Changes in cell size and DNA content in Sulfolobus cultures during dilution and temperature shift experiments

Stationary-phase cultures of different hyperthermophilic species of the arc haeal genus Sulfolobus were diluted into fresh growth medium and analyzed b y flow cytometry and phase-fluorescence microscopy, After dilution, cellula r growth started rapidly but no nucleoid partition, cell division, or chrom osome replication took place until the cells had been increasing in size fo r several hours, Initiation of chromosome replication required that the cel ls first go through partition and cell division, revealing a strong interde pendence between these key cell cycle events. The time points at which nucl eoid partition, division, and replication occurred after the dilution were used to estimate the relative lengths of the cell cycle periods. When expon entially growing cultures were diluted into fresh growth medium, there was an unexpected transient inhibition of growth and cell division, showing tha t the cultures did not maintain balanced growth. Furthermore, when cultures growing at 79 degrees C were shifted to room temperature or to ice-water b aths, the cells were found to "freeze" in mid-growth. After a shift back to 79 degrees C, growth, replication, and division rapidly resumed and the mo de and kinetics of the resumption differed depending upon the nature and le ngth of the shifts. Dilution of stationary-phase cultures provides a simple protocol for the generation of partially synchronized populations that may be used to study cell cycle-specific events.