Electrochemically Driven Photosynthetic Electron Transport in Cyanobacteria Lacking Photosystem II.

Light-activated photosystem II (PSII) carries out the critical step of splitting water in photosynthesis. However, PSII is susceptible to light-induced damage. Here, results are presented from a novel microbial electro-photosynthetic system (MEPS) that uses redox mediators in conjunction with an electrode to drive electron transport in live Synechocystis (ΔpsbB) cells lacking PSII. MEPS-generated, light-dependent current increased with light intensity up to 2050 µmol photons m-2 s-1, which yielded a delivery rate of 113 µmol electrons h-1 mg-chl-1 and an average current density of 150 A m-2 s-1 mg-chl-1. P700+ re-reduction kinetics demonstrated that initial rates exceeded wildtype PSII-driven electron delivery. The electron delivery occurs ahead of the cytochrome b6f complex to enable both NADPH and ATP production. This work demonstrates an electrochemical system that can drive photosynthetic electron transport, provides a platform for photosynthetic foundational studies, and has the potential for improving photosynthetic performance at high light intensities.

A Dedicated Type II NADPH Dehydrogenase Performs the Penultimate Step in the Biosynthesis of Vitamin K1 in Synechocystis and Arabidopsis.

Mutation of Arabidopsis thaliana NAD(P)H DEHYDROGENASE C1 (NDC1; At5g08740) results in the accumulation of demethylphylloquinone, a late biosynthetic intermediate of vitamin K1. Gene coexpression and phylogenomics analyses showed that conserved functional associations occur between vitamin K biosynthesis and NDC1 homologs throughout the prokaryotic and eukaryotic lineages. Deletion of Synechocystis ndbB, which encodes for one such homolog, resulted in the same defects as those observed in the cyanobacterial demethylnaphthoquinone methyltransferase knockout. Chemical modeling and assay of purified demethylnaphthoquinone methyltransferase demonstrated that, by virtue of the strong electrophilic nature of S-adenosyl-l-methionine, the transmethylation of the demethylated precursor of vitamin K is strictly dependent on the reduced form of its naphthoquinone ring. NDC1 was shown to catalyze such a prerequisite reduction by using NADPH and demethylphylloquinone as substrates and flavine adenine dinucleotide as a cofactor. NDC1 displayed Michaelis-Menten kinetics and was markedly inhibited by dicumarol, a competitive inhibitor of naphthoquinone oxidoreductases. These data demonstrate that the reduction of the demethylnaphthoquinone ring represents an authentic step in the biosynthetic pathway of vitamin K, that this reaction is enzymatically driven, and that a selection pressure is operating to retain type II NAD(P)H dehydrogenases in this process.

Redesigning photosynthesis to sustainably meet global food and bioenergy demand.

The world's crop productivity is stagnating whereas population growth, rising affluence, and mandates for biofuels put increasing demands on agriculture. Meanwhile, demand for increasing cropland competes with equally crucial global sustainability and environmental protection needs. Addressing this looming agricultural crisis will be one of our greatest scientific challenges in the coming decades, and success will require substantial improvements at many levels. We assert that increasing the efficiency and productivity of photosynthesis in crop plants will be essential if this grand challenge is to be met. Here, we explore an array of prospective redesigns of plant systems at various scales, all aimed at increasing crop yields through improved photosynthetic efficiency and performance. Prospects range from straightforward alterations, already supported by preliminary evidence of feasibility, to substantial redesigns that are currently only conceptual, but that may be enabled by new developments in synthetic biology. Although some proposed redesigns are certain to face obstacles that will require alternate routes, the efforts should lead to new discoveries and technical advances with important impacts on the global problem of crop productivity and bioenergy production.

The γ-aminobutyric acid shunt contributes to closing the tricarboxylic acid cycle in Synechocystis sp. PCC 6803.

