Coordination of replication initiation and cell growth in Escherichia coli

Like all organisms, the gut bacterium Escherichia coli must replicate its genome on average once per generation. This is easier said than done since E. coli have generation times ranging from 20 minutes to several hours. Furthermore, the time it takes to replicate the chromosome is longer than 20 minutes, putting immense pressure on coordinating growth with replication, specifically initiation. E. coli have solved this, illustrated by the narrow size range at which replication is initiated. The key protein in this process is DnaA. DnaA unwinds the origin of replication to allow the replisome to access the DNA. Only the active form of DnaA can initiate replication, and there are regulatory elements that activate and deactivate DnaA. DnaA can also bind to so-called DnaA boxes scattered throughout the chromosome. However, it is not known how all of these processes are coordinated. By fluorescently labelling different proteins involved in replication, we could determine the dynamics of initiation and its relationship to growth by growing cells in microfluidic chips and imaging them using time-lapse microscopy. With this approach, my colleagues and I tested different models for replication initiation control. Our results mostly agreed with a model based on the activation and deactivation of DnaA, but they also suggested that there exists one or more unknown regulatory elements involved in regulating DnaA. To this end, we developed a method to search for unknown regulatory elements. We created a transposon mutagenesis library to disrupt as many places in the genome as possible and imaged the library to determine which strains displayed a deviating phenotype. These strains were isolated from the library using an optical tweezer, and the transposon insertions were mapped to a genomic position. We found disrupted regions implicated in replication initiation control. However, we also found regions not previously associated with replication initiation control. These are potential candidates that have to be characterised further. The method can also be used to study other biological questions.