Site-directed mutagenesis of the basic residues 321K to321 G in the CP 47 protein of photosystem II alters the chloride requirement for growth and oxygen-evolving activity in Synechocystis 6803

CP 47, a component of photosystem II (PSII) in higher plants, algae and cyanobacteria, is encoded by the psbB gene. Site-specific mutagenesis has been used to alter a portion of the psbB gene encoding the large extrinsic loop E of CP 47 in the cyanobacterium Synechocystis 6803. Alteration of a lysine residue occurring at position 321 to glycine produced a strain with altered PSII activity. This strain grew at wild-type rates in complete BG-11 media (480 µM chloride). However, oxygen evolution rates for this mutant in complete media were only 60% of the observed wild-type rates. Quantum yield measurements at low light intensities indicated that the mutant had 66% of the fully functional PSII centers contained in the control strain. The mutant proved to be extremely sensitive to photoinactivation at high light intensities, exhibiting a 3-fold increase in the rate of photoinactivation. When this mutant was grown in media depleted of chloride (30 µM chloride), it lost the ability to grow photoautotrophically while the control strain exhibited a normal rate of growth. The effect of chloride depletion on the growth rate of the mutant was reversed by the addition of 480 µM bromide to the chloride-depleted BG-11 media. In the presence of glucose, the mutant and control strains grew at comparable rates in either chloride-containing or chloride-depleted media. Oxygen evolution rates for the mutant were further depressed (28% of control rates) under chloride-limiting conditions. Addition of bromide restored these rates to those observed under chloride-sufficient conditions. Measurements of the variable fluorescence yield indicated that the mutant assembled fewer functional centers in the absence of chloride. These results indicate that the mutation K321G in CP 47 affects PSII stability and/or assembly under conditions where chloride is limiting.     

Site-directed mutagenesis of glutamate residues in the large extrinsic loop of the photosystem II protein CP 43 affects oxygen-evolving activity and PS II assembly

The psbC gene encodes the intrinsic chlorophyll protein CP 43, a component of photosystem II in higher plants, green algae, and cyanobacteria. Oligonucleotide-directed mutagenesis was used to introduce mutations into the portion of psbC that encodes the large extrinsic loop E of CP 43 in the cyanobacterium Synechocystis 6803. Three mutations, E293Q, E339Q, and E352Q, each produced a strain with impaired photosystem II activity. The E293Q mutant strain grew photoautotrophically at rates comparable to the control strain. Immunological analyses of several PS II components indicated that this mutant accumulated normal quantities of PS II proteins. However, this mutant evolved oxygen to only 56% of control rates at saturating light intensities. Measurements of total variable fluorescence yield indicated that this mutant assembled approximately 60% of the fully functional PS II centers found in the control strain. The E339Q mutant grew photoautotrophically at a severely reduced rate. Both immunological analysis and variable fluorescence yield experiments indicated that E339Q assembled a normal complement of PS II centers. However, this mutant was capable of evolving oxygen to only 20% of control rates. Variable fluorescence yield experiments demonstrated that this mutant was inefficient at using water as an electron donor. Both E293Q and E339Q strains exhibited an increased (approximately 2-fold) sensitivity to photoinactivation. The E352Q mutant was the most severely affected. This mutant failed to grow photoautotrophically and exhibited essentially no capacity for oxygen evolution. Measurements of total variable fluorescence yield indicated that this mutant assembled no functional PS II centers. Immunological analysis of isolated thylakoid membranes from E352Q revealed a complete absence of CP 43 and reduced levels of both the D1 and manganese-stabilizing proteins. These results suggest that the mutations E293Q and E339Q each produce a defect associated with the oxygen-evolving complex of photosystem II. The E352Q mutation appears to affect the stability of the PS II complex. This is the first report showing that alteration of negatively charged residues in the CP 43 large extrinsic loop results in mutations affecting PS II assembly/function.     

Construction and characterization of cyanobacterial mutants lacking the manganese-stabilizing polypeptide of photosystem II.

