Direct single cell observation of a keyE. colicell cycle oscillator

Abstract

A long-standing hypothesis sees DNA replication control in E. coli as a central cell cycle oscillator at whose core is the DnaA protein. The consensus is that the activity of the DnaA protein, which is dependent on its nucleotide bound state, is an effector of initiation of DNA replication and a sensor of cell size. However, while several processes are known to regulate the change in DnaA activity, the oscillations in DnaA production and DnaA activity have never been observed at the single cell level, and their correlation with cell volume has yet to be established. Here, we measured the volume-specific production rate of a reporter protein under control of the dnaAP2 promoter in single cells. By a careful dissection of the effects of DnaA-ATP- and SeqA-dependent regulation of dnaAP2 promoter activity two distinct cell-cycle oscillators emerge. The first one, driven by both DnaA activity and SeqA repression, is strongly coupled to cell cycle and cell size, and its minima show the same "adder" behaviour as initiation events. The second, a reporter of DnaA activity in the absence of SeqA binding, is still coupled with cell size but not to the time of cell division, and its minima (corresponding to DnaA activity peaks) show a "sizer-like" behavior, hence deviating from actual initiations. These findings indicate that production of DnaA is tightly coupled to cell volume through the timing of gene duplication, positive and negative regulation by DnaA-ATP itself and SeqA repression, and that DnaA activity peaks are a necessary but not sufficient condition to trigger replication initiation, posing firmer quantitative bases for a mechanistic understanding of cell cycle progression in bacteria.