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.     

Inactivation of the open reading frame slr0399 in Synechocystis sp. PCC 6803 functionally complements mutations near the Q(A) niche of photosystem II. A possible role of Slr0399 as a chaperone for quinone binding.

The Synechocystis sp. PCC 6803 triple mutant D2R8 with V247M/A249T/M329I mutations in the D2 subunit of the photosystem II is impaired in Q(A) function, has an apparently mobile Q(A), and is unable to grow photoautotrophically. Several photoautotrophic pseudorevertants of this mutant have been isolated, each of which retained the original psbDI mutations of D2R8. Using a newly developed mapping technique, the site of the secondary mutations has been located in the open reading frame slr0399. Two different nucleotide substitutions and a deletion of about 60% of slr0399 were each shown to restore photoautotrophy in different pseudorevertants of the mutant D2R8, suggesting that inactivation of Slr0399 leads to photoautotrophic growth in D2R8. Indeed, a targeted deletion of slr0399 restores photoautotrophy in D2R8 and in other psbDI mutants impaired in Q(A) function. Slr0399 is similar to the hypothetical protein Ycf39, which is encoded in the cyanelle genome of Cyanophora paradoxa; in the chloroplast genomes of diatoms, dinoflagellates, and red algae; and in the nuclear genome of Arabidopsis thaliana. Slr0399 and Ycf39 have a NAD(P)H binding motif near their N terminus and have some similarity to isoflavone reductase-like proteins and to a subunit of the eukaryotic NADH dehydrogenase complex I. Deletion of slr0399 in wild type Synechocystis sp. PCC 6803 has no significant phenotypic effects other than a decrease in thermotolerance under both photoautotrophic and photomixotrophic conditions. We suggest that Slr0399 is a chaperone-like protein that aids in, but is not essential for, quinone insertion and protein folding around Q(A) in photosystem II. Moreover, as the effects of Slr0399 are not limited to photosystem II, this protein may also be involved in assembly of quinones in other photosynthetic and respiratory complexes.     

The role of the FtsH and Deg proteases in the repair of UV-B radiation-damaged Photosystem II in the cyanobacterium Synechocystis PCC 6803

The photosystem two (PSII) complex found in oxygenic photosynthetic organisms is susceptible to damage by UV-B irradiation and undergoes repair in vivo to maintain activity. Until now there has been little information on the identity of the enzymes involved in repair. In the present study we have investigated the involvement of the FtsH and Deg protease families in the degradation of UV-B-damaged PSII reaction center subunits, D1 and D2, in the cyanobacterium Synechocystis 6803. PSII activity in a DeltaFtsH (slr0228) strain, with an inactivated slr0228 gene, showed increased sensitivity to UV-B radiation and impaired recovery of activity in visible light after UV-B exposure. In contrast, in DeltaDeg-G cells, in which all the three deg genes were inactivated, the damage and recovery kinetics were the same as in the WT. Immunoblotting showed that the loss of both the D1 and D2 proteins was retarded in DeltaFtsH (slr0228) during UV-B exposure, and the extent of their restoration during the recovery period was decreased relative to the WT. However, in the DeltaDeg-G cells the damage and recovery kinetics of D1 and D2 were the same as in the WT. These data demonstrate a key role of FtsH (slr0228), but not the Deg proteases, for the repair of PS II during and following UV-B radiation at the step of degrading both of the UV-B damaged D1 and D2 reaction center subunits.     

The role of the FtsH and Deg proteases in the repair of UV-B radiation-damaged Photosystem II in the cyanobacterium Synechocystis PCC 6803

The photosystem two (PSII) complex found in oxygenic photosynthetic organisms is susceptible to damage by UV-B irradiation and undergoes repair in vivo to maintain activity. Until now there has been little information on the identity of the enzymes involved in repair. In the present study we have investigated the involvement of the FtsH and Deg protease families in the degradation of UV-B-damaged PSII reaction center subunits, D1 and D2, in the cyanobacterium Synechocystis 6803. PSII activity in a ΔFtsH (slr0228) strain, with an inactivated slr0228 gene, showed increased sensitivity to UV-B radiation and impaired recovery of activity in visible light after UV-B exposure. In contrast, in ΔDeg-G cells, in which all the three deg genes were inactivated, the damage and recovery kinetics were the same as in the WT. Immunoblotting showed that the loss of both the D1 and D2 proteins was retarded in ΔFtsH (slr0228) during UV-B exposure, and the extent of their restoration during the recovery period was decreased relative to the WT. However, in the ΔDeg-G cells the damage and recovery kinetics of D1 and D2 were the same as in the WT. These data demonstrate a key role of FtsH (slr0228), but not the Deg proteases, for the repair of PS II during and following UV-B radiation at the step of degrading both of the UV-B damaged D1 and D2 reaction center subunits.     

