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First published online June 24, 2005; 10.1104/pp.105.061549 Plant Physiology 138:1586-1595 (2005) © 2005 American Society of Plant Biologists FesM, a Membrane Iron-Sulfur Protein, Is Required for Cyclic Electron Flow around Photosystem I and Photoheterotrophic Growth of the Cyanobacterium Synechococcus sp. PCC 70021,[w]State Key Laboratory of Protein and Genetic Engineering, College of Life Science, Peking University, Beijing 100871, China
While it is known that cyclic electron flow around photosystem I is an important pathway of photosynthetic electron transfer for converting light energy to chemical energy, some components of cyclic electron flow remain to be revealed. Here, we show that fesM, encoding a novel membrane iron-sulfur protein, is essential to cyclic electron flow in the cyanobacterium Synechococcus sp. PCC 7002. The FesM protein is predicted to have a cAMP-binding domain, an NtrC-like domain, a redox sensor motif, and an iron-sulfur (4Fe-4S) motif. Deletion of fesM (fesM-D) led to an inability for Synechococcus cells to grow in the presences of glycerol and 3-(3,4-dichlorophenyl)-1,1-dimethylurea. Photoheterotrophic growth was restored by a complete fesM gene present on a replicable plasmid. A mutant fesM gene encoding a truncated FesM protein lacking the cAMP domain failed to restore the phenotype, suggesting this domain is important to the function of FesM. Measurements of reduction of P700+ and the redox state of interphotosystem electron carriers showed that cells had a slower rate of respiratory electron donation to the interphotosystem electron transport chain, and cyclic electron flow around photosystem I in fesM-D was impaired, suggesting that FesM is a critical protein for respiratory and cyclic electron flow. Phosphatase fusion analysis showed that FesM contains nine membrane-spanning helices, and all functional domains of FesM are located on the cytoplasmic face of the thylakoid membranes.
In plants and cyanobacteria, there are two types of photosynthetic electron transfer. Linear electron transfer results in the oxidation of water and reduction of NADP+ while generating a proton gradient across thylakoid membranes for ATP synthesis. Cyclic electron flow around PSI also generates a proton gradient across the thylakoid membranes. In addition, thylakoid membranes have a respiratory electron transport system that plays important roles in both plants and cyanobacteria (Schmitterer, 1994  ; Peltier and Cournac, 2002; Munekage et al., 2004). While the linear and cyclic photosynthetic electron transport systems share some common electron carriers, cyclic electron flow around PSI has its uniqueness in transporting electrons from the acceptor of PSI to P700 through the plastoquinone (PQ)/cytochrome b6f (Cytbf) complex (Allen, 2002; Joliot and Joliot, 2002). Some of the electron carriers involved in cyclic electron flow around PSI are still unknown, and details of this electron transfer pathway remain to be elucidated.
There are several pathways for electron donation to PQ/Cytbf on thylakoid membranes. In the cyanobacterium Synechococcus sp. PCC 7002, electrons from PSII account for more than 90% of the total electron donation to the PQ/Cytbf complex while NAD(P)H dehydrogenase (NDH) and PsaE-dependent cyclic electron transfer account for less than 10% of electron donation to Cytbf (Myers, 1987
While many thylakoid membrane proteins are organized into complexes, a significant number of proteins on thylakoid membranes do not form complexes or may only loosely interact with other proteins. These proteins may play important roles in photosynthetic electron transport and membrane organization. For example, the rubA gene product in Synechococcus sp. PCC 7002 is important for PSI assembly, but it is not tightly associated with the PSI complex (Shen et al., 2002
fesM Encodes a Membrane Protein Required for Photoheterotrophic Growth The complete genome sequence of Synechococcus sp. PCC 7002 has recently been determined (J. Zhao, unpublished data). In searching for proteins involved in cyclic electron flow, we constructed a mutant pool of Synechococcus sp. PCC 7002 through transposon mutagenesis and isolated a mutant that could not grow in the presences of DCMU and glycerol. The transposon was shown to be inserted into a previously unidentified open reading frame. To confirm that this phenotype was indeed due to the insertion, we reconstructed the mutation by deleting the entire coding region of the gene (fesM) and characterized the mutant strain. Search of databases