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Originally published In Press as doi:10.1074/jbc.M505976200 on July 18, 2005

J. Biol. Chem., Vol. 280, Issue 36, 31595-31602, September 9, 2005
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Photosystem II Assembly in CP47 Mutant of Synechocystis sp. PCC 6803 Is Dependent on the Level of Chlorophyll Precursors Regulated by Ferrochelatase*{boxs}

Roman Sobotka{ddagger}§, Josef Komenda{ddagger}§, Ladislav Bumba||, and Martin Tichy{ddagger}§

From the {ddagger}Institute of Physical Biology, University of South Bohemia, 373 33 Nove Hrady, the §Department of Autotrophic Microorganisms, Institute of Microbiology, Opatovicky mlyn, 379 71 Trebon, and the ||Institute of Plant Molecular Biology, Branisovska 31, 370 05 Ceske Budejovice, Czech Republic

Received for publication, June 1, 2005 , and in revised form, July 14, 2005.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Accumulation of chlorophyll and expression of the chlorophyll (Chl)-binding CP47 protein that serves as the core antenna of photosystem II are indispensable for the assembly of a functional photosystem II. We have characterized the CP47 mutant with an impaired photosystem II assembly and its two spontaneous pseudorevertants with their much improved photoautotrophic growth. The complementing mutations in these pseudorevertants were previously mapped to the ferrochelatase gene (1). We demonstrated that complementing mutations dramatically decrease ferrochelatase activity in pseudorevertants and that this decrease is responsible for their improved photoautotrophic growth. Photoautotrophic growth of the CP47 mutant was also restored by in vivo inhibition of ferrochelatase by a specific inhibitor. The decrease in ferrochelatase activity in pseudorevertants was followed by increased steady-state levels of Chl precursors and Chl, leading to CP47 accumulation and photosystem II assembly. Similarly, supplementation of the CP47 mutant with the Chl precursor Mg-protoporphyrin IX increased the number of active photosystem-II centers, suggesting that synthesis of the mutated CP47 protein is enhanced by an increased Chl availability in the cell. The probable role of ferrochelatase in the regulation of Chl biosynthesis is discussed.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
The photosystem-II (PSII)1 core complex of higher plants, algae, and cyanobacteria consists of the chlorophyll-binding proteins D1, D2, CP43, and CP47 and two subunits of cytochrome b559. These proteins are assembled into the PSII complex in an ordered manner, and the mutation or inactivation of a single protein frequently leads to PSII destabilization and its disappearance from the thylakoids (2, 3).

From several cofactors that are associated with the PSII core, chlorophyll (Chl) has been shown to be essential for PSII biogenesis. Both in vitro and in vivo analyses of expression and accumulation of PSII chlorophyll-binding proteins have suggested that cotranslational binding of Chl is required for correct apoprotein folding and stable integration into the thylakoid membrane (4–6). Mutation studies of Chl ligands and in vitro reconstitution of Chl-binding proteins indicate that, without Chl, apoproteins do not fold correctly and are recognized and degraded by specific proteases (7–9). However, the process of delivery and incorporation of Chl into apoproteins, as well as the role of Chl in protein folding, remain to be elucidated.

It is generally accepted that expression of Chl-binding proteins is synchronized with Chl biosynthesis to ensure formation of a proper amount of Chl for assembly of the photosynthetic apparatus and to avoid harmful accumulation of non-assembled (free) Chl and its precursors (10, 11). This regulation is rather complicated: because Chl shares a common biosynthetic pathway with heme, and demand for these cofactors varies considerably depending on environmental and growth conditions (reviewed in Ref. 12). Therefore, photosynthetic organisms have to balance fluxes through these two biosynthetic branches and coordinate them with apoprotein formation.

An important role in this process may be played by Mg- and Fe-chelatases lying at the branch point of the common tetrapyrrole pathway. Here, magnesium chelatase and ferrochelatase (FeCH) compete for the same substrate, protoporphyrin IX (Proto IX), to insert either magnesium for Chl biosynthesis or ferrous ion for heme, and in cyanobacteria also for phycobilin biosynthesis. To guarantee a balanced flow of precursors in the pathway, expression or activities of these chelatases are apparently controlled by light (13), diurnal and circadian rhythmicity (14, 15), and by the redox state of photosynthetic electron transport (16).

Recently, Tichy and Vermaas (1) described a C1.8 mutant of the cyanobacterium Synechocystis PCC 6803 carrying a mutation in the Chl-binding CP47 protein comprising the core antenna of PSII (see Fig. 1). The mutant exhibited very poor photoautotrophic growth, however fully autotrophic pseudorevertants were formed at high frequency. Interestingly, in two C1.8 pseudorevertants (P1.8.1 and P1.8.2), complementing mutations were mapped to the FeCH gene, and their sequencing revealed point mutations in this gene (see Fig. 1).

