INVESTIGATION OF C-PHYCOCYANIN FROM BLUE-GREEN-ALGA SPIRULINA-PLATENSIS WITH SCANNING TUNNELING MICROSCOPE

C-phycocyanin (C-PC) was isolated from blue-green alga spirulina platensis. A scanning tunneling microscope (STM) has been used to investigate its three-dimensional structure. The samples were dialyzed before the STM experiment, and then deposited on highly oriented pyrolytic graphite (HOPG). The measurement was carried out in ambient condition at room temperature. STM images showed that C-phycocyanin was uniformly distributed on solid-state substrate HOPG. The shape of C-phycocyanin is disklike with a channel in the center. It is concluded that STM has great potential to observe the structure of biliproteins and phycobilisomes.

Evolutionary analysis of phycobiliproteins: Implications for their structural and functional relationships

Phycobiliproteins, together with linker polypeptides and various chromophores, are basic building blocks of phycobilisomes, a supramolecular complex with a light-harvesting function in cyanobacteria and red algae. Previous studies suggest that the different types of phycobiliproteins and the linker polypeptides originated from the same ancestor. Here we retrieve the phycobilisome-related genes from the well-annotated and even unfinished cyanobacteria genomes and find that many sites with elevated d(N)/d(S) ratios in different phycobiliprotein lineages are located in the chromophore-binding domain and the helical hairpin domains (X and Y). Covariation analyses also reveal that these sites are significantly correlated, showing strong evidence of the functional-structural importance of interactions among these residues. The potential selective pressure driving the diversification of phycobiliproteins may be related to the phycobiliprotein-chromophore microenvironment formation and the subunits interaction. Sites and genes identified here would provide targets for further research on the structural-functional role of these residues and energy transfer through the chromophores.

三种红藻光合作用色素系统的比较研究

本研究分为三个部分：1.以坛紫菜（Porphyra haitanesis Chang et Zheng）的叶状体和丝状体为研究对象，比较坛紫菜叶状体和丝状体的光合色素、色素蛋白的组成，并提取纯化藻红蛋白、藻蓝蛋白、藻胆体及类囊体膜和光系统。研究结果表明坛紫菜叶状体和丝状体色素及色素蛋白的含量不同，藻红蛋白是主要的色素蛋白，坛紫菜叶状体和丝状体的藻红蛋白的含量分别为2.9mg藻红蛋白/g鲜重、4.2mg藻红蛋白/g鲜重，这表明坛紫菜叶状体和丝状体藻红蛋白含量丰富，是提取藻红蛋白很好的材料。藻胆体的性质差异不大，但类囊体膜差异显著，从坛紫菜叶状体中分离到了两种不同的类囊体膜带，光系统Ⅰ(PSⅠ)和PSⅡ分别结合在两条类囊体膜带上，但从坛紫菜丝状体中也分离到两条类囊体膜带，它们的光谱性质和蛋白组成相似，仅放氧速率和DCIP活性有差异，从坛紫菜丝状体中我们仅分离到PSⅡ。坛紫菜叶状体PSⅡ有5种外在蛋白(33、20、Cytc 550、15、12kDa蛋白)，而坛紫菜丝状体外在蛋白仅有4条，缺少12kDa蛋白。2. 以在中国江苏部分地区进行了大规模的商业化栽培的突变体条斑紫菜（Porphyra yezoensis Ueda）和野生型条斑紫菜为研究对象，比较其色素及色素蛋白组成、对不能光质的利用率及藻胆体的组成。条斑紫菜和突变型条斑紫菜对不同的光质利用效果有差异，在白光的照射下，野生型紫菜的放氧速率最大，而突变型紫菜在黄光照射下的放氧速率最大。条斑紫菜野生型与突变型色素含量上有明显的差异,突变型紫菜的藻红蛋白含量明显减少而藻蓝蛋白的含量增加。通过杂交的方法证实诱变所获得条斑紫菜突变体为细胞质突变，但是突变型紫菜却发生了由细胞核编码的γ亚基的缺失，这表明突变型紫菜藻红蛋白含量和性质发生了明显的变化。3. 为了找出淡水红藻-深紫美芒藻(Compsopogon coeruleus (Balbis) Montagne)分布狭窄及生物产量低的原因，本文对深紫美芒藻在不同的盐离子浓度下的放氧速率及藻胆体色素组成和结构上进行研究。结果显示：微量的NaCl（0.1mM）促进深紫美芒藻放氧，而深紫美芒藻在较高的NaCl（1、10mM）, NaH2PO4 （0.1、1、10mM）和 NH4NO3（0.1、1、10mM）溶液中却没有检测到氧气的产生。这与深紫美芒藻生长的环境一致即深紫美芒藻生活在低盐浓度、低营养的泉水中。深紫美芒藻的藻胆体是由藻红蛋白、藻蓝蛋白及别藻蓝蛋白组成，上面结合α、β和γ亚基，含有藻红胆素、藻篮胆素，但缺乏缺少藻尿胆素。

