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Journal of Bacteriology, May 2006, p. 3182-3191, Vol. 188, No. 9
0021-9193/06/$08.00+0     doi:10.1128/JB.188.9.3182-3191.2006
Copyright © 2006, American Society for Microbiology. All Rights Reserved.

Regulatory Roles for IscA and SufA in Iron Homeostasis and Redox Stress Responses in the Cyanobacterium Synechococcus sp. Strain PCC 7002{dagger}

Ramakrishnan Balasubramanian,1 Gaozhong Shen,1 Donald A. Bryant,1* and John H. Golbeck1,2*

Department of Biochemistry and Molecular Biology,1 Department of Chemistry, The Pennsylvania State University, University Park, Pennsylvania 168022

Received 6 December 2005/ Accepted 15 February 2006


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ABSTRACT
 
SufA, IscA, and Nfu have been proposed to function as scaffolds in the assembly of Fe/S clusters in bacteria. To investigate the roles of these proteins further, single and double null-mutant strains of Synechococcus sp. strain PCC 7002 were constructed by insertional inactivation of genes homologous to sufA, iscA, and nfu. Demonstrating the nonessential nature of their products, the sufA, iscA, and sufA iscA mutants grew photoautotrophically with doubling times that were similar to the wild type under standard growth conditions. In contrast, attempts to inactivate the nfu gene only resulted in stable merodiploids. These results imply that Nfu, but not SufA or IscA, is the essential Fe/S scaffold protein in cyanobacteria. When cells were grown under iron-limiting conditions, the iscA and sufA mutant strains exhibited less chlorosis than the wild type. Under iron-sufficient growth conditions, isiA transcript levels, a marker for iron limitation in cyanobacteria, as well as transcript levels of genes in both the suf and isc regulons were significantly higher in the iscA mutant than in the wild type. Under photosynthesis-induced redox stress conditions, the transcript levels of the suf genes are notably higher in the sufA and the sufA iscA mutants than in the wild type. The growth phenotypes and mRNA abundance patterns of the mutant strains contradict the proposed scaffold function for the SufA and IscA proteins in generalized Fe/S cluster assembly and instead suggest that they play regulatory roles in iron homeostasis and the sensing of redox stress in cyanobacteria.


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INTRODUCTION
 
In cyanobacteria and higher plants, Fe/S proteins function in a number of essential biological processes including electron transfer, catalysis, protein stabilization, and gene regulation (4). This diversity of functions is possible because of the wide variety of configurations, redox potentials, and chemical reactivities exhibited by protein-bound Fe/S clusters. There are three known systems for Fe/S cluster assembly (see references 11 and 12 for reviews). The nif (nitrogen fixation) operon in Azotobacter vinelandii is known to function in the specialized assembly of Fe/S clusters for the nitrogenase enzyme in nitrogen fixing organisms (11). The related isc (iron sulfur cluster) operon in A. vinelandii encodes proteins that function in the assembly of Fe/S clusters under non-nitrogen-fixing growth conditions (12). In 2002, the suf (sulfur utilization factor) operon was reported to encode a third Fe/S cluster assembly system (42). With the availability of genomic sequences of several cyanobacteria and a higher plant (Arabidopsis thaliana), genes with a possible function in Fe/S cluster assembly have been identified in cyanobacteria (Table 1). In cyanobacteria, the isc genes are scattered throughout the genome, and some are present in multiple copies (e.g., iscS1 and iscS2). In contrast, some of the suf genes (sufB-sufC-sufD-sufS) are organized in an operon, whereas others (sufA, sufE, and sufR) are located elsewhere in the genome (16). In nonphotosynthetic bacteria, the ISC system is proposed to play a housekeeping role in Fe/S cluster assembly (30, 50) whereas the SUF system is proposed to assume a supporting role in response to oxidative stress (25) and iron starvation (30). In cyanobacteria and in chloroplasts of higher plants, it has been reported that the SUF system is involved in the biogenesis of the Fe/S clusters for photosystem I (PS I) (37, 44).


