Photosynthesis re-wired on the pico-second timescale.

Photosystems II and I (PSII, PSI) are the reaction centre-containing complexes driving the light reactions of photosynthesis; PSII performs light-driven water oxidation and PSI further photo-energizes harvested electrons. The impressive efficiencies of the photosystems have motivated extensive biological, artificial and biohybrid approaches to 're-wire' photosynthesis for higher biomass-conversion efficiencies and new reaction pathways, such as H2 evolution or CO2 fixation1,2. Previous approaches focused on charge extraction at terminal electron acceptors of the photosystems3. Electron extraction at earlier steps, perhaps immediately from photoexcited reaction centres, would enable greater thermodynamic gains; however, this was believed impossible with reaction centres buried at least 4 nm within the photosystems4,5. Here, we demonstrate, using in vivo ultrafast transient absorption (TA) spectroscopy, extraction of electrons directly from photoexcited PSI and PSII at early points (several picoseconds post-photo-excitation) with live cyanobacterial cells or isolated photosystems, and exogenous electron mediators such as 2,6-dichloro-1,4-benzoquinone (DCBQ) and methyl viologen. We postulate that these mediators oxidize peripheral chlorophyll pigments participating in highly delocalized charge-transfer states after initial photo-excitation. Our results challenge previous models that the photoexcited reaction centres are insulated within the photosystem protein scaffold, opening new avenues to study and re-wire photosynthesis for biotechnologies and semi-artificial photosynthesis.

Is it realistic to use microbial photosynthesis to produce electricity directly?

It is possible to generate small amounts of electrical power directly from photosynthetic microorganisms-arguably the greenest of green energy. But will it have useful applications, and what are the hurdles if so?

Publisher Correction: Genetic tool development in marine protists: emerging model organisms for experimental cell biology.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Genetic tool development in marine protists: emerging model organisms for experimental cell biology.

Diverse microbial ecosystems underpin life in the sea. Among these microbes are many unicellular eukaryotes that span the diversity of the eukaryotic tree of life. However, genetic tractability has been limited to a few species, which do not represent eukaryotic diversity or environmentally relevant taxa. Here, we report on the development of genetic tools in a range of protists primarily from marine environments. We present evidence for foreign DNA delivery and expression in 13 species never before transformed and for advancement of tools for eight other species, as well as potential reasons for why transformation of yet another 17 species tested was not achieved. Our resource in genetic manipulation will provide insights into the ancestral eukaryotic lifeforms, general eukaryote cell biology, protein diversification and the evolution of cellular pathways.

3D-printed hierarchical pillar array electrodes for high-performance semi-artificial photosynthesis.

The rewiring of photosynthetic biomachineries to electrodes is a forward-looking semi-artificial route for sustainable bio-electricity and fuel generation. Currently, it is unclear how the electrode and biomaterial interface can be designed to meet the complex requirements for high biophotoelectrochemical performance. Here we developed an aerosol jet printing method for generating hierarchical electrode structures using indium tin oxide nanoparticles. We printed libraries of micropillar array electrodes varying in height and submicrometre surface features, and studied the energy/electron transfer processes across the bio-electrode interfaces. When wired to the cyanobacterium Synechocystis sp. PCC 6803, micropillar array electrodes with microbranches exhibited favourable biocatalyst loading, light utilization and electron flux output, ultimately almost doubling the photocurrent of state-of-the-art porous structures of the same height. When the micropillars' heights were increased to 600 µm, milestone mediated photocurrent densities of 245 µA cm-2 (the closest thus far to theoretical predictions) and external quantum efficiencies of up to 29% could be reached. This study demonstrates how bio-energy from photosynthesis could be more efficiently harnessed in the future and provide new tools for three-dimensional electrode design.

A dual compartment cuvette system for correcting scattering in whole-cell absorbance spectroscopy of photosynthetic microorganisms.