showed that cyanobacteria with genomes larger than 3 Mb, such as Synechocystis sp. PCC 6803 and Anabaena sp. PCC 7120, have fesM, while cyanobacteria with genomes smaller than 3 Mb, such as Synechococcus WH8102 and Prochlorococcus marinus SS120, do not have fesM. One exception is Prochlorococcus marinas MIT 9313, which also has a small genome. It has a small fesM gene encoding an FesM-like protein with an incomplete cAMP domain at the N terminus. No homolog of FesM is found in the Arabidopsis (Arabidopsis thaliana) and rice (Oryza sativa) genomes. A comparison of FesM sequences in various cyanobacteria shows that all domains of the protein are conserved, and the most conserved regions are the redox sensor and Fe-S motifs (Supplemental Fig. 1). The FesM protein from Synechococcus sp. PCC 7002 is predicted to be more closely related to FesM from heterocystous cyanobacteria than to FesM from unicellular cyanobacteria, based on analyses using the program ClustalW. Figure 1 shows a physical map of the fesM gene, the result of Southern hybridization and predicted domains of the gene product. The fesM gene encodes a protein with a cAMP-binding domain, an NtrC-like domain, and several membrane-spanning helices with motifs that could coordinate Fe-S centers and a redox sensor (Fig. 1B). Deletion of fesM by replacement with a kanamycin-resistant cassette in fesM-D was confirmed by Southern hybridization (Fig. 1C), which shows that the 3.2-kb band of the wild type was missing and replaced by a 2.0-kb band in the mutant strain (fesM-D) as predicted (Fig. 1A).
To test if fesM-D could grow photoheterotrophically, growth of both the wild type and fesM-D was measured. Figure 2A shows that fesM-D could grow normally under photoautotrophic conditions. When DCMU and glycerol were present in the growth medium for photoheterotrophic growth, fesM-D had virtually no growth with a doubling time >100 h (Fig. 2B), showing that the fesM gene was required for the normal photoheterotrophic growth. We constructed a complementary plasmid, pAQE-FesMs, which contained the entire fesM coding region plus 1000 bp upstream of the coding region. Transformation of fesM-D with pAQE-FesMs generated the complementary strain 323C, which could grow in the presence of DCMU and glycerol the same as the wild type (Fig. 2B). Strain 223C, which was constructed as strain 323C except that the predicted cAMP-binding domain at the N terminus of the fesM gene product was deleted, failed to grow photoheterotrophically, suggesting that the putative cAMP domain was essential to the function of FesM.
Table I compares fesM-D with wild-type PCC 7002 grown photoautotrophically in liquid medium. The PSI to PSII ratio of fesM-D cultured under various growth conditions was slightly lower than that of the wild type grown under the same conditions. Both strains showed a high PSI to PSII ratio when grown in air as compared with 1% CO2. The respiratory rate of fesM-D was 25% lower than that of the wild-type strain, an indication that the respiratory electron transfer chain was affected in fesM-D cells. The values of other parameters shown in Table I are nearly identical in wild-type and fesM-D cells.
Cyclic Electron Flow around PSI Is Impaired in fesM-D Cells The inability of the fesM-D cells to grow in the absence of PSII activity indicated that cyclic electron flow around PSI might be impaired. Light-induced redox changes of P700 in both the wild type and fesM-D were measured by monitoring the absorption changes of 820 to 860 nm, and the results are shown in Figure 3A. In the presence of DCMU, which blocked electron donation from PSII to the PQ pool, the rereduction P700+ of fesM-D was slower than that of the wild type. The apparent half-times (t1/2) were 330 and 890 ms for the wild type and fesM-D, respectively. Examination of the kinetics of both curves suggested that the rereduction of P700+ in both strains involved more than one component. Curve fitting was then performed to further analyze the kinetics of the P700+ rereduction. Figure 3B shows that the P700+ rereduction of the wild type contained two exponential components with decay half-times of 248 and 445 ms. Figure 3C shows that P700+ rereduction in fesM-D also contained two exponential components with half-times of 478 and 2804 ms. It seems likely that one of the components in P700+ rereduction in the wild type was replaced by the slow component (2804 ms) in fesM-D. The rereduction of P700+ in 323C is virtually identical to that of the wild type (Fig. 3A), confirming that fesM located on a plasmid could complement the fesM mutation.
