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ADP-ribosyltransferases use NAD.sup.+ to catalyse substrate ADP-ribosylation.sup.1, and thereby regulate cellular pathways or contribute to toxin-mediated pathogenicity of bacteria.sup.2-4. Reversible ADP-ribosylation has traditionally been considered a protein-specific modification.sup.5, but recent in vitro studies have suggested nucleic acids as targets.sup.6-9. Here we present evidence that specific, reversible ADP-ribosylation of DNA on thymidine bases occurs in cellulo through the DarT-DarG toxin-antitoxin system, which is found in a variety of bacteria (including global pathogens such as Mycobacterium tuberculosis, enteropathogenic Escherichia coli and Pseudomonas aeruginosa).sup.10. We report the structure of DarT, which identifies this protein as a diverged member of the PARP family. We provide a set of high-resolution structures of this enzyme in ligand-free and pre- and post-reaction states, which reveals a specialized mechanism of catalysis that includes a key active-site arginine that extends the canonical ADP-ribosyltransferase toolkit. Comparison with PARP-HPF1, a well-established DNA repair protein ADP-ribosylation complex, offers insights into how the DarT class of ADP-ribosyltransferases evolved into specific DNA-modifying enzymes. Together, our structural and mechanistic data provide details of this PARP family member and contribute to a fundamental understanding of the ADP-ribosylation of nucleic acids. We also show that thymine-linked ADP-ribose DNA adducts reversed by DarG antitoxin (functioning as a noncanonical DNA repair factor) are used not only for targeted DNA damage to induce toxicity, but also as a signalling strategy for cellular processes. Using M. tuberculosis as an exemplar, we show that DarT-DarG regulates growth by ADP-ribosylation of DNA at the origin of chromosome replication. Structural and mechanistic data of the ADP-ribosyltransferase DarT demonstrate the role of ADP-ribosylation of DNA by this enzyme in generating toxicity and regulating cellular signalling processes in bacteria., Author(s): Marion Schuller [sup.1] , Rachel E. Butler [sup.2] , Antonio Ariza [sup.1] , Callum Tromans-Coia [sup.1] , Gytis Jankevicius [sup.1] [sup.3] , Tim D. W. Claridge [sup.4] , Sharon [...]

Mycobacterium tuberculosis is able to use a variety of carbon sources in vivo and current knowledge suggests that cholesterol is used as a carbon source during infection. The catabolized cholesterol is used both as an energy source (ATP generation) and as a source of precursor molecules for the synthesis of complex methyl-branched fatty acids. In previous studies, we described a TetR-type transcriptional repressor, kstR, that controls the expression of a number of genes involved in cholesterol catabolism. In this study, we describe a second TetR-type repressor, which we call kstR2. We knocked this gene out in Mycobacterium smegmatis and used microarrays and quantitative RT-PCR to examine the effects on gene expression. We identified a palindromic regulatory motif for KstR2, showed that this motif is present in three promoter regions in mycobacteria and rhodococcus, and demonstrated binding of purified KstR2 to the motif. Using a combination of motif location analysis, gene expression analysis and the examination of gene conservation, we suggest that kstR2 controls the expression of a 15 gene regulon. Like kstR, kstR2 and the kstR2 regulon are highly conserved among the actinomycetes and studies in rhodococcus suggest a role for these genes in cholesterol catabolism. The functional significance of the regulon and implications for the control of cholesterol utilization are discussed. DOI 10.1099/mic.0.034538-0

A definitive transcriptome atlas for the non‐coding expressed elements of the members of the Mycobacterium tuberculosis complex (MTBC) does not exist. Incomplete lists of non‐coding transcripts can be obtained for some of the reference genomes (e.g., M. tuberculosis H37Rv) but to what extent these transcripts have homologues in closely related species or even strains is not clear. This has implications for the analysis of transcriptomic data; non‐coding parts of the transcriptome are often ignored in the absence of formal, reliable annotation. Here, we review the state of our knowledge of non‐coding RNAs in pathogenic mycobacteria, emphasizing the disparities in the information included in commonly used databases. We then proceed to review ways of combining computational solutions for predicting the non‐coding transcriptome with experiments that can help refine and confirm these predictions. [ABSTRACT FROM AUTHOR]

