Structure function analysis of ADP-dependent cyanobacterial phosphofructokinase reveals new phylogenetic grouping in the PFK-A family.

Dependent on the light conditions, photosynthetic organisms switch between carbohydrate synthesis or breakdown, for which the reversibility of carbohydrate metabolism, including glycolysis, is essential. Kinetic regulation of phosphofructokinase (PFK), a key-control point in glycolysis, was studied in the cyanobacterium Synechocystis sp. PCC 6803. The two PFK iso-enzymes (PFK- A1, PFK-A2), were found to use ADP instead of ATP, and have similar kinetic characteristics, but differ in their allosteric regulation. PFK-A1 is inhibited by 3- phosphoglycerate, a product of the Calvin-Benson-Bassham cycle, while PFK-A2 is inhibited by ATP, which is provided by photosynthesis. This regulation enables cyanobacteria to switch PFK off in light, and on in darkness. ADP dependence has not been reported before for the PFK-A enzyme family, and was thought to be restricted to the PFK-B ribokinase superfamily. Phosphate donor specificity within the PFK-A family could be related to specific binding motifs in ATP-, ADP-, and PPi-dependent PFK-As. Phylogenetic analysis revealed a distribution of ADP-PFK-As in cyanobacteria and in a few alphaproteobacteria, which was confirmed in enzymatic studies.

Hypothesis on the Synchronistic Evolution of Autotrophy and Heterotrophy.

All life on earth requires a source of energy and organic carbon. There has been a continuous debate on whether autotrophic or heterotrophic metabolism came first. A very similar discussion exists concerning the advent of oxygenic photosynthesis and aerobic respiration. I put forward the synchronistic evolution hypothesis supposing that all metabolic processes develop in a bidirectional manner from the very first. Bidirectionality is claimed to be intrinsic to the evolution of all metabolic processes as (i) all biochemical reactions and enzymes are per se bidirectional, (ii) substrates need to be regenerated, and (iii) flux regulation requires flexibility of direction. Autotrophy and heterotrophy are thus inherent to each other. A scenario for the synchronistic development of oxygenic photosynthesis and aerobic respiration is described.

GC/MS-based 13C metabolic flux analysis resolves the parallel and cyclic photomixotrophic metabolism of Synechocystis sp. PCC 6803 and selected deletion mutants including the Entner-Doudoroff and phosphoketolase pathways.

BACKGROUND: Cyanobacteria receive huge interest as green catalysts. While exploiting energy from sunlight, they co-utilize sugar and CO2. This photomixotrophic mode enables fast growth and high cell densities, opening perspectives for sustainable biomanufacturing. The model cyanobacterium Synechocystis sp. PCC 6803 possesses a complex architecture of glycolytic routes for glucose breakdown that are intertwined with the CO2-fixing Calvin-Benson-Bassham (CBB) cycle. To date, the contribution of these pathways to photomixotrophic metabolism has remained unclear. RESULTS: Here, we developed a comprehensive approach for 13C metabolic flux analysis of Synechocystis sp. PCC 6803 during steady state photomixotrophic growth. Under these conditions, the Entner-Doudoroff (ED) and phosphoketolase (PK) pathways were found inactive but the microbe used the phosphoglucoisomerase (PGI) (63.1%) and the oxidative pentose phosphate pathway (OPP) shunts (9.3%) to fuel the CBB cycle. Mutants that lacked the ED pathway, the PK pathway, or phosphofructokinases were not affected in growth under metabolic steady-state. An ED pathway-deficient mutant (Δeda) exhibited an enhanced CBB cycle flux and increased glycogen formation, while the OPP shunt was almost inactive (1.3%). Under fluctuating light, ∆eda showed a growth defect, different to wild type and the other deletion strains. CONCLUSIONS: The developed approach, based on parallel 13C tracer studies with GC-MS analysis of amino acids, sugars, and sugar derivatives, optionally adding NMR data from amino acids, is valuable to study fluxes in photomixotrophic microbes to detail. In photomixotrophic cells, PGI and OPP form glycolytic shunts that merge at switch points and result in synergistic fueling of the CBB cycle for maximized CO2 fixation. However, redirected fluxes in an ED shunt-deficient mutant and the impossibility to delete this shunt in a GAPDH2 knockout mutant, indicate that either minor fluxes (below the resolution limit of 13C flux analysis) might exist that could provide catalytic amounts of regulatory intermediates or alternatively, that EDA possesses additional so far unknown functions. These ideas require further experiments.

