Origins Left, Right, and Centre: Increasing the Number of Initiation Sites in the Chromosome.

The bacterium contains a single circular chromosome with a defined architecture. DNA replication initiates at a single origin called Two replication forks are assembled and proceed in opposite directions until they fuse in a specialised zone opposite the origin. This termination area is flanked by polar replication fork pause sites that allow forks to enter, but not to leave. Thus, the chromosome is divided into two replichores, each replicated by a single replication fork. Recently, we analysed the replication parameters in cells, in which an ectopic origin termed was integrated in the right-hand replichore. Two major obstacles to replication were identified: (1) head-on replication⁻transcription conflicts at highly transcribed operons, and (2) the replication fork trap. Here, we describe replication parameters in cells with ectopic origins, termed and , integrated into the left-hand replichore, and a triple origin construct with integrated in the left-hand and in the right-hand replichore. Our data again highlight both replication⁻transcription conflicts and the replication fork trap as important obstacles to DNA replication, and we describe a number of spontaneous large genomic rearrangements which successfully alleviate some of the problems arising from having an additional origin in an ectopic location. However, our data reveal additional factors that impact efficient chromosome duplication, highlighting the complexity of chromosomal architecture.