Impact of transcription and proteolysis in (p)ppGpp-dependent cell cycle control of Caulobacter crescentus

Résumé

Accumulating the hyperphosphorylated guanosine (p )ppGpp is a mechanism used by bacteria to face environmental stresses. Involved in regulation of many essential processes, (p )ppGpp is better known for its role in the stringent response allowing bacteria to face starving conditions. Upon starvation, an arsenal of cellular processes must indeed be modulated in order to adapt and survive. Interestingly, this alarmone controls the cell cycle of the dimorphic Caulobacter crescentus. By delaying both Gl-S and swarmer-to-stalked cell transitions, (p )ppGpp promotes the spread of a population experiencing starvation. As the RNA Polymerase (RNAP) has been described as the major target of (p)ppGpp in many bacteria, responsible of the transcriptional switch observed upon starvation, we checked whether RNAP might be the causative factor of the cell cycle delay in (p)ppGpp+ cells. To investigate a potential role of transcription in cell cycle control, we worked on (i) a RNAP mutant mimicking the state upon binding of (p )ppGpp and (ii) RNAP mutants unable to bind (p)ppGpp. Interestingly, we found rpoBT56JA mimics all the effects of RNAP bound by (p )ppGpp, i.e. displaying G 1 accumulation, higher motility and slower growth, even in a (p )ppGpp0 background. Unfortunately we did not have enough time to get the (p )ppGpp-blind RNAP mutants. However, the molecular tools to select them have been constructed and we are currently trying to introduce these mutations into C. crescentus. As an alternative mechanism to elongate G 1/swarmer lifetime, we characterized the effects (p )ppGpp has on the abundance of two cell cycle regulators, CtrA and DnaA. Indeed, the alarmone increases the level of CtrA that silences DNA replication initiation and at the same time clears the DNA replication initiator DnaA from the cell. Since these two proteins are regulated by proteolysis, we checked whether (p )ppGpp could regulate their proteolytic degradation rates and we found that (p )ppGpp strongly inhibits CtrA proteolysis whereas slightly stimulating DnaA degradation. Our work illustrates that the alarmone can regulate important cellular processes such as transcription and proteolysis

la date de réponse	2017
langue originale	Anglais
L'institution diplômante	Universite de Namur
Superviseur	Regis Hallez (Promoteur)