Rewiring cyanobacterial photosynthesis by the implementation of an oxygen-tolerant hydrogenase

Molecular hydrogen (H₂) is considered as an ideal energy carrier to replace fossil fuels in future. Biotechnological H₂ production driven by oxygenic photosynthesis appears highly promising, as biocatalyst and H₂ syntheses rely mainly on light, water, and CO₂ and not on rare metals. This biological process requires coupling of the photosynthetic water oxidizing apparatus to a H₂-producing hydrogenase. However, this strategy is impeded by the simultaneous release of oxygen (O₂) which is a strong inhibitor of most hydrogenases. Here, we addressed this challenge, by the introduction of an O₂-tolerant hydrogenase into phototrophic bacteria, namely the cyanobacterial model strain Synechocystis sp. PCC 6803. To this end, the gene cluster encoding the soluble, O₂-tolerant, and NAD(H)-dependent hydrogenase from Ralstonia eutropha (ReSH) was functionally transferred to a Synechocystis strain featuring a knockout of the native O₂ sensitive hydrogenase. Intriguingly, photosynthetically active cells produced the O₂ tolerant ReSH, and activity was confirmed in vitro and in vivo. Further, ReSH enabled the constructed strain Syn_ReSH⁺ to utilize H₂ as sole electron source to fix CO₂. Syn_ReSH⁺ also was able to produce H₂ under dark fermentative conditions as well as in presence of light, under conditions fostering intracellular NADH excess. These findings highlight a high level of interconnection between ReSH and cyanobacterial redox metabolism. This study lays a foundation for further engineering, e.g., of electron transfer to ReSH via NADPH or ferredoxin, to finally enable photosynthesis-driven H₂ production.     

The potential applications of cyanobacterial photosynthesis for clean technologies

Natural photosynthesis may be adapted to advantage in the development of clean energy technologies. Efficient biocatalysts that can be used in solar energy conversion technologies are the cyanobacteria. Photobioreactors incorporating cyanobacteria have been used to demonstrate (a) the production of hydrogen gas, (b) the assimilation of CO₂ with the production of algal biomass, (c) the excretion of ammonium, and (d) the removal of nitrate and phosphate from contaminated waters.Abbreviations Chl chlorophyll - DW dry weight - MSX L-methionine-D-L-sulphoximine - PAR photosynthetically active radiation - PU polyurethane - PV polyvinyl - PVC polyvinylchloride     

Toxicity of organotins towards cyanobacterial photosynthesis and nitrogen fixation

Abstract Inhibition of photosynthesis by a range of organotin compounds in Plectonema boryanum was concentration-dependent and decreased in the order tributyltin (Bu₃SnCl) > tripropyltin (Pr₃SnCl) ≥ dibutyltin (Bu₂SnCl₂) ≥ triphenyltin (Ph₃SnCl) > triethyltin (Et₃SnCl) > trimethyltin (Me₃SnCl) > monobutyltin (BuSnCl₃). IC₅₀ values were determined for the most toxic organotin species and varied from approximately 1.2 μM for Bu₃SnCl to approximately 13 μM for Ph₃SnCl. A similar order of inhibition of photosynthesis was observed in Anabaena cylindrica , although here IC₅₀ values were slightly lower (e.g. approximately 1 μM for Bu₃SnCl and 5 μM for Ph₃SnCl).Nitrogenase activity was generally more sensitive to inhibition by organotin compounds than photosynthesis in A. cylindrica and this was particularlyy evident for Bu₂SnCl₂; approximate IC₅₀ values for Bu₂SnCl₂ were 3 and 9 μM, as estimated by nitrogenase activity and photosynthesis, respectively. These results indicate that organotin compounds have the potential to inhibit cyanobacterial metabolism in aquatic systems.     

