A novel protein involved in the functional assembly of the oxygen-evolving complex of photosystem II in Synechocystis sp. PCC 6803

Mutation of Glu69 to Gln in the D2 protein of photosystem II is known to lead to a loss of photoautotrophic growth in Synechocystis sp. PCC 6803. However, second-site mutants (pseudorevertants) with restored photoautotrophic growth but still maintaining the E69Q mutation in D2 are easily obtained. Using a genomic mapping technique involving functional complementation, the secondary mutation was mapped to slr0286 in two independent mutants. The mutations in Slr0286 were R42M or R394H. To study the function of Slr0286, mutants of E69Q and of the wild-type strain were made that lacked slr0286. Deletion of slr0286 did not affect photoautotrophic capacity in wild type but led to a marked decrease in the apparent affinity of Ca(2+) to its binding site at the water-splitting system of photosystem II and to a reduced heat tolerance of the oxygen-evolving system, particularly in E69Q. Moreover, a small increase in the half-time for photoactivation of the oxygen-evolving complex of photosystem II for both wild type and the E69Q mutant was observed in the absence of Slr0286. The accumulation of photosystem II reaction centers, dark stability of the oxygen-evolving apparatus, stability of oxygen evolution, and the kinetics of charge recombination between Q(A)(-) and the donor side were not affected by deletion of slr0286. Slr0286 lacks clear functional motifs, and no homologues are apparent in other organisms, even not in other cyanobacteria. In any case, Slr0286 appears to help the functional assembly and stability of the water-splitting system of photosystem II.     

Targeted random mutagenesis to identify functionally important residues in the D2 protein of photosystem II in Synechocystis sp. strain PCC 6803

To identify important residues in the D2 protein of photosystem II (PSII) in the cyanobacterium Synechocystis sp. strain PCC 6803, we randomly mutagenized a region of psbDI (coding for a 96-residue-long C-terminal part of D2) with sodium bisulfite. Mutagenized plasmids were introduced into a Synechocystis sp. strain PCC 6803 mutant that lacks both psbD genes, and mutants with impaired PSII function were selected. Nine D2 residues were identified that are important for PSII stability and/or function, as their mutation led to impairment of photoautotrophic growth. Five of these residues are likely to be involved in the formation of the Q(A)-binding niche; these are Ala249, Ser254, Gly258, Ala260, and His268. Three others (Gly278, Ser283, and Gly288) are in transmembrane alpha-helix E, and their alteration leads to destabilization of PSII but not to major functional alterations of the remaining centers, indicating that they are unlikely to interact directly with cofactors. In the C-terminal lumenal tail of D2, only one residue (Arg294) was identified as functionally important for PSII. However, from the number of mutants generated it is likely that most or all of the 70 residues that are susceptible to bisulfite mutagenesis have been altered at least once. The fact that mutations in most of these residues have not been picked up by our screening method suggests that these mutations led to a normal photoautotrophic phenotype. A novel method of intragenic complementation in Synechocystis sp. strain PCC 6803 was developed to facilitate genetic analysis of psbDI mutants containing several amino acid changes in the targeted domain. Recombination between genome copies in the same cell appears to be much more prevalent in Synechocystis sp. strain PCC 6803 than was generally assumed.     

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.     

Four novel genes required for optimal photoautotrophic growth of the cyanobacterium Synechocystis sp. strain PCC 6803 identified by in vitro transposon mutagenesis

Four novel Synechocystis sp. strain PCC 6803 genes (sll1495, sll0804, slr1306, and slr1125) which encode hypothetical proteins were determined by transposon mutagenesis to be required for optimal photoautotrophic growth. Mutations were also recovered in ccmK4, a carboxysome coat protein homologue, and me, the decarboxylating NADP(+)-dependent malic enzyme. This is the first report that these known genes are required for optimal photoautotrophy.     

