Knockdown Experiment Reveals an Essential GTPase CgtA's Involvement in Growth, Viability, Motility, Morphology, and Persister Phenotypes in Vibrio cholerae.

CgtA is an essential bacterial GTPase consisting of a highly conserved N-terminal Spo0B-associated GTP-binding protein (Obg) domain, a central GTPase domain, and a variable C-terminal domain (CTD). This study reports global changes in the proteome and transcriptome of wild-type (Wt) versus full-length CgtA-depleted Vibrio cholerae in minimal media. Comparative transcriptome sequencing (RNA-Seq), followed by comparative proteomic analyses, revealed that the knockdown of cgtA significantly altered expressions of 311 proteins involved in diverse cellular activities, many of which are associated with the survival of V. cholerae. Various intracellular functional roles of CgtA in growth, viability, motility, morphology, and persister phenotypes in V. cholerae are revealed based on subsequent confirmatory experiments. Furthermore, a more sustained mRNA expression pattern of cgtA in a minimal medium than in a rich medium was also observed for Wt V. cholerae, where the highest level of mRNA expression of cgtA was observed during the logarithmic growth phase. Thereby, we propose that minimal medium-associated reduced growth rate coupled with cgtA depletion aggravates the intracellular stress in V. cholerae, interrupting vital cellular processes. The functional role of the CTD in V. cholerae is not fully understood. Hence, to specifically investigate the intracellular role of the 57-amino-acid-long CTD of CgtAVC, the CTD-only portion of CgtA was deleted. Subsequent proteomics studies revealed an altered expression of 240 proteins in the CgtA(ΔCTD) mutant, having major overlap with the full-length cgtA-deleted condition. Overall, our study reveals an alternative facet of the survival mechanism of V. cholerae during nutritional downshift as per the concomitant consequences of cgtA depletion. IMPORTANCE It is very important that we must find new drug target proteins from multidrug-resistant human-pathogenic organisms like V. cholerae. CgtA is among such potential candidates, and here, we are reporting about some newly identified cellular roles of it that are important for the survival of V. cholerae. Briefly, we knocked down the full-length cgtA gene, as well as did a partial deletion of this gene from the V. cholerae genome followed by RNA-Seq and proteomics studies. Results from our study revealed up- and downregulation of several known and unknown genes and proteins as the effect of the cgtA knockdown experiment. Also, we have presented some interesting observations that are linked with cgtA for growth, viability, motility, morphology, and persister phenotypes in V. cholerae. Our study enhances the importance of CgtA and paves the way for further exploration based on our provided data.

Effect of a single amino acid substitution G98D in a ribosome-associated essential GTPase, CgtA, on the growth and morphology of Vibrio cholerae.

CgtA, a highly conserved 50S ribosome-associated essential GTPase, acts as a repressor of the stringent stress response under nutrient-rich growth conditions to suppress basal levels of the alarmone ppGpp in V. cholerae. To further explore the in vivo functionality of CgtA, we introduced an amino acid substitution, i.e., Gly98Asp, in a conserved glycine residue in the N-terminal domain. The constructed V. cholerae mutant was designated CgtA(G98D). Comparison of cell sizes of the CgtA(G98D)mutant with its isogenic wild-type (Wt) strain N16961 under different phases of growth by Transmission Electron Microscopy (TEM) and statistical analysis suggests that CgtA may control the cell size of V. cholerae. The cell length is significantly reduced, corresponding to the delayed growth in the mid-logarithmic phase. The differences in the cell length of CgtA(G98D) and Wt are indistinguishable in the late logarithmic phase. During the stationary phase, marked by higher OD600, a sub-population of CgtA(G98D) cells outnumbered the Wt cells lengthwise. CgtA(G98D) cells appeared slenderer than Wt cells with significantly reduced cell width. However, the centerline curvature is preserved in CgtA(G98D) cells. We propose that in addition to its multitude of intracellular roles, CgtA may influence the cell size of V. cholerae.

Cold shock response and major cold shock proteins of Vibrio cholerae.

When exponentially growing Vibrio cholerae cells were shifted from 37 degrees C to various lower temperatures, it was found that the organism could adapt and grow at temperatures down to 15 degrees C, below which the growth was completely arrested. There was no difference between the patterns of the cold shock responses in toxinogenic and nontoxinogenic strains of V. cholerae. Gel electrophoretic analyses of proteins of cold-exposed cells revealed significant induction of two major cold shock proteins (Csps), whose molecular masses were 7.7 kDa (CspA(VC)) and 7.5 kDa (CspV), and six other Csps, most of which were much larger. We cloned, sequenced, and analyzed the cspV gene encoding the CspV protein of V. cholerae O139 strain SG24. Although CspA(VC) and CspV have similar kinetics of synthesis and down-regulation, the corresponding genes, cspA and cspV, which are located in the small chromosome, are not located in the same operon. A comparative analysis of the kinetics of synthesis revealed that the CspV protein was synthesized de novo only during cold shock. Although both CspA(VC) and CspV were stable for several hours in the cold, the CspV protein was degraded rapidly when the culture was shifted back to 37 degrees C, suggesting that this protein is probably necessary for adaptation at lower temperatures. Northern blot analysis confirmed that the cspV gene is cold shock inducible and is regulated tightly at the level of transcription. Interestingly, the cspV gene has a cold shock-inducible promoter which is only 12 nucleotides from the translational start site, and therefore, it appears that no unusually long 5' untranslated region is present in its mRNA transcript. Thus, this promoter is an exception compared to other promoters of cold shock-inducible genes of different organisms, including Escherichia coli. Our results suggest that V. cholerae may use an alternative pathway for regulation of gene expression during cold shock.