Key pointsBy sequencing 223 M. tuberculosis strains from Southern India, we expanded the studied genetic diversity of lineages 1 and 3. We observed local transmission of strains; unexplained resistance; potential novel resistance mutations; and that isoniazid resistance was gained first.

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Regulation of gene expression is one of the mechanisms of virulence in pathogenic organisms. In this context, we would like to understand the gene regulation of acetamidase enzyme of Mycobacterium smegmatis, which is the first reported inducible enzyme in mycobacteria. The acetamidase is highly inducible and the expression of this enzyme is increased 100-fold when the substrate acetamide is added. The acetamidase structural gene (amiE) is found immediately downstream of three predicted open reading frames (ORFs). Three of these genes along with a divergently expressed ORF are predicted to form an operon and involved in the regulation of acetamidase enzyme. Here we report expression, purification and functional characterization of AmiA which is one of these predicted ORFs. Electrophoretic mobility shift assays showed that AmiA binds to the region between the amiA and amiD near the predicted promoter (P2). Over-expression of AmiA significantly lowered the expression of acetamidase compared to the wild type as demonstrated by qRT-PCR and SDS-PAGE. We conclude that AmiA binds near P2 promoter and acts as a repressor in the regulation of acetamidase operon. The described work is a further step forward toward broadening the knowledge on understanding of the complex gene regulatory mechanism of Mycobacterium sp.

Background: Mycobacterium tuberculosis resides inside host macrophages during infection and adapts to resilient stresses generated by the host immune system. As a response, M. tuberculosis codes for short-chain dehydrogenases/ reductases (SDRs). These SDRs are nicotinamide adenine dinucleotide-reliant oxidoreductases involved in cell homeostasis. The precise function of oxidoreductases in bacteria especially M. tuberculosis were not fully explored. This study aimed to know the detail functional role of one of the oxidoreductase Rv0148 in M. tuberculosis. Results: In silico analysis revealed that Rv0148 interacts with Htdy (Rv3389) and the protein interactions were confirmed using far western blot. Gene knockout mutant of Rv0148 in M. tuberculosis was constructed by specialized transduction. Macrophage cell line infection with this knockout mutant showed increased expression of pro-inflammatory cytokines. This knockout mutant is sensitive to oxidative, nitrogen, redox and electron transport inhibitor stress agents. Drug susceptibility testing of the deletion mutant showed resistance to first-line drugs such as streptomycin and ethambutol and second-line aminoglycosides such as amikacin and kanamycin. Based on interactorme analysis for Rv0148 using STRING database, we identified 220 most probable interacting partners for Htdy protein. In the Rv0148 knockout mutants, high expression of htdy was observed and we hypothesize that this would have perturbed the interactome thus resulting in drug resistance. Finally, we propose that Rv0148 and Htdy are functionally interconnected and involved in drug resistance and cell homeostasis of M. tuberculosis. Conclusions:Our study suggests that Rv0148 plays a significant role in various functional aspects such as intermediatory metabolism, stress, homeostasis and also in drug resistance.