Function and Regulation of Ferredoxins in the Cyanobacterium, Synechocystis PCC6803: Recent Advances

Ferredoxins (Fed), occurring in most organisms, are small proteins that use their iron-sulfur cluster to distribute electrons to various metabolic pathways, likely including hydrogen production. Here, we summarize the current knowledge on ferredoxins in cyanobacteria, the prokaryotes regarded as important producers of the oxygenic atmosphere and biomass for the food chain, as well as promising cell factories for biofuel production. Most studies of ferredoxins were performed in the model strain, Synechocystis PCC6803, which possesses nine highly-conserved ferredoxins encoded by monocistronic or operonic genes, some of which are localized in conserved genome regions. Fed1, encoded by a light-inducible gene, is a highly abundant protein essential to photosynthesis. Fed2-Fed9, encoded by genes differently regulated by trophic conditions, are low-abundant proteins that play prominent roles in the tolerance to environmental stresses. Concerning the selectivity/redundancy of ferredoxin, we report that Fed1, Fed7 and Fed9 belong to ferredoxin-glutaredoxin-thioredoxin crosstalk pathways operating in the protection against oxidative and metal stresses. Furthermore, Fed7 specifically interacts with a DnaJ-like protein, an interaction that has been conserved in photosynthetic eukaryotes in the form of a composite protein comprising DnaJ- and Fed7-like domains. Fed9 specifically interacts with the Flv3 flavodiiron protein acting in the photoreduction of O2 to H2O.


Introduction
Ferredoxins (Fed) are small, mostly acidic, soluble proteins found ubiquitously in biological organisms. They possess a highly negative redox potential and use their iron-sulfur cluster to act as electron distributors in various metabolic pathways. The Fed proteins can be classified according to the nature of their iron-sulfur center ([2Fe-2S], [3Fe-4S] and [4Fe-4S]) and the organisms in which they were isolated for the first time [1]. Hence, the ferredoxins with a [2Fe-2S] cluster can be divided into plant-type or bacterial-type Feds. In plants, algae and cyanobacteria, i.e., prokaryotes with a plant-like oxygen-evolving photosynthetic apparatus, the most abundant ferredoxin, the plant-type [2Fe-2S] Fed, designated as Fed1, is recognized primarily as the protein that mediates electron transfer from iron-sulfur centers of photosystem I (PSI-C subunit) to ferredoxin NADP reductase, which, in turn, reduces NADP + for CO2 fixation [2]. Fed1 is also involved in other redox processes, such as cyclic photophosphorylation, nitrogen assimilation, biosynthesis of glutamate and chlorophyll, sulfite reduction, fatty acid metabolism and the modulation of the activity of various enzymes via the thioredoxin system [2]. Furthermore, a Fed1-like domain containing bacteriocin, named "peptocin M2", was recently shown to parasitize the cell entry and Fed-dependent iron acquisition system of the plant pathogens, Pectobacterium spp. [3].
Like other photosynthetic organisms, cyanobacteria possess various [2Fe-2S] and [4Fe-4S] Feds that have received less attention than Fed1, so far [2]. The interest in these ferredoxins has increased by the recent in vitro indications that one of these Fed can directly reduce NiFe-hydrogenase, which can produce hydrogen in some conditions [4]. In this review, we summarize what is known about the function and regulation of ferredoxins in cyanobacteria, emphasizing the unicellular model cyanobacterium, Synechocystis PCC6803, where the Fed proteins have been mostly studied.

The Nine Ferredoxins of Synechocystis Are Highly Conserved in Cyanobacteria
Synechocystis (hereafter Synechocystis) possesses a small sequenced genome [5], easily manipulable [6][7][8], which encodes nine Feds representative of the ferredoxin diversity. The fed1- 6 (Table 1). Furthermore a large number of the fed genes belong to well-conserved gene clusters (Figure 1), indicating that they operate in specific conserved functions relating to cyanobacterial life.
In contrast to fed1, the other fed genes of Synechocystis are weakly expressed under standard photoautotrophic conditions [9,11,13]. These observations are consistent with the fact that their products were undetected in Synechocystis protein extracts, unlike Fed1 [10]. Similarly to fed1, the eight other fed2-9 genes are regulated by environmental conditions (Table 2).

