Replication origins were mapped in hyperthermophilic crenarchaea, using 
high-throughput sequencing-based marker frequency analysis. We confirm previous 
origin mapping in Sulfolobus acidocaldarius, and demonstrate that the single 
chromosome of Pyrobaculum calidifontis contains four replication origins, the 
highest number detected in a prokaryotic organism. The relative positions of the 
origins in both organisms coincided with regions enriched in highly conserved 
(core) archaeal genes. We show that core gene distribution provides a useful 
tool for origin identification in archaea, and predict multiple replication 
origins in a range of species. One of the P. calidifontis origins was mapped in 
detail, and electrophoretic mobility shift assays demonstrated binding of the 
Cdc6/Orc1 replication initiator protein to a repeated sequence element, denoted 
Orb-1, within the origin. The high-throughput sequencing approach also allowed 
for an annotation update of both genomes, resulting in the restoration of open 
reading frames encoding proteins involved in, e.g., sugar, nitrate and energy 
metabolism, as well as in glycosylation and DNA repair.