The effect of Photosystem II inhibitors DCMU and BNT on the high-light induced D1 turnover in two cyanobacterial strains Synechocystis PCC 6803 and Synechococcus PCC 7942

The effect of the Photosystem II (PSII) inhibitors dichlorophenyldimethylurea (DCMU) and bromonitrothymol (BNT) on the rate of the high-light induced D1 protein turnover was studied in whole cells of two cyanobacterial strains Synechocystis PCC 6803 and Synechococcus PCC 7942. In Synechocystis the D1 degradation was slowed down to a similar extent in the presence of either inhibitor compared with control cells. This slower degradation corresponded with the retardation of Photosystem II photoinactivation (PSIIPI) measured as a decline of PS II activity in the illuminated cells treated with chloramphenicol (CAP). The ongoing D1 synthesis in the presence of both PS II inhibitors was confirmed by unchanging PS II activity and the steady-state level of D1 during illumination in the absence of CAP. In Synechococcus cells both DCMU and BNT blocked the turnover of the 'low-light' D1 form (D1:1) but did not prevent the exchange of the 'high-light' form D1:2 for the D1:1 form. The similar effect of both herbicides on the D1 exchange was in contrast with their influence on the rate of PSIIPI. While DCMU had a pronounced protective effect, BNT significantly increased the rate of PS II photodamage. The fast BNT-induced decline of PS II activity was also observed in Synechocystis cells treated with azide, an inhibitor of reactive oxygen species scavenging enzymes. Therefore, we assume that the distinct sensitivity of the two cyanobacterial strains to BNT can be caused by different content and/or activity of these enzymes in each strain.     

Differing involvement of sulfoquinovosyl diacylglycerol in photosystem II in two species of unicellular cyanobacteria.

Sulfoquinovosyl diacylglycerol (SQDG) is involved in the maintenance of photosystem II (PSII) activity in Chlamydomonas reinhardtii[Minoda, A., Sato, N., Nozaki, H., Okada, K., Takahashi, H., Sonoike, K. & Tsuzuki, M. et al. (2002) Eur. J. Biochem.269, 2353-2358]. To understand the spread of the taxa in which PSII interacts with SQDG, especially in cyanobacteria, we produced a mutant defective in the putative sqdB gene responsible for SQDG synthesis from two cyanobacteria, Synechocystis sp. PCC6803 and Synechococcus sp. PCC7942. The mutant of PCC6803, designated SD1, lacked SQDG synthetic ability and required SQDG supplementation for its growth. After transfer from SQDG-supplemented to SQDG-free conditions, SD1 showed decreased net photosynthetic and PSII activities on a chlorophyll (Chl) basis with a decrease in the SQDG content. Moreover, the sensitivity of PSII activity to 3-(3,4-dichlorophenyl)-1,1-dimethylurea and atrazine was increased in SD1. However, SD1 maintained normal amounts of cytochrome b559 and D1 protein (the subunits comprising the PSII complex) on a Chl basis, indicating that the PSII complex content changed little, irrespective of a decrease in the SQDG content. These results suggest that the role of SQDG is the conservation of the PSII properties in PCC6803, consistent with the results obtained with C. reinhardtii. In contrast, the SQDG-null mutant of PCC7942 showed the normal level of PSII activity with little effect on its sensitivity to PSII herbicides. Therefore, the difference in the SQDG requirement for PSII is species-specific in cyanobacteria; this could be of use when investigating the molecular evolution of the PSII complex.     

Expression of two nblA-homologous genes is required for phycobilisome degradation in nitrogen-starved Synechocystis sp. PCC6803

One of the responses exhibited by cyanobacteria when they are limited for an essential nutrient is the rapid degradation of their light-harvesting complex, the phycobilisome. Phycobilisome degradation is an ordered proteolytic process, visible by a color change of the cyanobacterial cell from blue-green to yellow-green (chlorosis). The small polypeptide NblA plays a key role in degradation of phycobilisomes in Synechococcus sp. PCC7942. Unlike Synechococcus, Synechocystis sp. PCC6803 has two nblA-homologous genes, nblA1 and nblA2, which are contiguous on the genome. Here we show that nblA1 and nblA2 are simultaneously expressed in Synechocystis 6803 upon nitrogen deprivation, and are both required for phycobilisome degradation.     

