DNA replication of the two Vibrio cholerae chromosomes
Final Report Abstract
Bacteria carry their genetic information on a single chromosome usually while for example human cells each carry 23 different chromosomes. The bacterium Vibrio cholerae and actually all Vibrio strains known to date carry two chromosomes which makes them an exception in the bacterial world in this respect. One fundamental scientific question that arises from this two-chromosome setup is how the replication of the two is coordinated. It has been found in the past that replication of a short DNA sequence on chromosome 1 (Chr1), called crtS, triggers the start of chromosome 2 (Chr2) replication. The goal of this project was to understand this inter-chromosomal communication better. One important goal was to define the important parts of the crtS site. To this end, we developed an entirely new methodology to test thousands of systematically varied crtS sequences testing functional from non-functional sequences. We could successfully establish the general procedure and show successful selection. However, the sequence library construction needs to be optimised to cover the entire sequence space. The position of the crtS on Chr1 is critical because it determines the timing of Chr2 replication start. To see how strict this localization is or how much deviation from the original is tolerated we developed a methodology to test this experimentally. To this end we designed, constructed and tested a synthetic transposable element with an implemented reporter to select for successful transposition events. We could show that the system works in general. However, the tuning of the transposase activity is critical in this setup and requires further optimization. To get an insight on the evolutionary conservation of the crtS site position we teamed up with colleagues from the German Strain Collection (DSMZ) in Braunschweig to sequence about 200 Vibrio genomes for which a sequence was missing. We found that the crtS position on Chr1 is consistently related to the size of the respective Chr2 in the respective strains consistant with a functional importance of the position. Our research topic is of high interest and multiple research groups around the world contribute to uncovering the underlying mechanisms. During the funding period of this project, a group of researchers in France used a technology we recently developed to do similar experiments to those we proposed within our application to determine the regulatory events of Chr2 replication in temporal resolution.