Mutants of the cyanobacterium Synechocystis sp. Pasteur Culture Collection (PCC) 6803 that specifically lack the extrinsic 33-kDa manganese-stabilizing polypeptide of the photosystem II oxygen-evolving complex have been constructed by two independent methods. Cartridge mutagenesis was used to insertionally inactivate the psbO gene of one mutant and completely delete the psbO gene of the other mutant. These mutants have no detectable manganese-stabilizing polypeptide, but they do accumulate steady-state levels of the intrinsic photosystem II polypeptides D1, D2, and CP-43 that are comparable to wild-type, as determined by immunoblot analysis. Measurement of the evolution of the relative quantum yields of chlorophyll fluorescence following actinic flash excitation indicates that though the concentration of reaction centers in mutant cells is comparable to that of wild-type cells, approximately 40% of these centers harbor a fluorescence-quenching species other than P680+. The mutants are capable of photoautotrophic growth at a slower rate than that of wild-type. Under conditions of Ca2+ depletion where wild-type growth is unaffected, the mutants are unable to grow at all. The manganese-stabilizing protein, therefore, enhances the binding of Ca2+ or protects the reaction center at low Ca2+ concentrations. The mutant evolve oxygen at approximately 70% of the wild-type rate, but are completely photoinactivated by high light intensities. Our results indicate that the manganese-stabilizing polypeptide is not absolutely required for photosystem II assembly or function in cyanobacteria, but its absence does lead to an enhanced sensitivity to photoinhibition.     

Insertional inactivation of the gene encoding subunit II of photosystem I from the cyanobacterium Synechocystis sp. PCC 6803

The cyanobacterium Synechocystis sp. PCC 6803 carries out oxygenic photosynthesis analogous to higher plants. Its photosystem I contains seven different polypeptide subunits. The cartridge mutagenesis technique was used to inactivate the psaD gene which encodes subunit II of photosystem I. A mutant strain lacking subunit II was generated by transforming wild type cells with cloned DNA in which psaD gene was interrupted by a gene conferring kanamycin resistance. The photoautotrophic growth of mutant strain is much slower than that of wild type cells. The membranes prepared from mutant cells lack subunit II of photosystem I. Studies on the purified photosystem I reaction center revealed that the complex lacking subunit II is assembled and is functional in P700 photooxidation but at much reduced rate. Therefore, subunit II of photosystem I is required for efficient function of photosystem I.     

Mutation of Phe-363 in the photosystem II protein CP47 impairs photoautotrophic growth, alters the chloride requirement, and prevents photosynthesis in the absence of either PSII-O or PSII-V in Synechocystis sp. PCC 6803

The deletion of the amino acids between Gly-351 and Thr-365 within the large, lumen-exposed, hydrophilic region (loop E) of the photosystem II (PSII) chlorophyll a-binding protein CP47 produced a strain of Synechocystis sp. PCC 6803 that failed to assemble stable PSII centers [Eaton-Rye, J. J., and Vermaas, W. F. J. (1991) Plant Mol. Biol. 17, 1165-1177]. The importance of two conserved Phe residues at positions 362 and 363 within this deletion has been investigated. The F363R strain had impaired photoautotrophic growth and an enhanced sensitivity to photoinactivation, demonstrating that Phe is required at position 363 for normal PSII function. In contrast, photoautotrophic growth in strains N361K and F362R was unaffected. Uniquely, among the mutant strains tested, F363R was unable to grow under chloride-limiting conditions, and this effect was reversed by replacing chloride with bromide. The removal of the manganese-stabilizing protein (PSII-O), the 12 kDa extrinsic protein (PSII-U), and cytochrome c-550 (PSII-V) was investigated in each mutant in vivo. In N361K and F362R, removal of PSII-V produced a more deleterious effect than the removal of PSII-O, but even so, all strains remained photoautotrophic. In contrast, the absence of PSII-V and PSII-O in F363R produced obligate photoheterotrophic strains. The removal of PSII-U increased the susceptibility of PSII to heat inactivation and further decreased the stability of PSII in F363R, demonstrating that PSII-U can contribute to the stabilization of mutations that have been introduced into CP47. The order of importance of the selective removal of the extrinsic proteins in strains carrying mutations in loop E of CP47 was found to be as follows: DeltaPSII-V >/= DeltaPSII-O > DeltaPSII-U.     