In this study we have characterized the Synechocystis C1.8 mutant and its photoautotrophic pseudorevertants in detail to explain how mutations in FeCH may functionally complement mutation in the CP47 protein. The results clearly indicate that, at least in this mutant, FeCH activity is controlling PSII assembly, most probably by modulation of chlorophyll availability in the cell.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Strains and Growth Conditions—The {Delta}CP47 deletion mutant, in which most of the psbB gene coding for CP47 is replaced by a spectinomycin-resistance cassette, was prepared by Eaton-Rye and Vermaas (29). The deletion mutant {Delta}1CP47 ({Delta}1), lacking four amino acid residues (Ile265–Phe268, Fig. 1) of the CP47 protein, has been prepared by Haag et al. (17). The poorly photoautotrophic C1.8 mutant has been constructed by combinatorial mutagenesis performed on {Delta}1 (1). Two photoautotrophic pseudorevertants originating from C1.8–P1.8.1 and P1.8.2 carry point mutations in the FeCH gene that cause amino acid substitution S50Y and S321F, respectively (1). Other PSII mutants used in this report are (name and inactivated genes): {Delta}CP43, psbC; {Delta}D1, psbA1, -2, and -3 (18); and {Delta}c559, psbEFLJ (19).

Synechocystis strains were grown in liquid BG-11 medium (20) supplemented by 10 mM TES at 30 °C and 50 µmol m–2 s–1 (normal light) or 5 µmol m–2 s–1 (low light) on the rotary shaker. Strains were grown on 5 mM glucose unless photoautotrophic conditions are indicated.

Oxygen Evolution—Light-saturated (3500 µmol m–2 s–1) steadystate rate of oxygen evolution was measured directly with samples taken from liquid cultures (OD730 = 0.5–0.7) using a Clark-type electrode in the presence of 1 mM potassium ferricyanate and 0.1 mM dimethyl-p-benzoquinone at 29 °C.

Determination of Chl and Carotenoids—Chl and carotenoids were extracted from cell pellets (50 ml, OD730 ~ 0.5) by 100% methanol. Chl content was measured spectrophotometrically on a Spectronic Unicam UV500 spectrophotometer (21). Carotenoids were separated by HPLC on a Vydac 201TP54 column (250 mm x 4.6 mm, C-18 reversed-phase silica gel). The column was eluted with a linear gradient of solvent B (methanol and acetone, 6:4, v/v) in solvent A (methanol and 0.5 M ammonium acetate, 8:2, v/v, pH 7.0) at a flow rate of 1.2 ml/min as follows: 0–80% in 3 min, 80–100% in the next 10 min, and then followed by 100% solvent B for 5 min. Carotenoid species were identified by their absorption spectra and by their typical retention times.

Electron Microscopy—Cells were washed twice in 50 mM phosphate buffer (pH 7.4) and fixed in a solution containing 2% glutaraldehyde in 50 mM phosphate buffer (pH 7.4) for 2 h. This was followed by three washes in phosphate buffer and post-fixated in 1% OsO4 in 50 mM phosphate buffer (pH 7.4) for 1 h. The cells were again washed three times, and the samples were dehydrated in acetone and embedded in Spurr's resin. Thin sections were cut with a diamond knife and post-stained for 30 min with 5% aqueous uranyl acetate followed by lead citrate for 80 s and examined with a Jeol 1010 electron microscope equipped with charge-coupled device video camera Lhesa 72WA and image acquisition software VideoTIP (Tescan).

Radiolabeling of the Cells, Membrane Preparation, and Protein Analysis Using Two-dimensional Native/SDS Electrophoresis and Immunoblotting—Radiolabeling of cells using a mixture of L-[35S]methionine and L-[35S]cysteine (>1000 Ci mmol–1, Tran35S-label, ICN, final activity, 400 µCi ml–1) and isolation of membranes was performed as described in a previous study (2). For two-dimensional analysis, isolated membranes were solubilized with n-dodecyl-{beta}-maltoside (n-dodecyl-{beta}-maltoside/Chl = 20, w/w), and the obtained complexes were separated by blue-native electrophoresis at 4 °C in 5–14% polyacrylamide gel according to a previous study (22) in the first dimension, and by electrophoresis in a denaturing 12–20% linear-gradient polyacrylamide gel containing 7 M urea in the second dimension (2). Proteins separated in the gel were transferred onto a polyvinylidene difluoride membrane. The membrane was incubated with specific primary antibodies and then with secondary antibody-horseradish peroxidase conjugate (Sigma). The primary antibodies used in the study were raised in rabbits against: (i) residues 58–86 of the spinach D1 polypeptide; (ii) 12 last residues of the Synechocystis D2 polypeptide; (iii) residues 380–394 of barley CP47; and (iv) the whole isolated CP43 (2). For autoradiography, the gel or the membrane with labeled proteins was exposed to x-ray film at laboratory temperature for 2–3 days.

