title: Slow changes in cytosolic free Ca2+ in Escherichia coli highlight two putative influx mechanisms in response to changes in extracellular calcium.
abstract: Free intracellular Ca2+ ([Ca2+]i) in Escherichia coli was measured using the bioluminescent protein aequorin. Overall, the bacteria maintained a tight control on their free [Ca2+]i. The results indicated a slow Ca2+ influx, the magnitude of the initial rise in free [Ca2+]i being dependent upon the concentrations of external Ca2+. This was followed by the slow removal of free Ca2+ until normal levels were restored. Specifically, addition of external Ca2+ (0.25-10 mM) resulted in a gradual rise in intracellular free Ca2+ from a basal level of approximately 272 nM, maximally reaching a peak of 0.85-5.4 microM within 30-40 min. This was followed by a slow fall over the next 30 min, culminating in an oscillatory pattern of free [Ca2+]i (range 0.3-0.7 microM for 0.25 mM external Ca2+). In the presence of EGTA, free [Ca2+]i was dramatically reduced. Neither the influx of Ca2+ nor restoration of intracellular free Ca2+ required protein synthesis. Moreover, preincubation with Ca2+ increased the rising phase of intracellular Ca2+ in response to further exposure to external Ca2+. This was further evidence against a specific adaptation process such as the synthesis of calcium exporters. A putative Ca2+ influx channel was demonstrated in stationary phase cells in particular, which could be blocked by La3+. This channel was consistent with the voltage-activated poly-3-hydroxybutyrate/polyphosphate Ca2+ channels previously detailed by Reusch et al. [23] Even in the presence of La3+, however, the free [Ca2+]i of log phase and stationary phase bacteria still increased two-fold over resting values in response to external Ca2+. This suggested the presence of at least two Ca2+ influx processes, one inhibited by La3+ and the other not.
authors.affiliation: Department of Medical Biochemistry, University of Wales College of Medicine, Cardiff, UK. joneshe3@cardiff.ac.uk
authors.forename: H E
authors.initials: HE
authors.lastname: Jones
authors.forename: I B
authors.initials: IB
authors.lastname: Holland
authors.forename: H L
authors.initials: HL
authors.lastname: Baker
authors.forename: A K
authors.initials: AK
authors.lastname: Campbell
mesh_terms: D000331:Aequorin / Q000378:metabolism; D001059:Apoproteins / Q000378:metabolism; D002118:Calcium / Q000378:metabolism* / Q000502:physiology; D020013:Calcium Signaling / Q000502:physiology*; D002455:Cell Division; D003600:Cytosol / Q000378:metabolism*; D004926:Escherichia coli / Q000502:physiology*; D008163:Luminescent Measurements; D011994:Recombinant Proteins / Q000378:metabolism; D013997:Time Factors
chemical_list: D001059:Apoproteins; D011994:Recombinant Proteins; C060014:apoaequorin; D000331:Aequorin; D002118:Calcium