The slow rereduction of P700+ (Fig. 3) and lower respiratory rate (Table I) of fesM-D cells suggested that the reduction of PQ/Cytbf was slower in fesM-D than that in the wild type. One method for determining the reduction level of PQ/Cytbf is the measurement of state transitions because state transitions are controlled by the redox state of interphotosystem electron carriers (Mullineaux and Allen, 1990 ). To measure the reduction of PQ/Cytbf, cells were incubated in the dark in the presence of 10 µM DCMU for 2 min before actinic light (500 µmol m–2 s–1) was turned on to bring the cells to state 1. Then the actinic light was turned off for various periods of time before a pulse (2-s duration time) of actinic light was given to determine the level of chlorophyll (Chl) fluorescence at 682 nm (Finit; Huang et al., 2003). High and low Finit levels indicate that the cells are in state 1 and state 2, respectively. As shown in Figure 4A, the Finit levels declined with increasing dark times in both strains, suggesting a transition from state 1 to state 2 that reflects a gradual reduction of PQ/Cytbf. However, the Finit in fesM-D was maintained at a higher level than that in the wild type, suggesting that there was impairment in the ability of fesM-D to reduce PQ/Cytbf. Figure 4B shows P700 oxidation by phycobilisome (PBS)-absorbed actinic light (630 nm) in dark-adapted cells of both strains. Incubation of the wild type in the dark caused cells to reside almost completely in state 2. The oxidation of P700 by PBS-absorbing light was near its maximum value immediately after the actinic light was turned on, followed by a decrease in the level of P700+ due to a transition from state 2 to state 1. In contrast, dark incubation of fesM-D did not drive cells completely into state 2, and oxidation of P700 by PBS-absorbing actinic light was not as complete as that in the wild type. However, a phase of rapid increase in the level of P700+ was observed after the initial P700 oxidation when continuous illumination by PBS-absorbing light was provided to the fesM-D cells. This rapid phase was an indication that more PBS-absorbed light energy was transferred to PSI during illumination, probably because of a state 2 transition due to electron donation from PSII to PQ/Cytbf.
Figure 5 shows percentages of the wild-type and fesM-D cells that survive when cultured in the dark in the absence of external organic carbon supply. The lack of a fesM gene apparently had a relatively strong effect on viability immediately after cells were transferred to darkness. The number of colony-producing cells in the culture of fesM-D started to decline within the first day after the culture was transferred to darkness. In contrast, the viability of wild-type cells only began to decline after 2 d in darkness. However, the rate of decline in viability was nearly the same for wild-type and fesM-D cells after the decline had begun.
Localization of FesM and Determination of Its Membrane Topology
Photosynthetic electron transfer in nearly all cyanobacteria, including cyclic electron flow around PSI, occurs on thylakoid membranes. Most components of respiratory electron transfer also occur on thylakoid membranes (Ohkawa et al., 2001
As shown in Figure 1, FesM is predicted to have multiple membrane-spanning helices. However, the exact number of the membrane spans could not be determined unambiguously with available computer programs. The FesM primary sequence that gives an ambiguous prediction of membrane spanning is the Fe-S motif region. Since this region is likely to be very important to the functions of FesM, we determined the topology of membrane-bound FesM using a phosphatase fusion system in E. coli (Manoil and Beckwith, 1986 ; Bibi et al., 1993). The phoA gene encoding E. coli phosphatase was fused to various parts of the fesM gene encoding N-terminal portions of FesM and expressed in E. coli. Thirteen fusion genes were constructed. Immunoblotting results showed that all the fusion genes with one exception were expressed, and the molecular masses of the gene products were as predicted (Fig. 7). The lane that showed only a weak cross-reaction with the phosphatase antibody was the construction that would overproduce a fusion protein between the first 417 residues of FesM and PhoA. Because the first 417 amino acid residues contained no membrane-spanning region, the reason for the weak expression of this fusion gene was not examined. We measured the extracellular phosphatase activities from the E. coli strains expressing the fusion genes, both on plates and in liquid, to determine which fusion constructions could target the phosphatase to the periplasmic space, and the results are shown in Table II. The results of on-plate assays and direct measurements of phosphatase activities agreed well: those fusion genes leading to blue bacterial colonies produced extracellular phosphatase activities and those leading to white colonies produced no extracellular phosphatase activity. Based on the information in Table II, the membrane topology of FesM should have the following features (Fig. 8): the N terminus of the protein, which includes the cAMP-binding domain and NtrC-like domain, is located on the stromal side of thylakoid membranes, while the C terminus of FesM is located on the lumenal side. There are nine membrane-spanning helices. Both the redox sensor domain and Fe-S motifs are located on the stromal side of thylakoid membranes. These domains and motifs are probably in close contact with the thylakoid membranes because of their hydrophobic nature.