Ferrochelatase Activity Assay—For the determination of FeCH activity in thylakoids, 50 ml of cells (OD730 ~ 0.5) were washed in the thylakoid buffer containing 100 mM Tris/HCl pH 8.0, 1 mM EDTA, 1 mM dithiothreitol, and 20% glycerol (v/v). The cell suspension was transferred into 0.5-ml microcentrifuge tubes, mixed with glass beads, and broken in a MiniBeadBeater. The homogenate was centrifuged at 32,000 x g for 20 min at 4 °C. Pelleted thylakoids were washed in EDTA-free thylakoid buffer and centrifuged at 32,000 x g. Finally, thylakoids were re-suspended in EDTA-free thylakoid buffer containing 1% Triton X-100 and frozen at –70 °C.

The standard assay was performed in a cuvette containing 1.5 ml of assay buffer (100 mM Tris/HCl, pH 8.0, 0.03% Tween 80, 0.1 µM ZnSO4, and 0.3 µM protoporphyrin IX) at 35 °C. The reaction was started with thylakoids and the formation of zinc-protoporphyrin IX recorded spectrofluorometrically using a Spectronic Unicam series 2 spectrofluorometer (23) and zinc-protoporphyrin IX (Sigma) as a standard.

Quantification of Chlorophyll Intermediates—For quantitative determination of protoporphyrin IX (Proto IX), Mg-protoporphyrin IX (Mg-proto IX) monomethyl ester, and protochlorophyllide (PChlide), pigments were extracted from cell pellets (150 ml, OD730 ~ 0.5) by three successive extractions with methanol containing 0.1% of NH4OH. Supernatants were combined, and the solvent was evaporated under vacuum to dryness. Extracts were dissolved in a small volume of basic methanol and immediately subjected to HPLC analysis with a Vydac 201TP54 column (250 mm x 4.6 mm, C-18 reversed-phase silica gel). The column was eluted with a linear gradient of solvent B (methanol and acetone, 9:1, v/v) in solvent A (methanol and 0.5 M ammonium acetate, 7:3, v/v, pH 7.0) at a flow rate of 1.5 ml/min. The 100% of solvent B was reached after 20 min. HPLC peaks corresponding to PChlide were identified from their absorption spectra. Positions corresponding to Proto IX and Mg-proto IX monomethyl ester were identified from comparison with authentic standards (Sigma and Frontier Scientific, Logan, UT).

HPLC fractions containing PChlide (retention time, 12.5–13.5 min), Mg-proto IX monomethyl ester (14–14.5 min), and Proto IX (17–17.8 min) were collected and concentrations of the corresponding compounds determined fluorometrically using a Spectronic Unicam series 2 spectrofluorometer.

Protoheme Determination—Protoheme was extracted from 0.5 liter of cells (OD730 ~ 0.5) by acidic acetone according to Ref. 24, and its concentration was determined from the reduced-minus-oxidized difference spectrum as described by Stillman and Gassman (25) with ammonium persulfate used for sample oxidation.

Chlorophyll Fluorescence Induction—A P.S.I. fluorometer (Photon System Instrument, Brno, Czech Republic) was used to record Chl fluorescence induction in the presence of 10 µM DCMU. PSII fluorescence was induced by a 60-ms pulse of light supplied by blue light-emitting diodes. Induction curves were measured directly in liquid cultures (OD730 = 0.5) after 2-min dark adaptation. The cells treated by Mg-proto IX were washed three times in BG-11 medium to eliminate traces of this compound.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
Mutant Characterization—The growth characteristics, Chl content and oxygen evolution of strains used in this study are listed in Table I. In normal light, all three CP47 mutants ({Delta}CP47, {Delta}1, and C1.8) exhibited slower growth rate and lower Chl accumulation than WT. Interestingly, growth impairment and decrease in Chl level were more pronounced in the {Delta}1 mutant with a short deletion in the E loop of the CP47 protein (Fig. 1A) than in the {Delta}CP47 strain lacking most of the CP47 gene. The growth rates and Chl levels were not affected in low light, indicating the light sensitivity of the CP47 mutants. This is supported by the significantly increased levels of carotenoids shown in normal light. Particularly myxoxanthophyll, important in defense against light stress (26), was highly increased in all CP47 mutants (see Supplemental Table S1). In contrast, carotenoid levels in both strains were comparable to WT in low light (data not shown). The phenotype of the poorly photoautotrophic C1.8 mutant was consistent with its original characterization (1).


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  		TABLE I
Growth rate, Chl content, and oxygen evolution of Synechocystis 6803 strains

WT, CP47 mutants, and pseudorevertants were grown photoautotrophically and photomixotrophically in normal and low light to OD730 0.4-0.5. Values given are the averages from three different determinations.

 
Transmission electron microscopy revealed that cells of {Delta}1 growing in normal light were much smaller than WT cells and displayed highly fragmented thylakoid membranes (Fig. 2). This was in contrast to the almost normal membrane organization in the corresponding {Delta}CP47 mutant (Fig. 2), confirming the strong adverse effect of the {Delta}1 mutation. Membrane organization in C1.8 was similar to that in the {Delta}CP47 mutant (not shown).



