**3. Iron uptake, transport and storage**

Siderophores are low-molecular-weight (generally <1000 Da) extracellular iron chelators produced by many prokaryotes and some eukaryotes including fungi, yeasts and plants. These secreted molecules often have a peptidic backbone, with modified amino acid side chains creating three main types of iron-coordinating ligands, that is hydroxamates, catecholates and carboxylates, which commonly form hexadentate octahedral complexes with one ferric ion [63, 64].

Most of the cyanobacterial siderophores appear to contain hydroxamate groups [65, 66], including the dihydroxamate siderophores schizokinen [65, 67] and synechobactin [68], though some species produce catecholate-type chelators such as anachelins [69, 70]. Hydroxamate-based siderophores are strong organic chelators showing a 1:1 stability constant with ferric iron of ~1030, something greater than that of the Fe3+-EDTA complex (~10<sup>25</sup>); however, ferric-catecholate siderophore complexes almost duplicate this affinity (~1049) [71]. Siderophores may coordinate other metals such as Zn2+, Cu2+, Ni2+, Pb2+, Cd2+, Mn3+, Co3+, Al3+, and Cr3+, playing significant roles in the biogeochemical cycling, biological uptake, and protection against deleterious exposure to high concentrations of these elements [72, 73]. In fact, the cyanobacterial siderophore schizokinen binds Cu2+ and contributes to alleviate copper toxicity under high environmental copper concentration. Secreted schizokinen sequesters extracellular Cu2+, but cupric-schizokinen is not recognized and internalized by cyanobacterial outer membrane transporters, thereby lowering the amount of copper taken up by the cells [74]. A similar detoxifying effect of cyanobacterial dihydroxamate siderophores has been observed with cadmium [75].

Among freshwater cyanobacteria, the model filamentous nitrogen-fixing heterocyst-forming cyanobacterium *Anabaena* sp. PCC 7120 as well as the bloom-forming, toxin-producing *A. flosaquae* synthesize schizokinen as their major siderophores [76]. Hydroxamate-based siderophore production has also been described in the paddy field cyanobacterium *A. oryzae* [75], and in nontoxic strains of the bloom-forming cyanobacterium *Microcystis aeruginosa* [77]. A novel group of cyanobacterial catecholate-type siderophores known as anachelins has been described in *A. cylindrica* [69]. In marine environments, only the coastal cyanobacterium *Synechococcus* sp. has been reported to produce siderophores. Notably, a distinct suite of dihydroxamate siderophores termed synechobactins is produced by *Synechococcus* sp. PCC 7002 [68]. In addition, xenosiderophore uptake (i.e., aerobactin and desferrioxamine B) has been documented in cyanobacteria [65], though the uptake of self-secreted siderophores is more efficient [78].

The routes of siderophore biosynthesis have not been extensively studied in cyanobacteria. Siderophore biosynthesis occurs in heterotrophic bacteria by two main pathways: one is directed by a large family of modular multienzymes called non-ribosomal peptide synthetases (NRPSs) and polyketide synthetases (PKS), while the other is known as the NRPS-independent siderophore (NIS) pathway [79]. Biosynthesis of hydroxamate-based siderophores with similar structures to schizokinen and synechobactins (e.g., aerobactin) takes place by the second route, involving four enzymes encoded by the gene cluster *iucABCD*, usually organized as an operon [80]. In *Anabaena* sp. PCC 7120, the outer membrane transports for ferric-schizokinen SchT (Alr0397) has been characterized [11], which showed a high amino acid sequence similarity with the ferric-aerobactin IutA transporter from *E. coli*. Near to the gene *alr0397*, a cluster of four open reading frames (*all0394*, *all0393*, *all0392*, *all0390*) show similarity with *iuc* genes, suggesting a role in the biosynthesis of schizokinen [11]. Since the defining characteristic of the NIS biosynthetic pathways is the presence of one or more nucleotide triphosphate-dependent synthetases responsible for condensation reactions during siderophore biosynthesis, this route has also been proposed for hydroxamate-based siderophore biosynthesis in *A. variabilis* and *Synechococcus* sp. PCC 7002 [81, 82].