Slr2013 is a novel protein regulating functional assembly of photosystem II in Synechocystis sp. strain PCC 6803

The Synechocystis sp. strain PCC 6803, which has a T192H mutation in the D2 protein of photosystem II, is an obligate photoheterotroph due to the lack of assembled photosystem II complexes. A secondary mutant, Rg2, has been selected that retains the T192H mutation but is able to grow photoautotrophically. Restoration of photoautotrophic growth in this mutant was caused by early termination at position 294 in the Slr2013 protein. The T192H mutant with truncated Slr2013 forms fully functional photosystem II reaction centers that differ from wild-type reaction centers only by a 30% higher rate of charge recombination between the primary electron acceptor, QA-, and the donor side and by a reduced stability of the oxidized form of the redox-active Tyr residue, YD, in the D2 protein. This suggests that the T192H mutation itself did not directly affect electron transfer components, but rather affected protein folding and/or stable assembly of photosystem II, and that Slr2013 is involved in the folding of the D2 protein and the assembly of photosystem II. Besides participation in photosystem II assembly, Slr2013 plays a critical role in the cell, because the corresponding gene cannot be deleted completely under conditions in which photosystem II is dispensable. Truncation of Slr2013 by itself does not affect photosynthetic activity of Synechocystis sp. strain PCC 6803. Slr2013 is annotated in CyanoBase as a hypothetical protein and shares a DUF58 family signature with other hypothetical proteins of unknown function. Genes for close homologues of Slr2013 are found in other cyanobacteria (Nostoc punctiforme, Anabaena sp. strain PCC 7120, and Thermosynechococcus elongatus BP-1), and apparent orthologs of this protein are found in Eubacteria and Archaea, but not in eukaryotes. We suggest that Slr2013 regulates functional assembly of photosystem II and has at least one other important function in the cell.     

The synechocystis sp PCC 6803 oxa1 homolog is essential for membrane integration of reaction center precursor protein pD1

Synechocystis sp PCC 6803 Slr1471p, an Oxa1p/Alb3/YidC homolog, is an essential protein for cell viability for which functions in thylakoid membrane biogenesis and cell division have been proposed. Using a fusion of green fluorescent protein to the C terminus of Slr1471p, we found that the mutant slr1471-gfp is photochemically inhibited when light intensities increase to 80 micromol x m(-2) x s(-1). We show that photoinhibition correlates with an increased redox potential of the reaction center quinone Q(A)(-) and a decreased redox potential of Q(B)(-). Analysis reveals that membrane integration of the D1 precursor protein is affected, leading to the accumulation of pD1 in the membrane phase. We show that Slr1471p interacts directly with the D1 protein and discuss why the accumulation of pD1 in two reaction center assembly intermediates is dependent on Slr1471p.     

Slr1122影响Hik12磷酸化并参与调节Synechocystis sp.PCC 6803的色素合成

Synechocystis sp.PCC 6803是一种良好的研究光合作用的模式生物,其中slr1122编码一个250个氨基酸的未知蛋白。据报道Slr1122可能与杂合传感激酶（hybrid sensory kinase）Sll1672（Hik12）相互作用,本研究通过复合物实验证实了Slr1122与Sll1672确实存在相互作用。利用32P标记证明,在加入Slr1122后Hik12的磷酸化受到了明显的影响,推测其可能参与该双组分系统的调控。通过同源双交换,用卡那霉素抗性基因替换slr1122,将slr1122从Synechocystis sp.PCC 6803中敲除,构建了slr1122的缺失体Δslr1122。研究发现在Δslr1122中,编码PSⅡ中核心蛋白D1亚基的slr1181（psbAI）的转录水平明显降低,使PSⅡ光合作用受到影响,导致Δslr1122的生长速率低于野生型（WT）。同时slr1122的缺失使得蓝细菌对光的敏感性增强,在弱光条件下,Δslr1122对光能的利用效率高于WT,其生长速率也较WT高,但与此相反,Δslr1122对强光的耐受力及生长速率则不及WT。Δslr1122体内的藻胆蛋白含量与色素含量均降低,尤其是类胡萝卜素,RT-PCR的结果也显示合成类胡萝卜素过程中的5个关键酶转录水平均下降。这可能是Δslr1122对氧化胁迫变得敏感的原因之一。总之,Slr1122影响杂合传感激酶Hik12磷酸化并参与调节Synechocystis sp.PCC 6803的光合色素合成。     