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  		FIG. 1.
Mutations in CP47 and FeCH genes in relevant strains. A, alignment of the amino acid sequences 260–271 of CP47 from WT, {Delta}1, C1.8, and two C1.8 pseudorevertants. The region probed by combinatorial mutagenesis is boxed. The approximate position of {Delta}1 mutation in CP47 protein is also shown. B, alignment of the amino acid sequence of FeCH showing the secondary mutations in P1.8.1 and P1.8.2 strains at positions 50 and 321, respectively.

 



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  		FIG. 2.
Transmission electron microscopy of Synechocystis 6803 cells. WT (A), {Delta}1 (B), {Delta}CP47 (C), and P1.8.1 cells (D–F). Atypical membrane structures in P1.8.1 cells are shown by arrows. Scale bar = 500 nm.

 
The rate of photoautotrophic growth and oxygen-evolving capacity of the pseudorevertants were comparable to that of WT (Table I), however, significantly higher levels of Chl, carotenoids and phycobilinoproteins accumulated in both strains (Table I and Supplemental Fig. S1). This concomitant increase of photosynthetic pigments in pseudorevertants suggests a higher abundance of photosystems and phycobilisomes in their cells. Indeed, electron microscopy revealed an atypical organization of thylakoid membranes in the cells of the P1.8.1 pseudorevertant. In addition to several concentric thylakoid membranes typical for Synechocystis 6803, new centric membranes were found in P1.8.1 (Fig. 2, C–E).

CP47 Accumulation and Photosystem II Assembly—We analyzed the effect of the mutation in the {Delta}1 and C1.8 strains on stability and accumulation of PSII core proteins and PSII assembly in CP47 mutants and pseudorevertants. To follow the accumulation of PSII core proteins, solubilized thylakoid membranes were analyzed by SDS-PAGE, the separated proteins blotted and exposed to a mix of antisera against CP47, CP43, D1, and D2. As shown in Fig. 3, the {Delta}1 mutant has no detectable amounts of CP47, similar to that of the deletion mutant {Delta}CP47. In the C1.8 mutant, about 20% of the WT steady-state level of CP47 was estimated, showing an improved CP47 translation/stability in comparison to {Delta}1. The accumulation of CP47 in pseudorevertants was comparable to WT (Fig. 3).

De novo synthesis and assembly of mutated CP47 into PSII was followed by a combination of protein pulse-chase radiolabeling and two-dimensional native/SDS electrophoresis. In thylakoid membranes of {Delta}1, no signal of CP47 was detected and the synthesis of labeled proteins was essentially identical to that of {Delta}CP47 (data not shown, for {Delta}CP47 see Ref. 2), indicating that the synthesis and/or incorporation of CP47 into the thylakoid membrane was completely disrupted.

In C1.8, only a very weak signal of labeled proteins assembled into the PSII complex and RC47 subcomplex (consisting of D1, D2, cytochrome b559, and CP47) was detected (Fig. 4). Instead, the D1-D2-cytochrome b559 subcomplex and free CP43 accumulated substantially, suggesting that PSII assembly was blocked at the CP47 insertion step. The very low level of unassembled CP47 indicates that the C1.8 mutation does not affect the assembly of CP47 into PSII.

In the pseudorevertants, the amount of synthesized CP47, as well as the rate of its assembly into PSII, were found to be very similar to WT. After a 30-min chase, the signal intensity of CP47 did not significantly decline, implying normal stability of the newly synthesized CP47 carrying the C1.8 mutation (Fig. 4). This indicates that the C1.8 strain is impaired at the level of CP47 synthesis. The mutated CP47 protein, once synthesized, is stable and is assembled normally into a functional PSII.

In Vitro FeCH Activity in C1.8 Strains—Because the effect of both complementing mutations on FeCH was not evident from the sequence alignments, and it was hypothesized that they may influence FeCH activity (1), we decided to compare in vitro FeCH activity in the CP47 mutants and both pseudorevertants. Because no FeCH activity was found in the soluble fraction, isolated thylakoid membranes were used for the FeCH assay using Proto IX and zinc as a substrate.

Surprisingly, in both the C1.8 and {Delta}1 mutants, FeCH activity was significantly higher than in WT (Fig. 5A). In the pseudorevertants, FeCH activity decreased dramatically below the WT level resulting in a 30- to 40-fold decrease in comparison to the C1.8 mutant (Fig. 5A). This indicates that both complementing mutations strongly inhibit FeCH activity. To determine whether the increase in FeCH activity observed in the {Delta}1 and C1.8 mutants is characteristic only of a mutation in this region of CP47, we also measured FeCH activity in other strains with impaired PSII assembly. In all PSII mutants tested, a significant increase in FeCH activity was observed, showing that up-regulation of FeCH activity is related to partly or completely destabilized PSII (Fig. 5B). To exclude the possibility that increased FeCH activity results from changes induced by photoheterotrophic metabolism in PSII mutants, FeCH activity was also measured in WT grown in the presence of a PSII inhibitor, DCMU. No significant increase in FeCH activity was observed under such conditions (Fig. 5B).