Absorption spectroscopy is widely used to determine absorption and transmission spectra of chromophores in solution, in addition to suspensions of particles, including micro-organisms. Light scattering, caused by photons deflected from part or all of the cells or other particles in suspension, results in distortions to the absorption spectra, lost information and poor resolution. A spectrophotometer with an integrating sphere may be used to alleviate this problem. However, these instruments are not universally available in biology laboratories, for reasons such as cost. Here, we describe a novel, rapid, and inexpensive technique that minimises the effect of light scattering when performing whole-cell spectroscopy. This method involves using a custom made dual compartment cuvette containing titanium dioxide in one chamber as a scattering agent. Measurements were conducted of a range of different photosynthetic micro-organisms of varying cell size and morphology, including cyanobacteria, eukaryotic microalgae and a purple non-sulphur bacterium. A concentration of 1 mg ml-1 titanium dioxide, using a spectrophotometer with a slit width of 5 nm, produced spectra for cyanobacteria and microalgae similar (1-4% difference) to those obtained using an integrating sphere. The spectrum > 520 nm was similar to that with an integrating sphere with the purple non-sulphur bacterium. This system produced superior results to those obtained using a recently reported method, the application of the diffusing agent, Scotch™ Magic tape, to the side of the cuvette. The protocol can be completed in an equivalent period of time to standard whole-cell absorbance spectroscopy techniques, and is, in principle, suitable for any dual-beam spectrophotometer.

Cytochrome c M Decreases Photosynthesis under Photomixotrophy in Synechocystis sp. PCC 6803.

Photomixotrophy is a metabolic state that enables photosynthetic microorganisms to simultaneously perform photosynthesis and metabolism of imported organic carbon substrates. This process is complicated in cyanobacteria, since many, including Synechocystis sp. PCC 6803, conduct photosynthesis and respiration in an interlinked thylakoid membrane electron transport chain. Under photomixotrophy, the cell must therefore tightly regulate electron fluxes from photosynthetic and respiratory complexes. In this study, we demonstrate, via characterization of photosynthetic apparatus and the proteome, that photomixotrophic growth results in a gradual inhibition of QA - reoxidation in wild-type Synechocystis, which largely decreases photosynthesis over 3 d of growth. This process is circumvented by deleting the gene encoding cytochrome c M (CytM), a cryptic c-type heme protein widespread in cyanobacteria. The ΔCytM strain maintained active photosynthesis over the 3-d period, demonstrated by high photosynthetic O2 and CO2 fluxes and effective yields of PSI and PSII. Overall, this resulted in a higher growth rate compared to that of the wild type, which was maintained by accumulation of proteins involved in phosphate and metal uptake, and cofactor biosynthetic enzymes. While the exact role of CytM has not been determined, a mutant deficient in the thylakoid-localized respiratory terminal oxidases and CytM (ΔCox/Cyd/CytM) displayed a phenotype similar to that of ΔCytM under photomixotrophy. This, in combination with other physiological data, and in contrast to a previous hypothesis, suggests that CytM does not transfer electrons to these complexes. In summary, our data suggest that CytM may have a regulatory role in photomixotrophy by modulating the photosynthetic capacity of cells.

Proteome Mapping of a Cyanobacterium Reveals Distinct Compartment Organization and Cell-Dispersed Metabolism.

Cyanobacteria are complex prokaryotes, incorporating a Gram-negative cell wall and internal thylakoid membranes (TMs). However, localization of proteins within cyanobacterial cells is poorly understood. Using subcellular fractionation and quantitative proteomics, we produced an extensive subcellular proteome map of an entire cyanobacterial cell, identifying ∼67% of proteins in Synechocystis sp. PCC 6803, ∼1000 more than previous studies. Assigned to six specific subcellular regions were 1,712 proteins. Proteins involved in energy conversion localized to TMs. The majority of transporters, with the exception of a TM-localized copper importer, resided in the plasma membrane (PM). Most metabolic enzymes were soluble, although numerous pathways terminated in the TM (notably those involved in peptidoglycan monomer, NADP+, heme, lipid, and carotenoid biosynthesis) or PM (specifically, those catalyzing lipopolysaccharide, molybdopterin, FAD, and phylloquinol biosynthesis). We also identified the proteins involved in the TM and PM electron transport chains. The majority of ribosomal proteins and enzymes synthesizing the storage compound polyhydroxybuyrate formed distinct clusters within the data, suggesting similar subcellular distributions to one another, as expected for proteins operating within multicomponent structures. Moreover, heterogeneity within membrane regions was observed, indicating further cellular complexity. Cyanobacterial TM protein localization was conserved in Arabidopsis (Arabidopsis thaliana) chloroplasts, suggesting similar proteome organization in more developed photosynthetic organisms. Successful application of this technique in Synechocystis suggests it could be applied to mapping the proteomes of other cyanobacteria and single-celled organisms. The organization of the cyanobacterial cell revealed here substantially aids our understanding of these environmentally and biotechnologically important organisms.

