The spatial organization of replication is determined by cell size independently of chromosome copy number

Abstract

AbstractWe have studied how Escherichia coli coordinates replication and division cycles by tracking replisomes in individual cells through thousands of division cycles. By analysing the replication and division of the cells at various growth rates and with different genetic perturbations, we have reached four major conclusions: (1) Initiation is not based on titration of DnaA to the chromosomal binding sites because initiation is still coordinated with the division cycle several generations after DnaA synthesis is turned off. (2) We can rule out that initiation is triggered by a simple DnaA-ADP/ATP switch mechanism because initiation is still triggered at a well-defined cell size in a DARS1/DARS2/datA triple knockout if DnaA is overexpressed. (3) From the spatial organization of replisomes at different growth rates, it appears as if initiation is triggered by termination, but this hypothesis is ruled out by an NGS-based marker frequency analysis. (4) Overlapping replication cycles do not affect the spatial organization of the replication machinery and it appears as if each replication center contains a fixed number of DnaN molecules independently of how many replication forks are being processed.