## 2 Second Messengers in Cyanobacteria
### Calcium, Ca\({}^{2+}\)
#### 2.1.2 The Role of Ca\({}^{2+}\) in Heterocyst Differentiation  
Calcium is required for the activity of the nitrogen-fixation enzyme nitrogenase _in vivo_ and is purported to have a role in protecting nitrogenase from oxygen inactivation [32; 33; 34; 35]. Moreover, the frequency of heterocysts found in a filament varies with different Ca\({}^{2+}\) concentrations in the growth medium, which indicates a Ca\({}^{2+}\)-regulated mechanism for determining heterocyst abundance and placement [35]. These early experiments provided evidence that Ca\({}^{2+}\) was involved in nitrogen fixation.  
The Ca\({}^{2+}\)-binding photoprotein aequorin can be used to measure intracellular levels of Ca\({}^{2+}\)[36]. Determining the levels of free cytosolic Ca\({}^{2+}\) is fundamental for establishing the role of Ca\({}^{2+}\) as second messenger. Expression of an exogenous aequorin in _Anabaena_ sp. PCC7120 (hereafter _Anabaena_) resulted in the detection of a distinct Ca\({}^{2+}\) transient after nitrogen deprivation [37]. Alteration of the amplitude or duration of the Ca\({}^{2+}\) transient using pharmacological treatments arrested heterocyst differentiation at an early stage [37]. Thus, proper regulation of the timing and amplitude of the transient promotes heterocyst differentiation [37]. Notably, an increase of Ca\({}^{2+}\) in the cell after nitrogen deficiency originated from an intracellular source of Ca\({}^{2+}\)[37].  
Cyanobacteria can regulate Ca\({}^{2+}\) homeostasis by using mechanosensitive ion channels and through protein-Ca\({}^{2+}\) complexes that may serve in Ca\({}^{2+}\) storage (Figure 2). The gene _hetR_ in _Anabaena_ encodes a calcium-stimulated protease essential for vegetative cells to differentiate into heterocysts [38]. In vegetative cells, intracellular free Ca\({}^{2+}\) levels are ten times lower than in mature heterocysts [39]. Overexpression of _ccbP_, which encodes a Ca\({}^{2+}\)-binding protein that is localized in vegetative cells, suppressed heterocyst formation, whereas a _ccbP_ mutant exhibited multiple contiguous heterocysts [39]. Thus, accumulation of CcbP in vegetative cells may contribute to sequestration of Ca\({}^{2+}\) in these cells and its absence in heterocysts likely allows the accumulation of Ca\({}^{2+}\) and associated induction of _hetR_ and heterocyst differentiation [31].  
Nitrogen deficiency is also an important signal mediated by Ca\({}^{2+}\) in the unicellular, non-diazotrophic cyanobacterium _Synechococcus elongatus_ PCC 7942 [40]. Cells under nitrogen deficiency produce 2-oxoglutarate (2-OG), an important biological compound involved in the carbon-nitrogen status signal. The anion 2-OG can trigger Ca\({}^{2+}\) accumulation transiently in _S. elongatus_ as an increase in 2-OG occurs in cells before observed changes in intracellular Ca\({}^{2+}\) levels [40]. In response to nitrogen starvation, _S. elongatus_ can degrade phycobilisomes to recyclenitrogen-rich amino acids through activating two transcriptional regulators, NtcA and NbIR [41, 42]. Under nitrogen deficiency, a transiently increased level of intracellular Ca\({}^{2+}\) is NtcA dependent [40]. NtcA, which is a member of the cAMP-receptor transcriptional regulator protein family, may contribute to transient accumulation of 2-OG followed by Ca\({}^{2+}\) in regulating the expression of NtcA-dependent genes involved in the process of phycobiliprotein degradation [40].