CyanoGate: A Modular Cloning Suite for Engineering Cyanobacteria Based on the Plant MoClo Syntax.

Recent advances in synthetic biology research have been underpinned by an exponential increase in available genomic information and a proliferation of advanced DNA assembly tools. The adoption of plasmid vector assembly standards and parts libraries has greatly enhanced the reproducibility of research and the exchange of parts between different labs and biological systems. However, a standardized modular cloning (MoClo) system is not yet available for cyanobacteria, which lag behind other prokaryotes in synthetic biology despite their huge potential regarding biotechnological applications. By building on the assembly library and syntax of the Plant Golden Gate MoClo kit, we have developed a versatile system called CyanoGate that unites cyanobacteria with plant and algal systems. Here, we describe the generation of a suite of parts and acceptor vectors for making (1) marked/unmarked knock-outs or integrations using an integrative acceptor vector, and (2) transient multigene expression and repression systems using known and previously undescribed replicative vectors. We tested and compared the CyanoGate system in the established model cyanobacterium Synechocystis sp. PCC 6803 and the more recently described fast-growing strain Synechococcus elongatus UTEX 2973. The UTEX 2973 fast-growth phenotype was only evident under specific growth conditions; however, UTEX 2973 accumulated high levels of proteins with strong native or synthetic promoters. The system is publicly available and can be readily expanded to accommodate other standardized MoClo parts to accelerate the development of reliable synthetic biology tools for the cyanobacterial community.

An essential pentatricopeptide repeat protein in the apicomplexan remnant chloroplast.

The malaria parasite Plasmodium and other apicomplexans such as Toxoplasma evolved from photosynthetic organisms and contain an essential, remnant plastid termed the apicoplast. Transcription of the apicoplast genome is polycistronic with extensive RNA processing. Yet little is known about the mechanism of apicoplast RNA processing. In plants, chloroplast RNA processing is controlled by multiple pentatricopeptide repeat (PPR) proteins. Here, we identify the single apicoplast PPR protein, PPR1. We show that the protein is essential and that it binds to RNA motifs corresponding with previously characterized processing sites. Additionally, PPR1 shields RNA transcripts from ribonuclease degradation. This is the first characterization of a PPR protein from a nonphotosynthetic plastid.

Photoelectrochemistry of Photosystem II in Vitro vs in Vivo.

Factors governing the photoelectrochemical output of photosynthetic microorganisms are poorly understood, and energy loss may occur due to inefficient electron transfer (ET) processes. Here, we systematically compare the photoelectrochemistry of photosystem II (PSII) protein-films to cyanobacteria biofilms to derive: (i) the losses in light-to-charge conversion efficiencies, (ii) gains in photocatalytic longevity, and (iii) insights into the ET mechanism at the biofilm interface. This study was enabled by the use of hierarchically structured electrodes, which could be tailored for high/stable loadings of PSII core complexes and Synechocystis sp. PCC 6803 cells. The mediated photocurrent densities generated by the biofilm were 2 orders of magnitude lower than those of the protein-film. This was partly attributed to a lower photocatalyst loading as the rate of mediated electron extraction from PSII in vitro is only double that of PSII in vivo. On the other hand, the biofilm exhibited much greater longevity (>5 days) than the protein-film (<6 h), with turnover numbers surpassing those of the protein-film after 2 days. The mechanism of biofilm electrogenesis is suggested to involve an intracellular redox mediator, which is released during light irradiation.