Site-directed mutagenesis of the CP 47 protein of photosystem II: 167W in the lumenally exposed loop C is required for photosystem II assembly and stability

The intrinsic chlorophyll-protein CP 47 is a component of photosystem II which functions in both light-harvesting and oxygen evolution. Using site-directed mutagenesis we have produced the mutant W167S which lies in loop C of CP 47. This strain exhibited a 75% loss in oxygen evolution activity and grew extremely slowly in the absence of glucose. Examination of normalized oxygen evolution traces indicated that the mutant was susceptible to photoinactivation. Analysis of the variable fluorescence yield indicated that the mutant accumulated very few functional PS II reaction centers. This was confirmed by immunoblotting experiments. Interestingly, when W167S was grown in the presence of 20 M DCMU, the mutant continued to exhibit these defects. These results indicate that tryptophan 167 in loop C of CP 47 is important for the assembly and stability of the PS II reaction center.     

The role of ΔpH-dependent dissipation of excitation energy in protecting photosystem II against light-induced damage in Arabidopsis thaliana

The Arabidopsis thaliana subunit PsbS of photosystem II (PSII) is essential for the non-photochemical quenching of chlorophyll fluorescence and thus for ΔpH-dependent energy dissipation (qE). As a result of the excision of an En-transposon, a frameshift mutation in the psbS gene was obtained, which results in the complete absence of the PsbS protein and of qE. Two-dimensional gel analyses of thylakoid membranes indicated that the depletion of PsbS has no effect on PSII composition, excluding a structural role for PsbS in the organization of the PSII antenna. The susceptibility of mutant plants to photoinactivation of PSII was significantly increased during exposure to high light for up to 8 h. Divergence of mutant plants from wild-type levels of photoinactivation were most pronounced during the first 2 h of illumination, while after longer exposure times the rate of PSII inactivation were similar in both genotypes. The increased PSII inactivation in the mutant was not accompanied by an increased rate of D1 protein degradation, and recovery of PSII activity in the mutant under low light was similar or even faster in comparison to wild-type plants. However, growth under high light intensities resulted in decreased growth rates of psbs mutant plants. We conclude that energy dissipation in PSII related to qE is not primarily required for the protection of PSII against light-induced destruction, but may rather be involved in reducing the electron pressure on the photosynthetic electron transport chain at saturating light intensities.     

Rye photosystem II. Nucleotide sequence of the psbC gene coding 43- kDa chlorophyll(a)-binding proteins

The structure of the rye chloroplast DNA, which contains psbC gene coding for 43-kDa chlorophyll(a)-binding subunit of photosystem II, is determined. The sequence of trnS (UGA) gene encoding tRNA Ser is located at a distance of 140 bp downstream from the stop codon of psbC gene on the opposite DNA strand. The 5'-terminal part of psbC gene, like in other plants, overlaps by 50 bp the 3'-terminal region of psbD gene coding for D2 protein of photosystem II. The amino acid sequence of the psbC gene product reveals common features with the structure of the psbB gene product (CPa-1 protein). The structural similarity of these two proteins seems to reflect their similar functions.     

Alterations of the oxygen-evolving apparatus induced by a 305Arg --> 305Ser mutation in the CP43 protein of photosystem II from Synechocystis sp. PCC 6803 under chloride-limiting conditions