Cell size reduction and/or morphological changes as response to iron starvation have also been described. For example, thylakoidal membranes and carboxysomes decrease as well as glycogen storage granules increase were observed in *A. nidulans* R2 by electron microscopy [26]. Iron limitation causes morphological changes in the thylakoid packing, promoting unpacking [59]. This phenomenon may be related with phosphorylation of light-harvesting chlorophyllbinding protein of PSII (LCHII) in barley induced by iron deficiency [60]. Iron deficiency causes in cyanobacteria a reduction of cell size [61, 26], sometimes related with growth rate [26*,* 62].

Siderophores are low-molecular-weight (generally <1000 Da) extracellular iron chelators produced by many prokaryotes and some eukaryotes including fungi, yeasts and plants. These secreted molecules often have a peptidic backbone, with modified amino acid side chains creating three main types of iron-coordinating ligands, that is hydroxamates, catecholates and carboxylates, which commonly form hexadentate octahedral complexes with one ferric

Most of the cyanobacterial siderophores appear to contain hydroxamate groups [65, 66], including the dihydroxamate siderophores schizokinen [65, 67] and synechobactin [68], though some species produce catecholate-type chelators such as anachelins [69, 70]. Hydroxamate-based siderophores are strong organic chelators showing a 1:1 stability constant with ferric iron of ~1030, something greater than that of the Fe3+-EDTA complex (~10<sup>25</sup>); however, ferric-catecholate siderophore complexes almost duplicate this affinity (~1049) [71]. Siderophores may coordinate other metals such as Zn2+, Cu2+, Ni2+, Pb2+, Cd2+, Mn3+, Co3+, Al3+, and Cr3+, playing significant roles in the biogeochemical cycling, biological uptake, and protection against deleterious exposure to high concentrations of these elements [72, 73]. In fact, the cyanobacterial siderophore schizokinen binds Cu2+ and contributes to alleviate copper toxicity under high environmental copper concentration. Secreted schizokinen sequesters extracellular Cu2+, but cupric-schizokinen is not recognized and internalized by cyanobacterial outer membrane transporters, thereby lowering the amount of copper taken up by the cells [74]. A similar detoxifying effect

of cyanobacterial dihydroxamate siderophores has been observed with cadmium [75].

nobacteria [65], though the uptake of self-secreted siderophores is more efficient [78].

Among freshwater cyanobacteria, the model filamentous nitrogen-fixing heterocyst-forming cyanobacterium *Anabaena* sp. PCC 7120 as well as the bloom-forming, toxin-producing *A. flosaquae* synthesize schizokinen as their major siderophores [76]. Hydroxamate-based siderophore production has also been described in the paddy field cyanobacterium *A. oryzae* [75], and in nontoxic strains of the bloom-forming cyanobacterium *Microcystis aeruginosa* [77]. A novel group of cyanobacterial catecholate-type siderophores known as anachelins has been described in *A. cylindrica* [69]. In marine environments, only the coastal cyanobacterium *Synechococcus* sp. has been reported to produce siderophores. Notably, a distinct suite of dihydroxamate siderophores termed synechobactins is produced by *Synechococcus* sp. PCC 7002 [68]. In addition, xenosiderophore uptake (i.e., aerobactin and desferrioxamine B) has been documented in cya-

**3. Iron uptake, transport and storage**

ion [63, 64].

114 Cyanobacteria

Another putative route of siderophore biosynthesis in *Anabaena* sp. PCC 7120 occurs presumably via a template-directed, nucleic acid-independent non-ribosomal mechanism which is mediated by the gene products of a cluster of nine open reading frames, from *all2641* to *all2649*, encoding seven NRPSs and two PKSs [83]. The expression of this NRPS-PKS gene cluster is transcriptionally repressed by the master regulator of iron homeostasis FurA [9], being induced under iron limitation or oxidative stress condition [83]. Since iron starvation induces oxidative stress in *Anabaena* sp. [7], maybe by dysfunction of the photosynthetic electron transport and some iron-containing antioxidant enzymes (e.g., SodB and DpsA), it has been postulated that release of siderophore biosynthesis to increase iron uptake during oxidative stress could restore both photosynthesis and ROS scavenging [83]. The protective effect of siderophores against oxidative stress has also been documented in heterotrophic bacteria [73].