This study describes how bkaR, a highly conserved mycobacterial Tet R-like transcriptional repressor, regulates a number of nearby genes that have associations with branched-chain keto-acid metabolism. bkaR ( MSMEG_4718) was deleted from the nonpathogenic species Mycobacterium smegmatis, and changes in global gene expression were assessed using microarray analysis and reporter gene studies. bkaR was found to directly control the expression of 10 genes in M. smegmatis, and its ortholog in Mycobacterium tuberculosis ( Rv2506) is predicted to control at least 12 genes. A conserved operator motif was identified, and binding of purified recombinant M. tuberculosis BkaR to the motif was demonstrated. Analysis of the stoichiometry of binding showed that BkaR binds to the motif as a dimer. [ABSTRACT FROM AUTHOR]

KstR2 (Rv3557c) is one of two TetR-family transcriptional repressors of cholesterol metabolism in Mycobacterium tuberculosis. The ability to degrade cholesterol fully is important for pathogenesis, and therefore this repressor was expressed, purified and crystallized. Crystals of KstR2 diffracted to better than 1.9 Å resolution and belonged to space group C2, with unit-cell parameters a = 72.3, b = 90.3, c = 49.7 Å, α = γ = 90, β = 128.2°. [ABSTRACT FROM AUTHOR]

Cholesterol can be a major carbon source for Mycobacterium tuberculosis during infection, both at an early stage in the macrophage phagosome and later within the necrotic granuloma. KstR is a highly conserved TetR-family transcriptional repressor (TFR) that regulates a large set of genes responsible for cholesterol catabolism. Many genes in this regulon, including kstR, are either induced during infection or are essential for survival of Mtb in vivo. In this study we identified two ligands for KstR, both of which are CoA thioester cholesterol metabolites with four intact steroid rings. A metabolite in which one of the rings was cleaved was not a ligand. We confirmed the ligand-protein interactions using instrinsic tryptophan fluorescence, and showed that ligand binding strongly inhibited KstR-DNA binding using surface plasmon resonance (IC50 for ligand = 25 nM). Crystal structures of the ligand-free form of KstR show variability in the position of the DNA-binding domain (DBD). In contrast, structures of KstR-ligand complexes are highly similar to each other and demonstrate a position of the DBD that is unfavourable for DNA binding. Comparison of ligand-bound and ligand-free structures identifies residues involved in ligand specificity and reveals a distinctive mechanism by which the ligand-induced conformational change mediates DNA release. [ABSTRACT FROM AUTHOR]

Abstract Tuberculosis (TB), caused by Mycobacterium tuberculosis (MTB), is a leading cause of infectious disease mortality. Animal infection models have contributed substantially to our understanding of TB, yet their biological and non-biological limitations are a research bottleneck. There is a need for more ethically acceptable, economical, and reproducible TB infection models capable of mimicking key aspects of disease. Here we demonstrate and present a basic description of how Galleria mellonella (the greater wax moth, Gm) larvae can be used as a low cost, rapid and ethically more acceptable model for TB research. This is the first study to infect Gm with the fully virulent MTB H37Rv, the most widely used strain in research. Infection of Gm with MTB resulted in a symptomatic lethal infection, the virulence of which differed from both attenuated Mycobacterium bovis BCG and auxotrophic MTB strains. The Gm-MTB model can also be used for anti-TB drug screening, although CFU enumeration from Gm is necessary for confirmation of mycobacterial load reducing activity of the tested compound. Furthermore, comparative virulence of MTB isogenic mutants can be determined in Gm. However, comparison of mutant phenotypes in Gm against conventional models must consider the limitations of innate immunity. Our findings indicate that Gm will be a practical, valuable and advantageous additional model to be used alongside existing models to advance tuberculosis research. [ABSTRACT FROM AUTHOR]