The results of this study show that the fesM gene, encoding an integral membrane protein with several distinct structural domains (Figs. 1 and 8), is required for photoheterotrophic growth of Synechococcus sp. PCC 7002 (Fig. 2). The inability of fesM-D to grow photoheterotrophically is a result of an impaired cyclic electron flow around PSI as demonstrated by the much slower rereduction of P700+ in the fesM-D strain (Fig. 3). Kinetic analysis of P700+ rereduction in the presence of DCMU (Fig. 3) reveals that there are two components in both the wild type and fesM-D. Previous measurements (Yu et al., 1993 ; Huang et al., 2003) suggest that in the wild type the fast reduction component may represent the NDH route, with the slow reduction component representing the PsaE-dependent route. In fesM-D, the fast and slow components in P700+ rereduction had half-times of 478 and 2804 ms, respectively. The similar P700+ reduction kinetics of the slow component of the wild type and the fast component of FesM-D (Fig. 3) suggest that the slow component in the fesM-D cells replaced the fast component of wild-type cells, although it cannot be ruled out that both components in P700+ rereduction were affected by the lack of FesM.
The electron donation to interphotosystem electron carriers in the dark is measured by room temperature fluorescence. In the presence of DCMU that closes all PSII, the level of room temperature fluorescence at 682 nm of cyanobacterial cells indicates how much light energy absorbed by PBS is distributed to PSII (state transitions) and reflects the redox state of interphotosystem electron carriers (Huang et al., 2003
Except for FesM from P. marinas MIT 9313, all structural domains of FesM are conserved. The cAMP-binding domain located at the N terminus of FesM is required for photoheterotrophic growth of Synechococcus sp. PCC 7002, suggesting that it is important to the functions of FesM. Since cAMP participates in the regulation of many cellular processes, we speculate that this domain could play an important role in the regulation of FesM activity. It would be interesting to measure cyclic electron flow in P. marinas MIT 9313, since the cAMP domain is incomplete in FesM of P. marinas MIT 9313 (Supplemental Fig. 1). The role of the NtrC-like domain in FesM is unknown. NtrC is a key protein that interacts with other proteins in regulation of nitrogen metabolism in many bacteria. This domain might be important for FesM interactions with other proteins or dimerization (Yang et al., 2004
The results presented in this study demonstrate that FesM plays an important role in respiratory and cyclic electron transport around PSI in Synechococcus sp. PCC 7002. The presence of a Fe-S motif and a redox sensor motif in FesM suggests that FesM could participate in the reduction of PQ/Cytbf in vivo. The NDH-1 complexes of Synechococcus sp. PCC 7002 have not been characterized in detail, and it is not known if the NDH-1 complexes in Synechococcus sp. PCC 7002 are equivalent to those of Synechocystis sp. PCC 6803 (Prommeenate et al., 2004
Strains, Culture Conditions, Mutagenesis, and DNA Cloning
Synechococcus sp. PCC 7002 wild-type and mutant cells were grown in A+ medium (Stevens et al., 1973
Spectroscopic Measurements
Cells in exponential growth were diluted with A+ medium to a Chl concentration of 5 µg mL–1 for room temperature fluorescence measurements. Room temperature fluorescence (682 nm) induction in the presence of DCMU was measured as follows according to Huang et al. (2003)