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.     

General distribution of the nitrogen control gene ntcA in cyanobacteria.

The ntcA gene from Synechococcus sp. strain PCC 7942 encodes a regulatory protein which is required for the expression of all of the genes known to be subject to repression by ammonium in that cyanobacterium. Homologs to ntcA have now been cloned by hybridization from the cyanobacteria Synechocystis sp. strain PCC 6803 and Anabaena sp. strain PCC 7120. Sequence analysis has shown that these ntcA genes would encode polypeptides strongly similar (77 to 79% identity) to the Synechococcus NtcA protein. Sequences hybridizing to ntcA have been detected in the genomes of nine other cyanobacteria that were tested, including strains of the genera Anabaena, Calothrix, Fischerella, Nostoc, Pseudoanabaena, Synechococcus, and Synechocystis.     

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.     

Inactivation of the clpP1 gene for the proteolytic subunit of the ATP-dependent Clp protease in the cyanobacterium Synechococcus limits growth and light acclimation

ClpP functions as the proteolytic subunit of the ATP-dependent Clp protease in eubacteria, mammals and plant chloroplasts. We have cloned a clpP gene, designated clpP1, from the cyanobacterium Synechococcus sp. PCC 7942. The monocistronic 591 bp gene codes for a protein 80% similar to one of four putative ClpP proteins in another cyanobacterium, Synechocystis sp. PCC 6803. The constitutive ClpP1 content in Synechococcus cultures was not inducible by high temperatures, but it did rise fivefold with increasing growth light from 50 to 175 µmol photons m^-2 s^-1. A clpP1 inactivation strain (clpP1) exhibited slower growth rates, especially at the higher irradiances, and changes in the proportion of the photosynthetic pigments, chlorophyll a and phycocyanin. Many mutant cells (ca. 35%) were also severely elongated, up to 20 times longer than the wild type. The stress phenotype of clpP1 when grown at high light was confirmed by the induction of known stress proteins, such as the heat shock protein GroEL and the alternate form of PSII reaction center D1 protein, D1 form 2. ClpP1 content also rose significantly during short-term photoinhibition, but its loss in clpP1 did not exacerbate the extent of inactivation of photosynthesis, nor affect the inducible D1 exchange mechanism, indicating ClpP1 is not directly involved in D1 protein turnover.     

A change in the sensitivity of elongation factor G to oxidation protects photosystem II from photoinhibition in Synechocystis sp. PCC 6803

The repair of photosystem II (PSII) after photodamage is particularly sensitive to oxidative stress and inhibition of such repair is associated with the oxidation of specific cysteine residues in elongation factor G (EF-G), a key translation factor, in the cyanobacterium Synechocystis sp. PCC 6803. Expression of mutated EF-G with a target cysteine residue replaced by serine in Synechocystis resulted in the protection of PSII from photoinhibition. This protection was attributable to the enhanced repair of PSII via acceleration of the synthesis of the D1 protein, which might have been due to reduced sensitivity of protein synthesis to oxidative stress.     

The deg proteases protect Synechocystis sp. PCC 6803 during heat and light stresses but are not essential for removal of damaged D1 protein during the photosystem two repair cycle

Members of the DegP/HtrA (or Deg) family of proteases are found widely in nature and play an important role in the proteolysis of misfolded and damaged proteins. As yet, their physiological role in oxygenic photosynthetic organisms is unclear, although it has been widely speculated that they participate in the degradation of the photodamaged D1 subunit in the photosystem two complex (PSII) repair cycle, which is needed to maintain PSII activity in both cyanobacteria and chloroplasts. We have examined the role of the three Deg proteases found in the cyanobacterium Synechocystis sp. PCC 6803 through analysis of double and triple insertion mutants. We have discovered that these proteases show overlap in function and are involved in a number of key physiological responses ranging from protection against light and heat stresses to phototaxis. In previous work, we concluded that the Deg proteases played either a direct or an indirect role in PSII repair in a glucose-tolerant version of Synechocystis 6803 (Silva, P., Choi, Y. J., Hassan, H. A., and Nixon, P. J. (2002) Philos. Trans. R. Soc. Lond. B Biol. Sci. 357, 1461-1467). In this work, we have now been able to demonstrate unambiguously, using a triple deg mutant created in the wild type strain of Synechocystis 6803, that the Deg proteases are not obligatory for PSII repair and D1 degradation. We therefore conclude that although the Deg proteases are needed for photoprotection of Synechocystis sp. PCC 6803, they do not play an essential role in D1 turnover and PSII repair in vivo.     