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  		TABLE 1. Assignment of Fe/S cluster biogenesis genes in Synechococcus sp. strain PCC 7002 and their homologs in Synechocystis sp. strain PCC 6803

Thus far, the most detailed and extensive information about the process of Fe/S cluster assembly has been obtained from the NIF system. NifS is a pyridoxal 5' phosphate-dependent cysteine desulfurase that provides sulfur in the form of a protein-bound persulfide (51, 52). NifU, a scaffold protein that acquires iron and sulfide, assembles an Fe/S cluster for delivery to the target apoproteins (13). Based on the sequence homology between IscU and NifU, a scaffold function has been ascribed to the former (1, 24). In cyanobacteria, there are no genes homologous to iscU. However, there is a gene, denoted nfu, which codes for a protein similar to the C-terminal domain of NifU (Table 1). Both SufA and IscA are homodimers, and the Fe/S clusters in these proteins are labile to oxygen and to chemical reduction (28, 46). IscA from Escherichia coli harbors a [2Fe-2S] cluster and has been shown to form a complex with ferredoxin (29). The labile [2Fe-2S] cluster in ISA, the IscA homolog from Schizosaccharomyces pombe, can be transferred in vitro to apoferredoxin (47). The recombinant protein of the slr1417 gene from Synechocystis sp. strain PCC 6803 is reported to form a homodimer and to harbor a [2Fe-2S] cluster (46). Slr1417, which is now annotated as SufA (Table 1), has been shown to transfer its labile Fe/S cluster to apoferredoxin as well as to biotin synthase, a [4Fe-4S] protein (46). The ability of IscA and SufA to transfer Fe/S clusters in vitro has led to their assignment as "alternative scaffold proteins."

In this report, we present results on the mutagenesis of the sufA, iscA, and nfu genes of the cyanobacterium Synechococcus sp. strain PCC 7002. The mRNA abundance of selected genes in the suf and isc regulons were compared by reverse transcription (RT)-PCR performed on total RNA isolated from cells of the wild type and mutants grown under standard, iron-limiting, and photosynthesis-specific redox stress conditions.


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MATERIALS AND METHODS
 
Mutagenesis of the sufA, iscA, and nfu genes in Synechococcus sp. strain PCC 7002. To generate a sufA null mutant, a 0.9-kb DNA fragment containing the sufA gene from Synechococcus sp. strain PCC 7002 was amplified by PCR and cloned into pUC19 (Fig. 1A). A 1.3-kb kanamycin resistance cartridge (aphII gene), or a 2-kb omega cartridge (aadA gene), conferring resistance to spectinomycin and/or streptomycin, was inserted at a unique SphI site (236 bases from the start codon) within sufA. These two constructs were used to transform wild-type cells of Synechococcus sp. strain PCC 7002. Antibiotic-resistant transformants were picked and streaked repeatedly on selective media to allow segregation of alleles.


Figure 1
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  		FIG. 1. Constructions for attempted inactivation of the sufA, iscA, and nfu genes and PCR analysis of transformants. (A) Physical map showing insertional inactivation of the sufA gene by a kanamycin resistance cartridge (aphII) and verification of transformants by PCR analysis. Arrows indicate gene orientations and primers used to check for segregation of alleles. Lane 1, wild type; lane 2, sufA mutant; lane 3, iscA mutant; and lane 4, sufA iscA mutant. (B) Physical map showing insertional inactivation of the iscA gene with a gentamicin resistance cartridge (aacC1) and verification of transformants by PCR analysis. Lanes are the same as for panel A. (C) Physical map showing the construction for the deletion of nfu and PCR analysis of transformants. PCR primers P3 and P4 were used to amplify the nfu loci in DNA isolated from the wild type (lane 1), a nfu transformant in the wild-type background (lane 2), and an nfu transformant in a PS I-less ({Delta}psaAB) background (lane 3).

To generate an iscA null mutant, a 1.1-kb DNA fragment encoding the iscA gene from Synechococcus sp. strain PCC 7002 was amplified by PCR and cloned into pUC19 (Fig. 1B). A 1.1-kb DNA fragment, encoding the aacC1 gene and conferring resistance to gentamicin, was inserted at the unique XbaI site (130 bases from the start codon). This construct was used to transform Synechococcus sp. strain PCC 7002 wild-type cells and the fully segregated sufA::aadA mutant. Gentamicin-resistant transformants of both strains were picked, streaked repeatedly on selective media, and screened by PCR amplification of the iscA locus.