Discovery of a Chlorophyll Binding Protein Complex Involved in the Early Steps of Photosystem II Assembly in Synechocystis

Efficient assembly and repair of the oxygen-evolving photosystem II (PSII) complex is vital for maintaining photosynthetic activity in plants, algae, and cyanobacteria. How chlorophyll is delivered to PSII during assembly and how vulnerable assembly complexes are protected from photodamage are unknown. Here, we identify a chlorophyll and β-carotene binding protein complex in the cyanobacterium Synechocystis PCC 6803 important for formation of the D1/D2 reaction center assembly complex. It is composed of putative short-chain dehydrogenase/reductase Ycf39, encoded by the slr0399 gene, and two members of the high-light-inducible protein (Hlip) family, HliC and HliD, which are small membrane proteins related to the light-harvesting chlorophyll binding complexes found in plants. Perturbed chlorophyll recycling in a Ycf39-null mutant and copurification of chlorophyll synthase and unassembled D1 with the Ycf39-Hlip complex indicate a role in the delivery of chlorophyll to newly synthesized D1. Sequence similarities suggest the presence of a related complex in chloroplasts.     

Identification of two genes, sll0804 and slr1306, as putative components of the CO2-concentrating mechanism in the cyanobacterium Synechocystis sp. strain PCC 6803

Insertional transposon mutations in the sll0804 and slr1306 genes were found to lead to a loss of optimal photoautotrophy in the cyanobacterium Synechocystis sp. strain PCC 6803 grown under ambient CO₂ concentrations (350 ppm). Mutants containing these insertions (4BA2 and 3ZA12, respectively) could grow photoheterotrophically on glucose or photoautotrophically at elevated CO₂ concentrations (50,000 ppm). Both of these mutants exhibited an impaired affinity for inorganic carbon. Consequently, the Sll0804 and Slr1306 proteins appear to be putative components of the carbon-concentrating mechanism in Synechocystis sp. strain PCC 6803.     

Type 2 NADH Dehydrogenases in the Cyanobacterium Synechocystis sp. Strain PCC 6803 Are Involved in Regulation Rather Than Respiration

Analysis of the genome of Synechocystis sp. strain PCC 6803 reveals three open reading frames (slr0851, slr1743, and sll1484) that may code for type 2 NAD(P)H dehydrogenases (NDH-2). The sequence similarity between the translated open reading frames and NDH-2s from other organisms is low, generally not exceeding 30% identity. However, NAD(P)H and flavin adenine dinucleotide binding motifs are conserved in all three putative NDH-2s in Synechocystis sp. strain PCC 6803. The three open reading frames were cloned, and deletion constructs were made for each. An expression construct containing one of the three open reading frames, slr1743, was able to functionally complement an Escherichia coli mutant lacking both NDH-1s and NDH-2s. Therefore, slr0851, slr1743, and sll1484 have been designated ndbA, ndbB, and ndbC, respectively. Strains that lacked one or more of the ndb genes were created in wild-type and photosystem (PS) I-less backgrounds. Deletion of ndb genes led to small changes in photoautotrophic growth rates and respiratory activities. Electron transfer rates into the plastoquinone pool in thylakoids in darkness were consistent with the presence of a small amount of NDH-2 activity in thylakoids. No difference was observed between wild-type and the Ndb-less strains in the banding patterns seen on native gels when stained for either NADH or NADPH dehydrogenase activity, indicating that the Ndb proteins do not accumulate to high levels. A striking phenotype of the PS I-less background strains lacking one or more of the NDH-2s is that they were able to grow at high light intensities that were lethal to the control strain but they retained normal PS II activity. We suggest that the Ndb proteins in Synechocystis sp. strain PCC 6803 are redox sensors and that they play a regulatory role responding to the redox state of the plastoquinone pool.     

