Cloning of the psbK gene from Synechocystis sp. PCC 6803 and characterization of photosystem II in mutants lacking PSII-K

We cloned and sequenced the psbK gene, coding for a small photosystem II component (PSII-K), from the transformable cyanobacterium, Synechocystis sp. PCC 6803, and determined the N-terminal sequence of mature PSII-K. The psbK gene product is processed by cleaving off eight amino acid residues from the N terminus. A mutant lacking psbK was constructed; this mutant grew photoautotrophically, but its growth rate was reduced. The number of photosystem II reaction centers on a chlorophyll basis was decreased by less than a factor of 2 in the psbK-deletion mutant. In Synechocystis sp. PCC 6803, the psbK gene is transcribed as a single gene and is not part of an operon. Single-site mutations were introduced into psbK leading to early termination or deletion of the presequence. The phenotype of these mutants strongly resembles that of the psbK deletion mutant, indicating that indeed the change in phenotype in the deletion mutant is directly correlated with PSII-K. PSII-K is not essential for photosystem II assembly or activity but is needed for optimal photosystem II function.     

The establishment of conditions to efficiently screen photosynthesis-deficient mutants of Synechocystis sp. PCC 6803 by nitrofurantoin treatment

A method based on the susceptibility of photosynthetic organisms to nitrofurantoin under illumination was established to screen mutants of Synechocystis sp. PCC 6803 deficient in the function of photosystem II, which were created by random PCR mutagenesis targeted to the psbAII gene coding for the D1 protein of the photosystem II reaction center. In this method, cyanobacterial colonies on a nitrocellulose membrane on a BG11 agar plate were treated with nitrofurantoin at 1.0 mM under white light at 40 microE x m(-2 ) x s(-1) for 2 h, and then kept under normal conditions without nitrofurantoin so that surviving cells could grow. This method was also shown to be useful for screening mutants deficient in the function of photosystem I.     

Deletion Mutagenesis of the Cytochrome b559 Protein Inactivates the Reaction Center of Photosystem II

In green plant-like photosynthesis, oxygen evolution is catalyzed by a thylakoid membrane-bound protein complex, photosystem II. Cytochrome b559, a protein component of the reaction center of this complex, is absent in a genetically engineered mutant of the cyanobacterium, Synechocystis 6803 [Pakrasi, H.B., Williams, J.G.K., and Arntzen, C.J. (1988). EMBO J. 7, 325-332]. In this mutant, the genes psbE and psbF, encoding cytochrome b559, were deleted by targeted mutagenesis. Two other protein components, D1 and D2 of the photosystem II reaction center, are also absent in this mutant. However, two chlorophyll-binding proteins, CP47 and CP43, as well as a manganese-stabilizing extrinsic protein component of photosystem II are stably assembled in the thylakoids of this mutant. Thus, this deletion mutation destabilizes the reaction center of photosystem II only. The mutant also lacks a fluorescence maximum peak at 695 nm (at 77 K) even though the CP47 protein, considered to be the origin of this fluorescence peak, is present in this mutant. We propose that the fluorescence at 695 nm originates from an interaction between the reaction center of photosystem II and CP47. The deletion mutant shows the absence of variable fluorescence at room temperature, indicating that its photosystem II complex is photochemically inactive. Also, photoreduction of QA, the primary acceptor quinone in photosystem II, could not be detected in the mutant. We conclude that cytochrome b559 plays at least an essential structural role in the reaction center of photosystem II.     

The carboxyl-terminal extension of the precursor D1 protein of photosystem II is required for optimal photosynthetic performance of the cyanobacterium Synechocystis sp. PCC 6803

