**2. CGP occurrence**

Most cyanobacteria, including unicellular and filamentous, as well as diazotrophic and nondiazotrophic groups are able to accumulate CGP (**Figure 1**).

In non-diazotrophic cyanobacteria, the amount of CGP is usually less than 1% of the cell dry mass during exponential growth. CGP accumulates conspicuously under unbalanced growth conditions including stationary phase, light stress or nutrient limitation (sulfate, phosphate or potassium starvation) that do not involve nitrogen starvation [9, 10]. Under such unbalanced conditions, the amount of CGP may increase up to 18% of the cell dry mass [10]. During the recovery from nitrogen starvation by the addition of a usable nitrogen source, CGP is transiently accumulated [11, 12].

In the unicellular diazotrophic cyanobacterium *Cyanothece* sp. ATCC 51142, nitrogen fixation and photosynthesis can coexist in the same cell, but temporarily separated. The nitrogenfixing enzyme, nitrogenase, is highly sensitive to oxygen. Nitrogen fixation occurs in dark periods and the fixed nitrogen is stored in CGP. In the light period, when photosynthesis is performed, the CGP is degraded to mobilize the fixed nitrogen [13]. Transient CPG accumulation during dark periods was also reported in the filamentous cyanobacterium *Trichodesmium* sp., which has a high abundance in tropical and subtropical seas and is an important contributor to global N and C cycling [14].

Furthermore, in heterocysts of diazotrophic cyanobacteria of the order *Nostocales,* polar nodules consisting of CGP are deposited at the contact site to adjacent vegetative cells [15] (**Figure 1**). The heterocystous CGP seems to be involved in transport of fixed nitrogen to the adjacent photosynthetically active vegetative cell. CGP catabolic enzymes are present at significantly higher levels in vegetative cells than in heterocysts. Moreover, CGP could serve as a sink for fixed nitrogen in the heterocyst to avoid feedback inhibition from soluble products of nitrogen fixation [16, 17]. In *Anabaena* sp. PCC 7120 and *Anabaena variabilis,* mutational studies have shown that strains lacking CGP synthetic genes are little affected in diazotrophic growth under standard laboratory conditions [15, 18]. However, a growth defect was observed under high light conditions [15]. Moreover, diazotrophic growth is significantly decreased in strains that are unable to degrade CGP [16, 18].

Akinetes are resting spore-like cells of a subgroup of heterocyst-forming cyanobacteria for surviving long periods of unfavorable conditions. During akinete development, the cells transiently accumulate storage compounds, namely glycogen, lipid droplets and CGP [19, 20] (**Figure 1**). CGP granules also appear during germination of dormant akinetes [21]. *Anabaena variabilis* akinetes lacking CGP granules were also able to germinate. This behavior agrees with early observations that CGP is not the direct nitrogen source for protein biosynthesis and

**Figure 1.** Light and electron microscopic pictures of CGP accumulating cyanobacteria. In light microscopic pictures, CGP was stained using the Sakaguchi reaction [10]. The intensity of the red color indicates the amount of arginine. Dark red to purple dots are CGP granules [CG]. (A) and (B) Phosphate starved *Synechocystis* sp. PCC 6803 in light and transmission electron microscopy, respectively. (C) *Cyanothece* sp. PCC 7424 cultivated in presence of nitrate and continuous light. (D) Filament of diazotrophic growing *Anabaena* sp. PCC 7120 with terminal heterocyst containing polar bodies [PB]. (E) Transmission electron micrographs of a heterocyst and adjacent vegetative cell from *Anabaena* sp. PCC 7120, showing a GCP consisting polar body [PB]. (F) *Oscillatoria* sp. cultivated with nitrate supplementation, showing small CGP granules. (G) Phosphate starved *Anabaena variabilis ATCC 29413* under nitrate supplemented growth. (H) *Nostoc punctiforme ATCC 29133* under phosphate starvation and nitrate supplementation. (I) and (J) Mature akinetes

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therefore not essential for akinete germination [21, 22].

of *Anabaena variabilis ATCC 29413* and *Nostoc punctiforme ATCC 29133*, respectively.

