**4. Regulation of iron homeostasis**

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 mem-

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

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

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

brane which delivers the iron-loaded siderophores to the citosol [1].

116 Cyanobacteria

iron deprivation as well as alterations in ferric-schizokinen uptake.

uptake of ferric-schizokinen in the filamentous cyanobacterium [78].

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 have been mainly identified and studied in *Synechocystis*, *Anabaena* and *Microcystis* [17, 110–112]. Cyanobacterial Fur proteins contain histidine rich motifs (HHXHXXCXXC) as potential metal binding sites, which share properties with Fur from other prokaryotes [113, 114]. In the classic model of operation for this transcriptional regulator, Fur functions as a repressor, using ferrous iron as a co-repressor. Under sufficient iron availability, a dimer of active Fe2+-Fur complex binds to *cis* regulatory elements in the promoter of target genes and thereby prevents transcription [115]. However, other regulatory mechanisms have been described indicating that Fur can also bind to specific promoters in its apo form repressing transcription. Even apo- and holo-Fur activations have been reported [113, 116]. In the cyanobacterial genomes, it is common to find diverse ORFs that encode different Fur homologs which perform several functions. In this sense, in *Synechococcus* 7002 or *Anabaena* sp. PCC 7120, three *fur*-type genes exist, but only one of them, denoted as *furA*, appears directly involved in upregulation of iron uptake genes under iron limitation [9, 117, 118]. Recent studies confirmed that FurA is an essential, well-conserved protein among cyanobacteria. A significant depletion of *furA* expression levels impaired the photoautotrophic growth of *Anabaena* sp. under standard culture conditions in both, solid and liquid media [14]. FurA is the master regulator of iron homeostasis in *Anabaena* sp. PCC 7120 [9] and presumably in many other cyanobacterial species [14]. FurA modulates not only the expression of the iron metabolism machinery, but also regulates directly or indirectly the transcription of a plethora of genes and operons involved in a variety of physiological processes including photosynthesis, respiration, response to oxidative stress, nitrogen fixation, heterocyst differentiation, cellular morphology, tetrapyrrole biosynthesis pathway, phycobilisome degradation, chlorophyll catabolism, programmed cell death, light sensing and response, signal transduction systems, exopolysaccharide biosynthesis, and cyanotoxin production, among others [15, 16, 94, 119].

Fur homologs, is part of a thiol/disulfide redox switch that determines FurA ability to bind the metal co-repressor [125]. Moreover, this residue belongs to a CXXC motif responsible of the disulfide reductase activity exhibited by *Anabaena* FurA, suggesting that Fur is involved in the cyanobacterial redox-signaling pathway. Apparently, Fur connects the response to

The Challenge of Iron Stress in Cyanobacteria http://dx.doi.org/10.5772/intechopen.76720 119

The amount of Fur is controlled in cyanobacteria by mechanisms present in the three levels of the flow of genetic information [123]. At the transcriptional level, the TetR family transcriptional regulator PfsR regulates *fur* transcription in *Synechocystis* PCC 6803. A *pfsR* deletion mutant displayed stronger tolerance to iron-limiting conditions as compared with the wild type. Moreover, the transcripts of *pfsR* were enhanced by iron limitation and inactivation of the gene affected pronouncedly expression of *furA* gene and genes involved in iron transport

At the post-transcriptional level, *cis*-encoded antisense RNAs regulate Fur expression in cyanobacteria [128]. In *Anabaena* sp. PCC 7120, a large dicistronic transcript encoding the putative membrane protein Alr1690 and a α-*furA* RNA transcript complementary to *furA* is involved in the control of the cellular levels of the protein [129]. Also, *cis* α-*furA* RNAs are present in *M.* 

Regulation of the Fur level and its activity also take place post-translationally by different mechanisms in cyanobacteria. It has been reported that the membrane cytoplasmic FtsH1/ FtsH3 protease heterocomplex, involved in the acclimation of cells to iron deficiency, controls the availability of *Synechocystis* sp. PCC 6803 Fur by degradation of apo-Fur in order to regulate transcription of iron responsive genes [131]. Moreover, cyanobacterial Fur can form a complex with heme that alters its ability to join to DNA. In particular, *Anabaena* sp. PCC 7120 FurA interacts strongly with heme in the micromolar range of concentration and inhibits the *in vitro* ability of this protein to bind to DNA [117]. The axial ligand of heme in the FurA-heme complex is a cysteine residue that belongs to a Cys-Pro motif (Heme regulatory motif) present in its primary sequence and the sequences of all cyanobacterial homologs but absent in most non cyanobacterial ones. The regulator undergoes a redox-dependent ligand switch so that heme could be involved in sensing redox variations within the cyanobacterial filament and