The D1 protein is an integral component of the photosystem II reaction center complex. In the cyanobacterium Synechocystis sp. PCC 6803, D1 is synthesized with a short 16-amino acids-long carboxyl-terminal extension. Removal of this extension is necessary to form active oxygen-evolving photosystem II centers. Our earlier studies have shown that this extension is cleaved by CtpA, a specific carboxyl-terminal processing protease. The amino acid sequence of the carboxyl-terminal extension is conserved among D1 proteins from different organisms, although at a level lower than that of the mature protein. In the present study we have analyzed a mutant strain of Synechocystis sp. PCC 6803 with a duplicated extension, and a second mutant that lacks the extension, to investigate the effects of these alterations on the function of the D1 protein in vivo. No significant difference in the growth rates, photosynthetic pigment composition, fluorescence induction, and oxygen evolution rates was observed between the mutants and the control strain. However, using long-term mixed culture growth analysis, we detected significant decreases in the fitness of these mutant strains. The presented data demonstrate that the carboxyl-terminal extension of the precursor D1 protein is required for optimal photosynthetic performance.     

Photosystem II function and integrity in spite of drastic protein changes in a conserved region of the D2 protein.

D1 and D2 are structurally related proteins forming the core of the photosystem II reaction center. The two proteins have several loop regions including an extended stroma-exposed loop between transmembrane helix D and parallel helix de. This loop (the D-de loop) is phylogenetically conserved in both proteins. The role of the D-de loop in photosystem II was studied in Synechocystis sp. PCC 6803 by constructing a chimeric D2 protein in which the stroma-exposed loop of D1 replaced that of D2. In one of the transgenic lines, a single-base deletion shifted the reading frame of the chimeric gene leading to loss of D2 accumulation and photosystem II assembly. Selection for spontaneous reversion to photoautotrophy yielded several suppressor mutants, five of which were analyzed. In all, further frameshifts in the inserted loop piece restored the original reading frame allowing readthrough to the normal carboxy terminus. However, the sequences in the restored D-de loop varied widely among the mutants. Changes ranged from a deletion of one amino acid residue to an insertion of 31, while the net charge of the D-de loop increased by up to 12 units. Mutant electron transfer rates and photoautotrophic growth were only mildly affected as compared to wild type. Nevertheless, in all mutants, the hydropathy profile of the stroma-exposed D-de loop region maintained its hydrophilic character including turns in similar locations. We conclude that the stroma-exposed, D-de loop of the D2 protein can accommodate drastic composition and size changes without extensive functional consequences in photosystem II.(ABSTRACT TRUNCATED AT 250 WORDS)     

Structure and targeted mutagenesis of the gene encoding 8-kDa subunit of photosystem I from the cyanobacterium Synechocystis sp. PCC 6803

Photosystem I reaction center of the cyanobacterium Synechocystis sp. PCC 6803 contains seven different polypeptide subunits. The subunit with a molecular mass of about 8 kDa was isolated, and the sequence of its amino-terminal residues was determined. Oligonucleotide probes corresponding to this sequence were used to isolate the gene encoding this subunit. The gene, termed as psaE, codes for a polypeptide with a mass of 8075 Da. It is present as a single copy in the genome and is transcribed as a monocistronic messenger. The amino acid sequence of the 8-kDa subunit deduced from the gene sequence shows high homology with the deduced amino acid sequence of subunit IV of photosystem I from spinach. The DNA fragment sequenced in these studies also contains two other unidentified major open reading frames. A stable deletion mutation for the psaE gene was generated by transforming Synechocystis sp. PCC 6803 with a cloned DNA in which the psaE gene for 8-kDa subunit was replaced by a gene conferring resistance to kanamycin. The mutant strain shows minor differences in growth under photoautotrophic conditions and in the photosystem I activity in comparison to the wild type.     

The BtpA protein stabilizes the reaction center proteins of photosystem I in the cyanobacterium Synechocystis sp. PCC 6803 at low temperature

Specific inhibition of photosystem I (PSI) was observed under low-temperature conditions in the cyanobacterium Synechocystis sp. strain PCC 6803. Growth at 20 degrees C caused inhibition of PSI activity and increased degradation of the PSI reaction center proteins PsaA and PsaB, while no significant changes were found in the level and activity of photosystem II (PSII). BtpA, a recently identified extrinsic thylakoid membrane protein, was found to be a necessary regulatory factor for stabilization of the PsaA and PsaB proteins under such low-temperature conditions. At normal growth temperature (30 degrees C), the BtpA protein was present in the cell, and its genetic deletion caused an increase in the degradation of the PSI reaction center proteins. However, growth of Synechocystis cells at 20 degrees C or shifting of cultures grown at 30 degrees C to 20 degrees C led to a rapid accumulation of the BtpA protein, presumably to stabilize the PSI complex, by lowering the rates of degradation of the PsaA and PsaB proteins. A btpA deletion mutant strain could not grow photoautotrophically at low temperature, and exhibited rapid degradation of the PSI complex after transfer of the cells from normal to low temperature.     