Oxidation of elongation factor G inhibits the synthesis of the D1 protein of photosystem II

Oxidative stress inhibits the repair of photodamaged photosystem II (PSII). This inhibition is due initially to the suppression, by reactive oxygen species (ROS), of the synthesis de novo of proteins that are required for the repair of PSII, such as the D1 protein, at the level of translational elongation. To investigate in vitro the mechanisms whereby ROS inhibit translational elongation, we developed a translation system in vitro from the cyanobacterium Synechocystis sp. PCC 6803. The synthesis of the D1 protein in vitro was inhibited by exogenous H2O2. However, the addition of reduced forms of elongation factor G (EF-G), which is known to be particularly sensitive to oxidation, was able to reverse the inhibition of translation. By contrast, the oxidized forms of EF-G failed to restore translational activity. Furthermore, the overexpression of EF-G of Synechocystis in another cyanobacterium Synechococcus sp. PCC 7942 increased the tolerance of cells to H2O2 in terms of protein synthesis. These observations suggest that EF-G might be the primary target, within the translational machinery, of inhibition by ROS.     

The rpaC gene product regulates phycobilisome-photosystem II interaction in cyanobacteria

State transitions in cyanobacteria are a physiological adaptation mechanism that changes the interaction of the phycobilisomes with the Photosystem I and Photosystem II core complexes. A random mutagenesis study in the cyanobacterium Synechocystis sp. PCC6803 identified a gene named rpaC which appeared to be specifically required for state transitions. rpaC is a conserved cyanobacterial gene which was tentatively suggested to code for a novel signal transduction factor. The predicted gene product is a 9-kDa integral membrane protein. We have further examined the role of rpaC by overexpressing the gene in Synechocystis 6803 and by inactivating the ortholog in a second cyanobacterium, Synechococcus sp. PCC7942. Unlike the Synechocystis 6803 null mutant, the Synechococcus 7942 null mutant is unable to segregate, indicating that the gene is essential for cell viability in this cyanobacterium. The Synechocystis 6803 overexpressor is also unable to segregate, indicating that the cells can only tolerate a limited gene copy number. The non-segregated Synechococcus 7942 mutant can perform state transitions but shows a perturbed phycobilisome-Photosystem II interaction. Based on these results, we propose that the rpaC gene product controls the stability of the phycobilisome-Photosystem II supercomplex, and is probably a structural component of the complex.     

Systematic analysis of the relation of electron transport and ATP synthesis to the photodamage and repair of photosystem II in Synechocystis

The photosynthetic machinery and, in particular, the photosystem II (PSII) complex are susceptible to strong light, and the effects of strong light are referred to as photodamage or photoinhibition. In living organisms, photodamaged PSII is rapidly repaired and, as a result, the extent of photoinhibition represents a balance between rates of photodamage and the repair of PSII. In this study, we examined the roles of electron transport and ATP synthesis in these two processes by monitoring them separately and systematically in the cyanobacterium Synechocystis sp. PCC 6803. We found that the rate of photodamage, which was proportional to light intensity, was unaffected by inhibition of the electron transport in PSII, by acceleration of electron transport in PSI, and by inhibition of ATP synthesis. By contrast, the rate of repair was reduced upon inhibition of the synthesis of ATP either via PSI or PSII. Northern blotting and radiolabeling analysis with [(35)S]Met revealed that synthesis of the D1 protein was enhanced by the synthesis of ATP. Our observations suggest that ATP synthesis might regulate the repair of PSII, in particular, at the level of translation of the psbA genes for the precursor to the D1 protein, whereas neither electron transport nor the synthesis of ATP affects the extent of photodamage.     

