ATP regulates calcium efflux and growth in E. coli.
Escherichia coli regulates cytosolic free Ca(2+) in the micromolar range through influx and efflux. Herein, we show for the first time that ATP is essential for Ca(2+) efflux and that ATP levels also affect generation time. A transcriptome analysis identified 110 genes whose expression responded to an increase in cytosolic Ca(2+) (41 elevated, 69 depressed). Of these, 3 transport proteins and 4 membrane proteins were identified as potential Ca(2+) transport pathways. Expression of a further 943 genes was modified after 1 h in growth medium containing Ca(2+) relative to time zero. Based on the microarray results and other predicted possible Ca(2+) transporters, the level of cytosolic free Ca(2+) 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 F(o)F(1) ATPase, as defective in Ca(2+) efflux. Seven other putative Ca(2+) transport proteins exhibited normal Ca(2+) handling. The defect in the DeltaatpD knockout cells could be explained by a 70% reduction in ATP. One millimolar glucose or 1 mM methylglyoxal raised ATP in the DeltaatpD knockout cells to that of the wild type and restored Ca(2+) efflux. One millimolar 2,4-dinitrophenol lowered the ATP in wild type to that in the DeltaatpD cells. Under these conditions, a similar defect in Ca(2+) efflux in wild type was observed in DeltaatpD cells. Ten millimolar concentration of Ca(2+) 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 Ca(2+) transporter, atoA, the beta subunit of acetate CoA-transferase likely to be involved in polyhydroxybutyrate synthesis, and ppk, encoding polyphosphate kinase, all indicated no defect in Ca(2+) efflux. We therefore propose that ATP is most likely to regulate Ca(2+) efflux in E. coli through an ATPase.
Naseem
Riffat
R
Department of Medical Biochemistry and Immunology, Cardiff University, Tenovus Building, Heath Park, Cardiff, Wales, UK.
Wann
Kenneth T
KT
Holland
I Barry
IB
Campbell
Anthony K
AK
D019297:2,4-Dinitrophenol / Q000494:pharmacology; D000255:Adenosine Triphosphate / Q000378:metabolism*; D002118:Calcium / Q000378:metabolism*; D004926:Escherichia coli / Q000254:growth & development / Q000378:metabolism / Q000502:physiology*; D029968:Escherichia coli Proteins / Q000235:genetics; D020869:Gene Expression Profiling; D015964:Gene Expression Regulation, Bacterial; D055786:Gene Knockout Techniques; D008565:Membrane Proteins / Q000172:deficiency / Q000235:genetics; D050505:Mutant Proteins / Q000235:genetics; D020411:Oligonucleotide Array Sequence Analysis; D006180:Proton-Translocating ATPases / Q000172:deficiency; D014157:Transcription Factors / Q000172:deficiency / Q000235:genetics; D014475:Uncoupling Agents / Q000494:pharmacology
D029968:Escherichia coli Proteins; D008565:Membrane Proteins; D050505:Mutant Proteins; D014157:Transcription Factors; D014475:Uncoupling Agents; C116245:atpD protein, E coli; D000255:Adenosine Triphosphate; D006180:Proton-Translocating ATPases; D019297:2,4-Dinitrophenol; D002118:Calcium