**5. The regulation of iron homeostasis is tightly connected to central metabolic pathways**

As mentioned previously, iron deficiency is one of the major causes of stress in cyanobacterial communities. Due to the occurrence of iron in most electron transport proteins conforming photosynthetic, respiratory and nitrogenase pathways, the adaptive strategies developed by the cyanobacteria are tightly related to the rearrangement and modulation of these processes. Furthermore, many of the different responses triggered by iron deprivation are aimed to prevent and alleviate oxidative stress and to the modulation of central metabolism.

Further transcriptomic studies evaluating the cyanobacterial response to iron deficiency unveiled that as a general trend, photosynthesis genes were repressed under low-iron conditions and induced upon the re-addition of iron. Many of those genes belonged to the *psa* and *psb* families, components of the phycobilisomes and genes involved in the synthesis of chlorophyll are also direct targets of FurA [14, 24, 141]. Furthermore, Fur is involved in the control of genes involved in carboxysome formation and Calvin cycle. Notably, a close relationship between light availability and iron requirements can be inferred from different studies, such as the differentially expressed genes in [142], the regulation of *furA* and the *alpha-furA* antisense RNA by light [143], or the need of an active photosynthetic electron transport chain for the expression of the *mcy* operon in *M. aeruginosa*, that in turn is controlled by FurA [124, 143, 144]. As *furA* from *M. aeruginosa*, the expression of the *Anabaena* sp. PCC7120 ortholog is controlled by an antisense RNA whose inactivation produces iron-deficient cells and severe structural disorders in the photosynthetic apparatus of *Anabaena*. Furthermore, disruption of the dicistronic message encoding the *alr1690-alpha-furA* tandem leads to lower photosynthetic performance indexes, unveiling that its expression is required for maintenance of a proper thylakoid arrangement, efficient regulation of iron uptake and optimal yield of the photosynthetic machinery [123, 145]. In addition, FurA modulates the transcription of the LexA regulator in *Anabaena* PCC7120. This regulator is critical to the survival of cyanobacterial cells facing inorganic carbon starvation, since most of the LexA-responsive genes were known to

The Challenge of Iron Stress in Cyanobacteria http://dx.doi.org/10.5772/intechopen.76720 121

be involved in carbon assimilation or controlled by carbon availability [146].

**5.3. Iron-responsive genes involved in cyanobacterial respiratory pathways**

wild-type strain has affected its respiratory activity [145].

**5.4. Cross-talk between iron and nitrogen metabolism**

In addition to the photosynthetic electron transport chains, cyanobacterial thylakoids contain multiple respiratory electron transport complexes [147]. Thus, photosynthesis and respiration are tightly related in cyanobacteria since both pathways share several components, such as a quinone/quinol pool [148], plastoquinone, cytochrome b6f and plastocyanin/cytochrome [148, 149]. Furthermore, the cyanobacteria contain a second complete respiratory chain present in the cell membrane that also uses the same mobile quinone pool mediating electrons in the photosynthetic and thylakoidal respiratory processes. Several studies evidence the relationship between the iron pool and the respiratory activity. The major oxidase in cyanobacteria, COX, is encoded by the *cox* operon (*coxBAC*) and FurA regulated though the modulation of *cox*B [15]. Similarly, the transcription of *alr0869* (*ndhF*) and the subunit 5 of NADH dehydrogenase encoded by *all1127* are regulated by FurA as response to iron availability [15]. Furthermore, iron starvation in *S. elongatus* causes upregulation of several cytochrome oxidases and the increase of respiratory electron transport [22, 150], while an *Anabaena* mutant lacking of the *alr1690-alpha-furA* message that exhibits a reduced iron pool with respect to the

