Geomicrobiology and microbial contributions to geochemical cycles

Coordinated transporter activity shapes high-affinity iron acquisition in cyanobacteria

Abstract

Iron bioavailability limits biological activity in many aquatic and terrestrial environments. Broad scale genomic meta-analyses indicated that within a single organism, multiple iron transporters may contribute to iron acquisition. Here, we present a functional characterization of a cyanobacterial iron transport pathway that utilizes concerted transporter activities. Cyanobacteria are significant contributors to global primary productivity with high iron demands. Certain cyanobacterial species employ a siderophore-mediated uptake strategy; however, many strains possess neither siderophore biosynthesis nor siderophore transport genes. The unicellular, planktonic, freshwater cyanobacterium, Synechocystis sp. PCC 6803, employs an alternative to siderophore-based uptake-reduction of Fe(III) species before transport through the plasma membrane. In this study, we combine short-term radioactive iron uptake and reduction assays with a range of disruption mutants to generate a working model for iron reduction and uptake in Synechocystis sp. PCC 6803. We found that the Fe(II) transporter, FeoB, is the major iron transporter in this organism. In addition, we uncovered a link between a respiratory terminal oxidase (Alternate Respiratory Terminal Oxidase) and iron reduction - suggesting a coupling between these two electron transfer reactions. Furthermore, quantitative RNA transcript analysis identified a function for subunits of the Fe(III) transporter, FutABC, in modulating reductive iron uptake. Collectively, our results provide a molecular basis for a tightly coordinated, high-affinity iron transport system.

Introduction

Iron limitation is a challenge common to both terrestrial and aquatic ecosystems. It is of particular importance to photosynthetic organisms who must maintain iron-rich photosynthetic machinery (Raven et al., 1999; Blaby-Haas and Merchant, 2012). Indeed, iron availability limits primary productivity in many aquatic habitats, including one-third of the world’s oceans (Martin et al., 1991; Boyd et al., 2007).

Iron bioavailability is determined by its concentration and speciation. In oxygenated environments, Fe(II) is rapidly oxidized to Fe(III). Unlike the readily soluble Fe(II), Fe(III) rapidly precipitates as poorly available ferrioxyhydroxides (for Fe(OH)3, Kso=10−37.5 M4 (Byrne and Kester, 1976)). The remaining dissolved fraction is dominated by organic complexation, where Fe-binding compounds help maintain iron in solution (Hunter and Boyd, 2007). Total dissolved Fe concentrations in aquatic environments are in the nanomolar to subnanomolar range (Johnson et al., 1997).

Cyanobacteria are Gram-negative photosynthetic prokaryotes that contribute significantly to global primary productivity (Falkowski, 1997). Cyanobacterial iron transport systems must contend with limited Fe bioavailability in order to meet their iron requirements (Shaked and Lis, 2012). As is the case with many other prokaryotes, a siderophore-mediated iron uptake strategy is commonly attributed to cyanobacteria. In this strategy, cells synthesize and secrete siderophores-low molecular weight compounds that are highly specific for Fe(III) (Neilands, 1981). Once bound to iron, the ferrisiderophore complex is transported through the outer and plasma membranes before the iron is released from the complex (Braun and Hantke, 2011). Siderophore biosynthesis and ferrisiderophore transporters were characterized in a range of cyanobacterial species (Goldman et al., 1983; Wilhelm, 1995; Ito and Butler, 2005; Stevanovic et al., 2012; Kranzler et al., 2013). However, many cyanobacteria, most notably open ocean strains, possess neither siderophore biosynthesis nor siderophore transport capabilities (Hopkinson and Morel, 2009), alluding to an alternative iron acquisition strategy.

Reductive iron uptake is a transport strategy in which iron substrates undergo reduction from Fe(III) to Fe(II) before transport. This pathway is well-characterized in eukaryotic phytoplankton (Jones et al., 1987; Soria-Dengg and Horstmann, 1995; Maldonado and Price, 2001; Shaked et al., 2005; Allen et al., 2007;Morrissey and Bowler, 2012) and involves plasma membrane reductases that promote the reductive dissociation of organically bound Fe(III). In contrast to substrate-specific siderophore-mediated iron uptake, this strategy grants phytoplankton access to a wide range of organic and inorganic Fe(III) complexes.

