A consenus chromosome origin of replication in alpha-proteobacteria /

Caulobacter crescenuts is a Gram negative bacterium with a defined cell cycle. It divides to yield two distincts cell types: a flagellated, non-replicating swarmer cell and a non-motile but replicative stalked cell. In order to initiate chromosome replication, the swarmer cell must differentiate into the stalked cell by ejecting the polar flagellum and acquiring a tubular stalk appendage. Therefore, chromosome replication is restricted to the stalked cell where chromosome replication initiates exactly once per cell cycle. A key feature of C. crescentus is the global response regulator CtrA that controls key events within the cell cycle including chromosomal replication. CtrA represses replication in the swarmer cell by binding to five specific sites on the C. crescentus chromosome origin of replication (Cori). To initiate replication, CtrA protein is removed from the stalked cell by proteolytic degradation. (C. crescentus represents the first example where a response regulator protein controls DNA synthesis.) We tested whether other replication origins from related bacteria also bind CtrA, and whether they also share other DNA elements implicated in replication control such as binding sites for the DnaA protein (DnaA boxes) and DNA methylation sites. We isolated potential replication origins from closely related freshwater and marine Caulobacter species by PCR amplification with degenerated primers that hybridize to conserved flanking genes. We amplified the origins of replication of 3 freshwater and 2 marine Caulobacter species, and we sequenced all of them. Further, we wanted to know whether we amplified the true origin of replication for the related bacteria or not. Therefore, we tested these clones by autonomous replication assays in C. crescentus. 4 out of 5 proposed replication origins supported autonomous plasmid replication in C. crescentus. The exception was the potential origin isolated from the most distantly related marine spe