Identification and Characterization of Replication Origins of MultipleChromosomes in Rhodobacter sphaeroides

DNA replication has been extensively studied in a number of bacterial species, which possess a unipartite genome consisting of a single circular chromosome. Approximately 10% of sequenced bacterial species have a multipartite genome structure, which is comprised of multiple chromosomes. However, the coordination and regulation of multi-chromosomal replication in bacteria remains poorly understood. Rhodobacter sphaeroides possesses a multipartite genome consisting of two chromosomes, the primary chromosome (CI) of approximately 3Mb and the secondary chromosome (CII) of 0.9 Mb. Z-curve and GC skew analyses revealed that CI and CII of R. sphaeroides exhibit three and five putative chromosomal origin regions, respectively. Then, the flanking regions of these putative regions were further analyzed in terms of gene conservation, gene density, and gene ratios between the corresponding forward and complement strands, previously identified near the biologically confirmed replicative origins of bacterial species that were closely related to R. sphaeroides. Subsequently, all the putative replicator regions were cloned into a pLO1 plasmid, a suicide vector in R. sphaeroides. The autonomous replication of these recombinant plasmids in R. sphaeroides was further examined using conjugation and molecular methods. Results demonstrated that CI and CII of R. sphaeroides have a single replication origin on its chromosomes, respectively, and this will provide the basis of future work on coordination and control of replication and segregation of multiple chromosomes in bacteria.