## 2 Second Messengers in Cyanobacteria
### Guanosine-3', 5'-(bis) Pyrophosphate, (p)ppGpp  
Guanosine-3', 5'-(bis) pyrophosphate, (p)ppGpp, was the first second messenger characterized in cyanobacteria \(\sim\)40 years ago. This molecule is involved in the stringent response, during which alterations in metabolism and gene expression occur due to limitation in the availability of amino acids or nutrition stress [39]. During the stringent response, resources are diverted away from growth towards amino acid synthesis to support survival until unfavorable conditions improve. During this process,ppGpp or pppGpp is synthesized from ATP and GTP/GDP by a (p)ppGpp synthetase, RelA [46], and can be degraded to GDP/GTP and pyrophosphate by a (p)ppGpp hydrolase, SpoT [46] (Figure 3). (p)ppGpp inhibits translation initiation to limit excessive protein synthesis during nutritional deficiencies [47]. Recently, more details of the regulation of (p)ppGpp and downstream processes emerged. In _E. coli_ (p)ppGpp binds the \(\beta\)' subunit of RNA polymerase and decreases the half-life of rRNA, resulting in decreased transcription [48]. (p)ppGpp also binds the \(\beta\)' subunit of the plastid RNA polymerase in chloroplasts of plants [49], suggesting that (p)ppGpp likely binds the \(\beta\)' subunit of the RNA polymerase in cyanobacteria, which are widely recognized as the progenitor of plastids [50].  
The (p)ppGpp homeostasis enzymes RelA/SpoT (PF04607) are present as bifunctional gene homologs, sometimes referred to as RSH (Rel Spo homologs) genes [47], in each of the 83 finished genomes present in the Integrated Microbial Genomes (IMG) database [51] (genomes listed in Table S1) suggesting a widespread utilization of (p)ppGpp as a signaling molecule in cyanobacteria (Table 1). _Cyanobium gracile_ PCC 6307, _Cyanothece_ sp. BH68 ATCC 51142, and _Nodularia spunigena_ CCY9414 possess two copies of the RSH genes annotated as RelA/SpoT (Table S2).  
_Anabaena_ possesses a single _relA/spoT_ homolog (_all1549_ or Ana-RSH) that regulates intracellular levels of (p)ppGpp in response to amino acid deprivation [52]. Ana-RSH was determined to be essential, as it could not be deleted from the genome [52]. Ana-RSH is maintained at a basal level in _Anabaena_ under non-stressful conditions and appears to be regulated at the enzymatic rather than at a transcriptional level under amino acid deprivation [52]. As noted above, nitrogen deprivation leads to transient increases in (p)ppGpp levels in some cyanobacteria [53,54]; thus, the role of Ana-RSH during  
Figure 3: External factors controlling intracellular levels of (p)ppGpp and phenotypes or processes that are controlled by (p)ppGpp in cyanobacteria. (p)ppGpp is synthesized from GDP or GTP together with ATP by RelA or SpoT proteins and degraded to GDP or GTP and the by-product pyrophosphate (PPi) by SpoT. Dashed lines denote hypothetical or suggested role of cyanophages under nutrient-deficient growth conditions in controlling internal (p)ppGpp levels. Green arrow indicates that increased synthesis of (p)ppGpp leads to noted phenotypes, whereas red arrow indicates that degradation of (p)ppGpp leads to noted phenotypes. \(+\), indicates process promoted by increased (p)ppGpp synthesis; \(-\), indicates process inhibited by increased (p)ppGpp levels.  
nitrogen deprivation and heterocyst development was explored. Although Ana-RSH was enriched in vegetative cells relative to heterocysts, the regulation of Ana-RSH or (p)ppGpp was not correlated with heterocyst formation and nitrogen fixation [52]. A more recent study, however, reported up-regulation of Ana-RSH under nitrogen deprivation and confirmed enrichment in vegetative cells [55]. These authors also were not able to delete Ana-RSH from the genome, but isolated a non-functional insertion mutant of Ana-RSH that exhibited reduced (p)ppGpp levels in response to amino acid starvation and failed to form heterocysts in nitrogen-limited conditions [55].