A traditional 2-oxoglutarate dehydrogenase complex is missing in the cyanobacterial tricarboxylic acid cycle. To determine pathways that convert 2-oxoglutarate into succinate in the cyanobacterium Synechocystis sp. PCC 6803, a series of mutant strains, Δsll1981, Δslr0370, Δslr1022 and combinations thereof, deficient in 2-oxoglutarate decarboxylase (Sll1981), succinate semialdehyde dehydrogenase (Slr0370), and/or in γ-aminobutyrate metabolism (Slr1022) were constructed. Like in Pseudomonas aeruginosa, N-acetylornithine aminotransferase, encoded by slr1022, was shown to also function as γ-aminobutyrate aminotransferase, catalysing γ-aminobutyrate conversion to succinic semialdehyde. As succinic semialdehyde dehydrogenase converts succinic semialdehyde to succinate, an intact γ-aminobutyrate shunt is present in Synechocystis. The Δsll1981 strain, lacking 2-oxoglutarate decarboxylase, exhibited a succinate level that was 60% of that in wild type. However, the succinate level in the Δslr1022 and Δslr0370 strains and the Δsll1981/Δslr1022 and Δsll1981/Δslr0370 double mutants was reduced to 20-40% of that in wild type, suggesting that the γ-aminobutyrate shunt has a larger impact on metabolite flux to succinate than the pathway via 2-oxoglutarate decarboxylase. (13) C-stable isotope analysis indicated that the γ-aminobutyrate shunt catalysed conversion of glutamate to succinate. Independent of the 2-oxoglutarate decarboxylase bypass, the γ-aminobutyrate shunt is a major contributor to flux from 2-oxoglutarate and glutamate to succinate in Synechocystis sp. PCC 6803.

The sll1951 gene encodes the surface layer protein of Synechocystis sp. strain PCC 6803.

Sll1951 is the surface layer (S-layer) protein of the cyanobacterium Synechocystis sp. strain PCC 6803. This large, hemolysin-like protein was found in the supernatant of a strain that was deficient in S-layer attachment. An sll1951 deletion mutation was introduced into Synechocystis and was easily segregated to homozygosity under laboratory conditions. By thin-section and negative-stain transmission electron microscopy, a ~30-nm-wide S-layer lattice covering the cell surface was readily visible in wild-type cells but was absent in the Δsll1951 strain. Instead, the Δsll1951 strain displayed a smooth lipopolysaccharide surface as its most peripheral layer. In the presence of chaotropic agents, the wild type released a large (>150-kDa) protein into the medium that was identified as Sll1951 by mass spectrometry of trypsin fragments; this protein was missing in the Δsll1951 strain. In addition, Sll1951 was prominent in crude extracts of the wild type, indicating that it is an abundant protein. The carotenoid composition of the cell wall fraction of the Δsll1951 strain was similar to that of the wild type, suggesting that the S-layer does not contribute to carotenoid binding. Although the photoautotrophic growth rate of the Δsll1951 strain was similar to that of the wild-type strain, the viability of the Δsll1951 strain was reduced upon exposure to lysozyme treatment and hypo-osmotic stress, indicating a contribution of the S-layer to the integrity of the Synechocystis cell wall. This work identifies the S-layer protein in Synechocystis and shows that, at least under laboratory conditions, this very abundant, large protein has a supportive but not a critical role in the function of the cyanobacterium.

Photosystem II component lifetimes in the cyanobacterium Synechocystis sp. strain PCC 6803: small Cab-like proteins stabilize biosynthesis intermediates and affect early steps in chlorophyll synthesis.

To gain insight in the lifetimes of photosystem II (PSII) chlorophyll and proteins, a combined stable isotope labeling (15N)/mass spectrometry method was used to follow both old and new pigments and proteins. Photosystem I-less Synechocystis cells were grown to exponential or post-exponential phase and then diluted in BG-11 medium with [15N]ammonium and [15N]nitrate. PSII was isolated, and the masses of PSII protein fragments and chlorophyll were determined. Lifetimes of PSII components ranged from 1.5 to 40 h, implying that at least some of the proteins and chlorophyll turned over independently from each other. Also, a significant amount of nascent PSII components accumulated in thylakoids when cells were in post-exponential growth phase. In a mutant lacking small Cab-like proteins (SCPs), most PSII protein lifetimes were unaffected, but the lifetime of chlorophyll and the amount of nascent PSII components that accumulated were decreased. In the absence of SCPs, one of the PSII biosynthesis intermediates, the monomeric PSII complex without CP43, was missing. Therefore, SCPs may stabilize nascent PSII protein complexes. Moreover, upon SCP deletion, the rate of chlorophyll synthesis and the accumulation of early tetrapyrrole precursors were drastically reduced. When [14N]aminolevulinic acid (ALA) was supplemented to 15N-BG-11 cultures, the mutant lacking SCPs incorporated much more exogenous ALA into chlorophyll than the control demonstrating that ALA biosynthesis was impaired in the absence of SCPs. This illustrates the major effects that nonstoichiometric PSII components such as SCPs have on intermediates and assembly but not on the lifetime of PSII proteins.

