**2.1. Rearrangement of photosynthetic electron transport chain under iron starvation conditions**

Many photosynthetic components are iron-containing proteins, and also iron is involved in chlorophyll synthesis. Chlorophyll level is affected by iron availability, so the photosynthetic machinery may be diminished or even dismantled if the deficiency occurs suddenly, as in laboratory experiments. In general, populations living in limiting environments adapt its chlorophyll synthesis to the bioavailability, and the chlorophyll per cell is lower. Iron deficiency adaptation implies a reduction of the linear photosynthetic electron transport and enhances respiratory electron transport [20, 21] as well as a concomitant increase of the cyclic photophosphorylation [22]. Moreover, under iron deficiency, several responses to oxidative stress have been described, evidencing the link between iron starvation and oxidative stress, with photosystems specially affected [7, 23]. Consistently, several photosynthetic and oxidative defense genes have been identified as regulated by iron availability [9, 14, 24]. Among the iron-induced genes, *isiAB* [13] and *idiAB* products are playing key roles in the adaptability of the photosynthetic machinery to optimize its function at low iron availability.

#### *2.1.1. IsiA and IsiB proteins*

concentration ranges from 10−9 to 10−18 M, virtually all living microorganisms require a minimum effective concentration of 10−8 M to live and growth, and at least 10−7 to 10−5 M to achieve

Iron limitation is a challenge of particular importance in cyanobacteria, being one of the main limiting factors of ocean primary productivity [2]. Cyanobacteria have an absolute dependence of iron for growth and optimal development of their major physiological processes, particularly photosynthesis and nitrogen fixation. Iron serves as a cofactor for every membrane-bound protein complex and other mobile electron carriers within the photosynthetic apparatus [3], which determines an iron quota about 10 times higher than that exhibited by a similarly sized nonphotosynthetic bacterium [4]. Additionally, diazotrophic cyanobacteria have significant further iron requirements compared with other phototrophs due to the abundance of iron-containing enzymes in the nitrogen-fixation machinery [5]. Although iron plays a key role in cyanobacterial physiology, an excess of free intracellular iron is extremely deleterious because it catalyzes the formation of reactive oxygen species (ROS) through Fenton reactions, leading to oxidative stress [6]. Likewise, iron starvation leads to significant increase in ROS and induces oxidative stress in cyanobacteria [7]. Hence, iron uptake and metabolism must be tightly regulated in order to ensure suitable supply maintaining the intracellular concentration within nontoxic levels [8, 9]. To cope with the usually frequent periods of iron starvation in nature, cyanobacteria have evolve efficient strategies which imply changes in the transcription of a plethora of genes, resulting among other changes in a deep rearrangement of the photosynthetic machinery [10] and the induction of the mechanisms involved in iron uptake. Thus, the transcription of genes coding for several TonB-dependent outer membrane transporters, periplasmic ferric-binding proteins, ATP-binding permeases as well as enzymes involved in siderophore biosynthesis

Since an effective balance between iron acquisition and protection against oxidative stress is crucial for cell survival, as occurs in most Gram-negative and several Gram-positive bacteria, in cyanobacteria iron homeostasis is controlled by a global transcriptional regulator known as Fur, which stands for ferric uptake regulator [9, 13, 14]. Fur typically acts as a transcriptional repressor, which senses intracellular free iron and modulates transcription in response to iron availability [1]. Fur not only controls the expression of iron acquisition and storage systems, but also a wide set of genes and operons belonging to a broad range of functional categories, thereby contributing to couple iron availability to major physiological processes in cyanobacteria [14–17]. In this chapter, we revise the strategies of these photosynthetic bacteria to face the challenge of iron starvation. We put special emphasis in the transcriptional and physiological changes triggered by iron starvation in this group of microorganisms. Details on cyanobacterial iron metabolism and control of iron homeostasis as well as their connections

optimal growth [1].

110 Cyanobacteria

will depend on iron availability [9, 11, 12].

with other cellular processes are discussed.

**2. Classical strategies to overcome iron starvation situations**

Cyanobacteria evolved very efficient mechanisms to cope with iron deficiency. Iron deprivation triggers a variety of responses that range from upregulation of the iron acquisition systems to In *Synechococcus* sp., the *isiAB* operon is transcriptionally regulated to be expressed under iron deficiency, and the monocistronic transcript of *isiA* is more abundant than the dicistronic one [25]. IsiA gene product was found to confer fitness of photosynthetic machinery under iron-limited environments. The product of *isiA* was described in iron-starved *Anacystis nidulans* as an induced chlorophyll-binding protein [26]. This protein was initially named CP43'due to its similarity to CP43, located at the photosystem (PS)II [25]. Initially, IsiA was proposed to play a role as an additional light-harvesting complex [27], and over the years, several functions have been suggested, summarized by Sun and Golbeck [28]: (i) IsiA is a chlorophyll storage protein for the rapid recovery of the cyanobacteria after stress [29]; (ii) it acts as an excitation energy dissipator, protecting PSs from photoinhibition [30]; (iii) it serves as a light-harvesting complex potentially for both PSs [27, 31] and (iv) IsiA replaces CP43 in PSII and permits a cyclic electron transfer pathway involving PSII and the cytochrome b6 f complex [32, 33].

