**6. Iron involvement in cyanotoxin production**

Metabolic plasticity of cyanobacteria includes the synthesis of a broad variety of secondary metabolites, some of them potentially toxic for eukaryotic organisms, the so-called cyanotoxins [163]. When toxins are synthetized, the cyanobacteria divert large amounts of carbon and nitrogen to this process so that it might be obvious to think that cyanotoxin synthesis gives them some adaptive advantage. Cyanotoxin production is not universal or constant even among those species and strains holding the necessary genes. The conditions that induce cyanotoxin production in capable species have not been elucidated. Under certain environmental conditions, cyanobacteria can proliferate to form blooms consisting of significant biomass and covering large areas in fresh or marine water. It is necessary to separate the phenomenon of blooms occurrence from the fact of toxicity, although obviously the problem is detected when the population of toxic cyanobacteria synthetizing toxins is high.

#### **6.1. Iron and blooms occurrence**

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].

122 Cyanobacteria

toxins is high.

**6. Iron involvement in cyanotoxin production**

Metabolic plasticity of cyanobacteria includes the synthesis of a broad variety of secondary metabolites, some of them potentially toxic for eukaryotic organisms, the so-called cyanotoxins [163]. When toxins are synthetized, the cyanobacteria divert large amounts of carbon and nitrogen to this process so that it might be obvious to think that cyanotoxin synthesis gives them some adaptive advantage. Cyanotoxin production is not universal or constant even among those species and strains holding the necessary genes. The conditions that induce cyanotoxin production in capable species have not been elucidated. Under certain environmental conditions, cyanobacteria can proliferate to form blooms consisting of significant biomass and covering large areas in fresh or marine water. It is necessary to separate the phenomenon of blooms occurrence from the fact of toxicity, although obviously the problem is detected when the population of toxic cyanobacteria synthetizing 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 competitive advantage is based on its ability to vertical migration [164].

#### **6.2. Iron and cyanotoxin production**

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 secondary metabolites are also synthesized as response to iron starvation.

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 could be another mechanism evolved by cyanobacteria related to iron homeostasis, on track to survive in iron-limited conditions. In agreement with this statement, it was shown that in *M. aeruginosa* PCC 7806, the *mcy* operon was regulated by Fur [124], and that the *mcy* operon transcription as well as microcystin content were enhanced under iron-limited conditions [172].

**Conflict of interest**

**Author details**

**References**

The authors declare no conflicts of interest.

\*Address all correspondence to: esevilla@unizar.es

Microbiology Reviews. 2003;**27**(2-3):215-237

1996;**383**(6600):495-501

Reviews. 2009;**33**(2):258-278

1994;**6**(2):199-210

Andrés González, María F. Fillat, María-Teresa Bes, María-Luisa Peleato and Emma Sevilla\*

The Challenge of Iron Stress in Cyanobacteria http://dx.doi.org/10.5772/intechopen.76720 125

Biocomputation and Physics of Complex Systems, University of Zaragoza, Zaragoza, Spain

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[3] Ferreira F, Straus NA. Iron deprivation in cyanobacteria. Journal of Applied Phycology.

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[6] Latifi A, Ruiz M, Zhang CC. Oxidative stress in cyanobacteria. FEMS Microbiology

[7] Latifi A, Jeanjean R, Lemeille S, Havaux M, Zhang CC. Iron starvation leads to oxidative stress in *Anabaena* sp. strain PCC 7120. Journal of Bacteriology. 2005;**187**(18):6596-6598 [8] Shcolnick S, Keren N. Metal homeostasis in cyanobacteria and chloroplasts. Balancing benefits and risks to the photosynthetic apparatus. Plant Physiology. 2006;**141**(3):805-810

[9] Gonzalez A, Bes MT, Valladares A, Peleato ML, Fillat MF. FurA is the master regulator of iron homeostasis and modulates the expression of tetrapyrrole biosynthesis genes in

*Anabaena* sp. PCC 7120. Environmental Microbiology. 2012;**14**(12):3175-3187

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and in situ from the North Atlantic. PLoS One. 2012;**7**(5):e35571

Department of Biochemistry and Molecular and Cell Biology and Institute for

Recently, microcystin ability to bind iron and other metals has been demonstrated using various experimental approaches [171], corroborating a possible role of this molecule in iron metabolism. A putative role of microcystin acting as iron chelator involved in iron acquisition has been recurrently suggested. The main problem associated to this theory is the fact that microcystin seems to be an endotoxin although the results showed in bibliography are contradictory. When radioactive inorganic carbon is supplied to *M. aeruginosa* and the fate of intracellular microcystin pool is followed, no export of microcystin was observed [173]. However, the *mcyH* gene included in the *mcy* operon encoded an ABC transporter reported to be essential for microcystin synthesis, suggesting a possible export of microcystin outside of the cell [174]. On the other hand, electron microscopy of immuno-gold labeled microcystin showed that the vast majority of intracellular microcystin is located around the thylakoids [175–177]; hence, a possible role in protecting the photosynthetic machinery to photo-oxidation has been proposed. Recently, it has been described that microcystin can perform metal-driven oligomerization. Some environmental stresses such as low iron or high light conditions cause oxidative stress in the cell which triggers photo-oxidation phenomena. In this scenario, the PSs can be disassembly and then, microcystin could perform oligomerization and capture of iron avoiding metal-dependent Fenton reactions [171]. Another proposed role is related with colony formation performed by *Microcystis* cells. Solid evidences linking microcystin presence and enhanced colony formation and size have been reported [178].
