Activation of RelA by pppGpp as the basis for its differential toxicity over ppGpp in Escherichia coli
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J Biosci (2020)45:28 DOI: 10.1007/s12038-020-9991-2
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Activation of RelA by pppGpp as the basis for its differential toxicity over ppGpp in Escherichia coli RAJESHREE SANYAL1,2 and RAJENDRAN HARINARAYANAN1* 1
Laboratory of Bacterial Genetics, Centre for DNA Fingerprinting and Diagnostics, Hyderabad, India 2
Manipal Academy of Higher Education, Manipal, India *Corresponding author (Email, [email protected])
MS received 24 June 2019; accepted 29 September 2019 The nucleotide derivatives (p)ppGpp, comprising ppGpp and pppGpp, are important signalling molecules that control various facets of gene regulation and protein synthesis in Escherichia coli. Their synthesis is catalysed by RelA (in response to amino acid limitation) and by SpoT (in response to the limitation of carbon source or fatty acids). SpoT is also a hydrolase for degradation of both ppGpp and pppGpp, while GppA catalyses the conversion of pppGpp to ppGpp. Here we provide evidence to show that pppGpp exerts heightened toxicity compared to that by ppGpp. Thus, gppA spoT double mutants exhibited lethality under conditions in which the single mutants were viable. The extent of RelA-catalysed (p)ppGpp accumulation in the gppA spoT strain was substantially greater than that in its isogenic gppA? derivative. The data is interpreted in terms of a model in which toxicity of pppGpp in the gppA spoT mutants is mediated by its activation of RelA so as to result in a vicious cycle of (p)ppGpp synthesis. Keywords.
(p)ppGpp; GppA; SpoT; RelA; stringent response
1. Introduction Nucleotide-based signalling molecules play a key role in the physiology of organisms by coordinating cellular processes with extracellular or intracellular signals (Pesavento and Hengge 2009). In eubacteria, the signalling molecules ppGpp and pppGpp [collectively referred to as (p)ppGpp] accumulate during starvation, switching the balance of metabolism from growth and cell division to survival and stress response (Chatterji and Kumar Ojha 2001; Braeken et al. 2006; Potrykus and Cashel 2008; Hauryliuk et al. 2015). The pair of molecules are synthesised by the transfer of a pyrophosphate moiety from ATP to GDP or GTP, respectively. The role of (p)ppGpp in bacterial physiology is well-studied in the gram-negative model bacterium Escherichia coli. In b- and c-proteobacteria, (p)ppGpp metabolism is driven by two proteins, RelA
and SpoT, which are members of the Rel/Spo homolog (RSH) family and share similar domain architecture (Mittenhuber 2001; Atkinson et al. 2011). The N-terminal half has the catalytic domain with the (p)ppGpp synthase and hydrolase functions in the case of SpoT and only synthase function in case of RelA. The C-terminal half of the proteins have the regulatory domains important for sensing stress and starvation signals. RelA is a ribosome-bound protein that is activated by the ‘hungry’ codons that appear following amino acid starvation and the consequent increase in the concentration of uncharged tRNA [Wendrich et al. (2002) and refer
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