Photosystem 2 effective fluorescence cross-section of cyanobacterium Synechocystis sp. PCC6803 and its mutants

The effective fluorescence cross-section of photosystem 2 (PS2) was defined by measurements of chlorophyll a fluorescence induction curves for the wild type of the unicellular cyanobacterium Synechocystis sp. PCC6803, C-phycocyanin deficient mutant (CK), and mutant that totally lacks phycobilisomes (PAL). It was shown that mutations lead to a strong decrease of the PS2 effective fluorescence cross-section. For instance, the effective fluorescence cross-section of PS2 for wild type, CK and PAL mutants excited at λ(ex)=655 nm were found to be 896, 220 and 83 ?(2) respectively. Here we present an estimation of energy transfer efficiency from phycobilisomes to the pigment-protein complexes of PS2. It was shown that the PS2 fluorescence enhancement coefficient reaches a maximum value of 10.7 due to the energy migration from phycobilisomes. The rate constant of energy migration was found to be equal to 1.04 × 10(10) s(-1).     

Photomovement of the Gliding Cyanobacterium Synechocystis sp. PCC 6803

Abstract— Using a computerized videomicroscope motion analysis system, we investigated the photomovements of two Synechocystis sp. (PCC 6803 and ATCC 27184). Synechocystis sp. PCC 6803 displays a relatively slow gliding motion. The phototactic and photokinetic speeds of this cyanobacterium in liquid media were 5μm/min and 15.8 μm/min, respectively, at 3μmol/m²/s of stimulant white light. Synechocystis sp. PCC 6803 senses light direction rather than intensity for phototaxis. Synechocystis sp. ATCC 27184 showed a weak photokinesis but no phototaxis. Analysis of Synechocystis sp. ATCC 27184 suggests that the loss of phototaxis results from spontaneous mutation during several years of subculture. When directional irradiation was applied, the cell population of Synechocystis sp. PCC 6803 began to deviate from random movement and reached maximum orientation at 5 min after the onset of stimulant white light. Synechocystis sp. PCC 6803 showed high sensitivity to the stimulant white light of fluence rates as low as 0.002 |unol/m²/s. Neither 1,3-dichlorophenyldimethyl urea nor cyanide affected phototactic orientation, whereas cyanide inhibited gUding speed. This result suggests that the phototaxis of Synechocystis sp. PCC 6803 is independent of photosynthetic phosphorylation and that its gliding movement is primarily powered by oxidative phosphorylation. In the visible wavelength region, 560 nm, 660 nm and even 760 nm caused positive phototaxis. However, 360 nm light induced strikingly negative phototaxis. Therefore, at least two independent photoreceptors may exist to control phototaxis. The photoreceptor for positive phototaxis appears likely to be a phytochrome-like tetrapyrrole rather than chlorophyll a .     

Function and association of CyanoP in photosystem II of Synechocystis sp. PCC 6803

The CyanoP protein is a cyanobacterial homolog of the PsbP protein, which is an extrinsic subunit of photosystem II (PSII) in green plant species. The molecular function of CyanoP has been investigated in mutant strains of Synechocystis but inconsistent results have been reported by different laboratories. In this study, we generated and characterized a Synechocystis mutant in which entire region of the CyanoP gene was eliminated. After repeated subculture in CaCl₂-depleted medium, growth retardation was clearly observed for a CyanoP knockout mutant of Synechocystis sp. PCC 6803 (?P). The PSII-mediated oxygen-evolving activity of the ?P cells was more susceptible to depletion of CaCl₂ than that of wild-type cells. The 77 K fluorescence emission spectra indicated that energy coupling between phycobilisome and PSII was perturbed in both wild-type and ?P cells under CaCl₂-depleted conditions, and was more evident for the ?P mutant. To examine the association of CyanoP with PSII complexes, we tested several detergents for solubilization of thylakoid membranes and showed that CyanoP was partly included in fractions containing large protein complexes in gel-filtration analysis. These results indicate that CyanoP constitutively stabilizes PSII functionality in vivo.     

