The mystery of bacterial replication initiation control - new methods to find the missing piece of the puzzle

For the past half-century, several models have been suggested to explain how bacteria can determine their size to trigger replication at a constant volume per chromosome irrespective of growth rate. The most promising model so far is based on titration of the key initiator DnaA. However, a recent study from my lab falsifies this scheme.

A model based on the cycling of DnaA from its active to its inactive form agrees better with the measurements but lacks at least one critical component.

In the first year, we will develop a new imaging-based screen of a deep mutation library to identify the missing component.

The library is loaded into a microfluidic growth chamber where 100.000 exponentially growing mutants can be phenotyped in high detail.

In year two, we will interrogate the impact of the mutations on initiation regulation in each cell by determining the spatiotemporal distribution of fluorescently labeled replisomes in real time.

When a deviation from the wildtype is detected, this mutant is isolated with an optical tweezer and sequenced to identify the disrupted locus.

This approach should provide us with the piece missing to make an integrated quantitative model that can be tested based on novel predictions in years three and four. The project will engage researchers already active in the group as well as a new postdoc.

The new imaging-based pooled screening method addresses a general problem in biology; how to connect a rare genotype to a complex dynamic phenotype.