巢湖蓝藻水华形成原因探索及"优势种光合假说"

为探索蓝藻水华的形成原因,从2007以来对巢湖西区浮游藻类种类、优势种季节变化、初级生产力、水质参数及优势种的光合生理生态学特性作了观测。关于蓝藻水华形成过程中迅猛发展的原因,近80a已提出了10种假说,但对解释巢湖形成的蓝藻水华,尚显不足。本文基于我们对蓝藻水华的了解,提出了如下“优势种光合假说”：（1）蓝藻水华包含各种藻类,蓝藻水华发生不仅与藻细胞浓度有关,还与水体初级生产力直接有关。巢湖中这两者在夏季最大,在冬季最小。但无定量关系。（2）水华藻类中生长最快、细胞密度最大的是优势种,含有多个优势种时可能随季节更替。巢湖几乎整年发生蓝藻水华,已检测出4种优势种都是蓝藻,从早春起先是水华鱼腥藻,以后有绿色微囊藻、惠氏微囊藻和铜绿微囊藻。（3）各种环境因子都影响优势种生长,其中少数主导因子影响较大。在巢湖富营养条件下,光强、温度和pH值是主导因子。（4）主导因子对优势种光合活性的影响,可决定其能否处于优势。巢湖的温度和pH值变化可能促进了惠氏微囊藻取代绿色微囊藻,铜绿微囊藻取代惠氏微囊藻,而光强变化可能调节冬季时水华鱼腥藻取代了绿色微囊藻,春季时正好是相反的取代。     

Diurnal and seasonal variations of nitrogen fixation and photosynthesis in cyanobacterial mats

In situ measurements of nitrogenase activity and photosynthesis were performed simultaneously in cyanobacterial mats of intertidal sand flats of the Southern North Sea. Two types of cyanobacterial mats, which differed in species composition and biomass content, were investigated. The measurements were done monthly during 3 years to detect seasonal variations of nitrogen fixation and photosynthesis. Diurnal variations were investigated as well. The results showed that (i) freshly colonized sediment with the cyanobacteriumOscillatoria limosa as the dominant organism revealed the highest specific nitrogenase activities (ii) nitrogenase activities were highest in spring and summer, when mat development was initiated and (iii) diurnal fluctuations of nitrogenase activity indicated that it occurred temporally separated from oxygenic photosynthesis.     

Effect of photosynthesis on pH variation in cyanobacterial biofilms from Roman catacombs

Cyanobacterial biofilms present on stone surfaces inRoman hypogea were studied with the aim of assessingtheir deteriogenic activity on the colonisedsubstrata. In order to achieve this, non-destructivemethods were developed and applied to measure pHvariation induced via photosynthesis and respirationin representative cyanobacteria from Roman catacombs.Amperometric and potentiometric microsensors were alsoused on Scytonema biofilms in culture in orderto measure photosynthesis and assess pH decreases andincreases during dark–light periods. Measurementsof pH showed that, starting with values slightly belowneutral, the pH in Scytonema biofilms increasedby 0.24–0.77 units in the transition from dark to1000 mol photon m^-2 s^-1 irradiance.Comparison of photosynthesis and pH curves recordedsimultaneously on the same artificial biofilm showeda maximum increase in pH value at irradiances higherthan those saturating photosynthesis. Alkalinisationof the substrate during illumination occurred to asufficient extent to induce precipitation of mineralcompounds, especially on calcareous substrates such asthose present in Roman hypogea.     

Interactions between nitrogen fixation and oxegenic photosynthesis in a marine cyanobacterial mat