*De novo* synthetized and re-used siderophores are secreted to the outside environment of bacterial cells by export systems which are not very well known in cyanobacteria. In *E. coli*, the export of enterobacterin siderophore involved different mechanisms comprising at least two components, the outer membrane channel tunnel protein TolC [84] which transports the siderophore from the periplasm to the outside, and several inner membrane transporters including the major facilitator superfamily (MFS) protein EntS [85] and the resistance nodulation cell division (RND) transport proteins AcrB, AcrD, AcrEF, MdtABC, and MdtEF [86]. In *Anabaena* sp. PCC 7120, the deletion mutant of the MFS-type inner membrane protein SchE (All4025) failed to secrete schizokinen siderophore to the external milieu [59]. Similar results were observed upon deletion of gene *hgdD* (*alr2887*) [59], encoding the only TolC-like protein in *Anabaena* sp. PCC 7120, termed HgdD, which is also required for protein and glycolipid secretion during heterocyst development [87] and secondary metabolite/antibiotic export [88]. Hence, hydroxamate siderophores appear to be exported in this model cyanobacterium through the mechanism SchE-HgdD.

Once bound to iron, ferric-siderophore complexes are efficiently taken up in Gram-negative bacteria through transport machinery which involves different outer and inner membraneassociated proteins as well as soluble periplasmic binding proteins [1, 12]. First, iron-loaded siderophores are recognized and translocated into the bacterial periplasm by TonB-dependent transporters (TBDTs) located in the outer membrane, in a process that is driven by the cytosolic membrane potential and mediated by the energy-transducing TonB-ExbB-ExbD system. Next, periplasmic binding proteins shuttle ferric-siderophores from the outer membrane transporter to ATP-binding cassette (ABC) permeases associated to the cytoplasmic membrane which delivers the iron-loaded siderophores to the citosol [1].

dominant picocyanobacterium *Prochlorococcus marinus* [82], and the open-ocean nitrogen fixers *Trichodesmium erythraeum* and *Crocosphaera watsonii* [82, 96]. However, some non-siderophoreproducing cyanobacteria express all the components of iron-siderophore uptake machinery, being capable of incorporate xenosiderophores [97]. Reductive iron uptake appears extended in many non-siderophore-producing cyanobacteria. In this strategy, reduction of free or complexed ferric iron (e.g., ferric-citrate) into its ferrous form takes place prior to the transport across the plasma membrane either by iron-reducing superoxide radicals secreted to the extracellular milieu as has been described in *Trichodesmium* and *Lyngbya* [96, 98], or through the action of plasma membrane-associated respiratory terminal oxidases as occurs in *Synechocystis* sp. PCC 6803 [95]. Given their small sizes, hydrophilic ferrous and unchelated ferric ions may passively diffuse to the periplasmic space through nonspecific outer membrane porins [95]. However, due to its frequent environmental low concentration, ferric iron uptake usually requires TonB-ExbB-ExbD-dependent active transport systems [99]. Once into the periplasm, high affinity ferric-binding soluble proteins bind ferric ions such as FutA1 and FutA2 and shuttle them to inner membrane ferric permeases such as FutB and FutC [100, 101]. Alternatively, ferric ions are reduced by any of the abovementioned mechanisms and cross the inner membrane through

The Challenge of Iron Stress in Cyanobacteria http://dx.doi.org/10.5772/intechopen.76720 117

