Slow changes in cytosolic free Ca2+inEscherichia colihighlight two putative influx mechanisms in response to changes in extracellular calcium

Abstract

Free intracellular Ca2+([Ca2+]i) inEscherichia coliwas 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+]ibeing 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 μM 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 μM for 0.25 mM external Ca2+). In the presence of EGTA, free [Ca2+]iwas 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+]iof 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.