Functional Expression of Gloeobacter Rhodopsin in Synechocystis sp. PCC6803

Proteorhodopsins are light‐driven proton pumps that occur widespread in Nature, where they function predominantly in environments with high incident irradiance. Their maximal absorbance is usually in the blue range, but can be extended into the (far)red range of the electromagnetic spectrum. Because they can be expressed heterologously, they may be exploited in studies aimed at increasing the efficiency of photosynthesis. Here we report further studies toward this goal, by comparing the expression of two different bacterial rhodopsins (Proteorhodopsin and Gloeobacter rhodopsin) in the model cyanobacterium Synechocystis sp. PCC6803. In particular, we investigated the pigments bound by the respective apo‐opsins, and the oligomeric state of the corresponding holo‐rhodopsins, both in Escherichia coli and in the cyanobacterial membranes. We conclude that the two proton‐pumping rhodopsins are predominantly present in an oligomeric state (hexamers for Proteorhodopsin and trimers for Gloeobacter rhodopsin). Furthermore, Gloeobacter rhodopsin is able to bind an antenna carotenoid (in addition to retinal) and has the highest pumping rate at given light intensity. However, its lower expression level will decrease its physiological effectiveness. It remains to be established which of these two bacterial rhodopsins is best in stimulating the growth rate of its cyanobacterial host.     

A synthetic DNA and fusion PCR approach to the ectopic expression of high levels of the D1 protein of photosystem II in Synechocystis sp. PCC 6803

A hybrid approach involving synthetic DNA, fusion PCR, and ectopic expression has been used to genetically manipulate the expression of the D1 protein of photosystem II (PSII) in the model cyanobacterium Synechocystis sp. PCC6803. Due to the toxicity of the full-length psbA gene in E. coli, a chimeric psbA2 gene locus was commercially synthesised and cloned in two halves. High-fidelity fusion PCR utilizing sequence overlap between the two synthetic gene halves allowed the production of a DNA fragment that was able to recombine the full-length psbA2 gene into the Synechocystis chromosome at an ectopic (non-native) location. This was accomplished by designing the synthetic DNA/fusion PCR product to have the psbA2 gene, with control sequences, interposed between chimeric sequences corresponding to an ectopic target chromosomal location. Additionally, a recipient strain of Synechocystis lacking all three psbA genes was produced by a combination of traditional marker replacement and markerless replacement techniques. Transformation of this multiple deletion strain by the synthetic DNA/fusion PCR product faithfully restored D1 expression in terms of its expression and PSII repair capacity. The advantages and potential issues for using this approach to rapidly introduce chimeric sequence characteristics as a general tool to produce novel genetic constructs are discussed.     

Calcium is involved in photomovement of cyanobacterium Synechocystis sp. PCC 6803

The unicellular cyanobacterium Synechocystis sp. PCC 6803 (Syn6803) exhibits photomovement through gliding motility. For a better understanding of photomovement in Syn6803, we examined the effects of Ca2+ on photoorientation and motility using a computer-assisted videomicroscope motion analysis system. When calcium ion was chelated from the basic motility medium by adding 0.5 mM ethylene glycol-bis-(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA), the photoorientation was completely inhibited, whereas the gliding motility remained approximately 70% of the control. Photoorientation impaired by EGTA was nearly recovered within 30 min upon addition of 1 mM Ca2+. The recovery of photoorientation by Ca2+ was mimicked by either Mn2+ or Mg2+ but not by Ba2+ or Sr2+. Lanthanum ion at 10 microM completely inhibited both phototactic orientation and gliding motility of Syn6803. Furthermore, pimozide (voltage-gated L-type calcium channel inhibitor), orthovanadate (calcium efflux blocker) and A23187 (calcium ionophore) partially inhibited phototactic orientation and gliding motility. Interestingly, photoorientation was prevented with increasing concentrations of calmodulin antagonist such as trifluoperazine (TFP) and chlorpromazine, but gliding motility was inhibited in proportion to the concentration of TFP. The results we present strongly indicate that Ca2+ plays a significant role in regulating the photomovement of Syn6803.     

