DNA topoisomerases and topology modulation

The bacterial genome is supercoiled, compacted and condensed into a nucleoid by the combined action of topoisomerases and  nucleoid associated proteins(NAPs). In my laboratory, a combination of genetics, biochemistry, structural and cell biology approaches are employed to study the function of both topoisomerases and NAPs of M. tuberculosis. Combined action of topoisomerases and NAPs ensure bacterial chromosome compaction to regulate all major DNA transaction processes. Some of the highlights of the work in our long- term studies:  development of peptide inhibitors specific to mycobacterial gyrase with a novel mechanism of action, elucidation of dual role of gyrase in supercoiling and decatenation, the molecular basis of switch from supercoiling to decatenation - binding of second DNA to GyrB subunit, demonstration of direct binding of fluoroquinolones to GyrA subunit and holoenzyme  apart from the established mode of action, action of gyrase modulatory protein, YacG, in regulating the supercoiling activity by inhibiting strand passage, importance of carboxyl terminal basic residues in strand passage activity of TopoI, development of inhibitory monoclonal antibodies against TopoI and first set of small molecule inhibitors, characterization of a membrane anchored NAP (Rv3852) involved in lipid metabolism, biofilm formation, direct physical interaction between HU and TopoI to enhance DNA relaxation activity of the enzyme by stimulating the strand passage, In addition to characterizing new mycobacteria specific NAPs, we have developed small molecule inhibitors of HU by structure based design. The inhibitors bind to the DNA binding cleft to displace DNA, de-compact the nucleoid and inhibit M. tuberculosis growth. Chemical probing using these inhibitors reveal the importance of HU regulon to the organism. HU is modified extensively by post- translational modifications, that include acetylation, phosphorylation, succinylation and methylation, influencing the bacterial gene expression. Some of these epigenetic modifiers impact the host genome dynamics and intracellular survival of the pathogen. One such bacterial effector- Rv2067 methyl transferase intercepts and reprograms host epigenetics and downstream signaling by multipronged approach ensuring the survival of the pathogen in the hostile intracellular environment.

Regulation of gene expression in mycobacteria

To study transcription initiation, elongation and termination, we developed a method for purification of optimized RNAP holoenzyme (with stoichiometric SigA). Promoter: polymerase interaction studies with this enzyme revealed different rate limiting steps at various promoters. Next, we elucidated the distinct mechanism of growth phase dependent regulation of gyr and rRNA promoters of Mtb. An interaction between promoter discriminator region and sigma 1.2 dictate the initiating NTP and pppGpp mediated regulation of transcription initiation. The mechanism of autoregulation of gyrase and topoisomerase I by RST and SST  and the connection between RST and reiterative transcription has been established. Both intrinsic and factor dependent transcription termination have been studied uncovering alternate paradigms and unusual characteristics. Understanding new facets of Rho function has led us to suggest it as the major regulator in the genus. Next, we have explored functional co-operation between intrinsic and Rho mediated termination. As a follow up, conditional knock down strains of key cellular players described above have been generated to investigate the topology - transcription connection in mycobacteria.