Once inside the cell, ferric iron is reduced to ferrous iron, which has a much lower affinity for the siderophore and spontaneously dissociates [1]. Due to poor bioavailability or iron and its frequent intermittent supply in nature, bacteria have evolved efficient iron storage mechanisms involved ubiquitously multi-subunit proteins termed ferritins and bacterioferritins [102]. These proteins can accommodate up to 4500 iron atoms into a central cavity in a form that is unlike to participate in ROS generation reactions [102, 103]. In *Synechocystis* sp. PCC 6803, bacterioferritins BfrA and BfrB are responsible for the storage up to 50% of intracellular iron content [104], while the DPS family ferritin MrgA plays a pivotal role in both the mobilization of the stored iron within the cell [105], and the coordination between iron homeostasis and oxidative stress response [4]. By contrast, little is known about the mechanisms of iron storage in *Anabaena* species. Only four nonheme-binding ferritin family genes have been identified in *Anabaena* sp. PCC 7120 [104], including *alr3808* [106] and *all1173* [107], encoding two DNA-binding protein homologs to DpsA from *Synechococcus* sp. PCC 7942 [108]. DpsA from *Synechococcus* displays a weak catalase activity *in vitro* and is presumably involved in peroxide-consuming mechanism located on the chromosomal DNA, conferring resistance to peroxide damage during oxidative stress conditions or long-term nutrient limitation [108]. According to the CyanoBase [109], the genomes of other environmentally relevant cyanobacteria such as *P. marinus*, *C. watsonii*, *T. erythraeum*,

and *M. aeruginosa* encode members of the ferritin/bacterioferritin superfamily.

Regulators of the Fur (Ferric uptake regulator) family constitute the primary mechanism in the maintenance of iron homeostasis in cyanobacteria. The first evidence of the existence of a Fur protein in cyanobacteria was the isolation of a *fur* gene in *Synechococcus* PCC 7942 through an *E. coli*-based *in vivo* repression assay [13]. Apart from *Synechococcus*, Fur homologs

ferrous iron transporters like FeoB [95].

**4. Regulation of iron homeostasis**

TBDTs are composed of a transmembrane β-barrel domain that encloses a globular plug domain, and a periplasmic exposed TonB box [89]. Bacteria often possess multiple TBDT receptors, each providing the bacterium with specificity for different siderophores [90], but also allowing uptake of other nutrients [89, 91, 92]. TBDTs involved in iron uptake are generally induced by iron starvation and usually are not present or poorly expressed under ironsufficient conditions [1]. Twenty-two TBDTs have been identified in the genome sequence of *Anabaena* sp. PCC 7120, most of them integrated into gene clusters or even putative operons containing genes coding for proteins involved in iron transport [93]. A TBDT receptor involved in schizokinen uptake, SchT (Alr0397), has been described in *Anabaena* sp. PCC 7120 [11]. The expression of this outer membrane ferric-siderophore transporter is induced under iron-limitation [11], and it is transcriptionally regulated by FurA [94]. SchT appeared not essential for cyanobacterial growth under iron-limited conditions, suggesting the occurrence of other iron transporters in *Anabaena* sp. [11]. A second TBDT termed IacT (All4026), involved in iron and copper uptake, has been characterized in *Anabaena* sp. PCC 7120. IacT is not a schizokinen transporter; it appears to function under conditions in which the copper concentration exceeds the concentration of iron and seems to transport iron as ferric-citrate [59]. Finally, a third TBDT also involved in ferric-schizokinen uptake, IutA2 (Alr2581), has been recently described [78]. The *iutA2* mutant showed significant growth impairment under iron deprivation as well as alterations in ferric-schizokinen uptake.