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  		FIG. 3.
Accumulation of PSII core proteins in Synechocystis 6803 mutants. Thylakoid membranes from analyzed strains were prepared, 3 µg of Chl was loaded for each sample, and amounts of CP47, CP43, D1, and D2 PSII proteins were determined by immunolabeling.

 



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  		FIG. 4.
PSII core protein expression and PSII assembly determined by two-dimensional PAGE. Cells were radiolabeled with [35S]methionine and their thylakoid membranes prepared. Analysis of thylakoid protein complexes and their protein composition was performed by a combination of blue-native and SDS-PAGE. iD1, intermediate D1; RCCII, PSII core complex; RC47, cyt559-D1-D2-CP47 complex; RC, cyt559-D1-D2 complex (2). Free CP43 protein is shown by black arrows and ATPase by asterisk.

 
In Vivo Inhibition of FeCH—To test whether decreased FeCH activity in the pseudorevertants is responsible for their photosynthetic phenotype, we used N-methylprotoporphyrin IX, which is a specific and potent inhibitor of FeCH (27). In the presence of 10 µM N-methylprotoporphyrin IX in the growth medium, photoautotrophic growth of C1.8 dramatically improved (doubling time changed from 65 to 16 h), whereas growth of WT was slightly inhibited (see Supplemental Fig. S2). No growth was observed in the {Delta}1 strain, even when the concentration of the inhibitor was increased to 50 µM (not shown).

To show that the improved photoautotrophic growth is due to increased PSII accumulation in the pseudorevertants, 77K fluorescence emission spectroscopy was used to monitor the accumulation of PSII in vivo. Upon Chl excitation at 435 nm, the presence of the 695-nm emission peak, which is characteristic of CP47 assembled into PSII, can be used to monitor the assembly of PSII core complexes (8). Absence of the 695-nm peak in the C1.8 strain corresponds to low levels of assembled PSII in this strain. After treatment of C1.8 with N-methylprotoporphyrin IX, significant 695-nm emission was observed (Fig. 6A). Moreover, appearance of the 695-nm emission peak was accompanied by increased oxygen-evolving capacity of the cells (Fig. 6B). The improved PSII activity was also accompanied by increased overall Chl accumulation in C1.8 cells (Fig. 6B). No changes in Chl accumulation were observed when 10 µM N-methylprotoporphyrin IX was added to the WT and {Delta}1 strains (not shown). These data show that the decreased FeCH activity in the C1.8 strain is necessary for PSII assembly and for the improved photoautotrophic growth.

Chlorophyll and Heme Precursor Levels—To determine the effect of CP47 and FeCH mutations on the Chl/heme biosynthetic pathway, levels of Proto IX, Mg-proto IX monomethyl ester, PChlide, and protoheme were quantified in the WT, {Delta}1, and C1.8 mutants, and the two pseudorevertants were grown in normal light.

The {Delta}1 and C1.8 mutations in CP47, which lead to the increased FeCH activity, caused about 5-fold decrease in the level of Proto IX, the last common intermediate of both Chl and heme biosynthesis, and ~2-fold decrease of the Chl precursors Mg-proto IX monomethyl ester and PChlide (Fig. 7). In contrast, in the pseudorevertants with decreased FeCH activity, precursor levels increased above WT levels (~3-fold increase in the level of Proto IX and ~2-fold increase in the level of Chl precursors). These data demonstrate that the decrease in FeCH activity in pseudorevertants resulted in a significant increase in the Chl precursor levels. Unexpectedly, drastic changes in FeCH activity had no effect on protoheme accumulation (Fig. 7).



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  		FIG. 5.
In vitro activity of FeCH determined by continuous spectrofluorometric assay. FeCH activity was determined in {Delta}1, C1.8 and C1.8 pseudorevertants (A) and WT treated by 10 µM DCMU (B) for 48 h and in several PSII mutants with inactivated PSII components: psbC ({Delta}CP43), psbB ({Delta}CP47), three psbA ({Delta}D1), or both subunits of cytochrome b559 ({Delta}c559). The values are the mean of three independent experiments.

 
To determine whether the increased accumulation of Chl precursors in the pseudorevertants is sufficient to restore PSII assembly in C1.8, the formation of the 695-nm Chl emission peak was followed after supplementation with Mg-protoporphyrin IX (Mg-proto IX), an early precursor of Chl. In C1.8 cultures supplemented with 200 µM Mg-proto IX for 2 days, the 695-nm peak became visible and was even more pronounced in cells supplemented with 400 µM Mg-proto IX (Fig. 6C). No such increase was detected in {Delta}1. No effect of Mg-proto IX on the FeCH activity was detected, suggesting that the improved PSII assembly is the direct effect of Mg-proto IX supplementation.

