The circular 4.6 megabase E. coli chromosome adopts defined geometries both in terms of genetic polarity (origin and terminus positions) and shape (compactness and three dimensional appearance). These geometries change dramatically and predictably during the cell cycle, and our group investigates the mechanism behind these cyclic changes as well as their effect on the major cellular synthetic processes, DNA replication and segregation, gene expression, and cell division.
One major limitation in studying chromosome dynamics is our inability to observe large-scale chromosome structure using standard fluorescence microscopy methods, which only label a discrete genetic position (focus). We are developing a novel chromosome painting technology to image individual domains within the entire chromosome in single cells. This method, inspired by in situ hybridization-based human karyotyping techniques, utilizes multi-color combinatorial labeling and high resolution three dimensional photography to generate whole genome maps of the chromosome. Our goal is to define the overall program of chromosome polarity and shape in E. coli using synchronized cell cultures produced by our baby cell machine (Bates et al, 2005).
DNA Replication and Cell Cycle Regulation
Analogously to cohesion in eukaryotic chromosomes, newly replicated E. coli DNA is transiently paired along homologous sequences (Joshi et al, 2011). We recently showed that cohesive linkages involve topological twisting of sister chromosomes (precatenanes) and are temporally mediated by a replication fork-tracking chromosome structure protein (Joshi et al, 2013). We are currently investigating how chromosome cohesion affects chromosome segregation, fork progression and DNA repair.
Our group is also very interested in mechanisms of DNA replication initiation control. One possible mechanism involves a transitory physical linkage of the chromosome to the cell division septum, which apparently precludes the replication origin from firing until cell division has completed. We are investigating this mechanism by creating inducible chromosome tethers elsewhere on the chromosome, and measuring the effects on chromosome replication by next generation sequencing (MiSeq) copy number analysis.
The basic mechanisms of chromosome replication are well conserved among all cells, and knowledge from the E. coli model will provide critical insight into analogous human mechanisms, defects in which lead to catastrophic diseases including many types of cancers and congenital disorders. The superb manipulability of the bacterial system makes rapid advances possible.