Effects of inactivating psbM and psbT on photodamage and assembly of photosystem II in Synechocystis sp. PCC 6803

PsbM and PsbT have been assigned to electron densities on both photosystem II (PSII) monomers at the PSII dimer interface in X-ray crystallographic structures from Thermosynechoccocus elongatus and T. vulcanus. Our results show that removal of either or both proteins from Synechocystis sp. PCC 6803 resulted in photoautotrophic strains but the DeltaPsbM:DeltaPsbT mutant did not form stable dimers. A CP43-less PSII monomer accumulated in both single mutants, although absence of PsbT destabilized PSII to a greater extent than removing PsbM. Additionally, DeltaPsbT cells exhibited slowed electron transfer between the plastoquinone electron acceptors, Q(A) and Q(B); however, S-state cycling in both mutants was similar to wild type. Oxygen evolution in these mutants rapidly inactivated following exposure to high light where recovery required protein synthesis and could proceed in the dark in DeltaPsbM cells but required light in DeltaPsbT cells. Interestingly, the extent of recovery of oxygen-evolving activity was greatest in the DeltaPsbM:DeltaPsbT strain. We also found recovery required Psb27 in DeltaPsbT cells although, under our conditions, the DeltaPsb27 strain remained similar to wild type. In contrast, the DeltaPsbM:DeltaPsb27 mutant could not assemble PSII beyond a CP43-minus intermediate. Our results suggest essential roles for Psb27 in biogenesis in the DeltaPsbM strain and for repair from photodamage in cells lacking PsbT.     

Psb29, a conserved 22-kD protein, functions in the biogenesis of Photosystem II complexes in Synechocystis and Arabidopsis

Photosystem II (PSII), the enzyme responsible for photosynthetic oxygen evolution, is a rapidly turned over membrane protein complex. However, the factors that regulate biogenesis of PSII are poorly defined. Previous proteomic analysis of the PSII preparations from the cyanobacterium Synechocystis sp PCC 6803 detected a novel protein, Psb29 (Sll1414), homologs of which are found in all cyanobacteria and vascular plants with sequenced genomes. Deletion of psb29 in Synechocystis 6803 results in slower growth rates under high light intensities, increased light sensitivity, and lower PSII efficiency, without affecting the PSII core electron transfer activities. A T-DNA insertion line in the PSB29 gene in Arabidopsis thaliana displays a phenotype similar to that of the Synechocystis mutant. This plant mutant grows slowly and exhibits variegated leaves, and its PSII activity is light sensitive. Low temperature fluorescence emission spectroscopy of both cyanobacterial and plant mutants shows an increase in the proportion of uncoupled proximal antennae in PSII as a function of increasing growth light intensities. The similar phenotypes observed in both plant and cyanobacterial mutants demonstrate that the function of Psb29 has been conserved throughout the evolution of oxygenic photosynthetic organisms and suggest a role for the Psb29 protein in the biogenesis of PSII.     

ggpS基因过表达对集胞藻PCC 6803甘油葡萄糖苷和甘油合成的影响

为了研究甘油葡萄糖苷磷酸合成酶(GgpS)在集胞藻PCC 803甘油葡萄糖苷和甘油合成中的作用,本研究在前期获得高产甘油葡萄糖苷藻株的基础上分别过量表达来自于集胞藻PCC 6803自身和聚球藻PCC7002的甘油葡萄糖苷磷酸合成酶基因ggpS,并测定了在不同浓度NaCl胁迫时突变藻株的甘油葡萄糖苷和甘油积累量。结果发现获得的突变株甘油葡萄糖苷合成没有提高,但是甘油合成显著增强。此外,当培养基NaCl浓度从600 mmol/L提高到900 mmol/L时,集胞藻PCC 6803自身ggpS过表达藻株的甘油合成进一步提高75%。这些结果显示了GgpS在将碳代谢流导入集胞藻甘油合成途径中的作用。研究成果也为进一步通过基因工程改造提高集胞藻甘油葡萄糖苷和甘油合成效率奠定了基础。