Abstract Cyanobacterial mats developed on fine sandy sediments of the upper littoral of the island of Mellum (North Sea). Freshly colonized sediment was dominated by the non-heterocystous, nitrogen-fixing cyanobacterium Oscillatoria limosa . Well established mats in which the cosmopolitan cyanobacterium Microcoleus chthonoplastes was the dominant organism also usually contained O. limosa as a minor component. This mat was about 1 mm thick and contained high biomass. Photosynthesis was maximal at about 150 μm depth and reached values of 280 μmol oxygen. 1⁻¹ · min⁻¹. On the other hand, in the dark, high respiratory activity turned the mat anaerobic within minutes. Freshly colonized sediment consisted of low cyanobacterial biomass loosely attached to the sand grains and present up to a depth of 2.5 mm. Respiratory activity was low and the sediment remained aerobic to a depth of 2 mm throughout the night. Nitrogen fixation (acetylene reduction) was measured during 24-h periods in both types of mats in order to elucidate interactions with oxygenic photosynthesis and oxygen concentration. Acetylene reduction in the mats showed very different diurnal patterns which depended on the type of mat investigated and the time of year. The results indicated that a temporary separation of oxygenic photosynthesis and nitrogen fixation occurred in the mat. Established mats fixed nitrogen predominantly during the transition from dark to light and vice versa, when oxygenic photosynthesis was reduced or absent. Freshly colonized sediment-fixed nitrogen throughout the night but often a stimulation was seen at dawn. The latter showed much higher specific activities than the established type. Also in spring, specific activities were much higher.     

Glycoengineering of cyanobacterial thylakoid membranes for future studies on the role of glycolipids in photosynthesis

The lipid composition of thylakoid membranes is conserved from cyanobacteria to angiosperms. The predominating components are monogalactosyl- and digalactosyldiacylglycerol. In cyanobacteria, thylakoid membrane biosynthesis starts with the formation of monoglucosyldiacylglycerol which is C4-epimerized to the corresponding galactolipid, whereas in plastids monogalactosyldiacylglycerol is formed at the beginning. This suggests that galactolipids have specific functions in thylakoids. We wanted to investigate whether galactolipids can be replaced by glycosyldiacylglycerols with headgroups differing in their epimeric and anomeric details as well as the attachment point of the terminal hexose in diglycosyldiacylglycerols. For this purpose putative glycosyltransferase sequences were identified in databases to be used for functional expression in various host organisms. From 18 newly identified sequences, four turned out to encode glycosyltransferases catalyzing final steps in glycolipid biosynthesis: two alpha-glucosyltransferases, one beta-galactosyltransferase and one beta-glucosyltransferase. Their functional annotation was based on detailed structural characterization of the new glycolipids formed in the transformant hosts as well as on in vitro enzymatic assays. The expression of alpha-glucosyltransferases in the cyanobacterium Synechococcus resulted in the accumulation of the new alpha-galactosyldiacylglycerol which is ascribed to epimerization of the corresponding glucolipid. The expression of the beta-glucosyltransferase led to a high proportion of new beta-glucosyl-(1-->6)-beta-galactosyldiacylglycerol almost entirely replacing the native digalactosyldiacylglycerol. These results demonstrate that modifications of the glycolipid pattern in thylakoids are possible.     

Characterization of cyanobacterial allophycocyanins absorbing far-red light

Photosynthesis Research - Phycobiliproteins (PBPs) are pigment proteins that comprise phycobilisomes (PBS), major light-harvesting antenna complexes of cyanobacteria and red algae. PBS core...     

Regulation of cyanobacterial photosynthesis determined from variable fluorescence yields of photosystem II

Abstract The cyanobacteria Fremyella diplosiphon 7601 and Synechocystis 6701 were grown in continuous cultures with monochromatic red light (680 nm). The distribution of light energy over photosystem I and II was determined from changes in PS II fluorescence at 685 nm. In both organisms, wavelengths absorbed primarily by chlorophyll a caused the high fluorescent state of PS II (State 1), while wavelengths absorbed by the phycobilisome led to low PS II fluorescence (State 2). Superimposing continuous light 2 on the excitation light yielded State 2 fluorescence patterns for Synechocystis 6701, while F. diplosiphon 7601 showed fluorescence patterns similar to state 1 → 2 transitions and changes in fluorescence yield were related to the intensity of the background light. Some ecological implications of energy (re)distribution in cyanobacterial photosynthesis are discussed.     