Targeted mutagenesis of the psbE and psbF genes blocks photosynthetic electron transport: evidence for a functional role of cytochrome b559 in photosystem II.

The genes encoding the two subunits (alpha and beta) of the cytochrome b559 (cyt b559) protein, psbE and psbF, were cloned from the unicellular, transformable cyanobacterium, Synechocystis 6803. Cyt b559, an intrinsic membrane protein, is a component of photosystem II, a membrane-protein complex that catalyzes photosynthetic oxygen evolution. However, the role of cyt b559 in photosynthetic electron transport is yet to be determined. A high degree of homology was found between the cyanobacterial and green plant chloroplastidic psbE and psbE genes and in the amino acid sequences of their corresponding protein products. Cartridge mutagenesis techniques were used to generate a deletion mutant of Synechocystis 6803 in which the psbE and psbF genes were replaced by a kanamycin-resistance gene cartridge. Physiological analyses indicated that the PSII complexes of the mutant were inactivated. We conclude that cyt b559 is an essential component of PSII.     

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.     

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.     

Role of the carboxy terminus of polypeptide D1 in the assembly of a functional water-oxidizing manganese cluster in photosystem II of the cyanobacterium Synechocystis sp. PCC 6803: assembly requires a free carboxyl group at C-terminal position 344.

The D1 polypeptide of the photosystem II (PSII) reaction center is synthesized as a precursor polypeptide which is posttranslationally processed at the carboxy terminus. It has been shown in spinach that such processing removes nine amino acids, leaving Ala344 as the C-terminal residue [Takahashi, M., Shiraishi, T., & Asada, K. (1988) FEBS Lett. 240, 6-8; Takahashi, Y., Nakane, H., Kojima, H., & Satoh, K. (1990) Plant Cell Physiol. 31, 273-280]. We show here that processing on the carboxy side of Ala344 also occurs in the cyanobacterium Synechocystis 6803, resulting in the removal of 16 amino acids. By constructing a deletion strain of Synechocystis 6803 that lacks the three copies of the psbA gene encoding D1, we have developed a system for generating psbA mutants. Using this system, we have constructed mutants of Synechocystis 6803 that are modified in the region of the C-terminus of the D1 polypeptide. Characterization of these mutants has revealed that (1) processing of the D1 polypeptide is blocked when the residue after the cleavage site is changed from serine to proline (mutant Ser345Pro) with the result that the manganese cluster is unable to assemble correctly; (2) the C-terminal extension of 16 amino acid residues can be deleted with little consequence either for insertion of D1 into the thylakoid membrane or for assembly of D1 into a fully active PSII complex; (3) removal of only one more residue (mutant Ala344stop) results in a loss of assembly of the manganese cluster; and (4) the ability of detergent-solubilized PSII core complexes (lacking the manganese cluster) to bind and oxidize exogenous Mn2+ by the secondary donor, Z+, is largely unaffected in the processing mutants (the Ser345Pro mutant of Synechocystis 6803 and the LF-1 mutant of Scenedesmus obliquus) and the truncation mutant Ala344stop. Our results are consistent with a role for processing in regulating the assembly of the photosynthetic manganese cluster and a role for the free carboxy terminus of the mature D1 polypeptide in the ligation of one or more manganese ions of the cluster.     