Synechococcus sp. PCC 7002 CpcB lyase null mutations alter phycocyanin chromophore function and, consequently, affect the redistribution of excitation energy via the light state transition /

Phycobilisomes are the major light harvesting complexes for cyanobacteria and phycocyanin is the primary phycobiliprotein of the phycobilisome rod. The phycocyanobilin lyases responsible for chromophorylating the phycocyanin p subunit (CpcB) have been recently identified in the cyanobacterium Synechococcus sp. PCC 7002. Surprisingly, mutants missing the CpcB lyases were nevertheless capable of producing pigmented phycocyanin. 10K absorbance measurements revealed that the energy states of the p phycocyanin chromophores were only subtly shifted; however, 77K steady state fluorescence emission spectroscopy showed excitation energy transfer involving the targeted chromophores to be highly disrupted. Such evidence suggests that phycobilin orientation within the binding domain is specifically modified. We hypothesized that alternate, less specific lyases are able to act on the p binding sites. A phycocyanin linker-polypeptide deficient mutant was similarly characterized. The light state transition, a short term adaptation of the photosynthetic light harvesting apparatus resulting in the redistribution of excitation energy among the photo systems, was shown to be dominated by the reallocation of phycocyanin-absorbed excitation energy. Treatment with a high M phosphate buffer effectively prevented the redistribution of both chlorophyll a- and phycobilisome- absorbed excitation energy, suggesting that the two effects are not strictly independent. The mutant strains required a larger redistribution of excitation energy between light states, perhaps to compensate for their loss in phycobilisome antenna function.

Characterization of the state transition in the cyanobacterium synechococcus sp. 7002 and a phycobilisome-less Mutant

ABSTRACT Photosynthetic state transitions were investigated in the cyanobacterium Synechococcus sp. PCC 7002 in both wild-type cells and mutant cells lacking phycobilisomes. Preillumination in the presence of DCMU (3(3,4 dichlorophenyl) 1,1 dimethyl urea) induced state 1 and dark adaptation induced state 2 in both wild-type and mutant cells as determined by 77K fluorescence emission spectroscopy. Light-induced transitions were observed in the wildtype after preferential excitation of phycocyanin (state 2) or preferential excitation of chlorophyll .a. (state 1). The state 1 and 2 transitions in the wild-type had half-times of approximately 10 seconds. Cytochrome f and P-700 oxidation kinetics could not be correlated with any current state transition model as cells in state 1 showed faster oxidation kinetics regardless of excitation wavelength. Light-induced transitions were also observed in the phycobilisomeless mutant after preferential excitation of short wavelength chlorophyll !l. (state 2) or carotenoids and long wavelength chlorophyll it (state 1). One-dimensional electrophoresis revealed no significant differences in phosphorylation patterns of resolved proteins between wild-type cells in state 1 and state 2. It is concluded that the mechanism of the light state transition in cyanobacteria does not require the presence of the phycobilisome. The results contradict proposed models for the state transition which require an active role for the phycobilisome.

Competing demands for a complex system : photosystem II repair, photoprotection and quantum yield