A novel layer of complexity of iron homeostasis regulation in cyanobacteria involves RNA molecules as IsaR1. When iron is scarce, IsaR1 affects the photosynthetic apparatus in three different ways: (1) directly, inhibiting the expression of proteins important in photosynthesis; (2) indirectly, by suppression of pigment production; (3) preventing the expression of proteins that contain iron-sulfur clusters. Homologs of IsaR1 are conserved throughout the cyanobacterial phylum [133]. Also, the SufA and IscA proteins, proposed to function as scaffolds in the assembly of Fe/S clusters in bacteria, seem to play regulatory roles in iron homeostasis in cyanobacteria, according to experiments performed on single and double null-mutant strains of *Synechococcus* sp. [134]. Even the three PchR regulators (PchR1, PchR2, PchR3) present in *Synechocystis* PCC 6803 seem to play a prominent role in the protection against iron stress,

changes in the intracellular redox state and iron management in cyanobacteria [126].

and storage among others [127].

*aeruginosa* PCC 7806 and *Synechocystis* sp. PCC 6803 [130].

alter the regulatory function of FurA [132].

among other stresses, in this cyanobacterium [135].

Cyanobacterial Fur regulators can function both as activator and repressor as observed in the transcriptional regulation by FurA of genes involved in the tetrapyrrole biosynthesis pathway in *Anabaena* sp. PCC 7120 [9]. In all these cases, regulation by Fur adapts the answer to provide iron in case of deficiency of this metal or to allow its storage or the use of proteins that depend on iron when this metal is sufficient [1]. Fur recognizes AT rich regions called Fur boxes located in the promoter region of iron responsive genes [120]. Although it is assumed that this regulator binds as a dimer to the promoter, a computational study of Fur proteins from *Synechocystis* sp. PCC 6803 proposed the binding of multimers of the Fur-like regulator onto its target DNA, which possesses internal repeats [121]. Lately, atomic force microscopy revealed the sequential binding of FurA to its own promoter boosted by DNA bending in *Anabaena* sp. PCC 7120 [122]. Cyanobacterial Fur-DNA recognition depends not only on metal levels. Apart from iron, a reduced form of FurA from *Anabaena* sp. PCC 7120 is required for *in vitro* optimal DNA-binding [112, 123]. Also, reduction of Fur from *M. aeruginosa* PCC 7806 increases the binding affinity to its target genes [124]. Cyanobacterial Fur homologs contain a variable number of cysteine residues in their primary sequence and the need for reducing power for this regulator to develop its function is based on the importance of the redox state of these residues. A cysteine mutational study of the five cysteines present in *Anabaena* sp. PCC 7120 Fur sequence revealed that C101, a residue conserved in most bacterial Fur homologs, is part of a thiol/disulfide redox switch that determines FurA ability to bind the metal co-repressor [125]. Moreover, this residue belongs to a CXXC motif responsible of the disulfide reductase activity exhibited by *Anabaena* FurA, suggesting that Fur is involved in the cyanobacterial redox-signaling pathway. Apparently, Fur connects the response to changes in the intracellular redox state and iron management in cyanobacteria [126].

have been mainly identified and studied in *Synechocystis*, *Anabaena* and *Microcystis* [17, 110–112]. Cyanobacterial Fur proteins contain histidine rich motifs (HHXHXXCXXC) as potential metal binding sites, which share properties with Fur from other prokaryotes [113, 114]. In the classic model of operation for this transcriptional regulator, Fur functions as a repressor, using ferrous iron as a co-repressor. Under sufficient iron availability, a dimer of active Fe2+-Fur complex binds to *cis* regulatory elements in the promoter of target genes and thereby prevents transcription [115]. However, other regulatory mechanisms have been described indicating that Fur can also bind to specific promoters in its apo form repressing transcription. Even apo- and holo-Fur activations have been reported [113, 116]. In the cyanobacterial genomes, it is common to find diverse ORFs that encode different Fur homologs which perform several functions. In this sense, in *Synechococcus* 7002 or *Anabaena* sp. PCC 7120, three *fur*-type genes exist, but only one of them, denoted as *furA*, appears directly involved in upregulation of iron uptake genes under iron limitation [9, 117, 118]. Recent studies confirmed that FurA is an essential, well-conserved protein among cyanobacteria. A significant depletion of *furA* expression levels impaired the photoautotrophic growth of *Anabaena* sp. under standard culture conditions in both, solid and liquid media [14]. FurA is the master regulator of iron homeostasis in *Anabaena* sp. PCC 7120 [9] and presumably in many other cyanobacterial species [14]. FurA modulates not only the expression of the iron metabolism machinery, but also regulates directly or indirectly the transcription of a plethora of genes and operons involved in a variety of physiological processes including photosynthesis, respiration, response to oxidative stress, nitrogen fixation, heterocyst differentiation, cellular morphology, tetrapyrrole biosynthesis pathway, phycobilisome degradation, chlorophyll catabolism, programmed cell death, light sensing and response, signal transduction systems, exopolysaccharide biosynthesis, and cyanotoxin production,