Phototaxis in the cyanobacterium Synechocystis sp. PCC 6803: role of different photoreceptors

The second cyanobacterial phytochrome Cph2 from Synechocystis sp. PCC 6803 was suggested as a part of a light-stimulated signal transduction chain inhibiting movement toward blue light. Cph2 has the two bilin binding sites, cysteine-129 and cysteine-1022, that might be involved in sensing of red/far-red and blue light, respectively. Here, we present data on wavelength dependence of the phototaxis inhibition under blue light, indicating that Cph2 itself is the photoreceptor for this blue light response. We found that inhibition of blue-light phototaxis in wild-type cells occurred below the transition point of about 470 nm. Substitution of cysteine-1022 with valine led to photomovement of the cells toward blue light (cph2(-) mutant phenotype). Analysis of mutants lacking cysteine-129 in the N-terminal chromophore binding domain indicated that this domain is also important for Cph2 function or folding of the protein. Furthermore, putative blue-light and phytochrome-like photoreceptors encoded by the Synechocystis sp. PCC 6803 genome were inactivated in wild-type and cph2 knockout mutant background. Our results suggest that none of these potential photoreceptors interfere with Cph2 function, although inactivation of taxD1 as well as slr1694 encoding a BLUF protein led to cells that reversed the direction of movement under blue light illumination in mutant strains of cph2.     

Tracking phototactic responses and modeling motility of Synechocystis sp. strain PCC6803

The unicellular cyanobacterium Synechocystis sp. Strain PCC 6,803 exhibits phototaxis by moving along a surface towards a light source. This process requires Type IV pili and a phytochrome-like photoreceptor coupled to a complex signal transduction pathway. Cells progress through different phases of interaction before the development of finger-like projections moving in the direction of the light that are characteristic of phototaxis. To probe the interaction between individual cells during the initial phase of phototaxis we tracked and analyzed a large number of cells. We observed that individual cells have limited motility, but when cells divide and/or aggregate to attain a certain minimal group size, enhanced motility and phototaxis is observed. At the later stages of motility, there is noticeable phototactic behavior which results in the appearance of the finger-like projections. Our results indicate that cells prefer to move over areas previously traversed by other cells and confine themselves to these areas and that cells alter local surface characteristics allowing for enhanced motility. Based on cell tracking data we present a preliminary random walk model showing the forces that might interact to create the typical phases of phototaxis and motility. In this model, we can simulate the formation of finger-like projections that are characteristic of phototaxis.     

Absorption and emission spectroscopic characterization of blue-light receptor Slr1694 from Synechocystis sp. PCC6803

The BLUF protein Slr1694 from the cyanobacterium Synechocystis sp. PCC6803 is characterized by absorption and emission spectroscopy. Slr1694 expressed from E. coli which non-covalently binds FAD, FMN, and riboflavin (called Slr1694(I)), and reconstituted Slr1694 which dominantly contains FAD (called Slr1694(II)) are investigated. The receptor conformation of Slr1694 (dark adapted form Slr1694(r)) is transformed to the putative signalling state (light adapted form Slr1694(s)) with red-shifted absorption and decreased fluorescence efficiency by blue-light excitation. In the dark at 22 degrees C, the signalling state recovers back to the initial receptor state with a time constants of about 14.2s for Slr1694(I) and 17s for Slr1694(II). Quantum yields of signalling state formation of approximately 0.63+/-0.07 for both Slr1694(I) and Slr1694(II) were determined by transient transmission measurements and intensity dependent steady-state transmission measurements. Extended blue-light excitation causes some bound flavin conversion to the hydroquinone form and some photo-degradation, both with low quantum efficiency. The flavin-hydroquinone re-oxidizes slowly back (time constant 5-9 min) to the initial flavoquinone form in the dark. A photo-cycle dynamics scheme is presented.     

