Packaging of genomic DNA by proteins and super coiling into chromatin and chromatin-like structures (in bacteria) influences nearly all nuclear process such as replication, transcription, repair, and recombination. A ubiquitous class of enzymes termed “DNA topoisomerases” pay key roles during these process. The reactions catalyzed by the members of the DNA topoisomerases family share a common chemistry, which involves phosphodiester bond breakage and re-joining, to bring about a change in the linking number of DNA. Nevertheless, the underlying mechanisms used by these enzymes differ significantly from another. Consequently, DNA topoisomerases are divided into type I and type II enzymes. The mechanism(s) by which DNA topoisomerases perform their functions, and act as targets for anti-bacterial and anti-neoplastic drugs, has attracted considerable interest. Based on these and other finding, I have chosen DNA gyrase from mycobacteria as the subject of my Ph.D. theses investigation. The prokaryotic enzyme, DNA gyrase, is unique amongst all topoisomerases being the only enzyme capable of introducing negative super coils in to duplex DNA. Since no equivalent enzymatic activity has been reported in humans, this essential enzyme has been exploited as a during target against many microbial infections including tuberculosis.DNA gyrase is a tetrameric protein, comprised of two pairs of subunits, encoded by gyrA and gyrB. Inhibitors of DNA gyrase know till date target either of the two subunits and are categorized broadly in to two class, viz. coumarins and quinolones. With the emergence of multiple-drug resistant strains of pathogenic bacteria such as Mycobacterium tuberculosis, which is a leading cause of death world-wide, there is a need to develops new lead molecules with novel mechanisms of inhibition. Towards this end, a new approach to inhibit the mycobacterial DNA gyrase using single-chain antibody has been explore in the present study. In addition to this, the differences in the catalytic properties of the subunits and assembly of the Mycobacterium smegmatis enzyme vis-à-vis Escherichia coli DNA gyrase have been examined. Further, the in vivo relationship of DNA gyrase with the transcription machinery of the cell has also been investigated, with an emphasis on the biology of mycobacteria.