The improved PSII assembly in the C1.8 mutant after Mg-proto IX supplementation was further confirmed by measurement of variable fluorescence of Chl (Fv), which is specific for photochemically active PSII. In untreated C1.8 cells, Fv reached only ~20% of that in WT (Fig. 6D), which is in agreement with the expected PSII accumulation in this strain. After 48-h incubation in 400 µM Mg-proto IX, Fv in C1.8 significantly increased, indicating a higher accumulation of PSII. No fluorescence induction was found in the {Delta}1 mutant (not shown).


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
Deletion of the {Delta}1 Region of CP47 Protein Disrupts Its Expression and Is Responsible for the Photosensitivity of the Mutant—The Chl-binding protein CP47 possesses six transmembrane domains with a large lumenal loop joining helices 5 and 6 (Fig. 1). The function of this loop has been extensively probed by partial deletions in which domains consisting of several amino acid residues have been removed (reviewed in Ref. 28). Deletion of four amino acid residues in the {Delta}1 domain resulted in a non-photoautotrophic phenotype (17) and the complete loss of CP47 protein from thylakoid membranes (Fig. 3). Interestingly, the phenotype of the {Delta}1 deletion mutant was different from that of the {Delta}CP47 mutant lacking most of the psbB gene (29). In contrast to {Delta}CP47, cells of {Delta}1 were severely damaged in normal light, although they accumulated high levels of carotenoids (Fig. 2 and Supplemental Table S1). It appears that the expression of the modified {Delta}1 CP47 protein is more deleterious for cell viability than its complete deletion. No full-length CP47 protein is detectable in thylakoid membranes of this strain, even by the highly sensitive radiolabeling technique, indicating that the {Delta}1 region is essential for the completion of CP47 translation or for the stable incorporation of CP47 into thylakoid membranes.

The mechanism of {Delta}1 photosensitivity is not clear. We have not seen increased levels of photoreactive Proto IX and Mg-porphyrins in this strain (Fig. 7). Possibly, Proto IX precursors can be slowly oxidized and also form photodynamically active compounds (30). Alternatively, non-assembled (free) Chl can be responsible for the {Delta}1 photosensitivity. As Chl molecules bind to the CP47 apoprotein cotranslationally (5), free Chl in {Delta}1 can originate from prematurely degraded uncompleted CP47. As will be shown later, we favor this explanation.

The Effect of C1.8 Mutation Is Suppressed by Decreased FeCH Activity—The C1.8 mutant with three substitutions in the {Delta}1 region (Fig. 1) exhibits very limited photoautotrophic growth due to the seriously impaired CP47 synthesis (Fig. 4). We have provided clear evidence that expression of the mutated CP47 and consequently the autotrophic capacity of C1.8 can be fully restored by lowering FeCH activity. The severe decrease in FeCH activity was observed in two spontaneous photoautotrophic C1.8 pseudorevertants with secondary mutations in the FeCH gene (Fig. 5). A proportional decrease in FeCH activity was observed in the mutant carrying the P1.8.2 point mutation in the FeCH gene in the WT background, showing that this mutation is solely responsible for the decrease in FeCH activity.2 Treatment with the specific inhibitor of FeCH resulted again in restored PSII assembly and photoautotrophic growth (Fig. 6, A and B, and Supplemental Fig. S2), demonstrating that the photosynthetic phenotype of the C1.8 pseudorevertants can be exclusively attributed to the lack of FeCH activity.

Decreased FeCH Activity in the C1.8 Pseudorevertants Increases Chl Accumulation and PSII Assembly—It is not immediately obvious how the decreased activity of the heme-producing enzyme can stabilize the expression of the Chl-binding CP47 protein. We have shown that the decrease in FeCH activity resulted in the increased accumulation of Proto IX, Mg-proto IX monomethyl ester, and PChlide (Fig. 7).

This is similar to FeCH-antisense plants, where decrease in FeCH activity resulted in accumulation of Proto IX (31). Moreover, a significant increase in PChlide and Chl levels was reported in plants genetically engineered for increased production of Proto IX (32, 33). A similar effect has been observed in other photosynthetic bacteria. In vivo inhibition of FeCH enhanced bacteriochlorophyll synthesis in the aerobic bacterium Erythrobacter longus (34) and led to the accumulation and excretion of Mg-proto IX in Rhodopseudomonas sphaeroides (35).



