Abstract
Haloarchaeal genomes are generally composed of multiple replicons, and each replicon has a single or multiple replication origin(s). The comparative genomic analysis of replication origins from closely related species can be used to reveal the evolutionary mechanisms that account for the development of multiple origin systems. Multiple replication origins have been in silico and experimentally investigated in Haloarcula hispanica, which raise the possibility for comparisons of multiple replication origins in Haloarcula species. Thus, we performed a comparison of H. hispanica replication origins with those from five additional Haloarcula species. We demonstrated that the multiple replication origins in the chromosome were evolved independently multiple times from the oriC1-dependent ancestral chromosome. Particularly, the two origins oriC1 and oriC2 were conserved in location, and both of them were adjacent to an rRNA operon, suggestive of correlations in replication and expression of surrounding genes that may promote the conservation of these two origins. Some chromosomal variable regions were used as hotspots for origin evolution in which replication origins were continually being acquired, lost, and disrupted. Furthermore, we demonstrated that autonomously replicating sequence plasmids with H. hispanica minichromosomal replication origins were extremely unstable. Because both organization and replication origins of minichromosomes were not conserved, we proposed an association between the evolution of extrachromosomal replicons and origin variation. Taken together, we provided insights into the evolutionary history of multiple replication origins in Haloarcula species, and proposed a general model of association between the dynamics of multiple replication origins and the evolution of multireplicon genome architecture in haloarchaea.