Gross morphological changes in thylakoid membrane structure are associated with photosystem I deletion in Synechocystis sp. PCC 6803.

Cells of Synechocystis sp. PCC 6803 lacking photosystem I (PSI-less) and containing only photosystem II (PSII) or lacking both photosystems I and II (PSI/PSII-less) were compared to wild type (WT) cells to investigate the role of the photosystems in the architecture, structure, and number of thylakoid membranes. All cells were grown at 0.5µmol photons m(-2)s(-1). The lumen of the thylakoid membranes of the WT cells grown at this low light intensity were inflated compared to cells grown at higher light intensity. Tubular as well as sheet-like thylakoid membranes were found in the PSI-less strain at all stages of development with organized regular arrays of phycobilisomes on the surface of the thylakoid membranes. Tubular structures were also found in the PSI/PSII-less strain, but these were smaller in diameter to those found in the PSI-less strain with what appeared to be a different internal structure and were less common. There were fewer and smaller thylakoid membrane sheets in the double mutant and the phycobilisomes were found on the surface in more disordered arrays. These differences in thylakoid membrane structure most likely reflect the altered composition of photosynthetic particles and distribution of other integral membrane proteins and their interaction with the lipid bilayer. These results suggest an important role for the presence of PSII in the formation of the highly ordered tubular structures.

ClpB1 overproduction in Synechocystis sp. strain PCC 6803 increases tolerance to rapid heat shock.

ClpB1 is a heat shock protein known to disaggregate large protein complexes. Constitutive, 16-fold ClpB1 overproduction in the cyanobacterium Synechocystis sp. strain PCC 6803 increased cell survival by 20-fold when cultures were heated quickly (1°C/s) to 50°C and delayed cell death by an average of 3 min during incubation at high temperatures (>46°C). Cooverexpression of ClpB1 and another heat shock protein, DnaK2, further increased cell survival. According to immunocytochemistry results, ClpB1 is dispersed throughout the cytoplasm but is concentrated in specific areas and is more prevalent near thylakoid membranes. However, ClpB1 overproduction does not lead to a change in the morphology, chlorophyll content, or photosystem ratio. Whereas electron microscopy demonstrated that apparent protein aggregation occurred after heat treatment in the control strain, protein aggregate size was maintained in the ClpB1 overexpresser. Constitutive ClpB1 overproduction allows an earlier response to heat shock and protects from rapid heating of cultures.

Polyhydroxybutyrate particles in Synechocystis sp. PCC 6803: facts and fiction.

Transmission electron microscopy has been used to identify poly-3-hydroxybutyrate (PHB) granules in cyanobacteria for over 40 years. Spherical inclusions inside the cell that are electron-transparent and/or slightly electron-dense and that are found in transmission electron micrographs of cyanobacteria are generally assumed to be PHB granules. The aim of this study was to test this assumption in different strains of the cyanobacterium Synechocystis sp. PCC 6803. Inclusions that resemble PHB granules were present in strains lacking a pair of genes essential for PHB synthesis and in wild-type cells under conditions that no PHB granules could be detected by fluorescence staining of PHB. Indeed, in these cells PHB could not be demonstrated chemically by GC/MS either. Based on the results gathered, it is concluded that not all the slightly electron-dense spherical inclusions are PHB granules in Synechocystis sp. PCC 6803. This result is potentially applicable to other cyanobacteria. Alternate assignments for these inclusions are discussed.

Carotenoid-triggered energy dissipation in phycobilisomes of Synechocystis sp. PCC 6803 diverts excitation away from reaction centers of both photosystems.

