Comparative analysis of signal transduction factors controlling cell cycle regulation in the alpha-proteobacteria – CASTACC

Due to the presence of multiple levels of regulation in the cell cycle circuit, the complete comprehension of the network comes only from a multidisciplinary approach. Genetic analysis of mutants, purification of proteins in order to reconstitute in vitro phosphorylation cascades, localization of factors in the cell using fluorescent molecule and structural biology are essential in this project to compare the regulatory circuits in the two species.

Preliminarily, CASTACC has revealed several unique features of S. meliloti cell cycle in comparison with C. crescentus. Moreover the cell cycle machinery of S. meliloti is also involved in the plants infection process opening interesting perspectives in the possibility to control this beneficial symbiotic process.

Cell cycle in living organisms is the most important task in order to produce new cells. The knowledge of this fundamental aspect of biology can be beneficial both to boost the growth of beneficial bacteria and kill pathogens during infections by new specific antibiotic design.

- Pini Francesco, De Nisco Nicole, Ferri Lorenzo, Penterman Jon, Fioravanti Antonella, Brilli Matteo, Mengoni Alessio, Bazzicalupo Marco, Viollier Patrick H, Walker Graham C, Biondi Emanuele G. (2015) Cell cycle control by the master regulator CtrA in Sinorhizobium meliloti. PLoS Genetics, 15;11(5):e1005232.
- Mohapatra SS, Fioravanti A, Biondi EG (2014) DNA methylation in Caulobacter and other Alphaproteobacteria during cell cycle progression. Trends in Microbiology, 22:528-535.
- Francesco Pini, Benjamin Frage, Lorenzo Ferri, Nicole J. De Nisco, Saswat S. Mohapatra, Lucilla Taddei, Antonella Fioravanti, Frederique Dewitte, Marco Galardini, Matteo Brilli, Vincent Villeret, Marco Bazzicalupo, Alessio Mengoni, Graham C. Walker, Anke Becker and Emanuele G. Biondi (2013) The essential DivJ/CbrA kinase and PleC phosphatase system controls DivK phosphorylation and symbiosis in Sinorhizobium meliloti. Mol. Micro, 2013. 90(1):54-71.
- Fioravanti A., Coralie Fumeaux, Saswat S. Mohapatra, Coralie Bompard, Matteo Brilli, Antonio Frandi, Vincent Castric, Vincent Villeret, Patrick H. Viollier, Emanuele G. Biondi (2013) DNA binding of the cell cycle transcriptional regulator GcrA depends on N6-adenosine methylation in Caulobacter crescentus and other Alphaproteobacteria PLOS Genetics, Accepted.
- Fioravanti A, Clantin B, Dewitte F, Lens Z, Verger A, Biondi EG, Villeret V. Structural insights into ChpT, an essential dimeric histidine phosphotransferase regulating the cell cycle in Caulobacter crescentus. Acta Crystallogr Sect F Struct Biol Cryst Commun. 2012 Sep 1;68(Pt 9):1025-9. doi: 10.1107/S1744309112033064. Epub 2012 Aug 29. PubMed PMID: 22949187; PubMed Central PMCID: PMC3433190.

The comprehension of the cell cycle control in cells is a fundamental task of biology. In bacteria, several model organisms have been studied in order to understand this complex regulation; among them, Caulobacter crescentus is definitely one of the most interesting cases. In this organism, the cell cycle progression is mainly controlled by two-component system proteins that, in bacteria, are the main players of the signal transduction processes. The essential master regulator of cell cycle in C. crescentus is the response regulator CtrA that is activated by the phosphorylation of the cascade CckA-ChpT. However, the activity of CtrA is repressed by the two-component system DivJ-DivK, probably by dynamic delocalization from cell pole. Also another system is inhibiting CtrA and is composed by the protease ClpPX and other factors, such as the response regulator CpdR. Both DivK-inhibition and proteolysis are controlled by CtrA, which activates transcription of divK itself and several genes of the proteolytic degradation, creating redundant negative feedbacks.
The conservation across organisms of this circuit has been studied in alpha-proteobacteria, where CtrA is present, using bioinformatic tools; this analysis revealed that the logic of the circuit is conserved across Caulobacter closely related bacteria but the architecture in each organism can significantly vary. The experimental analysis of the variability of cell cycle architecture in different organisms, together with a mechanistic molecular analysis of cell cycle regulation in C. crescentus and close bacteria, are still aspects that need a deeper investigation.
In this project I’m proposing to systematically analyze the core components of the regulatory network controlling bacterial cell cycle in Sinorhizobium meliloti, which is another model organism, belonging to alpha-proteobacteria and sharing the logic of cell cycle regulation with C. crescentus. The discoveries, generated by a multidisciplinary approach, will be experimentally compared with C. crescentus. In order to fill a gap of molecular knowledge of cell cycle machinery, the project will investigate factors and complexes that drive cell cycle progression using biochemical and structural biology tools.
More in details, the analysis of the principal cell cycle factors in S. meliloti will be carried out by the creation of mutants, fluorescence tagging of each gene and studying the biochemistry of each encoded protein. Moreover, the goal is to genetically compare the functionality of each single factor cross-complementing the deletion and each over-expression of each gene of S. meliloti with the orthologs taken from C. crescentus. This approach will give an indication about the conservation of factors across organisms and how their activity is regulated in different systems. In order to decipher mechanistic insights of the cell cycle in alpha-proteobacteria, the second goal will be to extend the knowledge of the biology of cell cycle factors in C. crescentus and S. meliloti; in particular, elucidating the structural biology of the core factors controlling cell cycle in C. crescentus and S. meliloti.