Progressive and Biased Divergent Evolution Underpins the Origin and Diversification of Peridinin Dinoflagellate Plastids.

Dinoflagellates are algae of tremendous importance to ecosystems and to public health. The cell biology and genome organization of dinoflagellate species is highly unusual. For example, the plastid genomes of peridinin-containing dinoflagellates encode only a minimal number of genes arranged on small elements termed "minicircles". Previous studies of peridinin plastid genes have found evidence for divergent sequence evolution, including extensive substitutions, novel insertions and deletions, and use of alternative translation initiation codons. Understanding the extent of this divergent evolution has been hampered by the lack of characterized peridinin plastid sequences. We have identified over 300 previously unannotated peridinin plastid mRNAs from published transcriptome projects, vastly increasing the number of sequences available. Using these data, we have produced a well-resolved phylogeny of peridinin plastid lineages, which uncovers several novel relationships within the dinoflagellates. This enables us to define changes to plastid sequences that occurred early in dinoflagellate evolution, and that have contributed to the subsequent diversification of individual dinoflagellate clades. We find that the origin of the peridinin dinoflagellates was specifically accompanied by elevations both in the overall number of substitutions that occurred on plastid sequences, and in the Ka/Ks ratio associated with plastid sequences, consistent with changes in selective pressure. These substitutions, alongside other changes, have accumulated progressively in individual peridinin plastid lineages. Throughout our entire dataset, we identify a persistent bias toward non-synonymous substitutions occurring on sequences encoding photosystem I subunits and stromal regions of peridinin plastid proteins, which may have underpinned the evolution of this unusual organelle.

Integration of plastids with their hosts: Lessons learned from dinoflagellates.

After their endosymbiotic acquisition, plastids become intimately connected with the biology of their host. For example, genes essential for plastid function may be relocated from the genomes of plastids to the host nucleus, and pathways may evolve within the host to support the plastid. In this review, we consider the different degrees of integration observed in dinoflagellates and their associated plastids, which have been acquired through multiple different endosymbiotic events. Most dinoflagellate species possess plastids that contain the pigment peridinin and show extreme reduction and integration with the host biology. In some species, these plastids have been replaced through serial endosymbiosis with plastids derived from a different phylogenetic derivation, of which some have become intimately connected with the biology of the host whereas others have not. We discuss in particular the evolution of the fucoxanthin-containing dinoflagellates, which have adapted pathways retained from the ancestral peridinin plastid symbiosis for transcript processing in their current, serially acquired plastids. Finally, we consider why such a diversity of different degrees of integration between host and plastid is observed in different dinoflagellates and how dinoflagellates may thus inform our broader understanding of plastid evolution and function.

Contribution of cyanobacterial alkane production to the ocean hydrocarbon cycle.

Hydrocarbons are ubiquitous in the ocean, where alkanes such as pentadecane and heptadecane can be found even in waters minimally polluted with crude oil. Populations of hydrocarbon-degrading bacteria, which are responsible for the turnover of these compounds, are also found throughout marine systems, including in unpolluted waters. These observations suggest the existence of an unknown and widespread source of hydrocarbons in the oceans. Here, we report that strains of the two most abundant marine cyanobacteria, Prochlorococcus and Synechococcus, produce and accumulate hydrocarbons, predominantly C15 and C17 alkanes, between 0.022 and 0.368% of dry cell weight. Based on global population sizes and turnover rates, we estimate that these species have the capacity to produce 2-540 pg alkanes per mL per day, which translates into a global ocean yield of ∼ 308-771 million tons of hydrocarbons annually. We also demonstrate that both obligate and facultative marine hydrocarbon-degrading bacteria can consume cyanobacterial alkanes, which likely prevents these hydrocarbons from accumulating in the environment. Our findings implicate cyanobacteria and hydrocarbon degraders as key players in a notable internal hydrocarbon cycle within the upper ocean, where alkanes are continually produced and subsequently consumed within days. Furthermore we show that cyanobacterial alkane production is likely sufficient to sustain populations of hydrocarbon-degrading bacteria, whose abundances can rapidly expand upon localized release of crude oil from natural seepage and human activities.