Cyanobacteria are capable of using dissipation of phycobilisome-absorbed energy into heat as part of their photoprotective strategy. Non-photochemical quenching in cyanobacteria cells is triggered by absorption of blue-green light by the carotenoid-binding protein, and involves quenching of phycobilisome fluorescence. In this study, we find direct evidence that the quenching is accompanied by a considerable reduction of energy flow to the photosystems. We present light saturation curves of photosystems' activity in quenched and non-quenched states in the cyanobacterium Synechocystis sp. PCC 6803. In the quenched state, the quantum efficiency of light absorbed by phycobilisomes drops by about 30-40% for both photoreactions-P700 photooxidation in the photosystem II-less strain and photosystem II fluorescence induction in the photosystem I-less strain of Synechocystis. A similar decrease of the excitation pressure on both photosystems leads us to believe that the core-membrane linker allophycocyanin APC-L(CM) is at or beyond the point of non-photochemical quenching. We analyze 77 K fluorescence spectra and suggest that the quenching center is formed at the level of the short-wavelength allophycocyanin trimers. It seems that both chlorophyll and APC-L(CM) may dissipate excess energy via uphill energy transfer at physiological temperatures, but neither of the two is at the heart of the carotenoid-binding protein-dependent non-photochemical quenching mechanism.

In vivo hyperspectral confocal fluorescence imaging to determine pigment localization and distribution in cyanobacterial cells.

Hyperspectral confocal fluorescence imaging provides the opportunity to obtain individual fluorescence emission spectra in small ( approximately 0.03-microm(3)) volumes. Using multivariate curve resolution, individual fluorescence components can be resolved, and their intensities can be calculated. Here we localize, in vivo, photosynthesis-related pigments (chlorophylls, phycobilins, and carotenoids) in wild-type and mutant cells of the cyanobacterium Synechocystis sp. PCC 6803. Cells were excited at 488 nm, exciting primarily phycobilins and carotenoids. Fluorescence from phycocyanin, allophycocyanin, allophycocyanin-B/terminal emitter, and chlorophyll a was resolved. Moreover, resonance-enhanced Raman signals and very weak fluorescence from carotenoids were observed. Phycobilin emission was most intense along the periphery of the cell whereas chlorophyll fluorescence was distributed more evenly throughout the cell, suggesting that fluorescing phycobilisomes are more prevalent along the outer thylakoids. Carotenoids were prevalent in the cell wall and also were present in thylakoids. Two chlorophyll fluorescence components were resolved: the short-wavelength component originates primarily from photosystem II and is most intense near the periphery of the cell; and the long-wavelength component that is attributed to photosystem I because it disappears in mutants lacking this photosystem is of higher relative intensity toward the inner rings of the thylakoids. Together, the results suggest compositional heterogeneity between thylakoid rings, with the inner thylakoids enriched in photosystem I. In cells depleted in chlorophyll, the amount of both chlorophyll emission components was decreased, confirming the accuracy of the spectral assignments. These results show that hyperspectral fluorescence imaging can provide unique information regarding pigment organization and localization in the cell.

Substrate specificities and availability of fucosyltransferase and beta-carotene hydroxylase for myxol 2'-fucoside synthesis in Anabaena sp. strain PCC 7120 compared with Synechocystis sp. strain PCC 6803.

To elucidate the biosynthetic pathways of carotenoids, especially myxol 2'-glycosides, in cyanobacteria, Anabaena sp. strain PCC 7120 (also known as Nostoc sp. strain PCC 7120) and Synechocystis sp. strain PCC 6803 deletion mutants lacking selected proposed carotenoid biosynthesis enzymes and GDP-fucose synthase (WcaG), which is required for myxol 2'-fucoside production, were analyzed. The carotenoids in these mutants were identified using high-performance liquid chromatography, field desorption mass spectrometry, and (1)H nuclear magnetic resonance. The wcaG (all4826) deletion mutant of Anabaena sp. strain PCC 7120 produced myxol 2'-rhamnoside and 4-ketomyxol 2'-rhamnoside as polar carotenoids instead of the myxol 2'-fucoside and 4-ketomyxol 2'-fucoside produced by the wild type. Deletion of the corresponding gene in Synechocystis sp. strain PCC 6803 (sll1213; 79% amino acid sequence identity with the Anabaena sp. strain PCC 7120 gene product) produced free myxol instead of the myxol 2'-dimethyl-fucoside produced by the wild type. Free myxol might correspond to the unknown component observed previously in the same mutant (H. E. Mohamed, A. M. L. van de Meene, R. W. Roberson, and W. F. J. Vermaas, J. Bacteriol. 187:6883-6892, 2005). These results indicate that in Anabaena sp. strain PCC 7120, but not in Synechocystis sp. strain PCC 6803, rhamnose can be substituted for fucose in myxol glycoside. The beta-carotene hydroxylase orthologue (CrtR, Alr4009) of Anabaena sp. strain PCC 7120 catalyzed the transformation of deoxymyxol and deoxymyxol 2'-fucoside to myxol and myxol 2'-fucoside, respectively, but not the beta-carotene-to-zeaxanthin reaction, whereas CrtR from Synechocystis sp. strain PCC 6803 catalyzed both reactions. Thus, the substrate specificities or substrate availabilities of both fucosyltransferase and CrtR were different in these species. The biosynthetic pathways of carotenoids in Anabaena sp. strain PCC 7120 are discussed.