To generate an nfu null mutant, a 1.2-kb EcoRI-HindIII fragment containing the nfu gene was amplified by PCR from Synechococcus sp. strain PCC 7002 and cloned into pUC19 (Fig. 1C). PCR was used to delete the nfu gene, which was replaced with a 1.3-kb kanamycin resistance cartridge (aphII); the deletion extended from the start codon of the nfu gene to a position 22 bp downstream of the nfu stop codon. This construct was used to transform the wild-type strain and a PS I-less strain of Synechococcus sp. strain PCC 7002 (36). Kanamycin-resistant transformants were picked and streaked repeatedly to allow segregation of alleles.

Cyanobacterial cell growth under normal, iron limitation, and redox stress conditions. The wild-type and mutant strains of Synechococcus sp. strain PCC 7002 were grown in A+ medium that was supplemented with 1 mg NaNO3 ml–1 (40). For growth of the mutant strains, the antibiotics kanamycin (100 µg ml–1), spectinomycin (100 µg ml–1), and/or gentamicin (50 µg ml–1) were added to the growth media as required. Liquid cultures were grown in 30-ml tubes at 38°C and were bubbled with air that was supplemented with 1% (vol/vol) CO2. Solid agar plates were prepared by addition of 3 g of Na2S2O3 and 15 g of Bacto agar per liter of A+ medium. Fluorescent light at an intensity of ~250 µmol photons m–2 s–1 was continuously provided for cell growth. Doubling times for the wild-type and mutant strains were calculated by monitoring the optical density of the cells at 730 nm during the exponential growth phase. To induce iron limitation, the wild type and the three mutant strains were serially subcultured three times in iron-depleted A+ medium (inoculation optical density at 730 nm [OD730 nm], 0.04). To induce photosynthesis-specific redox stress, the wild-type and mutant strains were grown to an OD730 nm of 1.0 in A+ medium to which either 2 µM methyl viologen or 2 µM DBMIB (2,5-dibromo-3-methyl-6-isopropylbenzoquinone) had been added. After 30 min of growth, the cells were harvested, frozen in liquid nitrogen, and stored at –80°C until used for RNA isolation.

Quantitation of pigments. Total pigments were extracted from cells of the wild-type and mutant strains with 100% methanol. The chlorophyll concentration was calculated by the methods of Mackinney (23) and Lichtenthaler (22). The carotenoid content was measured by the method of Cunningham et al. (8). Phycobiliprotein contents were calculated by the procedure described by Zhao et al. (49) by comparing the absorbance difference between untreated cells and cells that were incubated at 75°C for 5 min.

RNA isolation. Total RNA was isolated from the wild-type and mutant strains grown to mid-exponential phase using a High Pure RNA isolation kit (Roche Diagnostics, Indianapolis, IN) or a Mini to Midi RNA preparation kit (Invitrogen, Carlsbad, CA). To eliminate trace amounts of contaminating DNA, RNA samples were incubated with RNase-free DNaseI for 1 h at room temperature. DNase-treated RNA samples were repurified, using the RNA isolation/purification cartridges supplied by the manufacturer, before further use. The absence of DNA in the total RNA preparations was verified by PCR. The concentration of RNA was calculated from the absorbance of the sample at 260 nm. The concentration of RNA was also calculated from the fluorescence emission at 560 nm according to a method described by Schmidt and Ernst (33) using an SLM-Aminco spectrophotometer (model MC640).

RT-PCR analysis. RT-PCR analyses of total RNA isolated from wild-type and mutant strains were performed using a QIAGEN OneStep RT-PCR kit (Valencia, CA). The RT-PCRs were programmed as follows: 60 min reverse transcription reaction at 50°C; 15 min initial denaturation step at 95°C; and 30 cycles of a three-step program with 95°C denaturation for 30 sec, 55°C to 58°C primer annealing for 30 sec, and an extension time at 72°C that was set according to the size of the analyzed genes (at 45 sec kb–1). Based on the genomic sequence of Synechococcus sp. strain PCC 7002 (T. Li, G. Shen, J. Zhao, and D. A. Bryant, unpublished data), primers were designed to amplify specifically selected genes from the suf and isc regulons as well as genes involved in iron homeostasis (see Table S1 in the supplemental material). For each reaction, total RNA (5 ng) was used as the template. The results from RT-PCR analyses were analyzed by comparing the intensity of the product bands obtained following electrophoresis of the products on an agarose gel. The rimM gene, which codes for a 21-kDa protein essential for 16S RNA processing (16) and whose transcription is not expected to be affected by the loss of sufA or iscA, served as the loading control for the RT-PCRs. The transcript abundance data presented are typical of the results obtained from at least two completely independent experiments. It should be noted that the RNA samples used in Fig. 4 and 5 are derived from separate experiments.


