The halophilic archaeon Haloferax volcanii has a multireplicon genome, 
consisting of a main chromosome, three secondary chromosomes, and a plasmid. 
Genes for the initiator protein Cdc6/Orc1, which are commonly located adjacent 
to archaeal origins of DNA replication, are found on all replicons except 
plasmid pHV2. However, prediction of DNA replication origins in H. volcanii is 
complicated by the fact that this species has no less than 14 cdc6/orc1 genes. 
We have used a combination of genetic, biochemical, and bioinformatic approaches 
to map DNA replication origins in H. volcanii. Five autonomously replicating 
sequences were found adjacent to cdc6/orc1 genes and replication initiation 
point mapping was used to confirm that these sequences function as bidirectional 
DNA replication origins in vivo. Pulsed field gel analyses revealed that 
cdc6/orc1-associated replication origins are distributed not only on the main 
chromosome (2.9 Mb) but also on pHV1 (86 kb), pHV3 (442 kb), and pHV4 (690 kb) 
replicons. Gene inactivation studies indicate that linkage of the initiator gene 
to the origin is not required for replication initiation, and genetic tests with 
autonomously replicating plasmids suggest that the origin located on pHV1 and 
pHV4 may be dominant to the principal chromosomal origin. The replication 
origins we have identified appear to show a functional hierarchy or differential 
usage, which might reflect the different replication requirements of their 
respective chromosomes. We propose that duplication of H. volcanii replication 
origins was a prerequisite for the multireplicon structure of this genome, and 
that this might provide a means for chromosome-specific replication control 
under certain growth conditions. Our observations also suggest that H. volcanii 
is an ideal organism for studying how replication of four replicons is regulated 
in the context of the archaeal cell cycle.