Photosynthesis in general is a key biological process on Earth and Photo system II (PSII) is an important component of this process. PSII is the only enzyme capable of oxidizing water and is largely responsible for the primordial build-up and present maintenance of the oxygen in the atmosphere. This thesis endeavoured to understand the link between structure and function in PSII with special focus on primary photochemistry, repair/photodamage and spectral characteristics. The deletion of the PsbU subunit ofPSII in cyanobacteria caused a decoupling of the Phycobilisomes (PBS) from PSII, likely as a result of increased rates of PSII photodamage with the PBS decoupling acting as a measure to protect PSII from further damage. Isolated fractions of spinach thylakoid membranes were utilized to characterize the heterogeneity present in the various compartments of the thylakoid membrane. It was found that the pooled PSIILHCII pigment populations were connected in the grana stack and there was also a progressive decrease in the reaction rates of primary photochemistry and antennae size of PSII as the sample origin moved from grana to stroma. The results were consistent with PSII complexes becoming damaged in the grana and being sent to the stroma for repair. The dramatic quenching of variable fluorescence and overall fluorescent yield of PSII in desiccated lichens was also studied in order to investigate the mechanism by which the quenching operated. It was determined that the source of the quenching was a novel long wavelength emitting external quencher. Point mutations to amino acids acting as ligands to chromophores of interest in PSII were utilized in cyanobacteria to determine the role of specific chromophores in energy transfer and primary photochemistry. These results indicated that the Hl14 ligated chlorophyll acts as the 'trap' chlorophyll in CP47 at low temperature and that the Q130E mutation imparts considerable changes to PSII electron transfer kinetics, essentially protecting the complex via increased non-radiative charge Photosynthesis in general is a key biological process on Earth and Photo system II (PSII) is an important component of this process. PSII is the only enzyme capable of oxidizing water and is largely responsible for the primordial build-up and present maintenance of the oxygen in the atmosphere. This thesis endeavoured to understand the link between structure and function in PSII with special focus on primary photochemistry, repair/photodamage and spectral characteristics. The deletion of the PsbU subunit ofPSII in cyanobacteria caused a decoupling of the Phycobilisomes (PBS) from PSII, likely as a result of increased rates of PSII photodamage with the PBS decoupling acting as a measure to protect PSII from further damage. Isolated fractions of spinach thylakoid membranes were utilized to characterize the heterogeneity present in the various compartments of the thylakoid membrane. It was found that the pooled PSIILHCII pigment populations were connected in the grana stack and there was also a progressive decrease in the reaction rates of primary photochemistry and antennae size of PSII as the sample origin moved from grana to stroma. The results were consistent with PSII complexes becoming damaged in the grana and being sent to the stroma for repair. The dramatic quenching of variable fluorescence and overall fluorescent yield of PSII in desiccated lichens was also studied in order to investigate the mechanism by which the quenching operated. It was determined that the source of the quenching was a novel long wavelength emitting external quencher. Point mutations to amino acids acting as ligands to chromophores of interest in PSII were utilized in cyanobacteria to determine the role of specific chromophores in energy transfer and primary photochemistry. These results indicated that the Hl14 ligated chlorophyll acts as the 'trap' chlorophyll in CP47 at low temperature and that the Q130E mutation imparts considerable changes to PSII electron transfer kinetics, essentially protecting the complex via increased non-radiative charge.

Physiologische Veränderungen bei der Thylakoidmembranbiogenese und die Funktionsweise des Nukleotidaustauschfaktors GrpE in Cyanobakterien

Bei Wachstum im Dunkeln zeigten sich rudimentäre Thylakoidstrukturen, wobei nach dem Transfer ins Licht ein vollständiges Thylakoidmembransystem erneut ausgebildet wurde. Parallel stieg, der Chlorophyllgehalt pro Zelle und das Verhältnis von Phycobilisomen zu Chlorophyll verschob sich erneut auf die Seite des Chlorophylls. Das bei Wachstum im Dunkeln als Monomer vorliegende PS II, war nicht funktional. Nach dem Transfer ins Licht, war nach etwa acht bis zwölf Stunden ein aktives PS II zu detektieren. Das PS I lag nach der Inkubation im Dunkeln, in geringerer Konzentration aber aktiv als Trimer in den Zellen vor.rnZwei Typ I Signalpeptidasen aus Synechocystis sp. PCC 6803 zeigten Unterschiede im Bezug auf ihre intrazelluläre Lokalisation. Für die Untersuchungen der Lokalisation konnte ein neues System der Fluoreszenzmikroskopie entwickelt und erfolgreich eingesetzt werden. Das LepB1 zeigte einen (auto-) proteolytischen Abbau. Für Untersuchungen zur katalytischen Aktivität wurden Vorläuferproteine als Substrate für LepB2 identifiziert.rnDie Funktionsweise der GrpE-Proteine aus verwandten Cyanobakterien zeigt Unterschiede. Bei beiden GrpE-Proteinen erfolgt der reversible Übergang von einem Dimer hin zu einem Monomer innerhalb eines physiologisch relevanten Temperaturbereichs in einem Schritt. Bei dem Protein aus Synechocystis sp. ist der N-Terminus und bei dem Protein aus dem thermophilen Bakterium Thermosynechococcus der C-Terminus für die Dimerisierung essentiell. rn