Figure 4
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  		FIG. 4. Comparison of expression profiles of the genes in the suf and isc regulons in the wild-type and mutant strains when cells were grown under (lanes 1) normal photoautotrophic growth conditions and (lanes 2) iron-limiting photoautotrophic growth conditions. The rimM gene serves as the loading control.


Figure 5
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  		FIG. 5. Comparison of expression profiles of the genes in the suf and isc regulons in the wild-type and mutant strains when cells were grown under (lanes 1) normal photoautotrophic growth conditions and (lanes 2) redox stress conditions induced by addition of methyl viologen. The rimM gene serves as the loading control.


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RESULTS
 
Identification of sufA-like and iscA-like genes in cyanobacteria. All sequenced cyanobacterial genomes contain genes homologous to sufA and iscA. However, the scattered locations of the iscA and sufA genes, and the similar amino acid sequences of their gene products, make it difficult to identify the specific "A" gene associated with the isc and suf regulons in cyanobacteria. An important clue comes from the nuclear-encoded sufA gene of Arabidopsis thaliana. Figure 2 shows the amino acid sequence of processed SufA from A. thaliana, which contains five cysteine residues. The product of slr1417 in Synechocystis sp. strain PCC 6803 similarly contains five cysteine residues (labeled C1 through C5) (Fig. 2) located in comparable positions, whereas the slr1565 product contains only three cysteine residues that correspond to C2, C4, and C5. These three conserved cysteine residues are found in IscA orthologs from both photosynthetic and nonphotosynthetic organisms (Fig. 2). Because the SUF system is involved in the assembly of the Fe/S clusters in PS I (44, 48), and because the slr1417 gene product in cyanobacteria contains a longer N terminus than the slr1565 gene product, we designate slr1417 as sufA and slr1565 as iscA in Synechocystis sp. strain PCC 6803. It should be noted that the slr1417 gene was otherwise annotated as iscA in reference 46.


Figure 2
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  		FIG. 2. Sequence alignment of SufA and IscA proteins from cyanobacterium Synechococcus sp. strain PCC 7002, Synechocystis sp. strain PCC 6803, Arabidopsis thaliana, Escherichia coli, Erwinia chrysanthemi (SufA), and Azotobacter vinelandii (IscA). The conserved cysteine residues are highlighted and numbered C1 through C5 (2).

IscA and SufA are not essential in Synechococcus sp. strain PCC 7002. Synechococcus sp. strain PCC 7002 wild-type cells were transformed with the constructs shown in Fig. 1A and B. After selection and repeated streaking of transformants on selective media, segregation of alleles was verified by PCR analysis. A comparison of the results in Fig. 1A, lanes 1 and 2, shows that the sufA and sufA::aphII alleles have fully segregated in the transformant. Similarly, a comparison of Fig. 1B, lanes 1 and 3, shows that the iscA and iscA::aacC1 alleles have fully segregated in the transformant. Finally, the results shown in Fig. 1A and B, lane 4, establish that it is possible to inactivate both sufA and iscA simultaneously. These results unambiguously establish that SufA and IscA are not required for viability in Synechococcus sp. strain PCC 7002 under standard growth conditions.

Nfu is essential in Synechococcus sp. strain PCC 7002. The nfu gene from Synechococcus sp. strain PCC 7002 (the ortholog of ssl2667 in Synechocystis sp. strain PCC 6803) codes for a protein that is similar to the C-terminal domain of NifU of Azotobacter vinelandii. Nfu (denoted "SynNifU" in reference 27 and "NifU-like protein" in reference 34) contains a conserved CxxC motif and can ligate a [2Fe-2S] cluster (27). This Fe/S cluster can be efficiently transferred to apoferredoxin (27); hence, this protein is a candidate for a scaffold that assembles Fe/S clusters in cyanobacteria. To determine whether Nfu functions specifically in Fe/S cluster assembly for PS I, we attempted to replace the nfu gene with a kanamycin resistance cartridge in both a wild-type background and a psaAB deletion background. As shown in Fig. 1C, only stable merodiploids, in which both wild-type and mutant alleles were stably maintained, resulted from transformation with this nfu deletion construct into the wild type (Fig. 1C, lane 2) or the PS I-less strain (Fig. 1C, lane 3). These results contrast sharply with those for sufA and iscA, but they are similar to results reported for Synechocystis sp. strain PCC 6803 (34) and indicate that Nfu plays an essential role in cyanobacteria.

