**2.2. Siderophore synthesis and induction of high affinity transporters**

Derepression or induction of high affinity transporters to enhance iron acquisition as well as siderophore synthesis and cell surface enzymes production is a generalized response to iron starvation [1]. In cyanobacteria, siderophore-mediated iron uptake is thought to be an evolutionary advance that contributes to dominate iron-limited environments. Siderophores are strong Fe3+ chelators, and some of them synthetized by nonribosomal peptide synthetase systems. Siderophore production and secretion occurs, especially under iron starvation, when the intracellular iron concentration drops below a certain threshold required for functionality [58]. Siderophore-iron complexes are bound by outer membrane receptor proteins, the TonBdependent transporters (TBDTs). These outer membrane receptors are generally induced by iron starvation and usually are not present or poorly expressed under iron-sufficient conditions [1]. The iron uptake, transport and storage mechanisms in cyanobacteria are reviewed in detail in Section 3.

### **2.3. Retrenchment**

high-nutrient low-chlorophyll regions of the equatorial Pacific lead to the suggestion that the presence of this gene can be a natural biomarker for iron limitation in oceanic environments [34].

In most unicellular cyanobacteria downstream, *isiA* lies the *isiB* gene that encodes a small FMN-flavoprotein called flavodoxin. It is noticeable that, usually, in filamentous cyanobacteria, the flavodoxin gene is transcribed independently of *isiA* and lies in a different locus. Flavodoxin allows that the distribution of light energy as reducing power remains unaltered in iron deficient environments. When iron is not available, the synthesis of the iron-sulfur protein ferredoxin is repressed while flavodoxin is induced. Flavodoxin replaces ferredoxin as an electronic transporter in many of the reactions in which ferredoxin participates [35–39]; surprisingly, flavodoxin is not able to functionally replace heterocyst ferredoxin, even though electron transfer chain to nitrogenase is also an iron-dependent process [35]. Flavodoxin is not exclusive of cyanobacteria, and it may also be present in heterotrophic bacteria as well as in a few cases of algae [40]. Cyanobacteria which lack flavodoxin synthesis capability are particularly affected when iron is scarce, and ferredoxin downregulation under adverse conditions severely compromises survival [41]. Ferredoxin and flavodoxin are isofunctional proteins, but they do not share any significant similarity in primary, secondary or tertiary structures. These proteins can interact productively with the same redox partners [37, 38] and exhibit kinetics

constants in the same range even though flavodoxin is slightly less efficient [37].

enzymes as peroxiredoxins, Calvin cycle enzymes and NADP+

48] was used as iron-stress molecular marker.

*2.1.2. IdiA, IdiB and IdiC proteins*

112 Cyanobacteria

Flavodoxin expression is induced not only under iron deficiency but also under a wide range of several environmental stresses that result in ferredoxin downregulation [38, 42, 43], especially oxidative stress. Concerning the photosynthesis, flavodoxin behaved as an alternative intermediate for the photosynthetic electron transfer chain *in vivo*, acting, as ferredoxin does, as the main distributor of the reducing power [38, 44]. Under iron limitation, reduced flavodoxin also signals for the whole cell the presence of an active photosynthetic electron transfer chain through the thioredoxin electron transfer pathway. Reduced thioredoxins via thioredoxin reductase, regenerates, through reduction of their cysteine residues, the active forms of many target

others. Flavodoxin allows that this key process is still working under iron deficient conditions.

Since flavodoxin synthesis is one of the first responses to iron deficiency [45], flavodoxin was first proposed as an iron-deficiency biomarker in the marine diatom *Thalassiosira weissflogii* [46]. Similarly, in the green algae *Scenedesmus vacuolatus*, the ferredoxin/flavodoxin ratio [47,

In cyanobacteria under iron and manganese limitation, the *idiA* gene expresses the iron deficiency-induced protein, IdiA [49]; No counterpart seems to exist in green algae and higher plants [22]. The transcriptional regulator IdiB regulates the expression of *idiA*, in a response controlled by iron availability [50]. IdiA plays an important role in protecting the acceptor side of PSII against oxidative damage, especially under iron-limiting growth conditions [51]. IdiA shows considerable sequence similarity to a family of bacterial periplasmic ABC transporter complexes involved in iron import known as FutA, SfuA, FbpA or HitA (http://genome.


Retrenchment or downregulation of physiological rates is a progressive and reversible response, resulting in a modulation of the overall growth rate and changes in biochemical parameters. This mechanism is widely used in the adaptation of many organisms to adverse conditions. The most frequent response implies remodeling of bioenergetic pathways in response to iron availability (see Sections 2.1 and 5). As mentioned previously, low iron concentrations trigger a reduction in the level of iron-rich photosynthetic proteins in cyanobacteria while iron-rich mitochondrial proteins are preserved [22].

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].

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 sid-

The Challenge of Iron Stress in Cyanobacteria http://dx.doi.org/10.5772/intechopen.76720 115

erophore biosynthesis in *A. variabilis* and *Synechococcus* sp. PCC 7002 [81, 82].

heterotrophic bacteria [73].

through the mechanism SchE-HgdD.

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

*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
