Studies on the multifunctional GTPase protein CgtA in Mycobacterium smegmatis mc² 155

Singh, Rahul Kumar (2025) Studies on the multifunctional GTPase protein CgtA in Mycobacterium smegmatis mc² 155. PhD thesis, Indian Institute of Science Education and Research Kolkata.

Text (PhD thesis of Rahul Kumar Singh (15RS073)) 15RS073.pdf - Submitted Version Restricted to Repository staff only Download (9MB)

Abstract

CgtA or Obg is a highly conserved, multifunctional bacterial protein that is essential in many bacteria and is a potential drug target. The GTP binding protein CgtA shows association with the ribosome, DNA repair, cell division, and cell cycle. CgtA protein has three domains, i.e., N-terminal domain (NTD), central GTPase domain, and C-terminal domain (CTD). While the NTD and the GTPase domains are conserved the CTD is variable in length. The N-terminal domain has a specific structure called Obg fold which helps in binding with ribosome. The GTPase domain exhibits hydrolysis of GTP to GDP. However, the functions of CTD are not been very well studied so far. In Mycobacterium sp., the CgtA protein is not very well studied. Given its significance, an investigation of its mechanism of cellular activity specific to Mycobacterium smegmatis mc² 155 was undertaken and is reported in this thesis. In the first chapter four mutant strains have been created from the wild type strain of M. smegmatis: C-terminus merodiploid (CTD cgtA⁺ CTD cgtA::hygi), C-terminus knock out (ΔCTD cgtA::hygi), cgtA merodiploid (cgtA⁺cgtA::hygi), and cgtA knock out (ΔcgtA::hygr). The growth curve of mutant strains shows a significant decrease compared to the wild-type strain. The size, width, and area of mutants, including C-terminus merodiploid (CTD cgtA⁺ CTD cgtA::hygi), C-terminus knock out (ΔCTD cgtA::hygi), cgtA merodiploid (cgtA⁺cgtA::hygr), and cgtA knock out (ΔcgtA::hygr), are significantly reduced when compared to wild type cells. The reduction of biofilm formation in cells occurs in 7H10 agar medium following the knockout of cgtA and C-terminus cgtA gene strains. Biofilm formation of the full length cgtA and C-terminus cgtA gene knockout strains in broth medium does not occur. The second chapter of this thesis presents findings from biochemical studies aimed at exploring the effects of truncation mutation in CgtAms. The presence of 70S and 50S, along with an excess of GTP in the reaction, leads to an increase in specific GTPase activity. The activity of GTPase rises with the deletion of the C-terminal domain in CgtAms during the reaction. The binding assay of ribosome (70S and 50S) with the proteins (CgtAms and C-terminal domain deleted CgtA) conducted through ultracentrifugation density gradient indicates that the C-terminal domain of CgtAms enhances the interaction with CgtAms and the ribosome. The results indicate that the two domains of CgtAms, namely the Obg domain and the GTPase domain, work together, allowing for signal transmission from the Obg domain to the GTPase domain or the other way around. The third chapter illustrates the role of CgtA, primarily focused on the interaction between a DNA fragment and gDNA through chip technology. The full length of the cgtA ORF and its truncation mutations, specifically the C-terminal deleted cgtA, have been cloned into the pMnyT-kan plasmid vector. Following the successful cloning of the cgtA gene and the creation of truncation mutations, the proteins were expressed, and their expression was confirmed through western blot analysis. The validation of DNA-protein interactions has been accomplished by employing ChIP, followed by the isolation and sequencing of the interacting DNA. Analysis of isolated DNA reveals five different motifs and a coordinated ChIP binding network.

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