The electron carriers involved in nitrogen metabolism are also rich in iron, especially the proteins involved in nitrogen fixation. Nitrogenase and nitrogenase reductase complex harbor around 40 atoms of Fe2+ distributed between the iron-molybdenum cofactor (FeMo-co) and the [8Fe-7S] P-cluster present in NifDK nitrogenase, and the [4Fe-4S] cubane in the NifH dinitrogenase reductase. In addition, most of the proteins involved in the assembly

## **5.1. Iron availability and the oxidative stress response**

Oxidative stress is one of the many consequences of iron imbalance in cyanobacteria. Thus, the control of iron homeostasis is intimately linked to the regulation of many genes involved in the response to oxidative stress [4, 14, 24, 94]. Moreover, the master regulators involved in such processes in cyanobacteria, namely FurA and PerR/FurC, display a set of common targets [14, 136]. Furthermore, PerR/FurC is able to modulate *in vitro* FurA-DNA binding activity [117]. Transcriptomic analyses and differential proteomics focused on the definition of the FurA regulon in *Anabaena* PCC 7120 unveiled that around 13% of FurA targets with a known function were involved in detoxification of ROS [14]. Those FurA-regulated genes belong to different subcategories, such as electron transport proteins dedicated to restore oxidized thiols (*trxA*, *trxB*, the glutaredoxin-related protein *alr0799* and the glutathione S-transferases *alr3195* and *alr7354*, among others); detoxification of hydrogen peroxide (the Mn-catalase *katB* and the peroxiredoxins *all1541* and *alr4641*) or the protection of DNA (*dpsA*) [14, 106, 119, 137]. FurA also controls the expression of flavodoxin that is strongly induced under iron deficiency [13, 138]. Initially described as a substitute for ferredoxin I (Fd) in the photosynthetic electron transport to NADP+ [45, 138] (reviewed in Sections 2.1.1 and 5.2), flavodoxin is also a powerful scavenger of ROS. Interestingly, the expression of flavodoxin in chloroplasts of tobacco unveiled that this flavoprotein is able to effectively interact with several Fd-dependent oxidoreductive pathways, including thioredoxin reduction [139]. The expression of flavodoxin in plastids protected target enzymes of central metabolic pathways from oxidative inactivation, such as the Calvin cycle components fructose-1,6-bisphosphatase (FBPase) and phosphoribulokinase (PRK). Therefore, the expression of flavodoxin triggered by iron deficiency relieves the oxidative stress in the cyanobacteria and contributes to the reconstitution of the electron transport chains rich in iron-containing proteins whose iron-sulfur clusters are immediate targets of free radicals, minimizing the effect of the oxidative damage on the photosynthetic rates and the nitrogen metabolism, among other metabolic pathways [139].

### **5.2. Influence of iron availability in the control of photosynthetic genes**

As it has been shown previously, iron limitation has important consequences in the composition and performance of cyanobacterial photosystems. Several photosynthetic cyanobacterial specific genes induced under iron deficiency contribute to modify their photosynthetic machineries such as *isiA*, *isiB* (flavodoxin), *idiA*, *idiB* and *idiC* proteins (reviewed in Section 2.1.1). Fur controls the expression of *isiA* and isiB [13], whose transcription is induced by multiple stresses such as treatment with hydrogen peroxide or high salt [56, 136, 140].