The structure and reactivity of the HoxEFU complex from the cyanobacterium Synechocystis sp. PCC 6803.

Cyanobacterial Hox is a [NiFe] hydrogenase that consists of the hydrogen (H2)-activating subunits HoxYH, which form a complex with the HoxEFU assembly to mediate reactions with soluble electron carriers like NAD(P)H and ferredoxin (Fdx), thereby coupling photosynthetic electron transfer to energy-transforming catalytic reactions. Researchers studying the HoxEFUYH complex have observed that HoxEFU can be isolated independently of HoxYH, leading to the hypothesis that HoxEFU is a distinct functional subcomplex rather than an artifact of Hox complex isolation. Moreover, outstanding questions about the reactivity of Hox with natural substrates and the site(s) of substrate interactions and coupling of H2, NAD(P)H, and Fdx remain to be resolved. To address these questions, here we analyzed recombinantly produced HoxEFU by electron paramagnetic resonance spectroscopy and kinetic assays with natural substrates. The purified HoxEFU subcomplex catalyzed electron transfer reactions among NAD(P)H, flavodoxin, and several ferredoxins, thus functioning in vitro as a shuttle among different cyanobacterial pools of reducing equivalents. Both Fdx1-dependent reductions of NAD+ and NADP+ were cooperative. HoxEFU also catalyzed the flavodoxin-dependent reduction of NAD(P)+, Fdx2-dependent oxidation of NADH and Fdx4- and Fdx11-dependent reduction of NAD+ MS-based mapping identified an Fdx1-binding site at the junction of HoxE and HoxF, adjacent to iron-sulfur (FeS) clusters in both subunits. Overall, the reactivity of HoxEFU observed here suggests that it functions in managing peripheral electron flow from photosynthetic electron transfer, findings that reveal detailed insights into how ubiquitous cellular components may be used to allocate energy flow into specific bioenergetic products.

A Specific Glycogen Mobilization Strategy Enables Rapid Awakening of Dormant Cyanobacteria from Chlorosis.

Many organisms survive stressful conditions via entry into a dormant state that can be rapidly exited when the stressor disappears; this ability provides a strong selective advantage. In the cyanobacterium Synechocystis sp. PCC 6803, the exit from nitrogen chlorosis takes less than 48 h and is enabled by the impressive metabolic flexibility of these cyanobacteria, which pass through heterotrophic and mixotrophic phases before reentering photoautotrophic growth. Switching between these states requires delicate coordination of carbohydrate oxidation, CO2 fixation, and photosynthesis. Here, we investigated the contribution of the different carbon catabolic routes by assessing mutants of these pathways during nitrogen chlorosis and resuscitation. The addition of nitrate to nitrogen-starved cells rapidly starts the awakening program. Metabolism switches from maintenance metabolism, characterized by residual photosynthesis and low cellular ATP levels, to an initial heterotrophic phase, characterized by respiration and an immediate increase in ATP levels. This respiration relies on glycogen breakdown catalyzed by the glycogen phosphorylase GlgP2. In the following transient mixotrophic phase, photosynthesis and CO2 fixation restart and glycogen is consumed. During the mixotrophic phase, parallel operation of the oxidative pentose phosphate cycle and the Entner-Doudoroff pathway is required for resuscitation to proceed; the glycolytic route via the Embden-Meyerhof-Parnas pathway has minor importance. Our data suggest that, during resuscitation, only the Entner-Doudoroff and oxidative pentose phosphate pathways supply the metabolic intermediates necessary for the anabolic reactions required to reconstitute a vegetative cell. Intriguingly, the key enzymes for glycogen catabolism are already expressed during the preceding chlorotic phase, in apparent preparation for rapid resuscitation.