Among cyanobacteria, the analysis of both laboratory cultures and natural populations suggested the existence of a reductive iron uptake pathway (Rose and Waite, 2005; Lis and Shaked, 2009). In a previous work we demonstrated that the unicellular cyanobacterium, Synechocystis sp. PCC 6803 (henceforth Synechocystis 6803), employs a reductive iron uptake strategy for a range of substrates including Fe–siderophore complexes and free inorganic iron (Fe′) (Kranzler et al., 2011). Like many ecologically relevant Synechococcus and Prochlorococcus strains, it does not possess any known siderophore biosynthesis genes (Hopkinson and Morel, 2009).

Although a molecular pathway for siderophore-mediated iron uptake was characterized in the siderophore producer Anabaena sp. PCC 7120 (Stevanovic et al., 2012), the mechanism of iron transport in non-siderophore producers is not well-established. Several Synechocystis 6803 proteins were shown to have a role in iron transport through the plasma membrane. Katoh et al. (2001) identified the gene products of futA, futB and futC as the components of a FutABC transporter, involved in the transport of inorganic Fe(III) (Table 1). This transporter is made up of a soluble Fe(III)-binding periplasmic component (FutA), a permease (FutB) and a membrane associated ATPase (FutC) (Katoh et al., 2001). Two FutA homologs, FutA1 and FutA2, were identified and shown to bind Fe(III) (Katoh et al., 2001; Badarau et al., 2008). In addition, a protein homologous to the Escherichia coli Fe(II) transporter, FeoB (Kammler et al., 1993), was shown to be involved in Fe(II) acquisition (Table 1, Katoh et al., 2001).

Table 1 Mutant strains

The role of these transporters in the reductive iron uptake pathway employed by Synechocystis 6803 was not investigated. Furthermore, the source of reducing power required for reductive iron uptake is unknown. In this work we explore the molecular nature of iron reduction and transport. We present a detailed study of mutant strains for several of the aforementioned genes. On the basis of short-term iron transport and reduction assays complemented with quantitative RNA transcript analysis, we suggest a working model for the molecular mechanism of reductive iron uptake in Synechocystis 6803.

Materials and methods

Trace metal clean techniques

In order to avoid iron contamination of experimental solutions, all plastic and glass ware was washed in HCl. Stock solutions were prepared using analytical grade chemicals and double distilled water (18.2 mΩ). For short-term experiments, all preparation and experimental work was done in a clean room facility. For these experiments, all plastic ware was washed in HCl and in EDTA before use.

Growth conditions

Synechocystis 6803 was grown in YBG11 with constant shaking at 30 °C under 60 μmol photons m−2 s−1 (Kranzler et al., 2011). Culture growth was monitored in a Cary 300 spectrophotometer (Varian, Palo Alto, CA, USA) at 730 nm. As the correlation between optical density and cell numbers varied between wild-type and mutant strains, cell densities were determined using hemocytometer cell counts. Iron was added precomplexed with EDTA (in twofold excess) at 0.1 μM for Fe-deplete medium and 10 μM for Fe-replete medium. Iron was kept in solution by applying 16 μM of the trace metal buffer, EDTA, which prevents iron precipitation on cell surfaces and maintains a computable pool of free, inorganic Fe(III) (Fe′) through dissociation and complexation reactions. FeEDTA is not bioavailable to Synechocystis 6803 (Kranzler et al., 2011).

Before the transfer into Fe-deplete medium, cells were spun down and washed twice in YBGll medium with no added iron. Mutant strains for genes used in this work are described in Table 1.

Short-term experiments

In YBG11 medium, Fe′ is an excellent substrate for reduction by the cell and is transported with remarkable affinity (Km=0.27 nM) by a pathway that is upregulated by Fe limitation (Kranzler et al., 2011). Therefore, we chose to use an Fe′ experimental system (buffered by the Fe(III) chelator, EDTA) as a model with which to probe the molecular nature of iron reduction and transport processes. Assays were conducted with cultures in the logarithmic phase of growth, 2 days after transfer into Fe-deplete medium. This amount of time was sufficient to upregulate iron responsive genes in the wild-type (Singh et al., 2003; Hernandez-Prieto et al., 2012).