CGP was discovered in 1887 by the botanist Antonio Borzi during microscopic studies of filamentous cyanobacteria [3]. He observed opaque and light scattering inclusions by using light microscopy and created the name *cianoficina*. Early electron microscopic studies showed a strong structure variation of the CGP granules, depending on the fixatives and poststains used during electron microscopic examinations [4, 5]. This led to a controversy about the ultrastructure of these inclusions until the 1970s. Later, electron microscopic studies described CGP granules as membrane less, electron dense and highly structured cytoplasmic inclusions [6, 7]. With a C/N ratio of 2:1, CGP is extremely rich in nitrogen and consequently an excellent nitrogen storage compound. During the degradation of CGP and subsequent degradation of

Most cyanobacteria, including unicellular and filamentous, as well as diazotrophic and non-

In non-diazotrophic cyanobacteria, the amount of CGP is usually less than 1% of the cell dry mass during exponential growth. CGP accumulates conspicuously under unbalanced growth conditions including stationary phase, light stress or nutrient limitation (sulfate, phosphate or potassium starvation) that do not involve nitrogen starvation [9, 10]. Under such unbalanced conditions, the amount of CGP may increase up to 18% of the cell dry mass [10]. During the recovery from nitrogen starvation by the addition of a usable nitrogen source, CGP is tran-

In the unicellular diazotrophic cyanobacterium *Cyanothece* sp. ATCC 51142, nitrogen fixation and photosynthesis can coexist in the same cell, but temporarily separated. The nitrogenfixing enzyme, nitrogenase, is highly sensitive to oxygen. Nitrogen fixation occurs in dark periods and the fixed nitrogen is stored in CGP. In the light period, when photosynthesis is performed, the CGP is degraded to mobilize the fixed nitrogen [13]. Transient CPG accumulation during dark periods was also reported in the filamentous cyanobacterium *Trichodesmium* sp., which has a high abundance in tropical and subtropical seas and is an important contribu-

Furthermore, in heterocysts of diazotrophic cyanobacteria of the order *Nostocales,* polar nodules consisting of CGP are deposited at the contact site to adjacent vegetative cells [15] (**Figure 1**). The heterocystous CGP seems to be involved in transport of fixed nitrogen to the adjacent photosynthetically active vegetative cell. CGP catabolic enzymes are present at significantly higher levels in vegetative cells than in heterocysts. Moreover, CGP could serve as a sink for fixed nitrogen in the heterocyst to avoid feedback inhibition from soluble products of nitrogen fixation [16, 17]. In *Anabaena* sp. PCC 7120 and *Anabaena variabilis,* mutational studies have shown that strains lacking CGP synthetic genes are little affected in diazotrophic growth under standard laboratory conditions [15, 18]. However, a growth defect was observed under high light conditions [15]. Moreover, diazotrophic growth is significantly decreased in strains that are unable to

arginine, a function as energy source was also proposed [8].

diazotrophic groups are able to accumulate CGP (**Figure 1**).

**2. CGP occurrence**

86 Cyanobacteria

siently accumulated [11, 12].

tor to global N and C cycling [14].

degrade CGP [16, 18].

**Figure 1.** Light and electron microscopic pictures of CGP accumulating cyanobacteria. In light microscopic pictures, CGP was stained using the Sakaguchi reaction [10]. The intensity of the red color indicates the amount of arginine. Dark red to purple dots are CGP granules [CG]. (A) and (B) Phosphate starved *Synechocystis* sp. PCC 6803 in light and transmission electron microscopy, respectively. (C) *Cyanothece* sp. PCC 7424 cultivated in presence of nitrate and continuous light. (D) Filament of diazotrophic growing *Anabaena* sp. PCC 7120 with terminal heterocyst containing polar bodies [PB]. (E) Transmission electron micrographs of a heterocyst and adjacent vegetative cell from *Anabaena* sp. PCC 7120, showing a GCP consisting polar body [PB]. (F) *Oscillatoria* sp. cultivated with nitrate supplementation, showing small CGP granules. (G) Phosphate starved *Anabaena variabilis ATCC 29413* under nitrate supplemented growth. (H) *Nostoc punctiforme ATCC 29133* under phosphate starvation and nitrate supplementation. (I) and (J) Mature akinetes of *Anabaena variabilis ATCC 29413* and *Nostoc punctiforme ATCC 29133*, respectively.