The icfG Gene Cluster of Synechocystis sp. Strain PCC 6803 Encodes an Rsb/Spo-Like Protein Kinase, Protein Phosphatase, and Two Phosphoproteins

A set of open reading frames (ORFs) potentially encoding signal transduction proteins are clustered around icfG, a gene implicated in the regulation of carbon metabolism, in the genome of Synechocystis sp. strain PCC 6803. slr1860 is the ORF for icfG, whose predicted product resembles the protein phosphatases SpoIIE, RsbU, and RsbX from Bacillus subtilis. Bracketing slr1860/icfG are (i) ORF slr1861, whose predicted product resembles the SpoIIAB, RsbT, and RsbW protein kinases from B. subtilis, and (ii) ORFs slr1856 and slr1859, whose predicted products resemble the respective phosphoprotein substrates for the B. subtilis protein kinases: SpoIIAA, RsbS, and RsbV. In order to determine whether the protein products encoded by these ORFs possessed the functional capabilities suggested by sequence comparisons, each was expressed in Escherichia coli as a histidine-tagged fusion protein and analyzed for its ability to participate in protein phosphorylation-dephosphorylation processes in vitro. It was observed that ORF slr1861 encoded an ATP-dependent protein kinase capable of phosphorylating Slr1856 and, albeit with noticeably lower efficiency, Slr1859. Site-directed mutagenesis suggests that Slr1861 phosphorylated these proteins on Ser-54 and Ser-57, respectively. Slr1860 exhibited divalent metal ion-dependent protein-serine phosphatase activity. It catalyzed the dephosphorylation of Slr1856, but not Slr1859, in vitro.     

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。     

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.     

集胞藻PCC 6803类铁氧还蛋白Slr1205的功能研究

类铁氧还蛋白 (ferredoxin-like, Fd-like) 在高等植物中具有调控叶绿体发育等多种重要的生理功能,但在蓝藻中的生物功能尚未被发现。通过比较集胞藻PCC 6083编码Fd-like蛋白基因的敲除突变株Δslr1205与野生型 (WT) 在不同碳源和光周期条件下的生理生化表型,分析Slr1205在集胞藻中的功能。结果显示,在高CO2浓度自养、混合营养和光异养时,Δslr1205的生长速率低于WT,而在空气中自养条件下并无差异。与此相对应,混合营养和光异养时Δslr1205比WT的呼吸速率低,与呼吸作用密切相关的NDH-1L复合体的含量少。Δslr1205在所有测试的条件下有较高的类胡萝卜素以及偏黄的表型。这些数据表明,Fd-like蛋白Slr1205的缺失造成在碳源充足条件下的生长速率下降,这可能是由于呼吸作用下调导致供能不足。研究结果为今后深入研究蓝藻Fd-like蛋白奠定了基础,为开展光合作用和呼吸作用的调节机制研究探索了新方向。     

An intracellular modulator of the 3'5'-cGMP hydrolysis in growing Dictyostelium discoideum amoebae

A heat-stable and acid-stable macromolecular factor present in the cytosol of growing Dictyostelium discoideum amoebae affects specifically the intracellular cGMP phosphodiesterase. It decreases the V of the enzyme but does not alter its Km. It has no effect on the cAMP or cGMP hydrolysis catalyzed by the intracellular cAMP-cGMP phosphodiesterases or by the extracellular phosphodiesterase. It is also expressed in a mutant (HPX235), defective in the synthesis of the cAMP-cGMP phosphodiesterases but capable of intracellular transduction of the chemotactic signal. This factor is resistant to several nucleases, proteases and phospholipases, and has an apparent molecular weight between 3500-10000. In contrast, the protein phosphodiesterase inhibitor secreted by the amoebae exerts an opposite inhibition on the intracellular phosphodiesterases. These two inhibitory factors may regulate intracellular cGMP hydrolysis during the chemotactic response.     