Uptake and reduction assays

Short-term radioactive experiments were conducted in YBG11 medium supplemented with precomplexed 75–100 nM 55FeEDTA. Precomplexation reactions were performed with a twofold excess of EDTA and adjusted to pH 5–7 to ensure complex stability. Carrier FeEDTA was supplemented to a final concentration of 150 nM Fe, amounting to a total of 0.19 nM Fe′. EDTA was added to a final concentration of 16 μM. Medium was buffered at pH 7.8 (10 mM Hepes) in the absence of trace minerals in order to minimize iron contamination. Where indicated, the Fe(II)-specific chelator, Ferrozine (FZ),was added to a final concentration of 200 μM. Uptake and reduction experiments were run in the dark at 30 °C as described previously (Kranzler et al., 2011). Measurements were taken three to four times over the course of each experiment with a duplicate measurement at the first and final time points. When only three measurements were taken, the middle point was also measured in duplicates. Iron uptake and reduction rates were calculated from the linear regression of the data. Examples of raw data are presented in Figure 1.

Figure 1
figure1

Short-term radioactive iron uptake and reduction experiments. Representative uptake (a–d) and reduction (e–h) assays were conducted with strains: wild-type (a, e), ΔfutC (b, f), ΔfutA1 (c, g) and ΔfeoB (d, h). Medium contained free, inorganic Fe(III), supplied in the form of 55FeEDTA. Iron uptake and reduction rates reported in the manuscript were calculated from the linear regression of these data.

RNA extraction and reverse transcription

RNA was extracted from wild-type and mutant cultures 8 h after transfer to Fe-deplete medium (TRI Reagent, Molecular Research Center Inc., Cincinnati, OH, USA). Sampling time was selected based on the recent finding that maximal transcription of many Fe-limitation genes is reached after 12 h of Fe-limitation (Hernandez-Prieto et al., 2012). RNA was treated with TURBO DNAse-I, DNA-free (Ambion, Carlsbad, CA, USA). Reverse transcription reactions were performed with 0.5 μg RNA using the RevertAid M-Mul V R everse Transcriptase (Thermo Scientific, Rockford, IL, USA) and random hexamer primers (Thermo Scientific).

Quantitative PCR assays

Quantitative real-time PCR was performed using a Rotor-Gene 6000 Thermal Cycler (Corbett Research, Brisbane, Australia). Amplifications were carried out in triplicate using ABsolut Blue SYBR Green ROX (Thermo Scientific, ABgene, Rockford, IL, USA). Primer sequences used in this analysis are described in Supplementary Table S1. Primer amplification efficiency was determined using standard curve dilutions. A no-template reaction was used as a negative control. Melting curve analysis confirmed the specificity of each reaction. Quantitative real-time PCR was performed with RNA to verify that detectable DNA was removed from each sample. Transcript abundances were examined relative to the expression of the control gene rnpb.

Results

Analysis of Fe reduction and uptake rates in mutant strains

Our previous work demonstrated that Synechocystis 6803 employs a reductive iron uptake strategy (Kranzler et al., 2011). In this pathway, cell-mediated reduction of Fe(III) to Fe(II) occurs before transport through the plasma membrane. The Fe(II)-specific chelator, FZ, was applied in order to probe for the formation of an Fe(II) intermediate. When reductive iron uptake is at play, FZ binds biologically produced Fe(II). This results in the inhibition of Fe transport and enables the quantification of Fe(III) reduction rates (measured by Fe(II)FZ3 formation rates; Shaked et al., 2005). Reductive iron acquisition is deduced from a strong inhibitory effect of FZ on uptake rates, coupled to an Fe(III) reduction rate that corresponds to the degree of inhibition. We have used this approach to analyze wild-type and several mutant strains (Table 1) in order to identify the molecular components of the reductive iron uptake pathway.