Further transcriptomic studies evaluating the cyanobacterial response to iron deficiency unveiled that as a general trend, photosynthesis genes were repressed under low-iron conditions and induced upon the re-addition of iron. Many of those genes belonged to the *psa* and *psb* families, components of the phycobilisomes and genes involved in the synthesis of chlorophyll are also direct targets of FurA [14, 24, 141]. Furthermore, Fur is involved in the control of genes involved in carboxysome formation and Calvin cycle. Notably, a close relationship between light availability and iron requirements can be inferred from different studies, such as the differentially expressed genes in [142], the regulation of *furA* and the *alpha-furA* antisense RNA by light [143], or the need of an active photosynthetic electron transport chain for the expression of the *mcy* operon in *M. aeruginosa*, that in turn is controlled by FurA [124, 143, 144]. As *furA* from *M. aeruginosa*, the expression of the *Anabaena* sp. PCC7120 ortholog is controlled by an antisense RNA whose inactivation produces iron-deficient cells and severe structural disorders in the photosynthetic apparatus of *Anabaena*. Furthermore, disruption of the dicistronic message encoding the *alr1690-alpha-furA* tandem leads to lower photosynthetic performance indexes, unveiling that its expression is required for maintenance of a proper thylakoid arrangement, efficient regulation of iron uptake and optimal yield of the photosynthetic machinery [123, 145]. In addition, FurA modulates the transcription of the LexA regulator in *Anabaena* PCC7120. This regulator is critical to the survival of cyanobacterial cells facing inorganic carbon starvation, since most of the LexA-responsive genes were known to be involved in carbon assimilation or controlled by carbon availability [146].

## **5.3. Iron-responsive genes involved in cyanobacterial respiratory pathways**

**5. The regulation of iron homeostasis is tightly connected to central** 

vent and alleviate oxidative stress and to the modulation of central metabolism.

**5.1. Iron availability and the oxidative stress response**

As mentioned previously, iron deficiency is one of the major causes of stress in cyanobacterial communities. Due to the occurrence of iron in most electron transport proteins conforming photosynthetic, respiratory and nitrogenase pathways, the adaptive strategies developed by the cyanobacteria are tightly related to the rearrangement and modulation of these processes. Furthermore, many of the different responses triggered by iron deprivation are aimed to pre-

Oxidative stress is one of the many consequences of iron imbalance in cyanobacteria. Thus, the control of iron homeostasis is intimately linked to the regulation of many genes involved in the response to oxidative stress [4, 14, 24, 94]. Moreover, the master regulators involved in such processes in cyanobacteria, namely FurA and PerR/FurC, display a set of common targets [14, 136]. Furthermore, PerR/FurC is able to modulate *in vitro* FurA-DNA binding activity [117]. Transcriptomic analyses and differential proteomics focused on the definition of the FurA regulon in *Anabaena* PCC 7120 unveiled that around 13% of FurA targets with a known function were involved in detoxification of ROS [14]. Those FurA-regulated genes belong to different subcategories, such as electron transport proteins dedicated to restore oxidized thiols (*trxA*, *trxB*, the glutaredoxin-related protein *alr0799* and the glutathione S-transferases *alr3195* and *alr7354*, among others); detoxification of hydrogen peroxide (the Mn-catalase *katB* and the peroxiredoxins *all1541* and *alr4641*) or the protection of DNA (*dpsA*) [14, 106, 119, 137]. FurA also controls the expression of flavodoxin that is strongly induced under iron deficiency [13, 138]. Initially described as a substitute for ferredoxin I (Fd) in the photosynthetic electron

ful scavenger of ROS. Interestingly, the expression of flavodoxin in chloroplasts of tobacco unveiled that this flavoprotein is able to effectively interact with several Fd-dependent oxidoreductive pathways, including thioredoxin reduction [139]. The expression of flavodoxin in plastids protected target enzymes of central metabolic pathways from oxidative inactivation, such as the Calvin cycle components fructose-1,6-bisphosphatase (FBPase) and phosphoribulokinase (PRK). Therefore, the expression of flavodoxin triggered by iron deficiency relieves the oxidative stress in the cyanobacteria and contributes to the reconstitution of the electron transport chains rich in iron-containing proteins whose iron-sulfur clusters are immediate targets of free radicals, minimizing the effect of the oxidative damage on the photosynthetic

As it has been shown previously, iron limitation has important consequences in the composition and performance of cyanobacterial photosystems. Several photosynthetic cyanobacterial specific genes induced under iron deficiency contribute to modify their photosynthetic machineries such as *isiA*, *isiB* (flavodoxin), *idiA*, *idiB* and *idiC* proteins (reviewed in Section 2.1.1). Fur controls the expression of *isiA* and isiB [13], whose transcription is induced by mul-

rates and the nitrogen metabolism, among other metabolic pathways [139].

