Use of Recombinant Aequorin to Study Calcium Homeostasis and Monitor Calcium Transients in Response to Heat and Cold Shock in Cyanobacteria

Abstract

We investigated the possibility of Ca(2+) signaling in cyanobacteria (blue-green algae) by measuring intracellular free Ca(2+) levels ([Ca(2+)](i)) in a recombinant strain of the nitrogen fixing cyanobacterium Anabaena strain sp. PCC7120, which constitutively expresses the Ca(2+)-binding photoprotein apoaequorin. The homeostasis of intracellular Ca(2+) in response to increasing external Ca(2+) has been studied in this strain. The resting level of free Ca(2+) in Anabaena was found to be between 100 and 200 nM. Additions of increasing concentrations of external Ca(2+) gave a transient burst of [Ca(2+)](i) followed by a very quick decline, reaching a plateau within seconds that brought the level of [Ca(2+)](i) back to the resting value. These results indicate that Anabaena strain sp. PCC7120 is able to regulate its internal Ca(2+) levels. We also monitored Ca(2+) transients in our recombinant strain in response to heat and cold shock. The cell's response to both stresses was dependent on the way they were induced. The use of inhibitors suggests that heat shock mobilizes cytosolic Ca(2+) from both intracellular and extracellular sources, while the Ca(2+) source for cold shock signaling is mostly extracellular.