Phycobilin/chlorophyll excitation equilibration upon carotenoid-induced non-photochemical fluorescence quenching in phycobilisomes of the cyanobacterium Synechocystis sp. PCC 6803.

To determine the mechanism of carotenoid-sensitized non-photochemical quenching in cyanobacteria, the kinetics of blue-light-induced quenching and fluorescence spectra were studied in the wild type and mutants of Synechocystis sp. PCC 6803 grown with or without iron. The blue-light-induced quenching was observed in the wild type as well as in mutants lacking PS II or IsiA confirming that neither IsiA nor PS II is required for carotenoid-triggered fluorescence quenching. Both fluorescence at 660 nm (originating from phycobilisomes) and at 681 nm (which, upon 440 nm excitation originates mostly from chlorophyll) was quenched. However, no blue-light-induced changes in the fluorescence yield were observed in the apcE(-) mutant that lacks phycobilisome attachment. The results are interpreted to indicate that interaction of the Slr1963-associated carotenoid with--presumably--allophycocyanin in the phycobilisome core is responsible for non-photochemical energy quenching, and that excitations on chlorophyll in the thylakoid equilibrate sufficiently with excitations on allophycocyanin in wild type to contribute to quenching of chlorophyll fluorescence.

Functional implications of pigments bound to a cyanobacterial cytochrome b6f complex.

A highly purified cytochrome b(6)f complex from the cyanobacterium Synechocystis sp. PCC 6803 selectively binds one chlorophyll a and one carotenoid in analogy to the recent published structure from two other b(6)f complexes. The unknown function of these pigments was elucidated by spectroscopy and site-directed mutagenesis. Low-temperature redox difference spectroscopy showed red shifts in the chlorophyll and carotenoid spectra upon reduction of cytochrome b(6), which indicates coupling of these pigments with the heme groups and thereby with the electron transport. This is supported by the correlated kinetics of these redox reactions and also by the distinct orientation of the chlorophyll molecule with respect to the heme cofactors as shown by linear dichroism spectroscopy. The specific role of the carotenoid echinenone for the cytochrome b(6)f complex of Synechocystis 6803 was elucidated by a mutant lacking the last step of echinenone biosynthesis. The isolated mutant complex preferentially contained a carotenoid with 0, 1 or 2 hydroxyl groups (most likely 9-cis isomers of beta-carotene, a monohydroxy carotenoid and zeaxanthin, respectively) instead. This indicates a substantial role of the carotenoid - possibly for strucure and assembly - and a specificity of its binding site which is different from those in most other oxygenic photosynthetic organisms. In summary, both pigments are probably involved in the structure, but may also contribute to the dynamics of the cytochrome b(6)f complex.

Transformation of the cyanobacterium Synechocystis sp. PCC 6803 as a tool for genetic mapping: optimization of efficiency.

The cyanobacterium Synechocystis sp. PCC 6803 is transformable at high efficiency and integrates DNA by homologous double recombination. However, several genetic mapping procedures depend on the ability to generate transformants even with very small amounts of added DNA. This study is aimed at optimizing the transformation efficiency at limiting concentrations of exogenous DNA. The transformation efficiency showed little sensitivity to experimental conditions. Transformation with circular plasmid DNA was found to be no more than 30% more efficient than with linearized plasmid DNA. The efficiency of transformation remained essentially the same in the presence of competing DNA, indicating that the capacity of DNA uptake by the cells is not limiting. The incubation time of cells with DNA before plating (0-8 h) affected the transformation efficiency by up to 3-fold. Only minor changes in the efficiency were observed as a function of the presence of a membrane filter on the plate or the presence of TAE or TBE gel buffer residues in the transformation mixture. However, transformability of the host strain of Synechocystis sp. PCC 6803 was increased by two orders of magnitude if the sll1354 gene encoding the exonuclease RecJ was deleted. Therefore, the transformation efficiency of Synechocystis sp. PCC 6803 with exogenous DNA appears to be determined primarily by intracellular processes such as the efficiency of DNA processing and homologous recombination.