Efflux of antibiotics is an important survival strategy in bacteria. Mycobacterium tuberculosis has approximately sixty efflux pumps, but little is known about the role of each pump or the substrates they efflux. The putative efflux pump, EfpA, is a member of the major facilitator superfamily and has been shown to be essential by saturation transposon mutagenesis studies. It has been implicated in the efflux of isoniazid (INH), which is a first-line drug used to treat tuberculosis (TB). This is supported by evidence from transcriptional profiling showing that efpA is induced in response to INH exposure. However, its roles in the physiology and adaptation of M. tuberculosis to antibiotics have yet to be determined. In this study, we describe the repression of efpA in M. tuberculosis, using CRISPR interference (CRISPRi) to knockdown the expression of this essential gene and the direct effect of this on the ability of M. tuberculosis to survive exposure to INH over a 45-day time course. We determined that wild-type levels of efpA were required for recovery of M. tuberculosis following INH exposure and that, after 45 days of INH exposure, only a few viable colonies were recoverable from efpA -repressed M. tuberculosis . We conclude that EfpA is required for recovery of M. tuberculosis following INH exposure, which could reduce the efficacy of INH in vivo , and that EfpA may have a role in the development of resistance during drug therapy., Competing Interests: The authors declare no conflict of interest.

Background: Cross-species tuberculosis (TB) transmission between humans and animals has been reported for quite a long time in sub-Saharan Africa. Because humans and animals coexist in the same ecosystem, exploring their potential for cross-species transmission and the impact the disease may have on the health of humans, animals, and their products is critical., Objectives: This study aimed to identify risk factors for transmission of TB ( Mycobacterium tuberculosis ) and to assess the potential for zoonotic TB ( Mycobacterium bovis ) transmission in the Serengeti ecosystem where humans and animals are in intense contact. Our aim is to create a base for future implementation of appropriate control strategies to limit infection in both humans and animals., Methodology: We administered a semi-structured questionnaire to 421 self-reporting patients to gather information on risk factors and TB occurrence. In a parallel study, researchers screened sputum smears using Ziehl-Neelsen staining and confirmed by mycobacterial culture. We then performed descriptive statistics (Pearson's chi-square test) and logistic regression analysis to establish frequencies, association, and quantification of the risk factors associated with TB cases., Results: Our findings showed 44% (95% confidence interval [CI], 0.40-0.49) of the results were positive from sputum samples collected over a 1-year duration in areas with a high TB burden, particularly the Bunda district, followed by the Serengeti and Ngorongoro districts. Of the culture-positive patients who also had infections other than TB (43/187 patients), 21 (49%) were HIV positive. Contact with livestock products (odds ratio [OR] 6.0; 95% CI, 1.81-19.9), infrequent milk consumption (OR 2.5; 95% CI, 1.42-4.23), cigarette smoking (OR 2.9; 95% CI, 1.19-7.1.2), and alcohol consumption (OR 2.3; 95% CI, 1.22-4.23) were associated with a higher likelihood of TB infection., Conclusion: There was no evidence of direct cross-species transmission of either M tuberculosis or M bovis between humans and animals using the study methods. The absence of cross-species TB transmission could be due to limited chances of contact rather than an inability of cross-species disease transmission. In addition, not all people with presumptive TB are infected with TB, and therefore control strategies should emphasise confirming TB status before administering anti-TB drugs., Competing Interests: Competing Interests: None declared., (© The East African Health Research Commission 2017.)

The ability to select genes to knock out of mycobacterial genomes has greatly improved our understanding of mycobacteria. This chapter describes a method for doing this. The gene (including a 1-kb flanking region) is cloned into a pNIL series vector and disrupted by deletion or insertion of a cassette. A selection of marker genes obtained from the pGOAL series of vectors are inserted into the pNIL vector to create a suicide delivery system. This delivery vector is introduced into mycobacteria where the disrupted version of the gene replaces the wild-type version by a two-step homologous recombination process. The method involves selecting for a single crossover event followed by selection of double crossovers. Single crossovers have incorporated plasmid marker genes and are sucrose(S), kanamycin(R) and blue on media containing X-gal. Double crossovers have lost plasmid markers and are sucrose(R), kanamycin(S) and white on media containing X-gal.