We measured Fe′ uptake and reduction rates and tested the effect of FZ on uptake in each strain (Figures 1 and 2). Three distinct phenotypes were observed in the mutants analyzed in this study (Figure 1). (i) ΔFutC showed impaired Fe transport as compared with the wild-type (Figures 1a, b). ΔFutA2, ΔFutA1FutA2 and ΔARTO also displayed a similar phenotype (Supplementary Figure S1). (ii) ΔFutA1 showed higher uptake than the wild-type (Figure 1c). These phenotypes were reproducible in independent biological repeats (Supplementary Figure S1). As with the wild-type, strains exhibiting these two phenotypes were similarly inhibited by FZ (80–90%, Supplementary Figure S1 insert). Fe(III) reduction rates were measured in parallel (Figures 1e–g). Each of these strains exhibited an Fe reduction rate that was high enough to account for the inhibition of uptake by FZ (Figure 1). On the basis of these results, we suggest that these mutants employ a reductive iron uptake strategy under our experimental conditions. (iii) The third phenotype was found in ΔfeoB, which exhibited exceptionally slow uptake rates that were unaffected by FZ (Figure 1d). Interestingly, ΔfeoB displayed an Fe reduction rate comparable to that of the wild-type (Figure 1h).

Figure 2
figure2

Analysis of uptake and reduction rates. Fe uptake rates were plotted as a function of reduction rates. Because uptake and reduction rates sometimes vary, each set of experiments contained a wild-type culture for reference. The wild-type uptake rates were 1.1—3.8 × 10−20 mol Fe cell−1 h−1and the wild-type reduction rates were 1.3–4.9 × 10−20 mol Fe cell−1 h−1. For each experiment, mutant rates were normalized to internal wild-type reduction rates. Values are presented relative to the average wild-type reduction rate (2.7 × 10−20 mol Fe cell−1 h−1). Each point represents an independent biological repeat. Each mutant was tested at least twice. Two separate mutant strains for feoB were tested (n=4). ΔfutA1 results, that clusters away from the other strains, was tested an additional time (n=3). In all strains except ΔfeoB, uptake rates plot linearly as a function of reduction rates (y=0.62x+2.2 × 10−21, r2=0.88). Gray lines are 90% confidence bands enclosing the linear regression. Correlations between reduction and uptake rates in the data set are highlighted schematically (insert).

We found that mutants with faster or slower Fe reduction rates than the wild-type also had proportionally faster or slower uptake rates. The ratio between uptake and reduction rates remained constant in the wild-type and five of the six strains examined, as evident from the linear correlation in Figure 2. As Fe reduction occurs before transport through the plasma membrane (Kranzler et al., 2011), this finding suggests that the phenotypes observed in these mutants stem from changes in Fe reduction rather than Fe uptake capacity. It is important to note that all experiments were conducted at subsaturating iron concentrations (Kranzler et al., 2011), that is, the iron uptake capacities were higher than the measured iron uptake rates. Therefore, changes in reduction capacity will be reflected by higher or lower uptake rates. When following the trendline in Figure 2, data points above the wild-type have enhanced reduction capabilities, whereas those below the wild-type are impaired in these capabilities (Figure 2, insert). Only one mutant, ΔfeoB, falls below the trendline as its reduction rates were comparable to the wild-type but its uptake rates remained minimal.

The very slow iron uptake rates measured in ΔfeoB remained unaffected by FZ (Figure 1d, Supplementary Figure S1), suggesting that there is no transport of Fe(II) in this mutant despite its moderate to normal reduction capabilities (Figures 1h and 2). On the basis of these data, we suggest that FeoB functions as the major iron transporter under our experimental conditions and that its substrate is Fe(II).

Iron reduction: a role for respiration

We considered the cellular processes likely to be involved in iron reduction. As Fe′ uptake and reduction rates were unaffected by light (Kranzler et al., 2011), it is unlikely that photosynthetic electron flow is relevant. In addition, experiments with wild-type cells incubated with the photosynthetic inhibitor, 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU), did not affect Fe uptake rates but caused an increase in iron reduction rates (Supplementary Figure S2). As both photosynthetic and respiratory chains in cyanobacteria share a quinone/quinol pool (Schmetterer et al., 1994), the inhibition of photosynthesis may increase respiratory electron flow through Cytb6f. Does respiratory electron flow have a role in reductive iron uptake?

