Soutenance publique de thèse de doctorat en Sciences biologiques - Elie Marchand

Decrypting the complex PTSNtr-dependent regulation of (p)ppGpp levels under unstressed and stressful conditions in Caulobacter crescentus

Jury

• Prof. Xavier DE BOLLE (département de biologie, UNamur), Président
• Prof. Régis HALLEZ (département de biologie, UNamur), Secrétaire
• Prof. Kristina JONAS (The Wenner-Gren Institute, University of Stockholm)
• Prof. Boris GÖRKE (Max Perutz Labs, University of Vienna)
• Prof. Abel Garcia PINO (ULB

Résumé

The nitrogen-related phosphoenolpyruvate phosphotransferase system (PTS^Ntr) is a phosphorelay described to regulate diverse processes in bacteria. In the model organism Caulobacter crescentus, in response to nitrogen limitation the PTS^Ntr stimulates the synthesis of guanosine tetra- and penta-phosphate molecules [(p)ppGpp, also called alarmone] and inhibits its hydrolysis, thereby leading to a rapid increase of (p)ppGpp levels in nitrogen-depleted conditions. However, (p)ppGpp is also known to play a physiological role under replete conditions but the regulatory mechanism governing basal levels of alarmone accumulation remains unexplored.

First, a novel PTS^Ntr component in C. crescentus – EIIA^Man – was characterized.  EIIA^Man regulates the phosphorylation status of the PTS^Ntr in replete conditions thereby determining the basal (p)ppGpp levels in unstarved conditions. Indeed, we show that EIIA^Man in complex with HPr forms an active phosphatase that rapidly dephosphorylates the entire PTS^Ntr. Interestingly, we show that this phosphatase harbors a tripartite active site composed of residues brought by three different polypeptides: a dimer of EIIA^Man and a monomer of HPr. We also show that the basal levels of (p)ppGpp in unstressed condition are not constant but instead fluctuates along the cell cycle peaking at the G1 phase, that is the phase at which the alarmone has the most effects. Finally, we studied the mechanism leading to (p)ppGpp accumulation upon carbon starvation and demonstrate that although PTS^Ntr is involved, there is another – unknown – mechanism that participates to the carbon-dependent control of the alarmone. Our data suggest that that the respiratory chain might be involved.

In summary, my work focuses on the complex and intricated regulatory functions of the PTS^Ntr, and more specifically the uncharacterized actor EIIA^Man in Caulobacter crescentus.

 

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