Photosynthetic, respiratory and extracellular electron transport pathways in cyanobacteria.

Cyanobacteria have evolved elaborate electron transport pathways to carry out photosynthesis and respiration, and to dissipate excess energy in order to limit cellular damage. Our understanding of the complexity of these systems and their role in allowing cyanobacteria to cope with varying environmental conditions is rapidly improving, but many questions remain. We summarize current knowledge of cyanobacterial electron transport pathways, including the possible roles of alternative pathways in photoprotection. We describe extracellular electron transport, which is as yet poorly understood. Biological photovoltaic devices, which measure electron output from cells, and which have been proposed as possible means of renewable energy generation, may be valuable tools in understanding cyanobacterial electron transfer pathways, and enhanced understanding of electron transfer may allow improvements in the efficiency of power output. This article is part of a Special Issue entitled Organization and dynamics of bioenergetic systems in bacteria, edited by Conrad Mullineaux.

Distinguishing the Roles of Thylakoid Respiratory Terminal Oxidases in the Cyanobacterium Synechocystis sp. PCC 6803.

Various oxygen-utilizing electron sinks, including the soluble flavodiiron proteins (Flv1/3), and the membrane-localized respiratory terminal oxidases (RTOs), cytochrome c oxidase (Cox) and cytochrome bd quinol oxidase (Cyd), are present in the photosynthetic electron transfer chain of Synechocystis sp. PCC 6803. However, the role of individual RTOs and their relative importance compared with other electron sinks are poorly understood, particularly under light. Via membrane inlet mass spectrometry gas exchange, chlorophyll a fluorescence, P700 analysis, and inhibitor treatment of the wild type and various mutants deficient in RTOs, Flv1/3, and photosystem I, we investigated the contribution of these complexes to the alleviation of excess electrons in the photosynthetic chain. To our knowledge, for the first time, we demonstrated the activity of Cyd in oxygen uptake under light, although it was detected only upon inhibition of electron transfer at the cytochrome b6f site and in ∆flv1/3 under fluctuating light conditions, where linear electron transfer was drastically inhibited due to impaired photosystem I activity. Cox is mostly responsible for dark respiration and competes with P700 for electrons under high light. Only the ∆cox/cyd double mutant, but not single mutants, demonstrated a highly reduced plastoquinone pool in darkness and impaired gross oxygen evolution under light, indicating that thylakoid-based RTOs are able to compensate partially for each other. Thus, both electron sinks contribute to the alleviation of excess electrons under illumination: RTOs continue to function under light, operating on slower time ranges and on a limited scale, whereas Flv1/3 responds rapidly as a light-induced component and has greater capacity.

Hydrocarbons Are Essential for Optimal Cell Size, Division, and Growth of Cyanobacteria.

Cyanobacteria are intricately organized, incorporating an array of internal thylakoid membranes, the site of photosynthesis, into cells no larger than other bacteria. They also synthesize C15-C19 alkanes and alkenes, which results in substantial production of hydrocarbons in the environment. All sequenced cyanobacteria encode hydrocarbon biosynthesis pathways, suggesting an important, undefined physiological role for these compounds. Here, we demonstrate that hydrocarbon-deficient mutants of Synechococcus sp. PCC 7002 and Synechocystis sp. PCC 6803 exhibit significant phenotypic differences from wild type, including enlarged cell size, reduced growth, and increased division defects. Photosynthetic rates were similar between strains, although a minor reduction in energy transfer between the soluble light harvesting phycobilisome complex and membrane-bound photosystems was observed. Hydrocarbons were shown to accumulate in thylakoid and cytoplasmic membranes. Modeling of membranes suggests these compounds aggregate in the center of the lipid bilayer, potentially promoting membrane flexibility and facilitating curvature. In vivo measurements confirmed that Synechococcus sp. PCC 7002 mutants lacking hydrocarbons exhibit reduced thylakoid membrane curvature compared to wild type. We propose that hydrocarbons may have a role in inducing the flexibility in membranes required for optimal cell division, size, and growth, and efficient association of soluble and membrane bound proteins. The recent identification of C15-C17 alkanes and alkenes in microalgal species suggests hydrocarbons may serve a similar function in a broad range of photosynthetic organisms.

