The "baby machine" provides a means of generating synchronized cultures of 
minimally perturbed cells. We describe the use of this technique to establish 
the key cell-cycle parameters of hyperthermophilic archaea of the genus 
Sulfolobus. The 3 DNA replication origins of Sulfolobus acidocaldarius were 
mapped by 2D gel analysis to near 0 (oriC2), 579 (oriC1), and 1,197 kb (oriC3) 
on the 2,226-kb circular genome, and we present a direct demonstration of their 
activity within the first few minutes of a synchronous cell cycle. We also 
detected X-shaped DNA molecules at the origins in log-phase cells, but these 
were not directly associated with replication initiation or ongoing chromosome 
replication in synchronized cells. Whole-genome marker frequency analyses of 
both synchronous and log-phase cultures showed that origin utilization was close 
to 100% for all 3 origins per round of replication. However, oriC2 was activated 
slightly later on average compared with oriC1 and oriC3. The DNA replication 
forks moved bidirectionally away from each origin at approximately 88 bp per 
second in synchronous culture. Analysis of the 3 Orc1/Cdc6 initiator proteins 
showed a uniformity of cellular abundance and origin binding throughout the cell 
cycle. In contrast, although levels of the MCM helicase were constant across the 
cell cycle, its origin localization was regulated, because it was strongly 
enriched at all 3 origins in early S phase.