Physiological characterization of the wild-type and mutant strains. The photosynthetic competence of the iscA and sufA mutant strains was assessed by measuring the doubling times and pigment contents of the cells under photoautotrophic growth conditions (Table 2). Under normal growth conditions, the sufA, iscA, and sufA iscA mutant strains grew photoautotrophically with doubling times similar to the wild type. There was no significant alteration in the chlorophyll, carotenoid, or phycobilisome content in any of the mutant strains relative to the wild type (Table 2). These results indicate that sufA and iscA are not essential genes and hence cannot perform an essential role as a scaffold for Fe/S cluster assembly in cyanobacteria.


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  		TABLE 2. Comparison of chlorophyll, carotenoid, phycobiliprotein content, and the doubling time of the wild type and mutants of Synechococcus sp. strain PCC 7002d

When cells were grown under iron-limiting photoautotrophic growth conditions, the wild-type strain underwent chlorosis, but the iscA, sufA, and sufA iscA mutant strains remained blue-green, and the iscA mutant strain appeared as pigmented, or more so, than cells grown with normal levels of iron (see Fig. S1 in the supplemental material). This pattern was mirrored in the growth doubling times and pigment content of the cells grown under iron-limiting conditions (Table 2). The wild type had the longest doubling time, followed by the sufA iscA, sufA, and iscA mutant strains. The chlorophyll, carotenoid, and phycobilisome contents were also lowest in the wild type, followed in increasing order by the sufA iscA, sufA, and iscA mutants (Table 2). Thus, the mutant strains, especially the iscA mutant, are less severely affected by iron limitation than the wild-type strain (Table 2). A rapid and convenient method to approximate the ratio of PS I to PS II is to measure the low-temperature fluorescence emission from whole cells. Changes in the relative amounts of PS I to PS II can be inferred by changes in the 695 nm (PS II) and 715 nm (PS I) emissions when equal numbers of whole cells at 77 K are compared (see Fig. S2 in the supplemental material). When cells were grown under standard growth conditions, no obvious difference could be observed in the fluorescence emissions from cells of the wild type and the three mutant strains. However, as indicated by the higher chlorophyll fluorescence emission at 715 nm, it is clear that the iscA and sufA null-mutant strains (especially the iscA mutant) accumulate a relatively higher content of PS I per cell than the wild type under iron-limiting growth conditions. The phenotypes of the iscA and sufA mutant strains grown under iron-limiting conditions are reminiscent of the phenotype found earlier for the sufR mutant under similar growth conditions. In addition to enhanced growth under iron-limiting conditions, the mRNA levels of the sufB and sufS genes are higher in the sufR mutant in Synechococcus sp. strain PCC 7002 (44). This prompted us to measure the transcript levels of selected genes involved in Fe/S cluster assembly and iron homeostasis in the wild type and the three mutant strains by RT-PCR.

Expression of sufA and iscA in the mutant strains. As shown in Fig. 3, sufA transcripts were not detected in the sufA::aphII strain, iscA transcripts were not detected in the iscA::aacC1 strain, and neither sufA nor iscA transcripts were detected in the double mutant. Compared to the wild type, the expression level of iscA was not altered in the sufA mutant and the expression level of sufA was not altered in the iscA mutant. Thus, transcription of iscA does not increase to compensate for the absence of SufA, and transcription of sufA does not increase to compensate for the absence of IscA. The nfu transcript abundance was also similar in the wild type and mutants, indicating that it does not increase in the absence of both IscA and SufA. Moreover, the mRNA levels for ftn, a gene that codes for the iron storage protein ferritin, were similar in the wild type and the three mutant strains.


Figure 3
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  		FIG. 3. Expression profile of the genes in the suf and isc regulons. RT-PCR was performed on RNA isolated from the wild type (WT) (lane 1), the sufA mutant (lane 2), the iscA mutant (lane 3), and the sufA iscA mutant (lane 4), cells grown under normal photoautotrophic growth condition. The rimM gene serves as the loading control.