**5.2. Influence of iron availability in the control of photosynthetic genes**

tiple stresses such as treatment with hydrogen peroxide or high salt [56, 136, 140].

[45, 138] (reviewed in Sections 2.1.1 and 5.2), flavodoxin is also a power-

**metabolic pathways**

120 Cyanobacteria

transport to NADP+

In addition to the photosynthetic electron transport chains, cyanobacterial thylakoids contain multiple respiratory electron transport complexes [147]. Thus, photosynthesis and respiration are tightly related in cyanobacteria since both pathways share several components, such as a quinone/quinol pool [148], plastoquinone, cytochrome b6f and plastocyanin/cytochrome [148, 149]. Furthermore, the cyanobacteria contain a second complete respiratory chain present in the cell membrane that also uses the same mobile quinone pool mediating electrons in the photosynthetic and thylakoidal respiratory processes. Several studies evidence the relationship between the iron pool and the respiratory activity. The major oxidase in cyanobacteria, COX, is encoded by the *cox* operon (*coxBAC*) and FurA regulated though the modulation of *cox*B [15]. Similarly, the transcription of *alr0869* (*ndhF*) and the subunit 5 of NADH dehydrogenase encoded by *all1127* are regulated by FurA as response to iron availability [15]. Furthermore, iron starvation in *S. elongatus* causes upregulation of several cytochrome oxidases and the increase of respiratory electron transport [22, 150], while an *Anabaena* mutant lacking of the *alr1690-alpha-furA* message that exhibits a reduced iron pool with respect to the wild-type strain has affected its respiratory activity [145].

#### **5.4. Cross-talk between iron and nitrogen metabolism**

The electron carriers involved in nitrogen metabolism are also rich in iron, especially the proteins involved in nitrogen fixation. Nitrogenase and nitrogenase reductase complex harbor around 40 atoms of Fe2+ distributed between the iron-molybdenum cofactor (FeMo-co) and the [8Fe-7S] P-cluster present in NifDK nitrogenase, and the [4Fe-4S] cubane in the NifH dinitrogenase reductase. In addition, most of the proteins involved in the assembly of the metalloclusters embedded within the NifDK protein also contain diverse [Fe-S] centers [151, 152]. Thus, growing under nitrogen fixation conditions adds an additional iron stress to the cell. Therefore, optimal cyanobacterial performance requires a tight and coordinated regulation of iron and nitrogen metabolisms [137]. Nitrogen metabolism in cyanobacteria is controlled by the master regulator NtcA [153] that usually senses the C/N balance through the intracellular 2-oxoglutarate levels [154]. NtcA controls a wide regulon of genes involved in different functional categories [155, 156]. Among them, NtcA controls most steps required for nitrogen fixation in cyanobacteria, starting from heterocyst differentiation and development until *nif* genes expression. NtcA also controls key genes in nitrogen assimilation pathways in cyanobacteria [157]. Different studies evidence a tight relationship between iron and nitrogen metabolism. Interestingly, transcription of the *nif-HDK* operon and excision of the 11 kb DNA fragment required for heterocyst differentiation was observed in iron-starved *Anabaena*, even though cells grew in the presence of combined nitrogen [138]. Further studies showed that the expression of FurA is highly induced in the heterocyst [137]. FurA participates in the regulation of *nif* genes, and the levels of this regulator are critical for the modulation of heterocyst differentiation by controlling the expression of NtcA and vice versa [14, 16]. Thus, several iron-responsive genes in cyanobacteria, such as *nblA, petH*, *pkn41*, *pkn42*, among others, are also modulated by NtcA [137, 158–161]. Conversely, in *Synechocystis sp.* PCC 6803, the NtcA-regulated genes *bgtB, glnA* and *urtB* are highly upregulated under iron limitation [162]. Different studies focused on the identification of the FurA and NtcA regulons in different cyanobacterial strains support that FurA and NtcA are interactive regulators and corroborate that both transcription factors share an important number of targets mainly related to photosynthesis and respiration, iron uptake and incorporation, oxidative stress response and nitrogen metabolism [137]. However, given that both FurA and NtcA are global regulators, it is not surprising that the nitrogen starvation response involves a large number of genes not only related to iron metabolism but also to heavy metal and oxidative stress adaptation, reinforcing the interrelationship of those processes [162].