Cyanobacterial Fur regulators can function both as activator and repressor as observed in the transcriptional regulation by FurA of genes involved in the tetrapyrrole biosynthesis pathway in *Anabaena* sp. PCC 7120 [9]. In all these cases, regulation by Fur adapts the answer to provide iron in case of deficiency of this metal or to allow its storage or the use of proteins that depend on iron when this metal is sufficient [1]. Fur recognizes AT rich regions called Fur boxes located in the promoter region of iron responsive genes [120]. Although it is assumed that this regulator binds as a dimer to the promoter, a computational study of Fur proteins from *Synechocystis* sp. PCC 6803 proposed the binding of multimers of the Fur-like regulator onto its target DNA, which possesses internal repeats [121]. Lately, atomic force microscopy revealed the sequential binding of FurA to its own promoter boosted by DNA bending in *Anabaena* sp. PCC 7120 [122]. Cyanobacterial Fur-DNA recognition depends not only on metal levels. Apart from iron, a reduced form of FurA from *Anabaena* sp. PCC 7120 is required for *in vitro* optimal DNA-binding [112, 123]. Also, reduction of Fur from *M. aeruginosa* PCC 7806 increases the binding affinity to its target genes [124]. Cyanobacterial Fur homologs contain a variable number of cysteine residues in their primary sequence and the need for reducing power for this regulator to develop its function is based on the importance of the redox state of these residues. A cysteine mutational study of the five cysteines present in *Anabaena* sp. PCC 7120 Fur sequence revealed that C101, a residue conserved in most bacterial

among others [15, 16, 94, 119].

118 Cyanobacteria

The amount of Fur is controlled in cyanobacteria by mechanisms present in the three levels of the flow of genetic information [123]. At the transcriptional level, the TetR family transcriptional regulator PfsR regulates *fur* transcription in *Synechocystis* PCC 6803. A *pfsR* deletion mutant displayed stronger tolerance to iron-limiting conditions as compared with the wild type. Moreover, the transcripts of *pfsR* were enhanced by iron limitation and inactivation of the gene affected pronouncedly expression of *furA* gene and genes involved in iron transport and storage among others [127].

At the post-transcriptional level, *cis*-encoded antisense RNAs regulate Fur expression in cyanobacteria [128]. In *Anabaena* sp. PCC 7120, a large dicistronic transcript encoding the putative membrane protein Alr1690 and a α-*furA* RNA transcript complementary to *furA* is involved in the control of the cellular levels of the protein [129]. Also, *cis* α-*furA* RNAs are present in *M. aeruginosa* PCC 7806 and *Synechocystis* sp. PCC 6803 [130].

Regulation of the Fur level and its activity also take place post-translationally by different mechanisms in cyanobacteria. It has been reported that the membrane cytoplasmic FtsH1/ FtsH3 protease heterocomplex, involved in the acclimation of cells to iron deficiency, controls the availability of *Synechocystis* sp. PCC 6803 Fur by degradation of apo-Fur in order to regulate transcription of iron responsive genes [131]. Moreover, cyanobacterial Fur can form a complex with heme that alters its ability to join to DNA. In particular, *Anabaena* sp. PCC 7120 FurA interacts strongly with heme in the micromolar range of concentration and inhibits the *in vitro* ability of this protein to bind to DNA [117]. The axial ligand of heme in the FurA-heme complex is a cysteine residue that belongs to a Cys-Pro motif (Heme regulatory motif) present in its primary sequence and the sequences of all cyanobacterial homologs but absent in most non cyanobacterial ones. The regulator undergoes a redox-dependent ligand switch so that heme could be involved in sensing redox variations within the cyanobacterial filament and alter the regulatory function of FurA [132].

A novel layer of complexity of iron homeostasis regulation in cyanobacteria involves RNA molecules as IsaR1. When iron is scarce, IsaR1 affects the photosynthetic apparatus in three different ways: (1) directly, inhibiting the expression of proteins important in photosynthesis; (2) indirectly, by suppression of pigment production; (3) preventing the expression of proteins that contain iron-sulfur clusters. Homologs of IsaR1 are conserved throughout the cyanobacterial phylum [133]. Also, the SufA and IscA proteins, proposed to function as scaffolds in the assembly of Fe/S clusters in bacteria, seem to play regulatory roles in iron homeostasis in cyanobacteria, according to experiments performed on single and double null-mutant strains of *Synechococcus* sp. [134]. Even the three PchR regulators (PchR1, PchR2, PchR3) present in *Synechocystis* PCC 6803 seem to play a prominent role in the protection against iron stress, among other stresses, in this cyanobacterium [135].