It is interesting to note that *isiA* is not present in all cyanobacteria, and no homologs of *isiA* have been found in plants. In fact, the presence of *isiA* in cyanobacteria found in the iron-limited, 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].

microbedb.jp/cyanobase/). Although some IdiA-similar proteins have been found in the periplasm [52], IdiA is predominantly found associated to thylakoids [53], suggesting different functions for the distinct IdiA-similar proteins [52]. IdiA undergoes prominent structural changes upon iron deficiency and forms a tight and specific complex with dimeric PSII by interaction with CP43 and D1 [54], suggesting that IdiA protects the acceptor side of PSII, which is more

The Challenge of Iron Stress in Cyanobacteria http://dx.doi.org/10.5772/intechopen.76720 113

In the *idi* operon, IdiB positively regulates transcription of *idiA* under iron starvation. IdiB encoding a member of the Crp/Fnr transcriptional regulators family [55] is transcribed under iron limitation and oxidative stress and controlled itself by iron-responsive Fur family members [56]. A third iron-regulated gene is *idiC*, belonging to the thioredoxin-like (2Fe–2S) ferredoxin family. Even though IdiC synthesis is constitutive, iron limitation induces a strongly enhanced expression of *idiC*. IdiC is loosely attached to the thylakoid and to other membranes, and its expression is enhanced during conditions of iron starvation or during the late growth phase [57]. Even though its role is still unclear, based on the similarity of IdiC to NuoE of the respiratory *Escherichia coli* NDH-1 complex, it has been suggested that IdiC is a component of the NADH-1 complex in *Synechococcus elongatus* and, thus, has a function in the electron donation from NAD(P)H to plastoquinone. Under stress conditions, when PSII resulted damaged, IdiC would prevent or reduce the oxidative stress deviating electron transport via alternative dehydrogenases, increasing PSI cyclic flow interconnected with respiratory routes [57].

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

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 cyanobacte-

exposed under iron limitation due to ongoing phycobilisome degradation [54].

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

ria while iron-rich mitochondrial proteins are preserved [22].

in detail in Section 3.

**2.3. Retrenchment**

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].

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 enzymes as peroxiredoxins, Calvin cycle enzymes and NADP+ -malate dehydrogenase, among 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, 48] was used as iron-stress molecular marker.

#### *2.1.2. IdiA, IdiB and IdiC proteins*

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. microbedb.jp/cyanobase/). Although some IdiA-similar proteins have been found in the periplasm [52], IdiA is predominantly found associated to thylakoids [53], suggesting different functions for the distinct IdiA-similar proteins [52]. IdiA undergoes prominent structural changes upon iron deficiency and forms a tight and specific complex with dimeric PSII by interaction with CP43 and D1 [54], suggesting that IdiA protects the acceptor side of PSII, which is more exposed under iron limitation due to ongoing phycobilisome degradation [54].

In the *idi* operon, IdiB positively regulates transcription of *idiA* under iron starvation. IdiB encoding a member of the Crp/Fnr transcriptional regulators family [55] is transcribed under iron limitation and oxidative stress and controlled itself by iron-responsive Fur family members [56]. A third iron-regulated gene is *idiC*, belonging to the thioredoxin-like (2Fe–2S) ferredoxin family. Even though IdiC synthesis is constitutive, iron limitation induces a strongly enhanced expression of *idiC*. IdiC is loosely attached to the thylakoid and to other membranes, and its expression is enhanced during conditions of iron starvation or during the late growth phase [57]. Even though its role is still unclear, based on the similarity of IdiC to NuoE of the respiratory *Escherichia coli* NDH-1 complex, it has been suggested that IdiC is a component of the NADH-1 complex in *Synechococcus elongatus* and, thus, has a function in the electron donation from NAD(P)H to plastoquinone. Under stress conditions, when PSII resulted damaged, IdiC would prevent or reduce the oxidative stress deviating electron transport via alternative dehydrogenases, increasing PSI cyclic flow interconnected with respiratory routes [57].
