ATP Regulates Calcium Efflux and Growth in E. coli

SummaryEscherichia coli regulates cytosolic free Ca2+ in the micromolar range through influx and efflux. Herein, we show for the first time that ATP is essential for Ca2+ efflux and that ATP levels also affect generation time. A transcriptome analysis identified 110 genes whose expression responded to an increase in cytosolic Ca2+ (41 elevated, 69 depressed). Of these, 3 transport proteins and 4 membrane proteins were identified as potential Ca2+ transport pathways. Expression of a further 943 genes was modified after 1 h in growth medium containing Ca2+ relative to time zero. Based on the microarray results and other predicted possible Ca2+ transporters, the level of cytosolic free Ca2+ was measured in selected mutants from the Keio knockout collection using intracellular aequorin. In this way, we identified a knockout of atpD, coding for a component of the FoF1 ATPase, as defective in Ca2+ efflux. Seven other putative Ca2+ transport proteins exhibited normal Ca2+ handling. The defect in the ΔatpD knockout cells could be explained by a 70% reduction in ATP. One millimolar glucose or 1 mM methylglyoxal raised ATP in the ΔatpD knockout cells to that of the wild type and restored Ca2+ efflux. One millimolar 2,4-dinitrophenol lowered the ATP in wild type to that in the ΔatpD cells. Under these conditions, a similar defect in Ca2+ efflux in wild type was observed in ΔatpD cells. Ten millimolar concentration of Ca2+ resulted in a 30% elevation in ATP in wild type and was accompanied by a 10% reduction in generation time under these conditions. Knockouts of pitB, a potential Ca2+ transporter, atoA, the β subunit of acetate CoA-transferase likely to be involved in polyhydroxybutyrate synthesis, and ppk, encoding polyphosphate kinase, all indicated no defect in Ca2+ efflux. We therefore propose that ATP is most likely to regulate Ca2+ efflux in E. coli through an ATPase.