Effect of exogenous nucleotides on Ca2+ dependence and V antigen synthesis in Yersinia pestis

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.