The psbC gene encodes CP43, a component of Photosystem II (PSII) in higher plants, algae, and cyanobacteria. Previous work demonstrated that alteration of an arginine residue occurring at position 305 to serine produced a strain (R305S) with altered PSII activity (Knoepfle, N., Bricker, T. M., and Putnam-Evans, C. (1999) Biochemistry 38, 1582-1588). This strain grew at wild-type rates in complete BG-11 media (480 microM chloride) and evolved oxygen at rates that were 60-70% of the observed wild-type rates. The R305S strain assembled approximately 70-80% of the functional PSII centers contained in the control strain, and these PSII centers were very sensitive to photoinactivation at high light intensities. We recently observed that the R305S mutant exhibited a pronounced chloride effect. When this mutant was grown in media depleted of chloride (30 microM chloride), it exhibited a severely reduced photoautotrophic growth rate. The effect of chloride depletion on the growth rate of the mutant was reversed by the addition of 480 microM bromide to the chloride-depleted BG-11 media. Oxygen evolution rates for the mutant were further depressed to about 22% of that observed in control cells under chloride-limiting conditions. Addition of bromide restored these rates to those observed under chloride-sufficient conditions. The mutant exhibited a significantly lower relative quantum yield for oxygen evolution than did the control strain, and this was exacerbated under chloride-limiting conditions. Fluorescence yield measurements indicated that both the mutant and the control strains assembled fewer PSII reaction centers under chloride-limiting conditions. The reaction centers assembled by the mutant exhibited an enhanced sensitivity to photoinactivation under chloride-limiting conditions, with a t(1/2) of photoinactivation of 2.6 min under chloride-limiting conditions as compared to a t(1/2) of 4.7 min under normal growth conditions. The mutant also exhibited an enhanced stability of its S(2) state and increased number of centers in the S(1) state following dark incubation. These results indicate that the mutant R305S exhibits a defect in its ability to utilize chloride in support of efficient oxygen evolution in PSII. This is the first mutant of this type described in the CP43 protein.     

Isolation and characterization of photoautotrophic mutants of Chlamydomonas reinhardtii deficient in state transition.

In photosynthetic cells of higher plants and algae, the distribution of light energy between photosystem I and photosystem II is controlled by light quality through a process called state transition. It involves a reorganization of the light-harvesting complex of photosystem II (LHCII) within the thylakoid membrane whereby light energy captured preferentially by photosystem II is redirected toward photosystem I or vice versa. State transition is correlated with the reversible phosphorylation of several LHCII proteins and requires the presence of functional cytochrome b(6)f complex. Most factors controlling state transition are still not identified. Here we describe the isolation of photoautotrophic mutants of the unicellular alga Chlamydomonas reinhardtii, which are deficient in state transition. Mutant stt7 is unable to undergo state transition and remains blocked in state I as assayed by fluorescence and photoacoustic measurements. Immunocytochemical studies indicate that the distribution of LHCII and of the cytochrome b(6)f complex between appressed and nonappressed thylakoid membranes does not change significantly during state transition in stt7, in contrast to the wild type. This mutant displays the same deficiency in LHCII phosphorylation as observed for mutants deficient in cytochrome b(6)f complex that are known to be unable to undergo state transition. The stt7 mutant grows photoautotrophically, although at a slower rate than wild type, and does not appear to be more sensitive to photoinactivation than the wild-type strain. Mutant stt3-4b is partially deficient in state transition but is still able to phosphorylate LHCII. Potential factors affected in these mutant strains and the function of state transition in C. reinhardtii are discussed.     

PsbQ (Sll1638) in Synechocystis sp. PCC 6803 is required for photosystem II activity in specific mutants and in nutrient-limiting conditions

A PsbQ homologue has been found associated with photosystem II complexes in Synechocystis sp. PCC 6803 where it is involved in optimal photoautotrophic growth and water splitting under CaCl(2)-depleted conditions [Thornton, L. E., Ohkawa, H., Roose, J. L., Kashino, Y., Keren, N., and Pakrasi, H. B. (2004) Plant Cell 16, 2164-2175]. By inactivating psbQ in strains carrying photosystem II-specific mutations, we have identified stringent requirements for PsbQ in vivo. Whereas under nutrient-replete conditions the DeltaPsbQ mutant was similar to wild type, a strain lacking PsbQ and PsbV was not photoautotrophic, exhibiting decreased oxygen evolution and decreased photosystem II assembly compared to the DeltaPsbV mutant. Combining the removal of PsbU and PsbQ introduced an altered requirement for Ca(2+) and Cl(-), and photoautotrophic growth of the DeltaPsbQ strain was prevented in nutrient-limiting media depleted in Ca(2+), Cl(-), and iron. Unlike other photosystem II extrinsic proteins PsbQ did not participate in the acquisition of thermotolerance; however, photoautotrophic growth at elevated temperatures was impaired in this mutant. Growth of the DeltaPsbV:DeltaPsbQ mutant was restored at pH 10.0: in contrast, an additional deletion between Arg-384 and Val-392 in the CP47 protein of photosystem II prevented recovery at alkaline pH. When conditions prevented photoautotrophy in strains lacking PsbQ, photoheterotrophic growth was indistinguishable to wild type, indicating that photosystem II had been inactivated. These data substantiate a role for PsbQ in optimizing photosystem II activity in Synechocystis sp. PCC 6803 and establish an absolute requirement for the subunit under specific biochemical and physiological conditions.     

