The α-proteobacterium Caulobacter crescentus is one the best model organisms to study the temporal and the spatial regulation of the bacterial cell cycle, because it is easy to obtain a synchronized population of cells that are at the same stage of the cell cycle. This bacterium replicates its chromosome once and only once per cell cycle and its cell division is asymmetric, yielding two daughter cells, the swarmer cell and the stalked cell, with distinct morphological features and cell fates (Fig.1).
Figure 1: Schematic of the C. crescentus cell cycle and
the sequential accumulation of four master cell cycle regulators
Four essential master transcriptional regulators have been identified that control ~200 cell cycle-regulated genes in C. crescentus: CtrA, GcrA, DnaA, and CcrM. They oscillate out-of-phase temporally and spatially, so that each cell cycle-regulated gene is activated at its time of function during the cell cycle (Fig.1). Beside their functions as transcriptional regulators, CtrA and DnaA also directly bind to the origin of replication, to respectively inhibit and activate the initiation of chromosome replication. The CcrM DNA methyltransferase controls the transcription of several genes by methylating specific adenines in their promoters. Master regulators are interconnected with one another, forming a complex genetic network controlling the C. crescentus cell cycle (Fig.2).
Figure 2: Master regulators of the C. crescentus cell cycle are interconnected by transcriptional regulatory pathways
Our research addresses the following questions:
How is chromosome replication temporally and spatially regulated during the cell cycle?
(C. Fernández Fernández)
We wish to understand how chromosome replication is regulated, so that it occurs during the appropriate period of the cell cycle (Fig.1) and at the right sub-cellular localization (Fig.3). We propose to characterize the multiple, often redundant, regulatory pathways that control the timing of the initiation of DNA replication, and to study their localization in C. crescentus cells.
We recently identified an important regulator of the activity of the DnaA protein, named HdaA. This protein is spatially associated with the replisome (Fig.3) and it inactivates DnaA when functioning as the initiator of chromosome replication. This mechanism restricts the initiation of chromosome replication to only once per cell cycle.
Figure 3: HdaA co-localizes with the β-clamp of the DNA polymerase (DnaN) in live C. crescentus cells. Time lapse experiment using DnaN-RFP and HdaA-GFP-expressing cells analysed by DIC and fluorescence microscopy.
How is DNA replication coordinated with other cell cycle events? (Diego Gonzalez)
We expect that many mechanisms will be involved in the coordination of cell cycle events during the C. crescentus cell cycle. One of these may involve the essential CcrM DNA methyltransferase that changes the methylation state of the chromosome as a function of the cell cycle. We wish to understand why CcrM is essential for cell cycle progression and cell viability, and how DNA methylation regulates the transcription of diverse genes in C. crescentus.
How is the cell cycle influenced by metabolic cues and stresses? (Katharina Eich and Diego Gonzalez)
C. crescentus has extra-ordinary capacities to survive in nutrient-limited environments. We are characterizing several regulatory pathways adjusting the development of C. crescentus in response to such stresses or to metabolic changes.
How are the pathways involved in cell cycle regulation connected at the systems level?
Each of the above regulatory pathways will be characterized independently, but our ultimate goal is to combine all the results to analyse the cell cycle control circuit as an integrated system.
Nicolo De Coi (Master MLS, March 2011-January 2012)
Josip Mikulic (Master MLS, March 2011-January 2012)
Katharina Eich (PhD student, 2009-2012)
Colin Courvoisier (Master MLS, March 2012-January 2013)