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.