The Synechocystis 6803 genome contains three respiratory terminal oxidases. Two of these—a cytochrome c oxidase (Cox) and a quinol oxidase (Cyd)—are important in cellular respiration (Alge and Peschek, 1993; Schmetterer et al., 1994; Howitt and Vermaas, 1998). The function of the third, the Alternate Respiratory Terminal Oxidase (ARTO), or CtaII, remained unclear. Mutant strains defective in both cox and cyd genes exhibited very little respiratory activity, suggesting that ARTO does not reduce oxygen under the conditions tested (Howitt and Vermaas, 1998; Pils and Schmetterer, 2001). In order to determine whether ARTO is involved in the reductive iron uptake pathway, we measured iron uptake and reduction rates in the ΔARTO mutant strain.

ΔARTO displayed markedly impaired Fe reduction and uptake rates as compared with wild-type (Figure 2) indicating that ARTO is indeed involved in reductive iron uptake. As ΔARTO plots linearly with the wild-type and the other mutants (Figure 2), it is likely that its phenotype stems from diminished iron reduction capabilities.

FutABC and reductive iron uptake

The predominance of reductive iron uptake in Synechocystis 6803 raised questions regarding the function of the Fe(III) transporter, FutABC. ΔFutA2, ΔFutC and ΔFutA1ΔFutA2 displayed impaired iron reduction and uptake capabilities (Figure 2). The ΔFutA1 mutant exhibited significantly enhanced Fe reduction and uptake rates as compared with the wild-type (Figures 1 and 2). Interestingly, as with ΔARTO, these phenotypes also fall on the linear regression characteristic of the strains that use reductive iron uptake (Figure 2). What role might FutABC subunits have in this pathway?

We hypothesized that these subunits modulate Fe reduction capability. FutA2 is the most prominent Fe(III)-binding protein in the periplasm (Badarau et al., 2008). It is possible that formation of the Fe(III)FutA2 complex maintains the concentration gradient that drives external Fe′ into the periplasm. This gradient could not be established in ΔfutA2, decreasing the available substrate for reduction and uptake (Figure 2). FutC and FutA1 were both identified as intracellular proteins (Katoh et al., 2001; Tolle et al., 2002; Srivastava et al., 2005) rendering their location better suited for regulatory interactions (Tetsch and Jung, 2009; Ohashi et al., 2011; Richet et al., 2012).

Transcriptional changes in futA2 and feoB were measured in ΔFutA1 and ΔFutC mutants 8 h after transfer into Fe-deplete medium (Figure 3). Transcription of many iron transport-related genes is strongly induced by iron depletion within this time period. The transcript levels of futA2, futA1, futC and feoB are upregulated at least 2.5-fold following transition to iron limitation (Hernandez-Prieto et al., 2012). Quantitative PCR analysis showed that feoB transcript levels were higher in both strains as compared with wild-type (Figure 3). The effect was more pronounced in ΔFutC despite its impaired uptake and reduction rates (Figure 3 and Supplementary Figure S3). Interestingly, futA2 transcript abundance did correspond to Fe reduction and uptake rates in the different strains (Figure 3 and Supplementary Figure S3). Transcript levels were higher in ΔFutA1 and lower in ΔFutC as compared with wild-type (Figure 3 and Supplementary Figure S3). These data suggest that the observed phenotypes in ΔFutC and ΔFutA1 (Figures 1 and 2) stem from modifications of the regulatory network in the absence of these subunits.

Figure 3
figure3

Quantitative real-time PCR (qRT-PCR) analysis. futA2 and feoB transcription were quantified in wild-type, ΔfutA1 and ΔfutC 8 h after transfer into Fe-deplete medium. Transcript levels were internally normalized to the control gene, rnpB. The data are presented as compared with the wild-type. Error bars represent two biological repeats. Fe uptake and reduction rates in ΔfutA1 and ΔfutC are plotted as a function of futA2 and feoB transcript abundance.

Discussion

In this study, we examined the molecular pathway by which Fe is reduced and transported in the unicellular, non-siderophore-producing cyanobacterium, Synechocystis 6803. On the basis of our analysis of mutant strains (Figures 1) and the location of each protein (Table 1) we propose a working model for reductive iron uptake in Synechocystis 6803 (Figure 4).

