The DnaA cell cycle oscillator and its coordination with cell growth and division in Escherichia coli

Despite over 50 years of study, key questions on the bacterial cell cycle remain unanswered. In particular, the debate is open concerning the regulation of DNA replication initiation in E. coli and its role in the dynamics of cell growth and division. A key player in these processes is the DnaA protein, which is involved the initiation of DNA replication. DnaA is commonly believed to be a cell cycle oscillator and a cell size sensor, but neither of these facts have been firmly established.DnaA activity depends on its nucleotide bound state, the ATP bound form being the active one for origin recognition and activation. DnaA is also a transcription factor, a highly connected node in the network of genes coding for proteins required for DNA replication and the repair of DNA damage. The differential regulation of gene expression by the different DnaA nucleotide bound forms, including the regulation of its own promoter, is believed to be central in its role of cell-cycle oscillator and regulator. Indeed, because of its double role as a transcription factor and an activator of the initiation of the DNA replication process, the DnaA protein can act as the regulatory link between the timing and level of gene expression and the different phases of the bacterial cell cycle.This thesis addresses the problem of identifying the cell cycle oscillator related to DnaA activity, and relating it to the progression of the E. coli cell cycle, focusing in particular on cell-size sensing, individual cell growth rate, and cell division.One of the major challenges in this area has been to quantify the changes in DnaA-ATP activity in vivo in real time. This problem requires single-cell dynamic resolution, in order to relate DnaA levels and activity to key cell-cycle transitions. To address this problem, I have developed a set of reporters of gene expression using a gene for a fluorescent protein under control of a promoter that is regulated by DnaA-ATP, and deployed them in single-cell experiments coupling quantitative microscopy and microfluidics.To obtain robust and long-term single-cell tracking in steady growth conditions I have designed a dedicated experimental setup and data-analysis pipeline for studying the growth, size, and gene expression of E. coli in controlled environmental conditions.A careful analysis of these single cell data as a function of the cell cycle shows that E. coli growth is biphasic and it follows the expression of a constitutive ori-proximal promoter.Moreover, thanks to the analysis of different reporters, I was able to quantify for the first time the effect of the DnaA-dependent promoter regulatory elements. Specifically, I found that the volume-specific production rate of GFP from the DnaA promoter is a well-defined cell-cycle oscillator and that the signal from this oscillator can be related to key cell-cycle processes such as DNA replication and cell division.However, while the standard view of the cell cycle sees it as the result of a single oscillator, our data lead me to suggest that at least two coupled oscillators are needed to describe the processes that coordinate DNA replication, cell growth and cell-cycle progression. My approach also makes it possible to detect causality links between these different processes.These findings combine the use of mathematical models and single-cell dynamic data to pose firmer quantitative bases for a characterization of the mechanisms determining robust cell cycle progression in bacteria.