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  		FIG. 6.
Analysis of PSII assembly and Chl accumulation in C1.8 mutant treated by FeCH inhibitor or supplemented by Mg-proto IX. A, 77K fluorescence emission spectra of the C1.8 cells treated by 10 µM FeCH inhibitor N-methylprotoporphyrin IX for 0 h (solid line), 3 h (dotted line), 6 h (dashed line), and 12 h (dashed and dotted line). Control C1.8 cells after 12 h (circles). Excitation was at 435 nm, and fluorescence intensities were normalized to the fluorescence yield at 685 nm; a.u., arbitrary units. B, Chl content (circles) and oxygen-evolving activity (squares) in C1.8 mutant growing autotrophically (open symbols) and in the same strain treated by 10 µM FeCH inhibitor (closed symbols). C1.8 cells grown photomixotrophically were harvested, washed, and resuspended into autotrophic growth medium. After 12 h of autotrophic growth, 10 µM N-methylprotoporphyrin IX was added (0 h). C, 77 K fluorescence emission spectra of the WT (dotted line), C1.8 (solid line), and C1.8 cells treated by 200 µM Mg-proto IX (dash-dot-dot-dash line) and 400 µM Mg-proto IX (dash-dot-dash line) for 48 h. Mg-proto IX (Frontier Scientific, Logan, UT) was added to C1.8 growing at low light to reduce the effects of Mg-proto IX photoreactivity. Excitation was at 435 nm and fluorescence intensities were normalized to the fluorescence yield at 725 nm; a.u., arbitrary units. The inset shows emission of PSII-associated chlorophyll normalized to 685 nm. D, Chl fluorescence induction of Synechocystis cells in the presence of 10 µM DCMU. WT, control (open circles); WT treated by 400 µM Mg-proto IX (gray circles); C1.8, control (open triangles); C1.8 treated by 400 µM Mg-proto IX (black triangles). Curves were normalized to the F0.

 
We have shown that supplementation of the C1.8 mutant with Mg-proto IX also improved PSII accumulation (Fig. 6, C and D), indicating that the increased level of Chl precursors is the reason for PSII assembly in the C1.8 pseudorevertants. Surprisingly, increased Chl biosynthesis in the pseudorevertants was followed by the formation of extra thylakoid membranes with aberrant localization (Fig. 2). It looks like the increased pool of Chl leads to the accumulation of Chl-binding proteins and initiates the formation of extra thylakoid membranes. This suggests an important role of tetrapyrroles in the control of the accumulation of pigment-protein complexes and the proliferation of thylakoid membranes in the cyanobacterial cell.

Interestingly, a 10-fold decrease in FeCH activity had no effect on the protoheme level, and no symptoms of heme deficiency were observed. Although cyanobacteria utilize large amounts of heme for the biosynthesis of phycobilins (chromophores of the major light-harvesting structures), we have not observed any decrease in content of phycobilisomes in the cells (Supplemental Fig. S1). This is similar to our unsuccessful attempt to genetically inactivate FeCH, where no changes in phycobiliprotein levels were detectable until very late in the segregation process. A similar excess of FeCH capacity has been observed in other organisms: 10-fold decrease in FeCH activity in the purple bacterium R. sphaeroides, or 16-fold lower activity of FeCH in Bacillus subtilis, had no effect on cell viability (35, 36).



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  		FIG. 7.
Protoheme and Chl precursor levels in the CP47 mutants and pseudorevertants. Chl precursor pigments were extracted from cells by extraction with methanol and quantified by a combination of HPLC and spectrofluorometry. Protoheme was extracted by acidic acetone, and its concentration was determined from the reduced-minus-oxidized difference spectrum (see "Experimental Procedures"). The values are the mean of three independent experiments and are given as a percentage of WT.

 



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  		FIG. 8.
Model describing effect of the C1.8 mutation and its complementation by inactivation of FeCH. A, the C1.8 mutation prevents proper Chl binding to the CP47 apoprotein; an uncomplete holoprotein is degraded. Up-regulation of FeCH activity results in decreased levels of Chl precursors, further impairing synthesis of CP47. B, decreased FeCH activity in the pseudorevertants (or after treatment with FeCH inhibitor) increases Chl supply and results in the improved synthesis of mutated CP47 holoprotein that is stable and assembles normally into PSII.

 
Inversely, up-regulation of FeCH activity found in several PSII mutants (Fig. 5) may be responsible for the decreased accumulation of Chl precursors in these strains (Fig. 7). In Rhodobacter capsulatus, overexpression of FeCH led to a decrease of aminolevulinate synthase activity and to dramatically lowered levels of bacteriochlorophyll (37).

C1.8 Mutation Influences Synthesis of CP47 but Not Its Function or Assembly into PSII—During PSII assembly, CP47 is attached to the D1-D2-cytochrome b559 subcomplex followed by the insertion of CP43 (2). We have shown that in the C1.8 strain, PSII assembly is blocked at the CP47 insertion step most probably due to low levels of available CP47. Pseudorevertants carrying the same C1.8 mutation in CP47, but with an improved supply of Chl, synthesized the mutated CP47 protein with normal stability and assembled almost normal amounts of functional PSII complexes. This argues that C1.8 mutation influences mostly the synthesis of CP47 and not its final stability or functionality. The CP47 protein binds 16 Chl molecules, several of them having been shown to be critical for protein stability (3). By translation assays, it was revealed that Chl binding is required for the stability and membrane association of CP47 translation intermediates. Interestingly, ribosome pausing was observed after synthesis of helix 5 of the CP47 protein.3 This pausing could be related to Chl attachment and folding of the CP47 protein.