Figure 4
figure4

A working model for the reductive iron uptake pathway. In reductive iron uptake, Fe(III) is first transported through the outer membrane (OM) of the cell (yellow). FutA2 (blue) binds periplasmic Fe(III). The respiratory terminal oxidase, ARTO (orange), is involved in Fe(III) reduction before transport through the plasma membrane (PM). The resulting Fe(II) is transported through FeoB (green). Intracellular subunits FutA1 and FutC of the Fe(III) transporter, FutABC (blue), regulate the reductive iron uptake pathway.

Our data suggest that the ARTO in Synechocystis 6803 has a role in reductive iron uptake (Figures 1 and 2). It is interesting to note that it was suggested that ARTO functions under microaerobic conditions; directly upstream of the ARTO operon is a binding site for an oxygen responsive transcriptional regulator, responsible for activating anaerobic metabolism (Howitt and Vermaas, 1998). Dissimilatory metal reducing bacteria utilize extracellular Fe(III) as the terminal electron acceptor in anaerobic respiration (Schroder et al., 2003). In dissimilatory iron reduction electrons are transferred outside of the cell, in certain cases through pili structures (Shi et al., 2007; Richter et al., 2012). Although this mode of iron reduction might be the evolutionary progenitor of assimilatory reductive iron uptake, we were unable to identify homologs of the multiheme cytochromes that are involved in dissimilatory iron reduction in the Synechocystis 6803 genome.

It is likely that uptake-associated iron reduction occurs inside the periplasmic space in close proximity to electron sources and iron transporters. The Synechocystis 6803 ARTO is located exclusively in the plasma membrane (Pils and Schmetterer, 2001; Berry et al., 2002), a location that could facilitate the supply of electrons for the reduction of periplasmic Fe(III) (Figure 4). ARTO genes were also found in most cyanobacteria with multiple copies in certain diazotropic organisms (Hart et al., 2005).

In reductive iron uptake, Fe(III) is transported through the outer membrane of the cell (Figure 4). Some cyanobacteria have tonB-dependent transporters that shuttle iron through the outer membrane (Stevanovic et al., 2012; Kranzler et al., 2013), but there is no evidence of such a function for the tonB-dependent transporters identified in Synechocystis 6803 (Katoh et al., 2001). It is also possible that Fe′ diffuses through nonspecific porins in the outer membrane (Fujii et al., 2011). Upon entering the periplasm FutA2 binds Fe(III), generating the chemical gradient that facilitates the influx of Fe. This is consistent with the suggested role for FutA2 in metal partitioning in the periplasm (Waldron et al., 2007). On the basis of our findings (Figures 2 and 3), FutA2 is a major factor in determining iron reduction and subsequent transport. Given its abundance, the Fe(III)FutA2 complex may be a substrate for reduction. Structural analysis of the protein revealed that it is made up of two domains that clamp Fe(III) via five coordinating ligands (Badarau et al., 2008). FutA2 is a high-affinity Fe(III)-binding protein with only residual Fe(II) binding capabilities (Badarau et al., 2008). The reduction of Fe(III)FutA2 would therefore result in the release of Fe(II).

Following reduction, Fe(II) is transported through FeoB, the major iron transporter under our experimental conditions (Figure 4). In ΔfeoB, iron uptake rates are minimal but are nevertheless well within the detection limit (Figures 1 and 2). Katoh et al. (2001) found that ΔfeoB growth was similar to the wild-type when transferred from Fe-rich medium to medium without added iron. However, it is important to note that the transfer from Fe-rich medium to Fe-free conditions probes the internal iron storage that accumulated before the transfer rather than Fe transport capability under iron limitation (Keren et al., 2004; Shcolnick et al., 2007).

Furthermore, iron uptake rates in ΔfeoB were unaffected by FZ (Figure 1 and Supplementary Figure S1). This indicates the presence of an additional Fe uptake pathway that does not involve reduction, for which FutABC is a likely candidate. These results are consistent with the iron uptake experiments conducted by Katoh et al. (2001) that implicated FutABC in Fe(III) transport but also demonstrated that Fe(II) transport was significantly faster than Fe(III) transport. That work also reported severely impaired uptake of Fe(II) in ΔfeoB.