Akinetes are resting spore-like cells of a subgroup of heterocyst-forming cyanobacteria for surviving long periods of unfavorable conditions. During akinete development, the cells transiently accumulate storage compounds, namely glycogen, lipid droplets and CGP [19, 20] (**Figure 1**). CGP granules also appear during germination of dormant akinetes [21]. *Anabaena variabilis* akinetes lacking CGP granules were also able to germinate. This behavior agrees with early observations that CGP is not the direct nitrogen source for protein biosynthesis and therefore not essential for akinete germination [21, 22].

CGP was formally thought to be unique in cyanobacteria. In 2002, Krehenbrink et al. and Ziegler et al. discovered through evaluation of obligate heterotrophic bacteria genomes that many heterotrophic bacteria possess CGP synthetase genes [23, 24]. Genes of CGP metabolism occur in a wide range of different phylogenetic taxa and not closely related to cyanobacteria [25].

Simion [35]. The enzyme incorporates aspartate and arginine in an elongation reaction, which

ity, CphA1 needs a so far unknown CGP primer, as a starting point of the elongation reaction [35]. By using synthetically primers, Berg et al. could show that a single building block of CGP (β-Asp-Arg) does not serve as an efficient primer for CphA1 elongation reaction in vitro.

CphA1 activity [36]. Other peptides, like cell wall or other cellular components, have been suggested to serve as an alternative priming substance for the CphA1 reaction [37]. This could be an explanation for the functionality of CGP synthesis in recombinant bacteria, without the ability to produce native CGP primers [38]. Interestingly, the CphA1 of *Thermosynechococcus* 

Today, CphA1 enzymes from several bacteria, including cyanobacteria and heterotrophic bacteria, have been purified and characterized [33, 39–42]. The molecular mass of the characterized CphA1 enzymes ranges from 90 to 130 kDa. The active form of CphA1s from *Synechocystis* sp. PCC6308 and *Anabaena variabilis* PCC7937 is most likely homodimeric [33, 41], while the primer-independent CphA1 from *Thermosynechococcus elongatus* strain BP-1 forms a homotetramer [39]. The primary structure of cyanobacterial CphA1 can be divided into two regions [33]. The C-terminal region shows sequence similarities to peptide ligases that include murein ligases and folyl poly-γ-glutamate ligase. The N-terminal part of CphA1 shows sequence similarities with another superfamily of ATP-dependent ligases that include carboxylate-thiol

**Figure 2.** Schematic illustration of CGP metabolism in cyanobacteria. CGP is synthesized from aspartate and arginine by CGP synthetase (CphA1) in an ATP-depending elongation reaction using CGP primers, containing of at least three Asp-Arg building blocks. Intracellular CGP degradation is catalyzed by the CGPase (CphB). The β-Asp-Arg dipeptides resulting from cleavage of CGP are further hydrolyzed by isoaspartyl dipeptidase, releasing aspartate and arginine. In many nitrogen-fixing cyanobacteria, an additional CGP synthetase is present, termed CphA2. CphA2 can use β-aspartyl-

The primers need to consist of at least three Asp-Arg building blocks (β-Asp-Arg)<sup>3</sup>

*elongatus* strain BP-1 shows primer-independent CGP synthesis [39].

and a sulfhydryl reagent (β-mercaptoethanol or DTT). For its activ-

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to detect

89

requires ATP, KCl, MgCl2

arginine dipeptides to resynthesize CGP.
