Mycobacterium tuberculosis (Mtb) has successfully exploited macrophages as its primary niche in vivo. However, the bacterial requirements that promote its intracellular survival remain undefined. Our research interest is primarily organized to identify and unravel molecular mechanism of functioning of Mtb PhoP-PhoR system. Recent studies showing striking similarities between H37Ra and different Mtb phoP-phoR knock-out mutants in colony morphology, staining features and failure to grow as serpentine cords, led us to investigate whether MtbH37Ra expressed a functional PhoP-PhoR two-component system. As a beginning to understand the contribution of PhoP-PhoR system in Mtb physiology, a functional characterization of the phoP and phoR gene products was initiated in our laboratory. We investigate the origins of binding specificity in protein complexes, and probe the determinants of complex formation using biochemical assays. Our observations raise the possibility that PhoP may be capable of adopting different orientations as it binds to a vast array of genes to activate or repress transcription. Although much work remains to be done in identifying PhoP regulon in Mtb, our biochemical data on PhoP and its binding interactions(s) represents a significant advancement in beginning to understand the PhoPR system. Elucidation of the specific signal sensed by the Mtb PhoR sensor kinase is the next challenge to understand under which conditions and with which aim(s) PhoP function is regulated. Significant recognition: Awards, fellowships, international funding of distinction, technologies transferred/licensed etc.: Awarded Prof. B.K. Bachhawat travel grant for young scientist (2009) Awarded International travel grant by International S&T Affairs Division of Council of Scientific and Industrial Research (2011 Awarded International travel grant by Department of Science and Technology (2011) Selected for National Bioscience Award for Career Development (2011) Elected member of prestigious Guha Research Conference (GRC-2011) Awarded Raman Research Fellowship 2013-14 by Council of Scientific and Industrial Research (CSIR), Government of India Selected list of Publications and Patents: Bansal, R., Anil Kumar, V., Sevalkar, R. R., Singh, P. R. and Sarkar, D. (2017) Mycobacterium tuberculosis virulence regulator PhoP interacts with alternative sigma factor SigE during acid-stress response. Mol Microbiology 104, 400-411. Anil Kumar, V., Goyal, R., Bansal, R., Singh, N., Sevalkar, R.R., Kumar, A., and Sarkar, D. (2016) EspR-dependent ESAT-6 secretion of Mycobacterium tuberculosis requires the presence of virulence regulator PhoP. J Biol Chem 291, 19018-19030. Samuel, J.S., Kumar, D., Chodisetti, S.B., Agrewala, J.N., Singh, B., Guptasarma, P. and Sarkar D. (2015) Probing protease sensitivity of recombinant human erythropoietin reveals alpha 3- alpha 4 inter- helical loop as a stability determinant. Proteins 83, 1813-1822 Macdonald, R., Sarkar, D., Amer, B.R. and Clubb, R.T. (2015)Solution structure of the PhoP DNA binding domain from Mycobacterium tuberculosis. J Biomol NMR 63, 111-117. Singh, R., Anil Kumar, V., Das, A. K., Bansal, R. and Sarkar, D. (2014) A transcriptional co-repressor regulatory circuit controlling the heat-shock response of Mycobacterium tuberculosis. Mol Microbiology 94, 450-465. Das, A. K., Anil Kumar, V., Sevalkar, R. R., Bansal, R. and Sarkar, D. (2013) Unique N-terminal arm of Mycobacterium tuberculosis PhoP plays an unusual role in its regulatory function. J Biol Chem 288, 29182-29192. Goyal, R., Das, A. K., Singh, R., Singh, P. K., Korpole, S., and Sarkar, D. (2011) Phosphorylation of PhoP protein plays direct regulatory role in lipid biosynthesis of Mycobacterium tuberculosis. J Biol Chem 286, 45197-45208 Pathak, A., Goyal, R., Sinha, A., and Sarkar, D. (2010) Domain structure of virulence-associated response regulator PhoP of Mycobacterium tuberculosis: role of the linker region on regulator-promoter interaction(s). J Biol Chem 285, 34309-34318 Das, A., Pathak, A., Sinha, A., Datt, M., Singh, B., Karthikeyan, S., and Sarkar, D. (2010) A single amino acid substitution in the C terminus of PhoP determines DNA-binding specificity of the virulence-associated response regulator from Mycobacterium tuberculosis. J Mol Biol 398, 647-656 Gupta, S., Pathak, A., Sinha, A., and Sarkar, D. (2009) Mycobacterium tuberculosis PhoP recognizes two adjacent direct-repeat sequences to form head-to-head dimers. J Bacteriol 191, 7466-7576 Sinha, A., Gupta, S., Bhutani, S., Pathak, A., and Sarkar, D. (2008) PhoP-PhoP Interaction at Adjacent PhoP Binding Sites Is Influenced by Protein Phosphorylation. J Bacteriol 190, 1317-1328 Gupta, S., Sinha, A. and Sarkar, D. (2006) Transcriptional autoregulation by Mycobacterium tuberculosis PhoP involves recognition of novel direct repeat sequences in the regulatory region of the promoter. FEBS Letters 580, 5328-5338 Gupta, S., Chakrabarti, P. and Sarkar, D. (2005) Nucleotide induced conformational change in the catalytic subunit of the phosphate specific transporter from M. tuberculosis: implications for the ATPase Structure. Biochim Biophys Acta 1750, 112-21

Biochemistry and Molecular Microbiology