Understanding The Pleiotropic Effects Of CgtA, An Essential GTPase, In Vibrio cholerae

Das, Sagarika (2022) Understanding The Pleiotropic Effects Of CgtA, An Essential GTPase, In Vibrio cholerae. PhD thesis, Indian Institute of Science Education and Research Kolkata.

Text (PhD thesis of Sagarika Das (14RS084)) 14RS084.pdf - Submitted Version Restricted to Repository staff only Download (9MB)

Abstract

CgtA is a highly conserved 50S ribosome-associated essential Obg-GTPase protein that consists of three domains, N-terminal domain (NTD), GTPase domain, and C-terminal domain (CTD). The glycine-rich Obg-fold with finger-like projections in the conserved NTD makes CgtA unique from other GTPases. Extensive studies are a prerequisite to understanding its mode of action in vivo and in vitro. It is reported that in Vibrio cholerae, CgtA acts as a repressor of the stringent stress response only during growth under unstressed nutrient-rich media to suppress basal levels of ppGpp, alarmone for stringent response signaling. In order to explore the functionality of this protein in vivo, a total of three strains of V. cholerae were genetically engineered to incorporate one amino acid substitution mutation and two deletion mutations. The first chapter of the thesis deals with the construction and characterization of the full-length cgtA strain, and ΔC-terminal domain of CgtA (truncated) V. cholerae strains. Various biochemical technical techniques confirmed the strains. The growth pattern of the engineered strains was studied, the function of the uncharacterized C-terminal domain was characterized in vivo, the chemotactic motility pattern of the strains was observed, and the role of CgtA in persister cell formation of V. cholerae was implicated. In order to gain deeper insights into the cellular networks associated with CgtA GTPase, the enrichment of various interconnected pathways was achieved by using high-throughput techniques: Genome-wide transcriptome analysis by RNA-Seq and label-free comparative proteomics of the cgtA knockdown and C-terminal domain of CgtA truncated strains compared to the wild-type V. cholerae. In the second chapter, incorporating an amino-acid substitution CgtA(G98D) in a conserved glycine residue in the N-terminal domain of CgtA GTPase revealed some interesting morphological features, especially in the stationary phase. The correlation between CgtA and another stressor protein BolA (a morphogene), is highlighted. In the last chapter, deleting a stretch of the leucine-rich interdomain linker connecting the N-terminal domain and the GTPase domain highlighted the mechanistic aspects of the CgtA GTPase through biochemical studies.

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