Abstract
The stringent response is a general bacterial stress response that allows bacteria to adapt and survive adverse conditions. This reprogramming of cell physiology is caused by the accumulation of the alarmone (p)ppGpp which, in Escherichia coli, depends on the (p)ppGpp synthetase RelA and the bifunctional (p)ppGpp synthetase/hydrolase SpoT. Although conditions that control SpoT-dependent (p)ppGpp accumulation have been described, the molecular mechanisms regulating the switching from (p)ppGpp degradation to synthesis remain poorly understood. Here, we show that the protein YtfK promotes SpoT-dependent accumulation of (p)ppGpp in E. coli and is required for activation of the stringent response during phosphate and fatty acid starvation. Our results indicate that YtfK can interact with SpoT. We propose that YtfK activates the stringent response by tilting the catalytic balance of SpoT toward (p)ppGpp synthesis.
Highlights
The stringent response is a general bacterial stress response that allows bacteria to adapt and survive adverse conditions
To identify potential activators of SpoT-dependent (p)ppGpp synthesis, we selected for genes that, in multiple copies, would suppress the growth defect of an MG1655 E. coli K-12 ΔrelA strain on SMG agar plates
We pooled a collection of plasmids obtained from the ASKA library containing almost all E. coli K-12 genes[26], each cloned into the high-copy-number vector pCA24N downstream of the isopropyl β-D-1-thiogalactopyranoside (IPTG)-inducible PT5-lac promoter, and identified several putative candidate genes as described in the Methods section
Summary
The stringent response is a general bacterial stress response that allows bacteria to adapt and survive adverse conditions This reprogramming of cell physiology is caused by the accumulation of the alarmone (p)ppGpp which, in Escherichia coli, depends on the (p)ppGpp synthetase RelA and the bifunctional (p)ppGpp synthetase/hydrolase SpoT. We show that the protein YtfK promotes SpoT-dependent accumulation of (p)ppGpp in E. coli and is required for activation of the stringent response during phosphate and fatty acid starvation. The balance between both activities represents a critical point of regulation to control the intracellular level of (p)ppGpp. Long RSH proteins have two functional regions: the enzymatic N-terminal half (NTD), encompassing the synthetase (SYNTH) and the hydrolase (HD) domains, and the C-terminal regulatory half (CTD)[10,11]. YtfK is required to maintain elevated (p)ppGpp levels in response to phosphate and FA starvation, and enhances survival and promotes formation of antibiotic tolerant cells under nutritional stress conditions. We propose that YtfK acts by tilting the catalytic balance of SpoT toward (p)ppGpp synthesis rather than hydrolysis
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