Interaction of CPa-1 with the manganese-stabilizing protein of photosystem II: identification of domains on CPa-1 which are shielded from N-hydroxysuccinimide biotinylation by the manganese-stabilizing protein.

The structural organization of photosystem II proteins has been investigated by use of the amino group-labeling reagent N-hydroxysuccinimidobiotin (NHS-biotin) and calcium chloride-washed photosystem II membranes. We have previously shown that the presence of the extrinsic, manganese-stabilizing protein on photosystem II membranes prevents the modification of lysyl residues located on the chlorophyll protein CPa-1 (CP-47) by NHS-biotin [Bricker, T. M., Odom, W. R., & Queirolo, C. B. (1988) FEBS Lett. 231, 111-117]. Upon removal of the manganese-stabilizing protein by calcium chloride-washing, CPa-1 can be specifically modified by treatment with NHS-biotin. Preparative quantities of biotinylated CPa-1 were subjected to chemical cleavage with cyanogen bromide. Two major biotinylated peptides were identified with apparent molecular masses of 11.8 and 15.7 kDa. N-terminal sequence analysis of these peptides indicated that the 11.8-kDa peptide was 232G-330M and that the 15.7-kDa peptide was 360P-508V. The 15.7-kDa CNBr peptide was subjected to limited tryptic digestion. The two smallest tryptic fragments identified migrated at apparent molecular masses of 9.1 (nonbiotinylated) and 7.5 kDa (biotinylated). N-terminal sequence analysis and examination of the predicted amino acid sequences of these peptides suggest that the 9.1-kDa fragment was 422R-508V and that the 7.5-kDa fragment was 360P-421A. These results strongly suggest that two NHS-biotinylated domains, 304K-321K and 389K-419K, become exposed on CPa-1 when the manganese-stabilizing protein is removed by CaCl2 treatment. Both of these domains lie in the large extrinsic loop E of CPa-1.     

Tryptophan at position 181 of the D2 protein of photosystem II confers quenching of variable fluorescence of chlorophyll: implications for the mechanism of energy-dependent quenching.

The lumenal CD loop region of the D2 protein of photosystem II contains residues that interact with a reaction center chlorophyll and the redox-active Tyr(D). Using combinatorial mutagenesis, photoautotrophic mutants of Synechocystis sp. PCC 6803 have been generated with multiple amino acid changes in this region. The CD loop mutations were transferred into a photosystem I-less Synechocystis strain to facilitate characterization of photosystem II properties in the mutants. Most of the combinatorial photosystem I-less mutants obtained had a high yield of variable fluorescence, F(V). However, in three mutants, which shared a replacement of Phe181 by Trp, the F(V) yield was dramatically reduced although a high rate of oxygen evolution was maintained. A site-directed F181W D2 mutant shared similar properties. Picosecond time-resolved fluorescence measurements revealed that in the combinatorial F181W mutants the fluorescence lifetimes in closed and open photosystem II centers were essentially identical and were similar to the fluorescence lifetime in open centers of the control strain. These results are explained by quenching of variable fluorescence in the mutants by charge separation between Trp181 and excited reaction center chlorophyll. This reaction competes efficiently with fluorescence and nonradiative decay in closed photosystem II centers, where the lifetime of the excitation in the chlorophyll antenna is long. Thermodynamic considerations favor the formation of oxidized tryptophan and reduced chlorophyll in the quenching reaction, presumably followed by charge recombination. A possible role of tryptophan-chlorophyll charge separation in the mechanism of energy-dependent quenching of excitations in photosynthesis is discussed.     