Increased FeCH Activity in PSII Mutants: Possible Role of the C-terminal Extension of FeCH—The reasons for increased FeCH activity in PSII mutants are not known. We have shown by DCMU treatment that this increase is not caused by blocked photosynthetic electron transport or by the switch from photoautotrophic to photoheterotrophic growth. Interestingly, highly increased FeCH activity was found also in a PSI-less mutant,2 suggesting that FeCH activity is increased in mutants that may have a problem with chlorophyll placement. In higher plants, increase in FeCH activity has been reported as a response to environmental stresses such as wounding, oxidative stress, and viral infection (38). However, there are two different FeCH isozymes in plants (39) and only the FeCH-1 isozyme, ubiquitously expressed throughout the plant, was responsible for the described increase. In contrast, FeCH-2 localized in plastids, was repressed (38). Plant FeCH-2 is a homologue of cyanobacterial FeCH with a putative membrane-spanning region carrying the characteristic Chl-binding motif (40).

This C-terminal extension of FeCH, found only in organisms with oxygenic photosynthesis, is proposed as being involved in the modulation of FeCH activity and in the regulation of Chl/heme biosynthesis through Chl binding (30). Our data indirectly support this hypothesis. Firstly, point mutation in Ser-321 leading to a 40-fold decrease in FeCH activity in the P1.8.2 pseudorevertant is localized in the region connecting FeCH with this putative transmembrane region. Secondly, we demonstrated that changes in FeCH activity in C1.8 influence Chl biosynthesis, suggesting that changes in FeCH activity could regulate Chl biosynthesis even in WT. Lastly, our data indicate that the most photosensitive PSII– strains (in the order {Delta}1 > cyt559 >{Delta}CP47 >{Delta}CP43) have also the highest FeCH activity when grown in low light.2 We speculate that free Chl accumulating in PSII– strains as a consequence of degraded Chl-binding proteins may both induce photosensitivity of PSII– strains and up-regulate FeCH activity by binding to its transmembrane domain.

In the working model depicted in Fig. 8, the {Delta}1 region located near to the C-terminal of the CP47 protein is important for Chl-attachment during translation and for proper protein folding. In the C1.8 mutant, only a small fraction of translated CP47 proteins is completed due to the affected Chl binding to the CP47 protein. In pseudorevertants, the completion of properly folded CP47 is improved due to the increased Chl supply. Because the C1.8 mutation apparently only affects the synthesis of the CP47 protein and not its function or assembly into PSII, up-regulation of the Chl pathway allows the C1.8 mutant to grow fully photoautotrophically.

In conclusion, we have demonstrated that FeCH activity is controlling PSII assembly in the C1.8 mutant by the modulation of chlorophyll availability in the cell. The fact that FeCH activity can be easily regulated by a specific inhibitor makes the C1.8 strain a useful tool for studying tetrapyrrole metabolism and connection between Chl synthesis and the assembly of Chl-binding complexes. We are currently analyzing several C1.8 pseudorevertants in which secondary mutations did not map into the FeCH gene, to identify other proteins involved in the regulation of tetrapyrrole biosynthesis.


   FOOTNOTES
 
* This work was supported by the Grant Agency of the Czech Academy of Sciences (Project B5817301 to R. S.), by Institutional Research Concept AV0Z50200510 (to J. K. and M. T.), and by the Ministry of Education, Youth and Sports (Project MSM6007665808 to J. K. and M. T). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

{boxs} The on-line version of this article (available at http://www.jbc.org) contains supplemental Figs. S1 and S2 and Table S1. Back

To whom correspondence should be addressed: Tel.: 420-384-722-268; Fax: 420-384-721-246; E-mail: sobotka{at}alga.cz.

1 The abbreviations used are: PSII, photosystem II; WT, wild type; Chl, chlorophyll; DCMU, 3-(3,4-dichlorophenyl)-1,1-dimethylurea; TES, N-[tris(hydroxymethyl)methyl]-2-aminoethanesulfonic acid; FeCH, ferrochelatase; Proto IX, protoporphyrin IX; PChlide, protochlorophyllide; Mg-proto IX, Mg-protoporphyrin IX. Back

2 L. A. Eichacker, B. Müller, J. Kim, and J. E. Mullet, unpublished data. Back

3 R. Eichacker et al., unpublished data. Back


   ACKNOWLEDGMENTS
 
We thank Eva Prachova for her technical assistance, Michal Koblizek for reading the manuscript, Drs. P. Nixon, L. Eichacker, and R. Barbato for specific antisera, and Profs. W. Vermaas and H. Pakrasi for the PSII mutants.



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 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
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