The Nine Synechocystis Ferredoxins Play a Crucial Role in Photoautotrophic Growth or Tolerance to Environmental Stresses
To investigate the nine fed genes, we independently replaced each fed coding sequence (fed-CS) with a transcription terminator-less marker, Km r , for antibiotic selection, while maintaining their DNA flanking regions (about 300 bp) to serve for homologous recombinations mediating targeted gene replacement upon transformation to Synechocystis [16]. The resulting deletion cassettes (Δfed1::Km r , Δfed2::Km r , Δfed3::Km r , Δfed4::Km r , Δfed5::Km r , Δfed2::Km r , Δfed3::Km r , Δfed4::Km r and Δfed9::Km r ) were independently introduced by transformation in Synechocystis, which harbors about 10 chromosome copies per cell [16]. We verified through PCR and DNA-sequencing that the marker gene had been properly inserted in the Synechocystis chromosome, in place of the studied gene, and we assayed whether the segregation between wild-type (WT) and mutant (Δfed) chromosome copies was complete (the studied fed gene is dispensable to cell growth) or not (the studied gene is essential to cell viability).
The Δfed1::Km r /fed1 + mutant growing under photoautotrophic condition harbored both WT (fed1 + ) and mutant (Δfed1::Km r ) chromosomes, irrespective of the duration (≥100 generations) and dose (up to 300 µg·mL −1 ) of the Km r selection. This result showed that fed1 is essential to the photoautotrophic growth of Synechocystis [9], as observed in the obligate photoautotroph cyanobacterium, Synechococcus PCC7942 [17]. The Synechocystis fed1 gene was found to be also crucial in cells growing in the presence of glucose, which supports cell growth in the absence of photosynthesis [9].
Like fed1, the fed2, fed3, fed6 and fed8 genes appeared to be essential to the photoautotrophic growth of Synechocystis (Table 3). The cells depleted of either Fed3 or Fed8 were killed by a prolonged exposure to Km and could not be further studied, whereas the Fed2-depleted cells (Δfed2::Km r /fed2 + ) appeared to display an increased size as compared to WT or Fed1-depleted cells (Figure 2). The four fed mutants, Δfed4::Km r , Δfed5::Km r , Δfed7::Km r and Δfed9::Km r , retained no WT chromosome copies and grew healthily in photoautotrophic conditions. These findings indicate that the fed4, fed5, fed7 and fed9 genes are dispensable for Synechocystis growth, in agreement with previous reports on fed4 [4] and fed7 [15,18]. The complete absence of WT chromosomes in each mutant was also verified in cultures subsequently grown for about 100 generations in the absence of the Km antibiotic to stop counter-selecting the propagation of possibly remaining wild-type (WT) chromosome copies, prior to the PCR assays. The absence of WT chromosome copies confirmed that the fed4, fed5, fed7 and fed9 genes are dispensable for the viability of Synechocystis (Table 3).

