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  • CgtA is involved in stringent


    CgtA is involved in stringent response and it interacts with SpoT to maintain optimal (p)ppGpp/ppGpp quotient in bacteria. Depletion of CgtA results in increased ppGpp levels [8]. CgtA is also important for cellular development [12] and involved in synchronization of DNA replication initiation and chromosome partitioning. It has been reported that overexpression of CgtA significantly altered distribution of chromosomal DNA, thus hindered bacterial growth and led to enlargement of cells [13]. Apart from these, CgtA has a unique role in inducing multidrug tolerance in bacteria at single cell level, using (p)ppGpp stringent response alarmone and proceeds via regulatory mechanism of transcription of hokB-sokB type I toxin-antitoxin module. Increased CgtA levels cause induction in HokB expression, which in turn is the regulatory key of membrane potential. Elevated HokB levels are reflected via membrane depolarization that results ultimately in persistence [14]. On the structural part, first from Bacillus subtilis [15], then from Thermus thermophilus [16] and very recently from E. coli [17], crystal structures revealed that CgtA consists of three domains, N-terminal glycine rich Obg domain, middle Ras-like GTP hydrolysis domain, and C-terminal acidic domain. The GTP hydrolysis domain contains the conserved P-loop, switch I and switch II regions, which are involved in GTP hydrolysis. Although the role of GTPase domain is obvious, but the role played by Obg domain and the C-terminal domain is not very well defined and various related questions remain unaddressed. Thus, although plethora of information is available about CgtA, its mechanisms of action are yet to be elucidated. The Obg domain of the CgtA protein does not share any structural or sequential similarity with any known protein domains. Lagging similarity in amino norepinephrine bitartrate sequences and conserved domains, this domain contains only few surface-exposed nearly conserved amino acids, suggesting that the overall structure of the domain is more important than the sequence. The portion of the Obg domain shows some similarity to the α1 chain of collagen suggesting that it might have a similar role of anchoring the protein at a particular location of the cell [18]. Few temperature sensitive mutants of Obg domain have been studied (G79E and D84N, in B. subtilis) which clearly showed that the temperature sensitive phenotypes are not due to the impaired GTPase activity, but might be due to lack of interaction with the downstream proteins of the signalling pathway [19]. Recently, in E. coli, it was shown that mutation in the Obg domain inhibits its interaction with the ribosome [11]. Further insights are required into the structure-function correlation of Obg domain to elucidate its functional role and importance. The other uncharacterized portion in the CgtA protein i.e., the C-terminal domain (CTD) is known to vary largely in different organisms, both in amino acids content and length. CTD length varies from about 8 to 150 amino acids long and considered to be intrinsically unstructured [17]. The structural variations in the CTD across the species make the task of elucidating its generalized structural model and identifying its mechanism of action quite difficult. Increasing antibicrobial drug resistant varients of Vibrio cholerae makes it absolutely necessary to identify new drug targets to combat V. cholerae infection [[20], [21], [22]]. In this article, we have systematically dissected the CgtAvc (CgtA of V. cholerae) protein and revealed the functional roles of the aforesaid regions of CgtA, using Ribosome-CgtA GTPase activity as the major assay system. We believe our results will be effective in understanding how exactly this multifunctional CgtA protein works in bacteria in broader aspects, including V. cholerae.
    Materials and methods
    Experimental results
    Discussion Previously it was predicted that Obg domain interacts with other cellular protein partners or it is involved in anchoring the protein at a cellular location [18]. Recently it was shown that point mutations in the loops of Obg affect its ribosome binding ability [11]. We observed that deletion of the first loop caused a reduction in the GTPase activity compared to that of the wild type CgtAvc, whereas deletion of the second & third loop separately; deletion of the first and second loop together; and deletion of all the loops together; abolished the ribosome mediated augmentation of the GTPase activity of CgtAvc. This clearly implies that the Obg domain and the GTPase domain cross talk among themselves. Our results support previous MD simulations studies that exhibited inter-domain movements between Obg and GTPase domain during GTP hydrolysis through the inter-domain flexible linker region [30,31].