The Entner-Doudoroff pathway is an overlooked glycolytic route in cyanobacteria and plants.

Glucose degradation pathways are central for energy and carbon metabolism throughout all domains of life. They provide ATP, NAD(P)H, and biosynthetic precursors for amino acids, nucleotides, and fatty acids. It is general knowledge that cyanobacteria and plants oxidize carbohydrates via glycolysis [the Embden-Meyerhof-Parnas (EMP) pathway] and the oxidative pentose phosphate (OPP) pathway. However, we found that both possess a third, previously overlooked pathway of glucose breakdown: the Entner-Doudoroff (ED) pathway. Its key enzyme, 2-keto-3-deoxygluconate-6-phosphate (KDPG) aldolase, is widespread in cyanobacteria, moss, fern, algae, and plants and is even more common among cyanobacteria than phosphofructokinase (PFK), the key enzyme of the EMP pathway. Active KDPG aldolases from the cyanobacterium Synechocystis and the plant barley (Hordeum vulgare) were biochemically characterized in vitro. KDPG, a metabolite unique to the ED pathway, was detected in both in vivo, indicating an active ED pathway. Phylogenetic analyses revealed that photosynthetic eukaryotes acquired KDPG aldolase from the cyanobacterial ancestors of plastids via endosymbiotic gene transfer. Several Synechocystis mutants in which key enzymes of all three glucose degradation pathways were knocked out indicate that the ED pathway is physiologically significant, especially under mixotrophic conditions (light and glucose) and under autotrophic conditions in a day/night cycle, which is probably the most common condition encountered in nature. The ED pathway has lower protein costs and ATP yields than the EMP pathway, in line with the observation that oxygenic photosynthesizers are nutrient-limited, rather than ATP-limited. Furthermore, the ED pathway does not generate futile cycles in organisms that fix CO2 via the Calvin-Benson cycle.

PHB is Produced from Glycogen Turn-over during Nitrogen Starvation in Synechocystis sp. PCC 6803.

Polyhydroxybutyrate (PHB) is a polymer of great interest as a substitute for conventional plastics, which are becoming an enormous environmental problem. PHB can be produced directly from CO2 in photoautotrophic cyanobacteria. The model cyanobacterium Synechocystis sp. PCC 6803 produces PHB under conditions of nitrogen starvation. However, it is so far unclear which metabolic pathways provide the precursor molecules for PHB synthesis during nitrogen starvation. In this study, we investigated if PHB could be derived from the main intracellular carbon pool, glycogen. A mutant of the major glycogen phosphorylase, GlgP2 (slr1367 product), was almost completely impaired in PHB synthesis. Conversely, in the absence of glycogen synthase GlgA1 (sll0945 product), cells not only produced less PHB, but were also impaired in acclimation to nitrogen depletion. To analyze the role of the various carbon catabolic pathways (EMP, ED and OPP pathways) for PHB production, mutants of key enzymes of these pathways were analyzed, showing different impact on PHB synthesis. Together, this study clearly indicates that PHB in glycogen-producing Synechocystis sp. PCC 6803 cells is produced from this carbon-pool during nitrogen starvation periods. This knowledge can be used for metabolic engineering to get closer to the overall goal of a sustainable, carbon-neutral bioplastic production.

The Non-Coding RNA Ncr0700/PmgR1 is Required for Photomixotrophic Growth and the Regulation of Glycogen Accumulation in the Cyanobacterium Synechocystis sp. PCC 6803.