Beyond the TBDTs SchT and IutA2, the iron-loaded schizokinen uptake machinery in *Anabaena* sp. PCC 7120 appears to comprise, at least, the gene products of *tonB3* (*all5036*), *exbB3*/*exbD3* (*all5047*, *all5046*), and *fhuCDB* (*all0389*-*all0387*). Whereas several *tonB*-like genes, *exb* clusters, and permease systems (i.e., *fhu*, *fut*, *fec*) have been annotated in the *Anabaena* genome, only the expression of the abovementioned ORFs were induced under iron-limiting conditions and reduced at high iron concentrations [12]. Additionally, mutants of the periplasmic ferricsiderophore binding protein FecB1 (All2583), but not of its homolog FutA, showed a slightly reduced uptake rate of ferric-schizokinen [78]. The *Anabaena* sp. PCC 7120 siderophore uptake system SchT/FhuBCD appears to be also involved in ferric-aerobactin uptake; however, the uptake of this hydroxamate siderophore produced by *E. coli* was ~10 fold slower than the uptake of ferric-schizokinen in the filamentous cyanobacterium [78].

Whereas some cyanobacterial species produce siderophores to scavenge iron under iron-limiting conditions, many cyanobacteria do not possess this ability, including some environmentally relevant lineages such as the planktonic freshwater cyanobacterium *Synechocystis* sp. [95], the dominant picocyanobacterium *Prochlorococcus marinus* [82], and the open-ocean nitrogen fixers *Trichodesmium erythraeum* and *Crocosphaera watsonii* [82, 96]. However, some non-siderophoreproducing cyanobacteria express all the components of iron-siderophore uptake machinery, being capable of incorporate xenosiderophores [97]. Reductive iron uptake appears extended in many non-siderophore-producing cyanobacteria. In this strategy, reduction of free or complexed ferric iron (e.g., ferric-citrate) into its ferrous form takes place prior to the transport across the plasma membrane either by iron-reducing superoxide radicals secreted to the extracellular milieu as has been described in *Trichodesmium* and *Lyngbya* [96, 98], or through the action of plasma membrane-associated respiratory terminal oxidases as occurs in *Synechocystis* sp. PCC 6803 [95]. Given their small sizes, hydrophilic ferrous and unchelated ferric ions may passively diffuse to the periplasmic space through nonspecific outer membrane porins [95]. However, due to its frequent environmental low concentration, ferric iron uptake usually requires TonB-ExbB-ExbD-dependent active transport systems [99]. Once into the periplasm, high affinity ferric-binding soluble proteins bind ferric ions such as FutA1 and FutA2 and shuttle them to inner membrane ferric permeases such as FutB and FutC [100, 101]. Alternatively, ferric ions are reduced by any of the abovementioned mechanisms and cross the inner membrane through ferrous iron transporters like FeoB [95].

Once inside the cell, ferric iron is reduced to ferrous iron, which has a much lower affinity for the siderophore and spontaneously dissociates [1]. Due to poor bioavailability or iron and its frequent intermittent supply in nature, bacteria have evolved efficient iron storage mechanisms involved ubiquitously multi-subunit proteins termed ferritins and bacterioferritins [102]. These proteins can accommodate up to 4500 iron atoms into a central cavity in a form that is unlike to participate in ROS generation reactions [102, 103]. In *Synechocystis* sp. PCC 6803, bacterioferritins BfrA and BfrB are responsible for the storage up to 50% of intracellular iron content [104], while the DPS family ferritin MrgA plays a pivotal role in both the mobilization of the stored iron within the cell [105], and the coordination between iron homeostasis and oxidative stress response [4]. By contrast, little is known about the mechanisms of iron storage in *Anabaena* species. Only four nonheme-binding ferritin family genes have been identified in *Anabaena* sp. PCC 7120 [104], including *alr3808* [106] and *all1173* [107], encoding two DNA-binding protein homologs to DpsA from *Synechococcus* sp. PCC 7942 [108]. DpsA from *Synechococcus* displays a weak catalase activity *in vitro* and is presumably involved in peroxide-consuming mechanism located on the chromosomal DNA, conferring resistance to peroxide damage during oxidative stress conditions or long-term nutrient limitation [108]. According to the CyanoBase [109], the genomes of other environmentally relevant cyanobacteria such as *P. marinus*, *C. watsonii*, *T. erythraeum*, and *M. aeruginosa* encode members of the ferritin/bacterioferritin superfamily.