Fed7 and Fed9 Ferredoxins Plays a Prominent Role in the Tolerance to Oxidative and Metal Stresses, and the [2Fe-2S] Center of Fed7 Is Required for the Tolerance to Iron Starvation
As the mutants with fed4, fed5, fed7 or fed9 deleted grow well in standard photoautotrophic conditions, it was possible to investigate their tolerance to environmental stresses. In search of ferredoxin selectivity, we found that the absence of Fed7 or Fed9, but neither Fed4 nor Fed5, decreases the tolerance to oxidative and metal stresses (Figure 3). Furthermore, only Fed7 appeared to be involved in the protection against salt stress (Figure 3). Ten-microliter aliquots of exponentially growing cells (2.5 × 10 7 cells·mL −1 ) of the strains WT (wild-type), Δfed4 (fed4 null mutant), Δfed7 (fed7 null mutant) and Δfed9 (fed9 null mutant) were spotted onto solid mineral medium (MM) with or without the indicated concentration of the tested agents. The plates were subsequently incubated for 4-5 days in standard photoautotrophic conditions at 30 °C, prior to image acquisition. As it is indistinguishable from that of Δfed4, the phenotype of the Δfed5 mutant is not shown. These experiments were performed at least three times.
Because fed7 appeared to be induced by iron starvation (Table 2), we anticipated that Fed7 is required for protection against iron limitation. Indeed, the Δfed7::Km r null mutant appeared to be susceptible to iron limitation, and this phenotype could be rescued by plasmid complementation, as follows. The fed7 protein coding sequence (Table 1) was cloned into the pFC1 plasmid, which replicates autonomously in Synechocystis at the same copy number as the polyploid chromosome and expresses the studied genes proportionally to the growth temperature [8]. As expected, the moderate production of Fed7 (driven by the pFed7 plasmid) in cells incubated at 34 °C rescued the otherwise low tolerance to iron limitation of the Δfed7::Km r mutant back to the WT level ( Figure 4). Using the same strategy, we found that the cysteine to serine substitution at position 100 in the Fed7 amino-acid sequence did not impair the rescue complementation (compare the strains Δfed7 with or without the plasmids pFed7 or pFed7C100S). By contrast, the triple mutation of cysteines 53, 56 and 59, which coordinates the [2Fe2S] center of Fed7 (together with C96), abolished the complementation (compare the strains Δfed7 with or without the plasmids pFed7 or pFed7C53S,C56S,C59S), showing that the [2Fe2S] center of Fed7 is required for the tolerance to iron starvation. These data show that Fed7 operates in tolerance of iron limitation, likely by constituting a redox-responsive element. Typical growth of WT cells (black symbols) and mutant Δfed7 (red triangles), Δfed7 + pFed7 (green squares), Δfed7 + pFed7C53S56SC59S (blue circles) and Δfed7 + pFed7C100S (purple diamonds) cultivated for the indicated durations in standard liquid mineral medium (MM, which contains 17 µM Fe provided as ferric ammonium citrate) or in iron-limited medium (MM lacking ferric ammonium citrate, which contains only trace amounts of Fe) are shown. All experiments were performed at least three times at 34 °C to allow moderate expression of the various fed7 alleles from the replicating plasmid pFC1 of the fed7 (see above).

Analysis of the Selectivity/Redundancy of Ferredoxins: Identification of Fed-Interacting Proteins
To identify proteins that can physically interact with one or several Feds, we used the bacterial adenylate cyclase two-hybrid (BACTH) system [19], exactly as we described [18,20,21]. The full-length coding sequences of the Feds and possible redox partners were translationally fused to the intrinsically-inactive adenylate cyclase domains of the replication-compatible BACTH reporter plasmids, pKT25 and pUT18. The resulting pUT18 and pKT25 derivatives were doubly-transformed to the E. coli reporter strain, DHM1, to search for protein-protein interaction that reconstituted adenylate cyclase, which turned on β-galactosidase. Several of these Fed-partner interactions were verified through the identification of interaction-disruptive mutations in each protein partner (Table 4). Table 4. Identification and analysis of Fed-interacting proteins with the bacterial adenylate cyclase two-hybrid (BACTH) system.
The occurrence of physical interactions between the Feds and their partner proteins produced from the replication compatible pUT18 and pKT25 BACTH reporter plasmids co-transformed to E. coli was ascertained by measuring the β-galactosidase activity (1 β-Gal unit corresponds to the hydrolysis of 1 nmol of O-nitrophenyl-β-D-galactopyranoside; min −1 ·mg −1 of protein). The numbers are the mean value ± standard deviations of six assays (three measurements performed on two different cell extracts). The plasmids with or without the zip insert served as positive and negative controls, respectively [19]. The nature and position of amino-acid substitutions are written in subscripts. The presumed redox-active cysteines are indicated with superscripted asterisks. The name of the Feds proteins partners are as follows: DnaJ-domain-containing protein, sll1384; Flv3 (flavodiiron protein 3), sll0550; FTRc (ferredoxin-thioredoxin reductase catalytic chain), Sll0554.