The phoA gene from E. coli was amplified by PCR with the primers phoA1 and phoA2 (Table III). The fragment was digested by BamHI and XhoI and ligated into pET30a (Novagen, Madison, WI) digested by the same enzymes to obtain the plasmid pET-PhoA. To generate various fusion genes linking N-terminal portions of fesM with phoA, DNA fragments encoding the N-terminal portions of FesM were amplified by PCR with the primer L1 and the primers R1 through R13 (Table III). A total of 13 fragments were generated and digested by NdeI and BamHI. The digested fragments were then ligated into pET-phoA digested with the same enzymes. The generated plasmids pFesM1-phoA through pFesM13-phoA were transformed in BL21(DE3) for analysis of extracellular phosphatase activity. As a control, the entire fesM gene was amplified by PCR with primers LF and RF (Table III) and cloned into pET30a to generate pET-FesM. All constructions were verified by DNA sequencing. For analysis of the fusion gene expression, the transformed cells were first induced by 0.5 mM IPTG for 3 h at 37°C before centrifuged. The cell pellets were resuspended and treated with 2x SDS sample buffer for SDS-PAGE/immunoblotting analysis. A monoclonal antibody against E. coli alkaline phosphatase (Sigma-Aldrich, St. Louis) was used as primary antibody. The extracellular phosphatase activity of BL21(DE3) containing pFesM-phoA was determined both on plates and in liquid. For on-plate assays, the cells on agar plates containing 0.1 mM IPTG and 40 µg mL–1 XP (5-bromo-4-chloro-3-indolyl-phospate) were incubated overnight at 30°C, and those that generated blue colonies were recognized as having extracellular phosphatase activities. For measurements of extracellular phosphatase activity in liquid, the cells were first induced by 0.5 mM IPTG for 3 h at 37°C before the activity was determined by the method of Manoil and Beckwith (1986)
Chlorophyll concentration was determined according to MacKinney (1941) Upon request, all novel materials described in this publication will be made available in a timely manner for noncommercial research purposes, subject to the requisite permission from any third-party owners of all or parts of the material. Obtaining permission will be the responsibility of the requestor. Sequence data from this article have been deposited with the EMBL/GenBank data libraries under accession number AY849313.
We thank Dr. C.P. Wolk from Michigan State University for providing the plasmid pRL592 and Dr. X. Xu (Institute of Hydrobiology, Wuhan, China) for his help in constructing the mutant library of Synechococcus sp. PCC 7002. We also thank Dr. J.J. Brand (University of Texas, Austin) for his critical reading of the manuscript and valuable suggestions. Skillful technical assistance by Ms. C. Dong (Peking University, Beijing) is greatly appreciated. Received February 17, 2005; returned for revision March 28, 2005; accepted April 6, 2005.
1 This work was supported by the National Natural Science Foundation of China (30230040) and by the High Technology project from the Ministry of Science and Technology of China (2004AA626020). 
[w] The online version of this article contains Web-only data. Article, publication date, and citation information can be found at www.plantphysiol.org/cgi/doi/10.1104/pp.105.061549. * Corresponding author; e-mail jzhao{at}pku.edu.cn; fax 86–10–6275–1526.
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ABSTRACT
RESULTS
was used for all routine cloning, and strain BL21(DE3) was used for overproduction of recombinant proteins. All enzymes were purchased from Promega (Beijing) and used according to instructions. The plasmid pRL592 used for transposon mutagenesis of Synechococcus sp. PCC 7002 was a generous gift from Dr. P. Wolk (Michigan State University, East Lansing, MI). Plates of A+ solid medium with or without glycerol (10 mM) and DCMU (10 µM) were used in replicate to identify cells that could not grow photoheterotrophically. For construction of a fesM mutant (fesM-D), a DNA fragment containing fesM was amplified by PCR with primers P1 and P4 (
