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Control of Bacterial Multidrug Tolerance and Stress Response by Alarmone Synthetase SpoT

Periodic Reporting for period 3 - STRINGENCY (Control of Bacterial Multidrug Tolerance and Stress Response by Alarmone Synthetase SpoT)

Reporting period: 2020-01-01 to 2021-06-30

The Stringent Response is a general bacterial stress response induced by diverse nutritional and environmental stresses allowing bacteria to adapt and survive adverse conditions. This global resetting of bacterial cells physiology relies on the accumulation of two unusual phosphorylated derivatives of GTP and GDP collectively called (p)ppGpp which are synthetized by the RelA/SpoT homologue proteins. Importantly the Stringent Response has a key role in bacterial virulence and in the formation of antibiotic-tolerant cells. However, the molecular mechanisms by which environmental cues activate the stringent response are still largely unknown and represent one of the most fundamental, unsolved problems in prokaryotic molecular biology. Thus, there is a pressing need to understand how the activities of the enzymes responsible for synthesis and degradation of (p)ppGpp are regulated and to develop novel strategies to combat recalcitrante infections. Therefore, the STRINGENCY project aims to uncover the molecular mechanisms by which environmental stimuli trigger SpoT-dependent (p)ppGpp synthesis with special emphasis on its role in the formation of multidrug tolerant cells in Escherichia coli. The project lies on three main objectives: (i) To unravel how spoT expression is regulated (ii) To reveal how (p)ppGpp synthetase II activity encoded by spoT is mechanistically controlled and (iii) To decipher the physiological role of SpoT in multidrug tolerance. The program is ambitious and will provide fundamental insights into (p)ppGpp biology.
To gain further insights into the regulation of the bifunctional (p)ppGpp synthetase/hydrolase SpoT enzyme we have successfully developed genetic assays that allowed us to identify new protein candidates that modulate SpoT-dependent (p)ppGpp synthesis or degradation in E. coli. We have isolated five new candidates that either promotes accumulation or degradation of (p)ppGpp in vivo. Identification of these new protein partners provided us with a better understanding of (p)ppGpp regulation in response to a variety of stresses.
Among them we observed that YtfK, a small protein of unknown function, is required to sustain elevated (p)ppGpp levels in response to phosphate and fatty acid starvation, therefore ensuring cell survival under these stress conditions. We further observed that the SpoT/YtfK ratio governs the switching from (p)ppGpp degradation to synthesis and that SpoT synthetase activity is subjected to YtfK limitation in vivo. Interestingly YtfK is needed to produce antibiotic tolerant cells under nutritional stress conditions, further highlighting the important role of SpoT and the signalling molecule (p)ppGpp in controlling multidrug tolerance. Mechanistically we show that YtfK controls the stringent response via a direct and specific interaction with the N-terminal region of SpoT encompassing the hydrolase and synthetase domains. We further observed that YtfK modifies the protein surface of SpoT catalytic domains supporting conformational changes upon binding. Importantly, our work represents the first evidence that binding to the N-terminal catalytic domains of SpoT plays an important role in controlling the reciprocal regulation of the two enzymatic states. Finally we suggest that YtfK acts by tilting the catalytic balance of SpoT toward (p)ppGpp synthesis rather than hydrolysis.
The stringent response has been investigated for more than 50 years. Therefore, identification and the characterization of new protein partners that modulates SpoT activities represent important conceptual advances in the understanding of (p)ppGpp biology and bacterial survival mechanisms. The biochemical and structural analysis of SpoT activities with the different identified partners, which represent the major current research area, are likely to shed light on why cells have evolved several modes of (p)ppGpp regulation in order to respond to a variety of stresses. Therefore our work in the first half of the STRINGENCY project has pushed forward our understanding of the molecular mechanisms underlying the stringent response and we anticipate that it will open new perspectives for the development of novel drug-target discovery regimes to fight recurrent infections.
activation of SpoT by YtfK