CcbP, a calcium-binding protein from <i>Anabaena</i> sp. PCC 7120, provides evidence that calcium ions regulate heterocyst differentiation

<jats:p> Although it is known that calcium is a very important messenger involved in many eukaryotic cellular processes, much less is known about calcium's role in bacteria. CcbP, a Ca <jats:sup>2+</jats:sup> -binding protein, was isolated from the heterocystous cyanobacterium <jats:italic>Anabaena</jats:italic> sp. PCC 7120, and the <jats:italic>ccbP</jats:italic> gene was cloned and inactivated. In the absence of combined nitrogen, inactivation of <jats:italic>ccbP</jats:italic> resulted in multiple contiguous heterocysts, whereas overexpression of <jats:italic>ccbP</jats:italic> suppressed heterocyst formation. Calmodulin, which is not present in <jats:italic>Anabaena</jats:italic> species, could also suppress heterocyst formation in both <jats:italic>Anabaena</jats:italic> sp. PCC 7120 and <jats:italic>Anabaena variabilis</jats:italic> . HetR induction upon nitrogen step-down was slow in the strain overexpressing <jats:italic>ccbP</jats:italic> . The Ca <jats:sup>2+</jats:sup> reporter protein obelin was used to show that mature heterocysts had a high intracellular free Ca <jats:sup>2+</jats:sup> concentration {[Ca <jats:sup>2+</jats:sup> ] <jats:sub>i</jats:sub> }, and immunoblotting showed that CcbP was absent from heterocysts <jats:sub>.</jats:sub> A regular pattern of cells with higher [Ca <jats:sup>2+</jats:sup> ] <jats:sub>i</jats:sub> was established during heterocyst differentiation before the appearance of proheterocysts. A rapid increase of [Ca <jats:sup>2+</jats:sup> ] <jats:sub>i</jats:sub> could be detected 4 h after the removal of combined nitrogen, and this increase was suppressed by excessive CcbP. These results suggest that Ca <jats:sup>2+</jats:sup> ions play very important roles in <jats:italic>hetR</jats:italic> induction and heterocyst differentiation. </jats:p>