Soutenance publique de thèse de doctorat en Sciences biologiques - José Soares Maio

Unravelling (p)ppGpp signalling: From the control of Rel activities to metabolic and transcriptional targets in Caulobacter crescentus

Jury

Prof. Marc HENNEQUART (UNamur), Président
Prof. Régis HALLEZ (UNamur), Secrétaire
Dr Jade WANG (University of Wisconsin)
Dr Mohammad ROGHANIAN (Copenhagen University)
Dr Fabien LÉTISSE (Université de Toulouse)

Résumé

The stringent response is a ubiquitous regulatory mechanism across the bacterial kingdom, relying on the rapid intracellular accumulation of (p)ppGpp to ensure survival under adverse conditions. The levels of this signalling molecule are regulated by the highly conserved RelA/SpoT Homologue (RSH) protein family. Once accumulated, (p)ppGpp exerts a deep impact on bacterial physiology by remodelling the transcriptional

landscape, reconfiguring metabolism, inhibiting DNA replication, decreasing protein synthesis, and influencing cell cycle progression.

Despite the high conservation of the stringent response and the RSH protein family, the specific regulatory mechanisms and downstream targets of (p)ppGpp vary significantly across bacterial classes. Intriguingly, the regulation and effectors of (p)ppGpp remain less defined in the α-proteobacteria. Given the broad range of pleiotropic effects mediated by the alarmone, and the diverse cellular processes it regulates, it is essential

to understand how (p)ppGpp homeostasis is achieved, and to elucidate its specific effectors.

In this work, we set out to unravel the mechanisms regulating (p)ppGpp homeostasis and to elucidate the effectors targeted by the alarmone, in the α-proteobacterium Caulobacter crescentus. We demonstrated that the C. crescentus RSH protein - the bifunctional Rel - diverges from canonical models of RSH regulation; notably, it is not triggered by non-aminoacylated tRNAs or amino acid starvation, and does not appear to associate to stalled ribosomes. Beyond the reconfiguration of the "upstream" control of (p)ppGpp levels, our work has identified the PRPP synthetase, PrsA, as a novel target of the (p)ppGpp-mediated regulation. Our results showed that (p)ppGpp specifically binds to PrsA to inhibit its activity, thereby depleting intracellular PRPP pools. This results in the arrest of the developmental program of C. crescentus. Moreover, we also explored the interplay between (p)ppGpp and RNA polymerase. Crucially, we demonstrated that PRPP synthesis and transcription are major targets of the (p)ppGpp-mediated regulation, controlling cell cycle and cell differentiation in C. crescentus. Finally, we built into work exploring second messenger cross-talk, specifically between (p)ppGpp and c-di-GMP, which possess antagonistic activities. In particular, we demonstrated that the intracellular concentration of (p)ppGpp measured in vivo upon carbon exhaustion is compatible with the (p)ppGpp concentrations at which the diguanylate cyclase PleD

is inhibited, indicating that this (p)ppGpp-dependent inhibition of c-di-GMP synthesis can occur in physiological conditions.

In summary, this study contributes to the elucidation of the regulatory mechanisms within the (p)ppGpp network, which could constitute a divergent evolutionary reprogramming of the stringent response, possibly conserved among other oligotrophic bacteria.

 

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