Abstract
The stringent response, an adaptive response to nutrient limitation and exposure to xenobiotics in bacteria, is mediated by two intracellular signaling molecules, pppGpp and ppGpp, together called (p)ppGpp. The cellular level of (p)ppGpp in bacterial cells is controlled by the Rel/Spo family of proteins. In the cholera pathogen, Vibrio cholerae, (p)ppGpp metabolism is regulated by the products of at least three genes relA, spoT, and relV. In this study, we identify and characterize the function of the guanosine-5′-triphosphate 3′-diphosphate pyrophosphatase A (GppA) encoding gene gppA of V. cholerae. Genomic analysis indicates that the gppA locus is conserved in vibrios and organized as a bicistronic operon along with the rhlB gene. We engineered the genome of V. cholerae to develop different mutants devoid of GppA and/or other phosphate metabolic enzymes. Our findings indicate that in V. cholerae, GppA plays an important role in the conversion of pppGpp to ppGpp during amino acid deprivation but not during glucose starvation. Quantitative analyses of the gppA transcript level reveal its differential expression pattern at different growth phases and starvation conditions. It has been observed that the GppA deficiency during amino acid starvation condition could be complemented by overexpressing the exopolyphosphatase coding gene ppx of V. cholerae. By deletion analysis, we further demonstrate that the amino and carboxy terminal sequences flanking the Ppx-GppA motif of the GppA protein of V. cholerae are also important for its enzymatic function.
Highlights
The enteric pathogen Vibrio cholerae faces various physicochemical stresses while living within or outside of the human intestine
We found that unlike in E. coli, the gppA gene in the genome of V. cholerae is arranged in an operon along with rhlB (Figure 3)
At present no information is available about the function of RhlB of V. cholerae, the arrangement of the rhlB-gppA genes in an operon has raised a possibility that RhlB could be involved in the regulation of the stringent response (SR) in this pathogen, which needs investigation
Summary
The enteric pathogen Vibrio cholerae faces various physicochemical stresses while living within or outside of the human intestine. While in Escherichia coli, intracellular (p)ppGpp metabolism is controlled by two multidomain containing proteins RelA and SpoT, in V. cholerae, apart from these two enzymes, a small alarmone synthetase, called RelV, is involved in (p)ppGpp metabolism as shown in Figure 1 (Haralalka et al, 2003; Das and Bhadra, 2008; Das et al, 2009; Dasgupta et al, 2014). In E. coli, apart from RelA and SpoT, another well-characterized enzyme, called guanosine pentaphosphate 5 -phosphohydrolase A (GppA), is associated with the (p)ppGpp metabolic cycle (Somerville and Ahmed, 1979; Hara and Sy, 1983; Keasling et al, 1993; Mechold et al, 2013). The “Ppx-GppA” family of proteins share homology with both Ppx and GppA, and they may have the ability to hydrolyze both polyP and pppGpp as substrates (Kristensen et al, 2004, 2008; Choi et al, 2012; Malde et al, 2014; Song et al, 2019)
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