Proteomic study of the peripheral proteins from thylakoid membranes of the cyanobacterium Synechocystis sp. PCC 6803

Thylakoid membranes of cyanobacteria and plants contain enzymes that function in diverse metabolic reactions. Many of these enzymes and regulatory proteins are associated with the membranes as peripheral proteins. To identify these proteins, we separated and identified the peripheral proteins of thylakoid membranes of the cyanobacterium Synechocystis sp. PCC 6803. Trichloroacetic acid (TCA)-acetone extraction was used to enrich samples with peripheral proteins and to remove integral membrane proteins. The proteins were separated by two-dimensional electrophoresis (2-DE) and identified by peptide mass fingerprinting. More than 200 proteins were detected on the sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) gel that was stained with colloidal Coomassie blue. We analyzed 116 spots by peptide mass fingerprinting and identified 78 spots that were derived from 51 genes. Some proteins were found in multiple spots, indicating differential modifications resulting in charge differences. Therefore, a significant fraction of the peripheral proteins in thylakoid membranes is modified post-translationally. In our analysis, products of 17 hypothetical genes could be identified in the peripheral protein fraction. Therefore, proteomic analysis is a powerful tool to identify location of the products of hypothetical genes and to characterize complexity in gene expression due to post-translational modifications.     

Protective effects of mycosporine-like amino acids of Synechocystis sp. PCC 6803 and their partial characterization

In this work, mycosporine-like amino acids (MAAs) of Synechocystis sp. PCC 6803 were characterized and were investigated on UV induction and protective ability. High performance liquid chromatographic (HPLC) studies revealed three major compounds in the MAAs. By UV absorption and mass spectra analysis, one of the compounds was tentatively identified as mycosporine-tau (M-tau). One novel compound similar to usujirene was tentatively named as dehydroxylusujirene, and the other novel compound was named as M-343 according to its absorption maximum. In vivo experiments indicated that M-tau was induced by both UV-A and UV-B, while dehydroxylusujirene and M-343 were only induced by UV-A, suggesting that different chromophores were involved in MAAs synthesis in Synechocystis sp. PCC 6803. It was also indicated that M-343 could be photochemically synthesized from some precursors. Under both UV and oxidation stresses, M-343 was more stable than dehydroxylusujirene and M-tau. Considering the reaction with H2O2, M-tau and dehydroxylusujirene might be potential antioxidants in reaction with physiological reactive oxygen species in vivo. In protection experiments, the MAAs exhibited efficient protective ability towards UV-B and H2O2 stresses, with maximal protection rates of 30% and 21.5%, respectively. These results indicate that the MAAs in Synechocystis sp. PCC 6803 act as both UV-screen and antioxidant.     

Characterization of 1,4-dihydroxy-2-naphthoyl-coenzyme A synthase (MenB) in phylloquinone biosynthesis of Synechocystis sp. PCC 6803

The gene product Sll1127 is a predicted 1,4-dihydroxy-2-naphthoyl-CoA synthase catalyzing an intramolecular Claisen condensation in the phylloquinone biosynthesis of the cyanobacterium Synechocystis sp.PCC 6803.This predicted catalytic function has been verified and the enzyme has been characterized for the first time with kcat = 0.013 s-1 and KM = 9μM.Its catalytic activity is found to strictly depend on externally added bicarbonate with an apparent KD = 0.60 mM.In addition,this enzyme is inhibited by its 1,4-dihydroxy-2-naphthoyl-CoA product through high-affinity binding,which causes a 18 nm shift of the inhibitor absorption at 392 to 410 nm and engenders a new absorption peak at 345 nm.All these properties of the cyanobacterial enzyme are closely similar to those of the Escherichia coli orthologue from the menaquinone biosynthetic pathway.These results provide additional supporting evidence for the essential role of bicarbonate as a catalytic base in the enzymatic reaction and the eubacterial origin of the enzymes in the cyanobacterial biosynthesis of phylloquinone.     