Gene expression under standard growth conditions in the wild-type and mutant strains. When the sufA mutant was grown under standard growth conditions, the mRNA levels of the genes in the suf and isc regulons were similar to those of the wild type (Fig. 4, lanes 1). In contrast, in the iscA mutant, the mRNA levels for the above-mentioned genes in both the suf and isc regulons are much higher than in the wild type. Strikingly, the mRNA level for the isiA gene, a marker for iron limitation in cyanobacteria, was significantly elevated in the iscA strain, even in cells that were grown under standard iron-sufficient conditions (Fig. 4, lane 1). This unexpected phenotype indicates that the iscA mutant mistakenly senses iron deprivation when IscA is absent and that it accordingly responds by synthesizing the chlorophyll a-binding IsiA protein that forms rings around PS I under iron-limiting conditions (7, 10, 19, 20, 26, 31). These findings implicate IscA in the cascade of events that sense and/or respond to iron limitation in cyanobacteria. Under the nonstressed, standard growth conditions employed, the pattern of mRNA abundance in the sufA iscA double-mutant strain displays a profile more similar to that in the sufA mutant than in the iscA mutant. Notably, the up-regulation of the expression of genes in the isc and suf regulons seen in the iscA mutant is partially reduced by additionally inactivating the sufA gene. Thus, the cascade of events that lead to the (inappropriate) iron limitation response in the iscA mutant is ameliorated by additionally inactivating the sufA gene.

Gene expression under iron-limiting growth conditions in the wild-type and mutant strains. Expression of genes in the suf and isc regulons was examined using RT-PCR in cells grown under iron-limiting conditions. As expected (Fig. 4, lane 2), the mRNA level for the iron limitation marker gene isiA increased in the wild type, but the mRNA abundance of the essential gene nfu was nearly unchanged. In the sufA mutant grown under iron-limiting conditions, the mRNA levels of all genes in the suf regulon were much higher than under normal growth conditions. In the iscA mutant, the mRNA levels of genes in both the suf and isc regulons were higher than in the wild type grown under similar iron-limiting conditions, although the effect is more pronounced in the former than the latter. For the iscA mutant there was a significant decrease of the mRNA abundance of the isc genes in iron-limited cells compared to cells grown in normal growth media. Thus, iron limitation partially blocks the increase in mRNA levels of the isc genes that occurs in the iscA mutant under normal growth conditions. The transcript abundance of the isiA gene also increased significantly in the iscA strain. In the sufA iscA double mutant, the pattern of mRNA levels for the suf genes under iron limitation conditions was intermediate between those for the sufA or iscA single mutants and the wild type. Thus, under conditions of iron limitation, the transcript abundance for genes in the suf and isc regulons was increased regardless of whether sufA or iscA was interrupted.

The expression of high-affinity iron-binding siderophores is induced in some cyanobacteria that are grown under iron-limiting conditions (41, 45). The genes for siderophore biosynthesis in Synechococcus sp. strain PCC 7002 have recently been identified and are found in an operon containing eight genes (sidA to sidH) on the plasmid pAQ7 (T. Li, J. Zhao, and D. A. Bryant, unpublished results). RT-PCR was performed to check the mRNA abundance of representative genes from within this operon (here denoted as sidA and sidC) (Fig. 4). The data show that the pattern of siderophore gene expression is consistent with the observation that the mutant strains, especially the iscA mutant, exhibit an increased tolerance to iron limitation conditions. Such cells should have a higher apparent affinity for iron in the medium and would thus be able to grow better under iron-limiting concentrations (Table 2). The results suggest that IscA plays an important role in sensing and responding to the iron levels in the cell.

Gene expression under conditions of redox stress in wild-type and mutant strains. We previously proposed that the Fe/S cluster assembly in PS I was mediated by proteins in the SUF system (37, 44). DBMIB is an inhibitor of the cytochrome b6f complex, and methyl viologen is an inhibitor of photosynthetic electron transfer that blocks electron transfer on the acceptor side of PS I (14, 15). We studied the mRNA levels of selected genes in the suf and isc regulons in the wild type and the three null-mutant strains that had been treated with methyl viologen or DBMIB to induce photosynthesis-related redox stress. Similar to iron-limited cells, isiA transcript levels were higher in wild-type cells grown with methyl viologen for 30 min. Note that the expression of the nfu gene was again unaltered under conditions of redox stress compared to normal growth conditions.