D1' centers are less efficient than normal photosystem II centers

One prominent difference between the photosystem II (PSII) reaction center protein D1' in Synechocystis 6803 and normal D1 is the replacement of Phe-186 in D1 with leucine in D1'. Mutants of Synechocystis 6803 producing only D1', or containing engineered D1 proteins with Phe-186 substitutions, were analyzed by 77 K fluorescence emission spectra, chlorophyll a fluorescence induction yield and decay kinetics, and flash-induced oxygen evolution. Compared to D1-containing PSII centers, D1' centers exhibited a 50% reduction in variable chlorophyll a fluorescence yield, while the flash-induced O(2) evolution pattern was unaffected. In the F186 mutants, both the P680(+)/Q(A)(-) recombination and O(2) oscillation pattern were noticeably perturbed.     

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.     

Oxidative stress inhibits the repair of photodamage to the photosynthetic machinery

Absorption of excess light energy by the photosynthetic machinery results in the generation of reactive oxygen species (ROS), such as H2O2. We investigated the effects in vivo of ROS to clarify the nature of the damage caused by such excess light energy to the photosynthetic machinery in the cyanobacterium Synechocystis sp. PCC 6803. Treatments of cyanobacterial cells that supposedly increased intracellular concentrations of ROS apparently stimulated the photodamage to photosystem II by inhibiting the repair of the damage to photosystem II and not by accelerating the photodamage directly. This conclusion was confirmed by the effects of the mutation of genes for H2O2-scavenging enzymes on the recovery of photosystem II. Pulse labeling experiments revealed that ROS inhibited the synthesis of proteins de novo. In particular, ROS inhibited synthesis of the D1 protein, a component of the reaction center of photosystem II. Northern and western blot analyses suggested that ROS might influence the outcome of photodamage primarily via inhibition of translation of the psbA gene, which encodes the precursor to D1 protein.     

Relationship between excitation energy transfer, trapping, and antenna size in photosystem II

We present a systematic study of the effect of antenna size on energy transfer and trapping in photosystem II. Time-resolved fluorescence experiments have been used to probe a range of particles isolated from both higher plants and the cyanobacterium Synechocystis 6803. The isolated reaction center dynamics are represented by a quasi-phenomenological model that fits the extensive time-resolved data from photosystem II reaction centers and reaction center mutants. This representation of the photosystem II "trapping engine" is found to correctly predict the extent of, and time scale for, charge separation in a range of photosystem II particles of varying antenna size (8-250 chlorins). This work shows that the presence of the shallow trap and slow charge separation kinetics, observed in isolated D1/D2/cyt b559 reaction centers, are indeed retained in larger particles and that these properties are reflected in the trapping dynamics of all larger photosystem II preparations. A shallow equilibrium between the antennae and reaction center in photosystem II will certainly facilitate regulation via nonphotochemical quenching, and one possible interpretation of these findings is therefore that photosystem II is optimized for regulation rather than for efficiency.     

Accumulation of the D2 protein is a key regulatory step for assembly of the photosystem II reaction center complex in Synechocystis PCC 6803

Accumulation of monomer and dimer photosystem (PS) II reaction center core complexes has been analyzed by two-dimensional Blue-native/SDS-PAGE in Synechocystis PCC 6803 wild type and in mutant strains lacking genes psbA, psbB, psbC, psbDIC/DII, or the psbEFLJ operon. In vivo pulse-chase radiolabeling experiments revealed that mutant cells assembled PSII precomplexes only. In DeltapsbC and DeltapsbB, assembly of reaction center cores lacking CP43 and reaction center complexes was detected, respectively. In DeltapsbA, protein subunits CP43, CP47, D2, and cytochrome b559 were synthesized, but proteins did not assemble. Similarly, in DeltapsbD/C lacking D2, and CP43, the de novo synthesized proteins D1, CP47, and cytochrome b559 did not form any mutual complexes, indicating that assembly of the reaction center complex is a prerequisite for assembly with core subunits CP47 and CP43. Finally, although CP43 and CP47 accumulated in DeltapsbEFLJ, D2 was neither expressed nor accumulated. We, furthermore, show that the amount of D2 is high in the strain lacking D1, whereas the amount of D1 is low in the strain lacking D2. We conclude that expression of the psbEFLJ operon is a prerequisite for D2 accumulation that is the key regulatory step for D1 accumulation and consecutive assembly of the PSII reaction center complex.