Characterization of a cyanobacterial photosystem I complex

A simple procedure is described for the preparation of photosystem I (PSI) particles from Triton X-100-solubilized thylakoid membranes of the unicellular cyanobacterium Synechococcus 6301. The purified PSI complex contained the full complement of antenna chlorophylls, 130 +/- 5/P700, displayed the electron paramagnetic resonance signals characteristic of iron-sulfur centers X, A, and B, and had a protein/chlorophyll ratio of 2.9. Determination of the polypeptide composition, utilizing a uniformly 14C-labeled complex, showed that it contained polypeptides of 70, 18, 17.7, 16, and 10 kDa, in a molar ratio of 4.0:0.7:1.0:0.5:1.6. The relative amount of the lower molecular weight polypeptides showed progressive decrease with increase in Triton X-100 concentration and time of exposure to detergent. Consequently, it is proposed that in vivo the composition of the complex is [70 kDa]4 [18 kDa]1 [17.7 kDa]1 [16 kDa]1 [10 kDa]2. Relative to 130 mol of chlorophyll a, the PSI complex contained 16 mol of carotenoids, 13.7 +/- 1.0 g atoms of Fe, and 12.2 +/- 1.1 g atoms of labile sulfide. The properties of complexes fully depleted of the low-molecular weight polypeptides by treatment with sodium dodecyl sulfate or with proteinase K are also described.     

Structure and functional role of supercomplexes of IsiA and Photosystem I in cyanobacterial photosynthesis

Cyanobacteria express large quantities of the iron stress-inducible protein IsiA under iron deficiency. IsiA can assemble into numerous types of single or double rings surrounding Photosystem I. These supercomplexes are functional in light-harvesting, empty IsiA rings are effective energy dissipaters. Electron microscopy studies of these supercomplexes show that Photosystem I trimers bind 18 IsiA copies in a single ring, whereas monomers may bind up to 35 copies in two rings. Work on mutants indicates that the PsaF/J and PsaL subunits facilitate the formation of closed rings around Photosystem I monomers but are not obligatory components in the formation of Photosystem I-IsiA supercomplexes.     

An alternative explanation for cyanobacterial scum formation and persistence by oxygenic photosynthesis

The cause of persistent cyanobacteria scum formation in lakes is an unresolved subject. Scum refers to the event in which cyanobacteria are at the water surface of a lake. Factors like low turbulence levels, long day-light, high water temperatures and the buoyant capacity of cyanobacterial cells play a role in the occurrence of scums. However, they do not explain why scums are observed at periods during the day when according to theory they should have disappeared into the deeper water layers. In this study, we present an alternative explanation. The hypothesis we present here is that irreversible buoyancy of cyanobacteria colonies is created by the growth of gas bubbles on or within the mucilage of the colonies. These bubbles grow under oxygen super-saturated conditions. At low wind speed and high chlorophyll levels, the dissolved oxygen (DO) produced during photosynthesis by cyanobacteria, cannot escape sufficiently fast to the atmosphere hence a DO supersaturated condition arises in the water. At this stage, growth of oxygen bubbles may occur inside or attached to the mucilage. We present results of compression experiments to support our hypothesis. In a chamber, the pressure on lake water containing a natural cyanobacteria population is increased. At 3 × 10⁵ and 4 × 10⁵ Pa the cyanobacteria colonies were not able to float anymore and sank. This pressure is lower than the 10⁶ Pa needed to collapse all gas vacuoles inside the cyanobacteria cells (Walsby, 1994). The observed change from floating to sinking colonies due to increased water pressure suggests that gas bubbles were present inside the colonies. In lakes, these gas bubbles may lead to permanent buoyancy, i.e. a persistent scum.     

Characterization of photosystem II in salt-stressed cyanobacterial Spirulina platensis cells

PSII activity was inhibited after Spirulina platensis cells were incubated with different salt concentrations (0-0.8 M NaCl) for 12 h. Flash-induced fluorescence kinetics showed that in the absence of DCMU, the half time of the fast and slow components decreased while that of the middle component increased considerably with increasing salt concentration. In the presence of DCMU, fluorescence relaxation was dominated by a 0.6s component in control cells. After salt stress, this was partially replaced by a faster new component with half time of 20-50 ms. Thermoluminescence measurements revealed that S(2)Q(A)(-) and S(2)Q(B)(-) recombinations were shifted to higher temperatures in parallel and the intensities of the thermoluminescence emissions were significantly reduced in salt-stressed cells. The period-four oscillation of the thermoluminescence B band was highly damped. There were no significant changes in contents of CP47, CP43, cytochrome c550, and D1 proteins. However, content of the PsbO protein in thylakoid fraction decreased but increased significantly in soluble fraction. The results suggest that salt stress leads to a modification of the Q(B) niche at the acceptor side and an increase in the stability of the S(2) state at the donor side, which is associated with a dissociation of the PsbO protein.     