Regulation of the tetrapyrrole biosynthetic pathway leading to heme and chlorophyll in plants and cyanobacteria.

Photosynthetic organisms synthesize chlorophylls, hemes, and bilin pigments via a common tetrapyrrole biosynthetic pathway. This review summarizes current knowledge about the regulation of this pathway in plants, algae, and cyanobacteria. Particular emphasis is placed on the regulation of glutamate-1-semialdehyde formation and on the channelling of protoporphyrin IX into the heme and chlorophyll branches. The potential role of chlorophyll molecules that are not bound to photosynthetic pigment-protein complexes ('free chlorophylls') or of other Mg-containing porphyrins in regulation of tetrapyrrole synthesis is also discussed.

Lifetimes of photosystem I and II proteins in the cyanobacterium Synechocystis sp. PCC 6803.

The half-life times of photosystem I and II proteins were determined using (15)N-labeling and mass spectrometry. The half-life times (30-75h for photosystem I components and <1-11h for the large photosystem II proteins) were similar when proteins were isolated from monomeric vs. oligomeric complexes on Blue-Native gels, suggesting that the two forms of both photosystems can interchange on a timescale of <1h or that only one form of each photosystem exists in thylakoids in vivo. The half-life times of proteins associated with either photosystem generally were unaffected by the absence of Small Cab-like proteins.

Biological hydrogen production: prospects and challenges.

Hydrogen gas provides exceptional value as an energy carrier and industrial feedstock, but currently is produced entirely by reforming fossil fuels. Biological hydrogen production (BioH(2)), which offers the possibility of being renewable and carbon neutral, can be achieved by photosynthesis, fermentation, and microbial electrolysis cells. This review introduces the principles, advantages and challenges of each approach to BioH(2). Photosynthetic BioH(2) is the ultimate renewable source, since it directly uses inexhaustible resources: sunlight energy and electrons from H(2)O. However, it presents major technical challenges, particularly due to oxygen sensitivity. Fermentative BioH(2) offers a high production rate, but poor conversion efficiency from the organic substrate to H(2). The microbial electrolysis cell can achieve high conversion efficiency, but is an emerging technology.

Sll0254 (CrtL(diox)) is a bifunctional lycopene cyclase/dioxygenase in cyanobacteria producing myxoxanthophyll.

Upon depletion of Sll0254 in Synechocystis sp. strain PCC 6803, cyclized carotenoids were replaced by linear, relatively hydrophilic carotenoids, and the amount of the two photosystems decreased greatly. Full segregants of the sll0254 deletion in Synechocystis were not obtained, implying that this gene is essential for survival, most likely to allow normal cell division. The N-terminal half of Sll0254 has limited similarity to the family of lycopene cyclases, has an additional dehydrogenase motif near the N terminus, and is followed by a Rieske 2Fe-2S center sequence signature. To test whether Sll0254 serves as a lycopene cyclase in Synechocystis, the corresponding gene was expressed in Escherichia coli strains that can produce lycopene or neurosporene. In the presence of Sll0254 these linear carotenoids were converted into cyclized, relatively hydrophilic pigments, with masses consistent with the introduction of two hydroxyl groups and with spectra indicative of only small changes in the number of conjugated double bonds. This suggests that Sll0254 catalyzes formation of oxygenated, cyclized carotenoids. We interpret the appearance of the hydroxyl groups in the carotenoids to be due to dioxygenase activity involving the Rieske 2Fe-2S center and the additional dehydrogenase domain. This dioxygenase activity is required in the myxoxanthophyll biosynthesis pathway, after or concomitant with cyclization on the other end of the molecule. We interpret Sll0254 to be a dual-function enzyme with both lycopene cyclase and dioxygenase activity and have named it CrtL(diox).