In the sufA mutant cells that were grown in the presence of methyl viologen for 30 min, the mRNA levels for all of the genes in the suf regulon were higher (Fig. 5 lanes 2) than under standard growth conditions. In the iscA mutant grown in the presence of methyl viologen, the transcript levels for the genes in the isc regulon were comparable to those in cells grown under normal growth conditions. Interestingly, transcript levels for all the genes in the sufBCDS operon were lower than for cells grown under normal growth conditions. This is in sharp contrast to the results obtained from the iscA mutant strain when grown under iron-limiting conditions, where mRNA levels were lower for all of the isc genes. In the sufA iscA double mutant grown in the presence of methyl viologen, the mRNA levels of the suf genes increased strongly, more so than for the wild type and similar to those of the single-mutant strains. Thus, under conditions of redox stress, the loss of SufA alone is sufficient to cause an increase in mRNA levels of the suf genes.

The mRNA levels of the suf and isc genes in the wild-type and mutant cells grown in the presence of DBMIB, an inhibitor of the cytochrome b6f-Rieske iron-sulfur protein complex, were similar to those for the same genes in cells grown in the presence of methyl viologen (data not shown).


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DISCUSSION
 
We undertook the task of attempting to create null mutants of iscA, sufA, and nfu in Synechococcus sp. strain PCC 7002, an organism that possesses good visual markers for iron limitation. The high demand for Fe/S clusters associated with PS I in cyanobacteria makes it possible to correlate growth, pigment content, and photosynthetic competence with the expression levels of genes involved in the Fe/S cluster assembly.

The role of Nfu in Fe/S cluster assembly in cyanobacteria. It is not possible to inactivate the nfu gene in Synechococcus sp. strain PCC 7002 (this work) or in Synechocystis sp. strain PCC 6803 (34). Additionally, RT-PCR analysis on RNA isolated from wild-type cells shows that nfu expression is not strongly modulated under conditions of iron limitation and methyl viologen-induced redox stress. These results, taken together with the sequence similarity of Nfu to the C terminus of NifU, point to an important and indispensable role for Nfu in the Fe/S cluster biogenesis in cyanobacteria. Similar to the iscA and sufA single-mutant strains, the sufA iscA double mutant can be completely segregated, and the resulting strain grows photoautotrophically at rates similar to the wild type. This finding eliminates the possibility that one of these proteins substitutes for the other as a scaffold protein for housekeeping (general) Fe/S cluster assembly. Thus, Nfu, and not SufA or IscA, is likely the essential scaffold protein that is involved in Fe/S cluster assembly in cyanobacteria.

The role of IscA in Fe/S cluster assembly in cyanobacteria. Under conditions of iron limitation, the wild-type strain of Synechococcus sp. strain PCC 7002 grows slowly and undergoes chlorosis; in contrast, the three mutant strains grow better photoautotrophically and do not undergo chlorosis to the same extent as the wild type. We show here that higher mRNA levels for genes in both the suf and isc regulons accompany the enhanced tolerance to iron limitation. This result is similar to that found in E. coli, in which the suf and isc regulons responded in a similar manner to iron limitation (the addition of {alpha}-{alpha}-dipyridyl to the medium) and oxidative stress (the addition of H2O2 to the medium) (30). In light of the strong response of the single-mutant strains, the attenuated response of the suf regulon in the double-mutant strain is striking. The highly modulated expression of genes in the suf regulon in the three mutant strains in response to iron limitation implies that additional layers of regulatory control probably exist in cyanobacteria.

The expression of the isiA gene is normally suppressed under normal, iron-sufficient growth conditions in cyanobacteria. However, the expression of the isiA gene is induced under iron limitation and peroxide stress conditions (21, 39). IsiA is a chlorophyll-binding protein which forms a ring, under iron limitation conditions, around the PS I trimer in cyanobacteria (5, 6, 26). As expected, under conditions of iron limitation, the mRNA levels for the isiA gene are high in the wild type and the three mutant strains. Expression of the isiA gene is highly induced in the iscA mutant even under standard growth conditions. The mRNA levels of the genes involved in siderophore biosynthesis are also higher in the iscA mutant than in the wild type, under both normal and iron limitation conditions. The increased mRNA levels for the suf genes and the additional light-harvesting capability due to the presence of IsiA are additionally likely to contribute to the higher growth rate of the sufA and iscA mutants in the iron-limiting growth conditions. These results suggest that IscA is involved in the regulation of iron metabolism or iron homeostasis in cyanobacteria. This is also supported by the high affinity (3 x 1019 M–1) of IscA for iron in E. coli (9).