Fed1, Fed7 and Fed9 Belong to a Ferredoxin-Glutaredoxin-Thioredoxin Crosstalk Pathway Operating in Stress Resistance
Using a combination of methods (two-hybrid, GST pull-down, western blotting, enzymatic assays and gene deletion and plasmid-rescue complementation in Synechocystis), we showed that Fed7 belongs to a complex redox pathway [18]. This pathway sequentially transfers the photosynthetic electrons to Fed1, FTRc (the ferredoxin-thioredoxin reductase catalytic chain), Fed7 and glutaredoxin 2. In addition, glutaredoxin 2 can also receive electrons from the NAD(P)H-thioredoxin reductase-glutaredoxin 1 pathway. The resulting crosstalk pathway plays a crucial role in the protection against hydrogen peroxide and selenate [18].
Similarly to Fed1 and Fed7, Fed9 appeared to interact with FTRc (Table 4). From this Fed9-FTRc interaction and the following lines of evidence, we propose that Fed1 and Fed9 interact with the same face of FTRc to reduce it, whereas both Fed7 and thioredoxin (Trx) interact with the other face of FTRc to be reduced by it ( Figure 5). First, knowing that electrons are transferred from the most to the least electronegative proteins, it is worth noting that the approximate redox potentials are −420 mV for both Fed1 and Fed9; −350 mV for FTRc; −400 mV or −150 mV for the [4Fe-4S] or the [3Fe-4S] forms of Fed7; and −270 mV for Trx (thioredoxin). Second, it has been shown that Fed1 and Trx bind on opposite sites of the disc-shaped FTRc protein, to form the Fed1-FTRc-TrxA pathway, which transfers electrons in that order [2,22]. Third, the Fed9-FTRc interaction was abolished by the D80A mutation in Fed9 and by the C88S mutation in FTRc, which did not impair the FTRc-Fed7 interaction (Table 4). Fourth, the FtrC-Fed7 interaction was abolished by the C100S mutation in Fed7 and by the C58S mutation in FTRc, which did not alter the Fed9-FTRc interaction (Table 4). By analogy with the Fed1-FTRc-TrxA redox pathway [22], we propose that in the case of oxidative stress, the [4Fe4S] cluster of Fed7 is converted into a [3Fe4S] center, thereby liberating the C56 cysteine that normally operates in the coordination of the [4Fe4S] cluster. The liberated C56 cysteine forms a disulfide bridge with the C100 cysteine, thereby turning the [3Fe4S] form of Fed7 into a TrxA-like protein ( Figure 5). The FtrC protein and its [4Fe-4S] center are represented by the green form and the red cube, respectively, while Fed1 (and Fed9) are represented by the red circle and TrxA (and Fed7) by the yellow form.

Identification of Proteins Selectively Interacting with Either Fed7 or Fed9, but Not Both
As a step towards the identification of selective functions of Fed7 or Fed9, we noticed that Fed7, but not Fed9, physically interacts with Sll1384, a DnaJ-like protein (Table 4), which is dispensable to cell life [23], like Fed7 [15,18]. Our finding is consistent with the occurrence in photosynthetic eukaryotes, such as Chlamydomonas reinhardtii, of a DnaJ-Fed composite protein comprising a DnaJ domain (similar to Sll1384) fused to a Fed domain (similar to Fed7, the closest homolog of this Fed domain in Synechocystis). Together, these data support the proposal that eukaryotic DnaJ-Fed composite proteins evolved from independent, but physically-interacting DnaJ-like and Fed7-like cyanobacterial proteins [15,24].

Conclusions
It is important to analyze the selectivity/redundancy of ferredoxins in cyanobacteria, because these enzymes play crucial roles in the growth and/or tolerance to environmental stresses of these fascinating organisms, which produce a large part of the oxygen and biomass for the food chain and also have high biotechnological interest. So far, most of what we know concerning cyanobacterial ferredoxins came from the analysis of the nine ferredoxins of the model strain, Synechocystis PCC6803, which are highly conserved in cyanobacteria. However, it is important to prolong and extend those studies by analyzing the ferredoxins of other cyanobacteria that colonize different biotopes (marine waters, desert soils) or perform processes not accomplished by Synechocystis (nitrogen fixation, multicellularity and differentiation of specialized cells, such as akinetes and/or heterocysts).