**6.1. Iron and blooms occurrence**

**6.2. Iron and cyanotoxin production**

Iron availability and biolimitation by iron of the phytoplankton are important subjects discussed for many years. After IronExII [2], it was definitively established that iron availability limits rates of cell division, as well as abundance and production of phytoplankton of the equatorial Pacific and likely in other "high nutrient, low chlorophyll regions" [55]. There is broad agreement that nutrient over-enrichment of freshwater and marine ecosystems promote cyanobacterial blooms. Phosphorus and nitrogen have traditionally been considered the key nutrients limiting primary productivity and algal biomass. But based on such accessibility (and light and temperature suitable for cyanobacterial growth), iron availability could be suggested to be the switch that triggers a bloom. Cyanobacteria compete very efficiently with other phytoplankton species for iron resources and often end up dominating the population. In addition to all, the adaptive strategies previously mentioned, in some cases, their competi-

The Challenge of Iron Stress in Cyanobacteria http://dx.doi.org/10.5772/intechopen.76720 123

Cyanotoxins are a heterogenous group of molecules that include hepatotoxins, neurotoxins, dermatotoxins and cytotoxins, with diverse chemical nature such as cyclic peptides: cyclic peptides, alkaloids, non-proteic amino acids. The synthesis of most toxins is inducible, and the genes involved in its biosynthesis have been identified during these last years [165]. The genes conforming biosynthetic pathways, its regulation and the molecular mechanisms involved in toxicity are in each case different. However, NRPS are present in all the described toxic operons, involved in cyanotoxin synthesis. Many NRPS present in many bacteria are iron regulated [166, 167]. A substantial variety of siderophore structures, toxins and antimicrobial molecules with toxic effects are produced from similar NRPS assembly lines [167], and a large number of

Among cyanotoxins, microcystins are the most ubiquitous toxins causing several environmental and health problems. They are a family of cyclic heptapeptides, synthesized by a mixed PKS-NRPS system called microcystin synthetase encoded in *mcy* operon [168]. The role of microcystins in cyanobacteria is still unclear, but there are evidences that could confer to the toxic strains advantages for survival in iron-limited conditions. The microcystin synthesis has been linked to iron metabolism for many years. Lyck and colleagues [169] showed that during iron depletion, toxic strains of *Microcystis* maintained cell vitality much longer than the nontoxic strains. Moreover, Utkilen and Gjolme [170] found that toxic strains exhibited higher rates of iron uptake than nontoxic strains. They proposed that microcystin could be an intracellular chelator of Fe+2, as well as predicted that the synthesis of the toxin would be controlled by the amount of free iron present in the cells. Structural similarities between microcystin and bacterial siderophores [167] led also to propose a putative role as an extracellular iron-scavenging molecule. Recently, it was shown that while the microcystin producing strain *M. aeruginosa* PCC 7806 and its close strain, the non-producing *M. aeruginosa* PCC 7005 grew similarly in BG11 in the presence of 17 μM iron, under severe iron deficient conditions (0.05 μM), the toxigenic strain grew slightly less than in iron-replete conditions, while the non-producing microcystin strain was not able to grow [171]. Taking together all these data suggest that microcystin production

tive advantage is based on its ability to vertical migration [164].

secondary metabolites are also synthesized as response to iron starvation.