The disruption of FutABC genes yielded mutant phenotypes that retained the ratio between iron reduction and uptake rates observed in the wild-type (Figure 2). Fe reduction and transport rates combined with transcriptional profiles in ΔFutC and ΔFutA1 suggest a regulatory function for these intracellular subunits (Figure 4). There is evidence to suggests that ABC transporters are used as co-sensors for signal transduction in bacteria (Tetsch and Jung, 2009). For example, the maltose transporter MalEFGK sequesters the MalT transcriptional regulator (Richet et al., 2012). In cyanobacteria, the ATP-binding subunit of the NRT-ABC nitrate/nitrite transporter is required for the regulation of ammonium promoted inhibition of transport (Ohashi et al., 2011). Furthermore, a similar function for a FutABC subunit may have been retained in vascular plant chloroplasts. FutC is the only remaining subunit of the cyanobacterial iron transport system in plants. Inactivation of a FutC ortholog (atNAP14) in Arabidopsis thaliana resulted in impaired iron homeostasis (Shimoni-Shor et al., 2010). Although it is likely that FutABC is a functional Fe(III) transporter, our findings demonstrate a role for FutABC subunits in the modulation of reductive iron uptake.

Siderophore synthesis and transport genes are absent from many cyanobacterial genomes (Hopkinson and Morel, 2009) raising the possibility of additional iron transport pathways. Reductive iron uptake constitutes an alternative to siderophore-mediated uptake that is advantageous in dilute heterogeneous environments (Volker and Wolf-Gladrow, 1999).

Our experimental conditions singled out the reductive iron uptake pathway and enabled the identification of its components in Synechocystis 6803. The composition of Fe transporters analyzed here is not unique. Many microorganisms possess the genetic potential for multiple iron transporters (Rivers et al., 2009; Desai et al., 2012; Hopkinson and Barbeau, 2012; Morrissey and Bowler, 2012). Bioinformatic analyses of iron transport genes revealed that fut genes are common in cyanobacteria (Rivers et al., 2009; Hopkinson and Barbeau, 2012, with additional analysis included in Supplementary Figure S4). Feo genes were identified in freshwater and coastal cyanobacteria but were notably absent from marine picocyanobacteria (Palenik et al., 2006; Rivers et al., 2009; Desai et al., 2012; Hopkinson and Barbeau, 2012; Supplementary Figure S4). However, many of the marine picocyanobacteria strains did possess a broad-specificity divalent metal transporter, the natural resistance-associated macrophage protein (Hopkinson and Barbeau, 2012), suggesting the potential for Fe(II) transport. An additional divalent metal transporter, ZIP (ZRT-, IRT-like protein), was also identified (Hopkinson and Barbeau, 2012). Both natural resistance-associated macrophage protein and ZIP have important roles in the acquisition of a wide range of divalent metals including Zn(II), Co(II), Fe(II), Mn(II) and Cd(II) (Nevo and Nelson, 2006; Taudte and Grass, 2010). These transporters were also identified in some freshwater and coastal cyanobacteria (Supplementary Figure S4).

The widespread genetic potential for Fe(II) transport suggests that reductive iron uptake may be employed by other cyanobacteria. The occurrence of several distinct transporters in a single organism necessitates coordination in order to acquire iron efficiently. The reductive iron uptake mechanism elucidated here in Synechocystis 6803 provides a model for coordinated transporter function.

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Acknowledgements

We thank Terou Ogawa for generously sharing mutant strains with us. This work was supported by the Israeli Science Foundation (grant no. 806/11). GS acknowledges financial support from ‘Projekt Chlorophyll’.

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Correspondence to Nir Keren.

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Kranzler, C., Lis, H., Finkel, O. et al. Coordinated transporter activity shapes high-affinity iron acquisition in cyanobacteria. ISME J 8, 409–417 (2014). https://doi.org/10.1038/ismej.2013.161

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Keywords

  • cyanobacteria
  • iron
  • transport

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