Carbohydrate metabolism is a tightly regulated process in photosynthetic organisms. In the cyanobacterium Synechocystis sp. PCC 6803, the photomixotrophic growth protein A (PmgA) is involved in the regulation of glucose and storage carbohydrate (i.e. glycogen) metabolism, while its biochemical activity and possible factors acting downstream of PmgA are unknown. Here, a genome-wide microarray analysis of a ΔpmgA strain identified the expression of 36 protein-coding genes and 42 non-coding transcripts as significantly altered. From these, the non-coding RNA Ncr0700 was identified as the transcript most strongly reduced in abundance. Ncr0700 is widely conserved among cyanobacteria. In Synechocystis its expression is inversely correlated with light intensity. Similarly to a ΔpmgA mutant, a Δncr0700 deletion strain showed an approximately 2-fold increase in glycogen content under photoautotrophic conditions and wild-type-like growth. Moreover, its growth was arrested by 38 h after a shift to photomixotrophic conditions. Ectopic expression of Ncr0700 in Δncr0700 and ΔpmgA restored the glycogen content and photomixotrophic growth to wild-type levels. These results indicate that Ncr0700 is required for photomixotrophic growth and the regulation of glycogen accumulation, and acts downstream of PmgA. Hence Ncr0700 is renamed here as PmgR1 for photomixotrophic growth RNA 1.

The bidirectional NiFe-hydrogenase in Synechocystis sp. PCC 6803 is reduced by flavodoxin and ferredoxin and is essential under mixotrophic, nitrate-limiting conditions.

Cyanobacteria are able to use solar energy for the production of hydrogen. It is generally accepted that cyanobacterial NiFe-hydrogenases are reduced by NAD(P)H. This is in conflict with thermodynamic considerations, as the midpoint potentials of NAD(P)H do not suffice to support the measured hydrogen production under physiological conditions. We show that flavodoxin and ferredoxin directly reduce the bidirectional NiFe-hydrogenase of Synechocystis sp. PCC 6803 in vitro. A merodiploid ferredoxin-NADP reductase mutant produced correspondingly more photohydrogen. We furthermore found that the hydrogenase receives its electrons via pyruvate:flavodoxin/ferredoxin oxidoreductase (PFOR)-flavodoxin/ferredoxin under fermentative conditions, enabling the cells to gain ATP. These results strongly support that the bidirectional NiFe-hydrogenases in cyanobacteria function as electron sinks for low potential electrons from photosystem I and as a redox balancing device under fermentative conditions. However, the selective advantage of this enzyme is not known. No strong phenotype of mutants lacking the hydrogenase has been found. Because bidirectional hydrogenases are widespread in aquatic nutrient-rich environments that are capable of triggering phytoplankton blooms, we mimicked those conditions by growing cells in the presence of increased amounts of dissolved organic carbon and dissolved organic nitrogen. Under these conditions the hydrogenase was found to be essential. As these conditions close the two most important sinks for reduced flavodoxin/ferredoxin (CO2-fixation and nitrate reduction), this discovery further substantiates the connection between flavodoxin/ferredoxin and the NiFe-hydrogenase.

hypD as a marker for [NiFe]-hydrogenases in microbial communities of surface waters.

Hydrogen is an important trace gas in the atmosphere. Soil microorganisms are known to be an important part of the biogeochemical H2 cycle, contributing 80 to 90% of the annual hydrogen uptake. Different aquatic ecosystems act as either sources or sinks of hydrogen, but the contribution of their microbial communities is unknown. [NiFe]-hydrogenases are the best candidates for hydrogen turnover in these environments since they are able to cope with oxygen. As they lack sufficiently conserved sequence motifs, reliable markers for these enzymes are missing, and consequently, little is known about their environmental distribution. We analyzed the essential maturation genes of [NiFe]-hydrogenases, including their frequency of horizontal gene transfer, and found hypD to be an applicable marker for the detection of the different known hydrogenase groups. Investigation of two freshwater lakes showed that [NiFe]-hydrogenases occur in many prokaryotic orders. We found that the respective hypD genes cooccur with oxygen-tolerant [NiFe]-hydrogenases (groups 1 and 5) mainly of Actinobacteria, Acidobacteria, and Burkholderiales; cyanobacterial uptake hydrogenases (group 2a) of cyanobacteria; H2-sensing hydrogenases (group 2b) of Burkholderiales, Rhizobiales, and Rhodobacterales; and two groups of multimeric soluble hydrogenases (groups 3b and 3d) of Legionellales and cyanobacteria. These findings support and expand a previous analysis of metagenomic data (M. Barz et al., PLoS One 5:e13846, 2010, http://dx.doi.org/10.1371/journal.pone.0013846) and further identify [NiFe]-hydrogenases that could be involved in hydrogen cycling in aquatic surface waters.

