercular natural products (NPs), including the re-isolation of the compound named PK-B, reported to be produced by S. speibonae PK-BlueT . Efforts were made to re-isolate the compound named PK-B. However, the results could not be replicated and focus was shifted to the bioassay-guided isolation of antimycobaterial compounds from the three actinobacteria. Culture conditions were optimised for antimycobacterial activity of S. speibonae PK-BlueT extracts by testing against the MTB analogue Mycobacterium aurum A+ (a non-pathogenic, fast-growing mycobacterium that has a similar antibiotic susceptibility to Mycobacterium tuberculosis). Bioactive crude extracts were prioritised for liquid-liquid partitioning and dereplication by LR LC-MS, followed by prefractionation and purification using a combination of HPLC and benchtop column chromatography. Structure elucidation of isolated compounds was achieved using NMR, LCMS and GC-MS data. Bioassays against M. aurum A+ were implemented at every stage of the isolation process to make sure that the isolated compounds had antimycobacterial activity. This strategy led to the isolation of four known compounds, the alkaloid N-phenylpyridin-2- aminium (3.1), a 1:1 mixture of the long-chain fatty acids (LCFAs) n-hexadecanoic acid (3.2) and 14-methylpentadecanoic acid (3.3), and the isoflavone 7-hydroxy-3-(4-hydroxyphenyl)- 4H-chromen-4-one (3.4), from S. speibonae PK-BlueT cultures. Of the four isolated compounds, only 3.1 has not previously been reported from a natural source. The approach of using M. aurum A+ in searching for anti-tubercular compounds was vindicated as 3.1, the mixture of 3.2 and 3.3, and 3.4, in addition to inhibiting the growth of M. aurum A+, exhibited activity against M. tuberculosis H37RvT with MIC90 values of 135 μM, 26 μM and 195 μM, respectively. Additionally, the effect of different growth media on the chemical diversity of S. speibonae PK-BlueT , S. africanus CPJVR-H T and S. pharetrae CZA14T extracts was demonstrated by bioassay where the extracts were screened against a panel of test organisms. HR LC-MS dereplication was used to identify a list of 11 suggested molecular formulae of potentially novel, bioactive NPs in liquid-liquid partitioned extracts of theses three filamentous actinobacteria. Unfortunately, these compounds could not be investigated further due to low biomass and time limitations.
- 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 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 AccessVisualising the Mycobacterial Mutasome(2018) Reiche, Michael Anton; Warner, DigbyAn SOS-inducible DNA repair system has been linked to transient hyper-mutation and the development of drug resistance in Mycobacterium tuberculosis. Previous work has established that this “mycobacterial mutasome” comprises the specialist DNA polymerase, DnaE2, and accessory factors of unknown function, ImuA′ and ImuB. However, the molecular interactions and sub-cellular recruitment dynamics enabling mutasome function remain poorly understood. Here, a panel of fluorescent strains of M. smegmatis was developed to investigate expression and subcellular localization of ImuA′ and ImuB in live mycobacteria exposed to genotoxic agents. Using fluorescence microscopy, it was observed that, during prolonged genotoxic stress, single M. smegmatis cells exhibited an elongated cell phenotype and apparent aneuploidy – potentially providing an environment for recombination between differentially mutated chromosomes. Furthermore, ImuB was seen to associate with the dnaNencoded β clamp in discrete foci during mutagenic DNA repair. In contrast, ImuA′ did not exhibit similar localization and instead appeared to diffuse throughout the bacillus. A mutant ImuB protein deficient in the β clamp-binding motif failed to colocalize with the β clamp, reinforcing the inferred essentiality of the ImuB-β clamp protein-protein interaction for mutasome recruitment and induced mutagenesis. Additionally, exposure of M. smegmatis to griselimycin, a novel β clamp-targeting natural product antibiotic, prevented ImuB-β clamp co-localization during SOS induced mutagenesis, an observation confirmed by superresolution, threedimensional interferometric photo-activated light microscopy. These results establish the capacity of griselimycin to inhibit DNA replication as well as prevent DNA damage-induced mutagenesis by disrupting mutasome assembly and activity. Notably, this differentiates griselimycin from other inhibitors of DNA metabolic function which carry the often-unavoidable liability of accelerating drug-resistance by inducing mutagenic DNA repair. In turn, it suggests the potential application of griselimycin as an anti-evolution agent in novel therapeutic regimens designed to protect existing tuberculosis drugs.
- 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.