Exogenous Spermidine Alleviates UV-Induced Growth Inhibition of Synechocystis sp. PCC 6803 via Reduction of Hydrogen Peroxide and Malonaldehyde Levels

Effects of exogenously added spermidine (Spd) to UV-treated Synechocystis sp. PCC 6803 cultures on their growth, intracellular pigments, hydrogen peroxide (H₂O₂), malonaldehyde (MDA) contents, and antioxidant enzymes were investigated. Growth inhibition of cells subjected to 1-h UV-A, UV-B, and UV-C irradiation was abolished in culture added with 0.5 mM Spd. Both chlorophyll a and carotenoid contents were decreased under UV radiations in cells grown in BG₁₁ medium. However, the contents of these two pigments were slightly increased under UV radiations in Spd-supplemented cells with the consequence of enhanced oxygen evolution. Intracellular levels of H₂O₂ and MDA generated during 1-h UV irradiation were decreased when the culture medium contained 0.5 mM Spd. The antioxidative enzymes, catalase, and superoxide dismutase had a little or no response towards Spd supplementation under UV irradiation except for some increase in superoxide dismutase activity under UV-C. Total intracellular polyamines were decreased during Spd supplementation under UV stress; however, the cells showed a drastic increase in the amount of Put under this condition. Altogether, exogenous Spd is likely a potential compound that enables Synechocystis cells to cope with UV stress.     

The first solution structure of a paramagnetic copper(II) protein: the case of oxidized plastocyanin from the cyanobacterium Synechocystis PCC6803.

The NMR solution structure of oxidized plastocyanin from the cyanobacterium Synechocystis PCC6803 is here reported. The protein contains paramagnetic copper(II), whose electronic relaxation times are quite unfavorable for NMR solution studies. The structure has been solved on the basis of 1041 meaningful NOESY cross-peaks, 18 1D NOEs, 26 T(1) values, 96 dihedral angle constraints, and 18 H-bonds. The detection of broad hyperfine-shifted signals and their full assignment allowed the identification of the copper(II) ligands and the determination of the Cu-S-C-H dihedral angle for the coordinated cysteine. The global root-mean-square deviation from the mean structure for the solution structure family is 0.72 +/- 0.14 and 1.16 +/- 0.17 A for backbone and heavy atoms, respectively. The structure is overall quite satisfactory and represents a breakthrough, in that it includes paramagnetic copper proteins among the metalloproteins for which solution structures can be afforded. The comparison with the available X-ray structure of a triple mutant is also performed.     

The [NiFe]-hydrogenase of the cyanobacterium Synechocystis sp. PCC 6803 works bidirectionally with a bias to H2 production

Protein film electrochemistry (PFE) was utilized to characterize the catalytic activity and oxidative inactivation of a bidirectional [NiFe]-hydrogenase (HoxEFUYH) from the cyanobacterium Synechocystis sp. PCC 6803. PFE provides precise control of the redox potential of the adsorbed enzyme so that its activity can be monitored under changing experimental conditions as current. The properties of HoxEFUYH are different from those of both the standard uptake and the "oxygen-tolerant" [NiFe]-hydrogenases. First, HoxEFUYH is biased toward proton reduction as opposed to hydrogen oxidation. Second, despite being expressed under aerobic conditions in vivo, HoxEFUYH is clearly not oxygen-tolerant. Aerobic inactivation of catalytic hydrogen oxidation by HoxEFUYH is total and nearly instantaneous, producing two inactive states. However, unlike the Ni-A and Ni-B inactive states of standard [NiFe]-hydrogenases, both of these states are quickly (<90 s) reactivated by removal of oxygen and exposure to reducing conditions. Third, proton reduction continues at 25-50% of the maximal rate in the presence of 1% oxygen. Whereas most previously characterized [NiFe]-hydrogenases seem to be preferential hydrogen oxidizing catalysts, the cyanobacterial enzyme works effectively in both directions. This unusual catalytic bias as well as the ability to be quickly reactivated may be essential to fulfilling the physiological role in cyanobacteria, organisms expected to experience swings in cellular reduction potential as they switch between aerobic conditions in the light and dark anaerobic conditions. Our results suggest that the uptake [NiFe]-hydrogenases alone are not representative of the catalytic diversity of [NiFe]-hydrogenases, and the bidirectional heteromultimeric enzymes may serve as valuable models to understand the diverse mechanisms of tuning the reactivity of the hydrogen activating site.     