Molecular cloning and targeted mutagenesis of the gene psaF encoding subunit III of photosystem I from the cyanobacterium Synechocystis sp. PCC 6803

Photosystem I is one of the two multisubunit pigment-protein complexes in the thylakoid membranes of cyanobacteria. Subunit III of photosystem I complex was isolated from a mutant of the cyanonbacterium Synechocystis sp PCC 6803, which lacks subunit II. The sequence of its NH2-terminal residues was determined and corresponding oligonucleotide probes were used to isolate the gene encoding this subunit. The gene, designated as psaF, codes for a mature protein of 15705 Da that is synthesized with a 23-amino acid extension. The deduced amino acid sequence is homologous to subunit III from spinach and Chlamydomonas reinhardtii. The presequence of subunit III shows characteristics typical of bacterial presequences and exhibits remarkable amino acid identity around the proteolytic processing site when compared to corresponding regions from the precursors of eukaryotic subunit III. There are two conserved hydrophobic regions in the mature subunit III which may cross or interact with thylakoid membrane. The gene psaF exists as a single copy in the genome and is expressed as a monocistronic RNA. A stable mutant strain in which the gene psaF was replaced by a gene conferring resistance to kanamycin was generated by targeted mutagenesis. Photoautotrophic growth of the mutant strain was comparable with that of the wild type suggesting that function of subunit III is dispensable for photosynthesis in Synechocystis sp. PCC 6803. Addition of more MgSO4 to BG11 medium enhanced growth of the mutant strain but not of the wild type cells.     

Involvement of the SppA1 peptidase in acclimation to saturating light intensities in Synechocystis sp. strain PCC 6803

The sll1703 gene, encoding an Arabidopsis homologue of the thylakoid membrane-associated SppA peptidase, was inactivated by interposon mutagenesis in Synechocystis sp. strain PCC 6803. Upon acclimation from a light intensity of 50 to 150 microE m(-2) s(-1), the mutant preserved most of its phycobilisome content, whereas the wild-type strain developed a bleaching phenotype due to the loss of about 40% of its phycobiliproteins. Using in vivo and in vitro experiments, we demonstrate that the DeltasppA1 strain does not undergo the cleavage of the L(R)(33) and L(CM)(99) linker proteins that develops in the wild type exposed to increasing light intensities. We conclude that a major contribution to light acclimation under a moderate light regime in cyanobacteria originates from an SppA1-mediated cleavage of phycobilisome linker proteins. Together with changes in gene expression of the major phycobiliproteins, it contributes an additional mechanism aimed at reducing the content in phycobilisome antennae upon acclimation to a higher light intensity.     

Molecular analysis of a mutant defective in photosynthetic oxygen evolution and isolation of a complementing clone by a novel screening procedure.

Photosynthesis-defective mutants of the transformable cyanobacterium Synechocystis 6803 have been isolated following nitrosoguanidine mutagenesis. The photosystem II- phenotype of one of these mutants is shown by DNA sequencing to be attributable to a short deletion in psbC, the gene encoding the 44-kd, chlorophyll-binding protein of photosystem II. Although not a component of the reaction center of photosystem II, the 44-kd protein is none the less shown to be essential in vivo for photosystem II activity. The deletion in psbC also results in greatly diminished levels of D-2 (a component of the reaction center of photosystem II) indicating that the loss of the product of the psbC gene affects the assembly or stability of the photosystem II reaction center. The isolation of a clone capable of restoring both photosystem II activity and photoautotrophy to the mutant cells was aided by the observation that restriction fragments or cloned Synechocystis 6803 DNA applied in liquid or in melted agarose directly onto a lawn of Synechocystis 6803 will lead to the transformation of the cells. This in situ 'dot' transformation procedure provides a convenient method for the rapid identification of fractions or clones containing complementing Synechocystis 6803 DNA.