Evolution of chloroplast transcript processing in Plasmodium and its chromerid algal relatives.

It is well understood that apicomplexan parasites, such as the malaria pathogen Plasmodium, are descended from free-living algae, and maintain a vestigial chloroplast that has secondarily lost all genes of photosynthetic function. Recently, two fully photosynthetic relatives of parasitic apicomplexans have been identified, the 'chromerid' algae Chromera velia and Vitrella brassicaformis, which retain photosynthesis genes within their chloroplasts. Elucidating the processes governing gene expression in chromerid chloroplasts might provide valuable insights into the origins of parasitism in the apicomplexans. We have characterised chloroplast transcript processing pathways in C. velia, V. brassicaformis and P. falciparum with a focus on the addition of an unusual, 3' poly(U) tail. We demonstrate that poly(U) tails in chromerids are preferentially added to transcripts that encode proteins that are directly involved in photosynthetic electron transfer, over transcripts for proteins that are not involved in photosynthesis. To our knowledge, this represents the first chloroplast transcript processing pathway to be associated with a particular functional category of genes. In contrast, Plasmodium chloroplast transcripts are not polyuridylylated. We additionally present evidence that poly(U) tail addition in chromerids is involved in the alternative processing of polycistronic precursors covering multiple photosynthesis genes, and appears to be associated with high levels of transcript abundance. We propose that changes to the chloroplast transcript processing machinery were an important step in the loss of photosynthesis in ancestors of parasitic apicomplexans.

Diversity of transcripts and transcript processing forms in plastids of the dinoflagellate alga Karenia mikimotoi.

Plastids produce a vast diversity of transcripts. These include mature transcripts containing coding sequences, and their processing precursors, as well as transcripts that lack direct coding functions, such as antisense transcripts. Although plastid transcriptomes have been characterised for many plant species, less is known about the transcripts produced in other plastid lineages. We characterised the transcripts produced in the fucoxanthin-containing plastids of the dinoflagellate alga Karenia mikimotoi. This plastid lineage, acquired through tertiary endosymbiosis, utilises transcript processing pathways that are very different from those found in plants and green algae, including 3' poly(U) tail addition, and extensive substitutional editing of transcript sequences. We have sequenced the plastid transcriptome of K. mikimotoi, and have detected evidence for divergent evolution of fucoxanthin plastid genomes. We have additionally characterised polycistronic and monocistronic transcripts from two plastid loci, psbD-tRNA (Met)-ycf4 and rpl36-rps13-rps11. We find evidence for a range of transcripts produced from each locus that differ in terms of editing state, 5' end cleavage position, and poly(U) tail addition. Finally, we identify antisense transcripts in K. mikimotoi, which appear to undergo different processing events from the corresponding sense transcripts. Overall, our study provides insights into the diversity of transcripts and processing intermediates found in plastid lineages across the eukaryotes.

Exploiting algal NADPH oxidase for biophotovoltaic energy.

Photosynthetic microbes exhibit light-dependent electron export across the cell membrane, which can generate electricity in biological photovoltaic (BPV) devices. How electrons are exported remains to be determined; the identification of mechanisms would help selection or generation of photosynthetic microbes capable of enhanced electrical output. We show that plasma membrane NADPH oxidase activity is a significant component of light-dependent generation of electricity by the unicellular green alga Chlamydomonas reinhardtii. NADPH oxidases export electrons across the plasma membrane to form superoxide anion from oxygen. The C. reinhardtii mutant lacking the NADPH oxidase encoded by RBO1 is impaired in both extracellular superoxide anion production and current generation in a BPV device. Complementation with the wild-type gene restores both capacities, demonstrating the role of the enzyme in electron export. Monitoring light-dependent extracellular superoxide production with a colorimetric assay is shown to be an effective way of screening for electrogenic potential of candidate algal strains. The results show that algal NADPH oxidases are important for superoxide anion production and open avenues for optimizing the biological component of these devices.