| dc.description.abstract | In the current dissertation, efforts have been made to probe the in vivo role of DNA gyrase to determine its importance in the growth, physiology and gene expression in mycobacteria. In this dissertation, the role of DNA gyrase in gene expression were explored. The thesis is divided into four chapters. In Chapter I, a general introduction to DNA topology, genome supercoiling, DNA topoisomerases, their mechanism of action
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and regulation is provided. It covers a description of the central player- DNA gyrase followed by its functions in vitro and various factors involved in the regulation of supercoiling. Further, the regulation of topoisomerase activity and the role in gene regulation has been described. In Chapter 2, the studies are aimed at generation and characterization of conditional gyrase mutant in M. smegmatis. Depletion of gyrase level beyond the 50% threshold drastically impaired cell growth and viability indicating the minimal gyrase level is required for cell sustenance. Various pleiotropic effects, altered colony morphology, elongated cells and diffused nucleoid were observed in the gyrase-depleted cells. The perturbation in the gyrase level resulted in a reduction of FtsZ levels in elongated cells suggesting the link between gyrase and cell division. Further, transcript analysis indicated gyrase as a global regulator modulating the expression of the genes involved in encoding topology modulators, transcription and core DNA transaction processes. Altered transcription pattern was a result of impaired occupancy of RNAP at the promoters and coding sequences. The gyrase knockdown strain acquired increased sensitivity to drugs used in TB treatment demonstrating its utility to screen new anti-tubercular drugs. The study thus establishes the essentiality of gyrase for mycobacterial growth, physiology and illustrates the consequences of perturbing intracellular gyrase levels on gene expression in mycobacteria. In Chapter 3, studies have been carried out to validate the in vivo functionality of the MtGyrBA fusion protein. Since gyrB and gyrA of M. tuberculosis are separated by a 34 bp stretch and transcription is controlled by the presence of 3 promoters, efforts have been directed towards constructing the fusion gyrase by linking the gyrB and gyrA genes together with a 6 bp linker sequence. The gyrBA fusion has been expressed in a mycobacterial vector
under an inducible promoter. MtGyrBA rescued the growth defect showed by the M. smegmatis gyrase-depleted cells and partially complemented the E. coli ts mutants. The utility of the complemented strain has been tested to screen for drugs that target the M. tuberculosis gyrase in the background of fast growing M. smegmatis. Chapter 4 of the thesis focuses on the transcriptomic landscape of M. tuberculosis gene expression using novobiocin as an agent to bring about the relaxation of the genome at 6 hr and restoration of supercoiling due to relaxation-stimulated transcription (RST) at 24 hr of treatment. Treatment with the inhibitor changed the expression of a large number of genes. 53% of the genome exhibited relaxation-dependent transcription while 41% showed supercoiling-sensitive transcription. Genes with altered expression are distributed as large clusters across the chromosome with a distinct pattern observed during chromosome relaxation. The presence supercoil-sensitive genes interspersed between the clusters were also detected. The downregulated genes had higher AT percentage both upstream and downstream of transcription start site. Three major groups of supercoiling-sensitive genes have been identified throughout the genome: up-regulated (U), down-regulated (D) and less-responsive (LR). Thus, organization of supercoil-sensitive genes in the M. tuberculosis genome reveals DNA supercoiling as a major controller of gene expression in pathogenic mycobacteria. In conclusion, the results presented in this dissertation indicate a close connection between transcription regulation and topology by DNA gyrase mediated supercoiling | en_US |
