Impact of DivK dimerization on Caulobacter crescentus cell cycle

Promoter

Prof. Jean-Yves MATROULE, UNamur, Research Unit in Biology of Microorganisms (URBM)

Jury

Jean-Yves Matroule (Supervisor), Xavier De Bolle (President), Justine Collier (Jury), Laurence Van Melderen (Jury) & Pierre Cornelis (Jury)

Summary

Throughout evolution, bacteria have set up very efficient signaling pathways in order to quickly adapt to constantly changing environmental conditions or to regulate energy-consuming processes such as DNA replication or cell division. The CtrA pathway allows Caulobacter crescentus to tightly coordinate cell cycle progression with DNA replication. The CtrA activity is directly dependent on the phosphorylation state of the single-domain response regulator DivK. A previous study has demonstrated that in vitro and under non-reducing conditions, DivK is able to dimerize (Guillet et al. 2002). However, the relevance of this dimerization in vivo and its impact on C. crescentus cell cycle were not investigated.

During the first part of this thesis, we have confirmed by several methods that DivK could be able to dimerize in vivo. In order to evaluate the impact of the DivK conformation on cell cycle homeostasis, we have mutated several key residues in the DivK structure. The substitution of the tyrosine 102 by a phenylalanine seemed to alter DivK conformation and subsequently cell cycle homeostasis by lowering the CtrA phosphorylation level. The potential modification of the DivKY102F conformation likely resulted in an increased affinity between DivK and DivL, which might trigger the subsequent decrease of CtrA phosphorylation.

Since the CtrA pathway architecture is conserved among alpha-proteobacteria, the second part of this thesis was dedicated to assess the interaction between DivK and DivL in Sinorhizobium meliloti and Agrobacterium tumefaciens and to confirm this interaction in Caulobacter crescentus and Brucella abortus via a yeast-two-hybrid assay. Interestingly, the DivK-DivL affinity seems variable in the 4 investigated alpha-proteobacteria, suggesting a differential regulation of the CtrA activity by the DivJ-PleC-DivK two-component system among alpha-proteobacteria.