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A 37 but not 26 degrees C virulent Yersinia pestis is known to require at least 2.5 mM Ca2+ for growth; this requirement is potentiated by Mg2+. After shift of log-phase cells (doubling time of 2 h) from 26 to 37 degrees C in Ca2+-deficient medium, shutoff of net ribonucleic acid synthesis preceded that of protein and cell mass. With 2.5 mM Mg2+, about two doublings in cell mass and number occurred before restriction with synthesis of sufficient deoxyribonucleic acid to account for initiation and termination of two postshift rounds of chromosome replication. Temperature shift with 20 mMMg2+ resulted in a single doubling of cell mass and number with one round of chromosome replication. Subsequent to shutoff of ribonucleic acid accumulation, ribonucleoside but not deoxyribonucleoside triphosphate pools became reduced to about 50% of normal values and the adenylate energy change fell from about 0.8, typical of growing cells, to about 0.6. Excretion of significant concentrations of adenine nucleotides under both permissive and restrictive conditions was observed. Only trace levels (less than 0.01 microM ol/g [dry weight]) of guaninosine 5'-diphosphate 3'-diphosphate accumulated under restrictive or permissive conditions; guanosine 5'-triphosphate 3'-diphosphate was not detected. Return of fully restricted cells from 37 to 26 degrees C with Ca2+ resulted in prompt growth, whereas addition of Ca2+ at 37 degrees C was ineffective. This finding indicates that the observed temperature-sensitive lesion in ribonucleic acid synthesis that results in restriction can be prevented but not reversed by cultivation with Ca2+.

Cells of Yersinia pestis strain EV76 are known to cease growth after a shift from 26 to 37 degrees C in neutral Ca2+-deficient medium; this effect is potentiated by Mg2+. With 2.5 mM Mg2+ and no added Ca2+, restriction was relaxed at elevated pH at which maximum cell yields occurred in the presence of 20 mM exogenous ATP. This ATP-dependent growth was inhibited by Ca2+ or 20 mM Mg2+; the nucleotide was neither transported into the organism nor hydrolyzed extracellularly. With strain EV76, ATP could be replaced by GTP but not other nucleotides, nucleosides, free bases, or pyrophosphate. CTP and UTP also promoted growth of strain KIM, in which limited division also occurred with nucleoside di- and monophosphates. Intracellular V antigen was detected 1 h after temperature shift in Ca2+-deficient medium containing 20 mM Mg2+, a time corresponding to the earliest known events associated with restriction (shutoff of stable RNA synthesis and reduction of adenylate energy charge). Maximum yield of V was obtained 2 h later when cell division ceased; the titer of the antigen remained constant thereafter. The specific activity of V in cells grown with ATP was significantly reduced, especially at elevated pH. These results would be expected if exogenous nucleotides promote growth by sequestering sufficient Mg2+ to prevent restriction of cell division mediated by V antigen.