The malic enzyme is required for optimal photoautotrophic growth of Synechocystis sp. strain PCC 6803 under continuous light but not under a diurnal light regimen

A mutation was recovered in the slr0721 gene, which encodes the decarboxylating NADP(+)-dependent malic enzyme in the cyanobacterium Synechocystis sp. strain PCC 6803, yielding the mutant 3WEZ. Under continuous light, 3WEZ exhibits poor photoautotrophic growth while growing photoheterotrophically on glucose at rates nearly indistinguishable from wild-type rates. Interestingly, under diurnal light conditions (12 h of light and 12 h of dark), normal photoautotrophic growth of the mutant is completely restored.     

Slr1293 in Synechocystis sp. strain PCC 6803 Is the C-3',4' desaturase (CrtD) involved in myxoxanthophyll biosynthesis

When grown at high light intensity, more than a quarter of the total carotenoids in the unicellular cyanobacterium Synechocystis consists of myxoxanthophyll, a polar carotenoid glycoside. The biosynthetic pathway of myxoxanthophyll is unknown but is presumed to involve a number of enzymes, including a C-3',4' desaturase required to add one double bond to generate 11 conjugated double bonds in the monocyclic myxoxanthophyll. A candidate for this desaturase is Slr1293, which was identified by genome similarity searching. To determine whether Slr1293 is a desaturase recognizing neurosporene and lycopene, slr1293 was expressed in Escherichia coli strains accumulating neurosporene or lycopene. Confirming such a desaturase function for Slr1293, these E. coli strains accumulated 3',4'-didehydroneurosporene and 3',4'-didehydrolycopene, respectively. Indeed, deletion of slr1293 in Synechocystis provides further evidence that Slr1293 is a desaturase recognizing neurosporene: In the slr1293 deletion mutant, neurosporene was found to accumulate and was further processed to produce neurosporene glycoside. Neurosporene hereby becomes a primary candidate to be the branch point molecule between carotene and myxoxanthophyll biosynthesis in this cyanobacterium. The slr1293 gene was concluded to encode a C-3',4' desaturase that is essential for myxoxanthophyll biosynthesis, and thus it was designated as crtD. Furthermore, as Slr1293 appears to recognize neurosporene and to catalyze the first committed step on the myxoxanthophyll biosynthesis pathway, Slr1293 plays a pivotal role in directing a portion of the precursor pool for carotenoid biosynthesis toward myxoxanthophyll biosynthesis in Synechocystis sp. strain PCC 6803.     

Late assembly steps and dynamics of the cyanobacterial photosystem I

The dynamics of photosystem I assembly in cyanobacteria have been addressed using in vivo pulse-chase labeling of Synechocystis sp. PCC 6803 proteins in combination with blue native polyacrylamide gel electrophoresis. The analyses indicate the existence of three different monomeric photosystem I complexes and also the high stability of photosystem I trimers. We show that in addition to a complete photosystem I monomer, containing all 11 subunits, we detected a PsaK-less monomer and a short-lived PsaL/PsaK-less complex. The latter two monomers were missing in the ycf37 mutant of Synechocystis sp. PCC 6803 that accumulates also less trimers. Pulse-chase experiments suggest that the three monomeric complexes have different functions in the biogenesis of the trimer. Based on these findings we propose a model where PsaK is incorporated in the latest step of photosystem I assembly. The PsaK-less photosystem I monomer may represent an intermediate complex that is important for the exchange of the two PsaK variants during high light acclimation. Implications of the presented data with respect to Ycf37 function are discussed.     

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.