The heme oxygenase gene (pbsA) in the red alga Rhodella violacea is discontinuous and transcriptionally activated during iron limitation

Heme oxygenase (HO) catalyzes the opening of the heme ring with the release of iron in both plants and animals. In cyanobacteria, red algae, and cryptophyceae, HO is a key enzyme in the synthesis of the chromophoric part of the photosynthetic antennae. In an attempt to study the regulation of this key metabolic step, we cloned and sequenced the pbsA gene encoding this enzyme from the red alga Rhodella violacea. The gene is located on the chloroplast genome, split into three distant exons, and is presumably expressed by a trans-splicing mechanism. The deduced polypeptide sequence is homologous to other reported HOs from organisms containing phycobilisomes (Porphyra purpurea and Synechocystis sp. strain PCC 6803) and, to a lesser extent, to vertebrate enzymes. The expression is transcriptionally activated under iron deprivation, a stress condition frequently encountered by algae, suggesting a second role for HO as an iron-mobilizing agent in photosynthetic organisms.

Requirement of phosphatidylglycerol for photosynthetic function in thylakoid membranes

To investigate the role of phosphatidylglycerol (PG) in photosynthesis, we constructed a mutant defective in the CDP-diacylglycerol synthase gene from a cyanobacterium, Synechocystis sp. PCC6803. The mutant, designated as SNC1, required PG supplementation for growth. Growth was repressed in PG-free medium concomitantly with the decrease in cellular content of PG. These results indicate that PG is essential, and that SNC1 is defective in PG synthesis. Decrease in PG content was accompanied by a reduction in the cellular content of chlorophyll, but with little effect on the contents of phycobilisome pigments, which showed that levels of chlorophyll–protein complexes decreased without alteration of those of phycobilisomes. Regardless of the decrease in the PG content, CO2-dependent photosynthesis by SNC1 was similar to that by the wild type on a chlorophyll basis, but consequently became lower on a cell basis. Simultaneously, the ratio of oxygen evolution of photosystem II (PSII) measured with p-benzoquinone to that of CO2-dependent photosynthesis, which ranged between 1.3 and 1.7 in the wild type. However, it was decreased in SNC1 from 1.3 to 0.4 during the early growth phase where chlorophyll content and CO2-dependent photosynthesis were little affected, and then finally to 0.1, suggesting that PSII first lost its ability to reduce p-benzoquinone and then decreased in its level and actual activity. These results indicate that PG contributes to the accumulation of chlorophyll–protein complexes in thylakoid membranes, and also to normal functioning of PSII.

A response regulator of cyanobacteria integrates diverse environmental signals and is critical for survival under extreme conditions

Microorganisms must sense their environment and rapidly tune their metabolism to ambient conditions to efficiently use available resources. We have identified a gene encoding a response regulator, NblR, that complements a cyanobacterial mutant unable to degrade its light-harvesting complex (phycobilisome), in response to nutrient deprivation. Cells of the nblR mutant (i) have more phycobilisomes than wild-type cells during nutrient-replete growth, (ii) do not degrade phycobilisomes during sulfur, nitrogen, or phosphorus limitation, (iii) cannot properly modulate the phycobilisome level during exposure to high light, and (iv) die rapidly when starved for either sulfur or nitrogen, or when exposed to high light. Apart from regulation of phycobilisome degradation, NblR modulates additional functions critical for cell survival during nutrient-limited and high-light conditions. NblR does not appear to be involved in acclimation responses that occur only during a specific nutrient limitation. In contrast, it controls at least some of the general acclimation responses; those that occur during any of a number of different stress conditions. NblR plays a pivotal role in integrating different environmental signals that link the metabolism of the cell to light harvesting capabilities and the activities of the photosynthetic apparatus; this modulation is critical for cell survival.

Ultraviolet-B photodestruction of a light-harvesting complex.

Cyanobacteria are important contributors to global photosynthesis in both marine and terrestrial environments. Quantitative data are presented on UV-B-induced damage to the major cyanobacterial photosynthetic light harvesting complex, the phycobilisome, and to each of its constituent phycobiliproteins. The photodestruction quantum yield, phi295 nm, for the phycobiliproteins is high (approximately 10(-3), as compared with approximately 10(-7) for visible light). Energy transfer on a picosecond time scale does not compete with photodestruction. Photodamage to phycobilisomes in vitro and in living cells is amplified by causing dissociation and loss of function of the complex. In photosynthetic organisms, UV-B damage to light-harvesting complexes may significantly exceed that to DNA.