A mammalian cell utilizes DNA methylation to modulate gene expression in response to environmental changes during development and differentiation. Aberrant DNA methylation changes as a correlate to diseased states like cancer, neurodegenerative conditions and cardiovascular diseases have been documented. Here we show genome-wide DNA methylation changes in macrophages infected with the pathogen M. tuberculosis. Majority of the affected genomic loci were hypermethylated in M. tuberculosis infected THP1 macrophages. Hotspots of differential DNA methylation were enriched in genes involved in immune response and chromatin reorganization. Importantly, DNA methylation changes were observed predominantly for cytosines present in non-CpG dinucleotide context. This observation was consistent with our previous finding that the mycobacterial DNA methyltransferase, Rv2966c, targets non-CpG dinucleotides in the host DNA during M. tuberculosis infection and reiterates the hypothesis that pathogenic bacteria use non-canonical epigenetic strategies during infection.

Calcium is a very important second messenger, whose concentration in various cellular compartments is under tight regulation. A disturbance in the levels of calcium in these compartments can play havoc in the cell, as it regulates various cellular processes by direct or indirect mechanisms. Here, we have investigated the functional importance of a calcium transporting P2A ATPase, CtpF of Mycobacterium tuberculosis (Mtb) in the pathogen's interaction with the host. Among its uncanny ways of dealing with the host with umpteen strategies for survival and persistence in humans, CtpF is identified as a new player. The levels of ctpF are upregulated in macrophage stresses like hypoxia, high nitric oxide levels and acidic pH. Using confocal microscopy and fluorimetry, we show that CtpF effluxes calcium in macrophages in early stages of Mtb infection. Downregulation of ctpF expression by conditional knockdown resulted in perturbation of host calcium levels and consequent decreased activation of mTOR. We present a mechanism how calcium efflux by the pathogen inhibits mTOR-dependent autophagy and enhances bacterial survival. Our work highlights how Mtb engages its metal efflux pumps to exploit host autophagic process for its proliferation.

Among the nucleoid-associated proteins (NAPs), HU is the most conserved in eubacteria, engaged in overall chromosome organization and regulation of gene expression. Unlike other bacteria, HU from Mycobacterium tuberculosis (MtHU), has a long carboxyl terminal domain enriched in basic amino acids, resembling eukaryotic histone N-terminal tails. As with histones, MtHU undergoes post-translational modifications and we have previously identified interacting kinases, methyltransferases, an acetyltransferase and a deacetylase. Here we show that Rv0802c interacts and succinylates MtHU. Although categorized as a succinyltransferase, we show that this GNAT superfamily member can catalyse both succinylation and acetylation of MtHU with comparable kinetic parameters. Like acetylation of MtHU, succinylation of MtHU caused reduced interaction of the NAP with DNA, determined by electrophoretic mobility shift assay and surface plasmon resonance. However, in vivo expression of Rv0802c did not significantly alter the nucleoid architecture. Although such succinylation of NAPs is rare, these modifications of the archetypal NAP may provide avenues to the organism to compensate for the underrepresentation of NAPs in its genome to control the dynamics of nucleoid architecture and cellular functions.

Known common single-nucleotide polymorphisms in MTRR and BHMT genes may not be significant risk factors for CAD. The functional impact of these polymorphisms on enzyme activity is still unknown. Before additional epidemiologic studies are done, the functional impact of these polymorphisms, if any, should be established.

Gre, one of the conserved transcription factors in bacteria, modulates RnA polymerase (RnAp) activity to ensure processivity and fidelity of RNA synthesis. Gre factors regulate transcription by inducing the intrinsic-endonucleolytic activity of RnAp, allowing the enzyme to resume transcription from the paused and arrested sites. While Escherichia coli and a number of eubacteria harbor GreA and GreB, genus mycobacteria has a single Gre (GreA). to address the importance of the GreA in growth, physiology and gene expression of Mycobacterium smegmatis, we have constructed a conditional knockdown strain of GreA. the GreA depleted strain exhibited slow growth, drastic changes in cell surface phenotype, cell death, and increased susceptibility to front-line anti-tubercular drugs. transcripts and 2D-gel electrophoresis (2D-PAGE) analysis of the GreA conditional knock-down strain showed altered expression of the genes involved in transcription regulation. Among the genes analysed, expression of RnAp subunits (β, β' and ω), carD, hupB, lsr2, and nusA were affected to a large extent. Severe reduction in the expression of genes of rRnA operon in the knock-down strain reveal a role for GreA in regulating the core components of the translation process. Transcription is the central process in the cell. It is regulated by a variety of proteins at different stages. Many of these regulators control transcription by modulating the RNAP activity. The movement of RNAP along the template often gets interrupted by pauses resulting in backtracked RNAP. During backtracking, RNAP slides backwards along the DNA and the 3′ end of the newly synthesized RNA is disengaged from the active centre 1,2. In order to continue RNA extension from the 3′ end, bacteria have evolved strategies that serve to assist the backtracked RNAP to reinitiate elongation. The newly synthesized 3′ end of RNA is subjected to intrinsic cleavage by RNAP itself 3-6. Gre factors bind to the RNAP and assist transcript-cleavage 1,7-12. The absence of Gre would prolong or even prevent rescuing of backtracked RNAP complexes leading to a pause or arrest of transcription. This would limit overall rate and processivity of transcription and hence cell survival. Although Gre factors or their homologues are conserved in all forms of life, present understanding of their in vivo role in bacteria other than in E. coli is limited. Even in E. coli, a complete understanding of the effect of Gre mutants is hindered because of the presence of two Gre (GreA and GreB) and also the partial redundancy in function by the other secondary channel binding proteins such as DksA, Rnk and TraR 13-15. While GreA cleaves 2-3 nucleotide from 3′ end of RNA, GreB cleaves up to 9 nucleotide fragments. However, how the other secondary channel binding proteins complement their function is not clearly understood. It has been shown that GreA, GreB and DksA are mutually competing and exhibit functional redundancy 15. In E. coli, a double knockout of greA and greB did not show lethality, although colonies were s...