Characterization of photosynthesis of Populus euphratica grown in the arid region

Net photosynthetic rate (P _N), stomatal conductance (g _s), intercellular CO₂ concentration (C _i), transpiration rate (E), water use efficiency (WUE), and stomatal limitation (L_s) of Populus euphratica grown at different groundwater depths in the arid region were measured. g _s of the trees with groundwater depth at 4.74 m (D₄) and 5.82 m (D₅) were lower and a little higher than that at 3.82 m (D₃), respectively. Compared with C _i and L_s of the D₃ trees, C _i decreased and L_s increased at 4.74 m, however, Ci increased and L_s decreased at D₅. Hence photosynthetic reduction of P. euphratica was attributed to either stomatal closure or non-stomatal factors depending on the groundwater depths in the plant locations. P _N of the D₃ trees was significantly higher than those at D₄ or D₅. The trees of D₄ and D₅ did not show a significant difference in their P _N, indicating that there are mechanisms of P. euphratica tolerance to mild and moderate drought stress.     

Characterization of cyanobacterial cells synthesizing 10-methyl stearic acid

Photosynthesis Research - Recently, microalgae have attracted attention as sources of biomass energy. However, fatty acids from the microalgae are mainly unsaturated and show low stability in...     

Further evidence that activation of net photosynthesis by dry cyanobacterial lichens requires liquid water

Abstract: In contrast to green algal lichens, cyanobacterial species of different families, growth forms and habitats proved to be unable to attain positive net CO₂ assimilation when the dry thalli were treated with air of high relative humidity; they needed liquid water for the reactivation of their photosynthetic apparatus. Identical behaviour is shown by all of the 47 lichen species with cyanobacterial photobionts, from six different genera, studied so far. This suggests a widely distributed, if not general, characteristic of cyanobacterial lichens. The difference in performance between both groups of photobionts was maintained when the lichen thallus was macerated. Furthermore, cultures of Chroococcidiopsis were unable to make use of water vapour hydration for positive net photosynthesis, and were similar in this respect to some free-living aerophilic cyanohacteria tested earlier. Possible physiological implications as well as ecological consequences for water-relation-dependent habitat selection of green-algal and cyanobacterial lichens are discussed.     

Characterization of photosynthesis in Arabidopsis ER-to-plastid lipid trafficking mutants

Vascular plants use two pathways to synthesize galactolipids, the predominant lipid species in chloroplasts—a prokaryotic pathway that resides entirely in the chloroplast, and a eukaryotic pathway that involves assembly in the endoplasmic reticulum. Mutants deficient in the endoplasmic reticulum pathway, trigalactosyldiacylglycerol (tgd1-1 and tgd2-1) mutants, had been previously identified with reduced contents of monogalactosyldiacylglycerol and digalactosyldiacylglycerol, and altered lipid molecular species composition. Here, we report that the altered lipid composition affected photosynthesis in lipid trafficking mutants. It was found that proton motive force as measured by electrochromic shift was reduced by ~40 % in both tgd mutants. This effect was accompanied by an increase in thylakoid conductance attributable to ATPase activity and so the rate of ATP synthesis was nearly unchanged. Thylakoid conductance to ions also increased in tgd mutants. However, gross carbon assimilation in tgd mutants as measured by gas exchange was only marginally affected. Rubisco activity, electron transport rate, and photosystem I and II oxidation status were not altered. Despite the large differences in proton motive force, responses to heat and high light stress were similar between tgd mutants and the wild type.