Functional in situ evaluation of photosynthesis-protecting carotenoids in mutants of the cyanobacterium Synechocystis PCC6803

Cyanobacteria possess different carotenoids as scavengers of reactive oxygen species. In Synechocystis PCC6803, zeaxanthin, echinenone, beta-carotene and myxoxanthophyll are synthesized. By disruption of the ketolase and hydroxylase genes, it was possible to obtain mutants devoid of either zeaxanthin, echinenone, or a combination of both carotenoids. With these mutants, their function in protecting photosynthetic electron transport under high light stress as well as chlorophyll and carotenoid degradation after initiation of singlet oxygen or radical formation was analyzed. Wild type Synechocystis is very well protected against high light-mediated photooxidation. Absence of echinenone affects photosynthetic electron transport to only a small extent. However, complete depletion of zeaxanthin together with a modification of myxoxanthophyll resulted in strong photoinhibition of overall photosynthetic electron transport as well as the photosystem II reaction. In the double mutant lacking both carotenoids the effects were additive. The light saturation curves of photosynthetic electron transport of the high light-treated mutants exhibited not only a lower saturation value but also smaller slopes. Using methylviologen or methylene blue as a radical or singlet oxygen generators, respectively, massive degradation of chlorophyll and carotenoids, indicative of photooxidative destruction of the photosynthetic apparatus, was observed, especially in the mutants devoid of zeaxanthin.     

D1 protein turnover is involved in protection of Photosystem II against UV-B induced damage in the cyanobacterium Arthrospira (Spirulina) platensis

By using two strains of Arthrospira (Spirulina)platensis, an economically important filamentous cyanobacterium, we compared the impairment of PSII activity and loss of D1 protein content under UV-B radiation. Our study showed that UV-B radiation induced a gradual loss of the oxygen-evolving activity to about 56% after 180 min UV-B irradiation both in strains 439 and D-0083, which have been kept under indoor and an outdoor culturing conditions, respectively for a prolonged period of time. The loss of oxygen evolution was accelerated in both strains in the presence of lincomycin, an inhibitor of protein synthesis, and the amount of D1 protein showed a decrease comparable to that of oxygen evolution during the UV-B exposure. However, the UV-B induced loss of oxygen-evolving activity and D1 protein amount was largely prevented when A. platensis cells were exposed to UV-B irradiance supplemented with visible light. Comparison of the two strains also showed a smaller extent of D1 protein synthesis dependent PSII repair in the indoor strain. Our results show that turnover of the D1 protein is an important defense mechanism to counteract the UV-B induced damage of PSII in A. platensis, and also that visible light plays an important role in maintaining the function of PSII under simultaneous exposure to UV-B and visible light.     

Two intermediate states I and J trapped at low temperature in the photocycles of two BLUF domain proteins of cyanobacteria Synechocystis sp. PCC6803 and Thermosynechococcus elongatus BP-1

We identified the two intermediate states, I and J, that are common in the photocycles of the cyanobacterial BLUF (sensor of Blue Light Using Flavin) domain proteins of Slr1694 of Synechocystis sp. PCC6803 and Tll0078 of Thermosynechococcus elongatus BP-1 by analyzing the absorption spectra at 5 K. Illumination at 5 K accumulated intermediate forms (designated as I5 and I9), which showed 5 and 9 nm redshifts of the absorption bands of flavin in the Tll0078 and Slr1694 proteins, respectively. I5 (I9) was converted into the next intermediate, which have 11 nm (14 nm) red-shifted absorption bands J11 (J14) after dark annealing at 230 K (240 K). Further dark annealing at 280 K (270 K) of J11 (J14) produced the signal-transmitting final form F490 (F495), with a small increase in the absorption at around 490 nm (495 nm). The results indicate that the BLUF proteins of Tll0078 and Slr1694 exhibit the common photocycle of D471 (D467) --> I5 (I9) --> J11 (J14) --> F490 (F495) at low temperature. The transition temperatures for these intermediate forms differ for two proteins. The amount of I5 (I9) accumulated at 5 K was small and increased at a higher temperature, suggesting heterogeneity of the protein structure that determines the reaction pathway.