The role of SufA in the Fe/S cluster assembly in cyanobacteria. In the studies reported here, the mRNA levels of selected suf and isc genes increase in the sufA mutant under redox stress conditions induced by methyl viologen or DBMIB. This indicates that in cyanobacteria, SufA plays a role in the cascade of events that leads to the sensing and enhanced expression of the suf genes under photosynthesis-related, redox stress conditions. This pattern is consistent with the proposal that the SUF system is induced to repair Fe/S clusters in proteins that are damaged as a result of redox or oxidative stress. Although it is unclear whether SufA participates in redox regulation in nonphotosynthetic bacteria, the cyanobacterial SufA homologs possess a unique feature that may additionally allow this protein to sense redox stress. Cyanobacterial SufA homologs contain two additional, highly conserved cysteine residues (C1 and C3 in Fig. 2) that are absent in the SufA homologs of nonphotosynthetic bacteria. One possible function for these cysteines is to serve as a redox-sensitive regulatory switch by reversible disulfide bond formation, as in the bacterial transcription factor OxyR (18, 32).

Model for the regulatory roles of IscA and SufA in cyanobacteria. A working model for the regulatory roles of IscA and SufA in sensing iron homeostasis and redox poise in photosynthetic organisms is illustrated in Fig. 6. In cyanobacteria and higher plants, the SUF system plays an essential role in the biogenesis of Fe/S clusters (37). We propose that Nfu performs the role of the Fe/S scaffold protein, which delivers Fe/S clusters to various target proteins including SufA and IscA. SufA and IscA in cyanobacteria are proposed to play regulatory roles in the biogenesis of Fe/S proteins and iron metabolism. IscA may play a role in sensing the iron status in cells, and SufA may play a role in sensing redox/oxidative stress. It is possible that the labile Fe/S centers associated with SufA and IscA sense the capacity of cells to produce Fe/S clusters and thereby transmit the information as an intact Fe/S cluster to a regulatory protein, e.g., SufR, that either directly or indirectly controls the expression of genes in the suf and isc regulons. Further, the strong increase in mRNA levels for the suf genes in the iscA mutant under photoautotrophic conditions shows that the suf and isc regulons are not independently regulated. It is possible that this proposed mechanism of action of SufA and IscA is unique to cyanobacteria because of the need to control the high throughput of Fe/S clusters for PS I. However, based on the high degree of sequence similarity and on the similarities in molecular properties between IscA and SufA from different organisms, it is possible that the functions we propose here may also occur in diverse, nonphotosynthetic bacteria.


Figure 6
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  		FIG. 6. A model for the regulation and assembly of Fe/S clusters in cyanobacteria. Solid lines indicate known processes, while dashed lines indicate proposed processes for the model. The SUF system plays an important role in the Fe/S cluster assembly. SufS and SufE mediate the release of sulfur from cysteine. SufB, SufC, and SufD may play a role in the iron mobilization and Fe/S cluster formation. The Nfu protein may function as a scaffold protein and plays an essential role in the transient assembly and delivery of Fe/S clusters to target proteins. We propose that Fe/S cluster homeostasis in the cell is maintained by a mechanism that involves the assembly of Fe/S clusters on SufA and IscA for the purpose of sensing and transmitting information about the cluster status of the cell to regulatory proteins like SufR. apo, protein with no Fe/S clusters; SH, reduced form of cysteine.


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ACKNOWLEDGMENTS
 
This work is supported in part by USDA contract no. 2005-35318-15284 (to J.H.G. and D.A.B.) and by NSF grants MCB-0117079 to J.H.G. and MCB-0077586 to D.A.B.


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FOOTNOTES
 
* Corresponding author. Mailing address: Department of Biochemistry and Molecular Biology, The Pennsylvania State University, University Park, PA 16802. Phone for Donald A. Bryant: (814) 865-1992. Fax: (814) 863-7024. E-mail: dab14{at}psu.edu. Phone for John H. Golbeck: (814) 865-1163. Fax: (814) 863-7024. E-mail: jhg5{at}psu.edu. Back

{dagger} Supplemental material for this article may be found at http://jb.asm.org/. Back


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Journal of Bacteriology, May 2006, p. 3182-3191, Vol. 188, No. 9
0021-9193/06/$08.00+0     doi:10.1128/JB.188.9.3182-3191.2006
Copyright © 2006, American Society for Microbiology. All Rights Reserved.



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