Involvement of an FtsH homologue in the assembly of functional photosystem I in the cyanobacterium Synechocystis sp. PCC 6803

The Synechocystis sp. PCC 6803 genome encodes four putative homologues of the AAA protease FtsH, two of which (slr0228 and sll1463) have been subjected to insertional mutagenesis in this study. Disruption of sll1463 had no discernible effect but disruption of slr0228 caused a 60% reduction in the abundance of functional photosystem I, without affecting the cellular content of photosystem II or phycobilisomes. Fluorescence and immunoblotting analyses show reductions in PS I polypeptides and possible structural alterations in the residual PS I, indicating an important role for slr0228 in PS I biogenesis.     

蓝细菌PCC6803染色体上的毒素蛋白Slr0664与抗毒素蛋白Ssr1114的相互作用

【目的】证明蓝细菌PCC6803染色体上的毒素-抗毒素系统(TA,toxin-antitoxin system)ssr1114/slr0664中毒素蛋白Slr0664与抗毒素蛋白Ssr1114之的相互作用。【方法】构建在大肠杆菌(Escherichia coli)BL21(DE3)中诱导表达H6-Ssr1114或共表达H6-Ssr1114和Slr0664的重组质粒,诱导表达后借助亲和捕捉技术在不同的条件下纯化H6-Ssr1114或共纯化重组蛋白H6-Ssr1114和Slr0664,并通过肽谱分析共纯化的重组蛋白,证明H6-Ssr1114与Slr0664之间存在相互作用。【结果】诱导Slr0664表达对细胞产生毒性作用导致生长抑制或细胞死亡,诱导H6-Ssr1114和Slr0664共表达时细胞能能正常生长,在非变性条件下可纯化共表达的重组蛋白H6-Ssr1114和Slr0664,在变性条件下仅H6-Ssr1114被纯化,肽谱分析结果表明共纯化的的重组蛋白为H6-Ssr1114和Slr0664。【结论】ssr1114/slr0664TA系统中抗毒素蛋白Ssr1114与毒素蛋白Slr0664之间存在相互作用。     

Functional Characterization of the slr1944 Gene of Cyanobacterium Synechocystis sp. PCC 6803

The properties of Slr1944 protein encoded by the slr1944 gene and participating in the metabolism of lipophilic compounds in a cyanobacterium Synechocystis were under study. Located in the periplasm, this protein comprises a conserved pentapeptide G-X-S-X-G characteristic of lipases, acetylcholinesterases, and thioesterases. An attempt to delete the gene from the cyanobacterial genome failed; this fact presumes an essential function of Slr1944 protein under the optimum growth conditions. Expression of the slr1944 gene in Escherichia coli cells demonstrated a high affinity of the product for lipophilic compounds. An enhanced slr1944 expression deprived Synechocystis cells of the ability to restore the activity of the photosynthetic electron-transport chain following photoinactivation. The authors believe that Slr1944 participates in the biogenesis of the lipophilic components of photosynthetic complexes.     

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.     

Molecular characterization of a novel gene from Synechocystis sp. PCC 6803

A novel gene slr2049 was identified in Synechococcus sp. PCC7002 by homologous alignment. The features and possible functions of slr2049 gene were predicted by bioinformatics analysis. The function of slr2049 was analyzed in vitro with a heterologous Escherichia coli system with plasmids conferring biosynthesis of phycocyanobilin (PCB) and of the acceptor proteins, β-phycocyanin (CpcB). The resulting products were evaluated with SDS-PAGE and absorption spectra. The function of slr2049 was further analyzed via site-directed mutations. Two mutants, slr2049 (W14L) and slr2049 (Y132S) were generated. The results showed that Slr2049 could catalyze the chromophorylation of CpcB. Compared to wild type, mutant Slr2049 (W14L) had red-shifted absorbance maxima and was not highly fluorescent as the wild-type. However, mutant Slr2049 (Y132S) was almost the same as the wild-type. In conclusion, our study suggests that we have cloned a novel gene and this gene may play an important role in attachment of the chromophores to the apo-proteins.