Structural and biophysical properties of a [4Fe4S] ferredoxin-like protein from Synechocystis sp. PCC 6803 with a unique two domain structure.

Electron carrier proteins (ECPs), binding iron-sulfur clusters, are vital components within the intricate network of metabolic and photosynthetic reactions. They play a crucial role in the distribution of reducing equivalents. In Synechocystis sp. PCC 6803, the ECP network includes at least nine ferredoxins. Previous research, including global expression analyses and protein binding studies, has offered initial insights into the functional roles of individual ferredoxins within this network. This study primarily focuses on Ferredoxin 9 (slr2059). Through sequence analysis and computational modeling, Ferredoxin 9 emerges as a unique ECP with a distinctive two-domain architecture. It consists of a C-terminal iron­sulfur binding domain and an N-terminal domain with homology to Nil-domain proteins, connected by a structurally rigid 4-amino acid linker. Notably, in contrast to canonical [2Fe2S] ferredoxins exemplified by PetF (ssl0020), which feature highly acidic surfaces facilitating electron transfer with photosystem I reaction centers, models of Ferredoxin 9 reveal a more neutral to basic protein surface. Using a combination of electron paramagnetic resonance spectroscopy and square-wave voltammetry on heterologously produced Ferredoxin 9, this study demonstrates that the protein coordinates 2×[4Fe4S]2+/1+ redox-active and magnetically interacting clusters, with measured redox potentials of -420 ± 9 mV and - 516 ± 10 mV vs SHE. A more in-depth analysis of Fdx9's unique structure and protein sequence suggests that this type of Nil-2[4Fe4S] multi-domain ferredoxin is well conserved in cyanobacteria, bearing structural similarities to proteins involved in homocysteine synthesis in methanogens.

Evidence for Electron Transfer from the Bidirectional Hydrogenase to the Photosynthetic Complex I (NDH-1) in the Cyanobacterium Synechocystis sp. PCC 6803.

The cyanobacterial bidirectional [NiFe]-hydrogenase is a pentameric enzyme. Apart from the small and large hydrogenase subunits (HoxYH) it contains a diaphorase module (HoxEFU) that interacts with NAD(P)+ and ferredoxin. HoxEFU shows strong similarity to the outermost subunits (NuoEFG) of canonical respiratory complexes I. Photosynthetic complex I (NDH-1) lacks these three subunits. This led to the idea that HoxEFU might interact with NDH-1 instead. HoxEFUYH utilizes excited electrons from PSI for photohydrogen production and it catalyzes the reverse reaction and feeds electrons into the photosynthetic electron transport. We analyzed hydrogenase activity, photohydrogen evolution and hydrogen uptake, the respiration and photosynthetic electron transport of ΔhoxEFUYH, and a knock-out strain with dysfunctional NDH-1 (ΔndhD1/ΔndhD2) of the cyanobacterium Synechocystis sp. PCC 6803. Photohydrogen production was prolonged in ΔndhD1/ΔndhD2 due to diminished hydrogen uptake. Electrons from hydrogen oxidation must follow a different route into the photosynthetic electron transport in this mutant compared to wild type cells. Furthermore, respiration was reduced in ΔhoxEFUYH and the ΔndhD1/ΔndhD2 localization of the hydrogenase to the membrane was impaired. These data indicate that electron transfer from the hydrogenase to the NDH-1 complex is either direct, by the binding of the hydrogenase to the complex, or indirect, via an additional mediator.

CP12 fine-tunes the Calvin-Benson cycle and carbohydrate metabolism in cyanobacteria.

The regulatory protein CP12 can bind glyceraldehyde 3-phosphate dehydrogenase (GapDH) and phosphoribulokinase (PRK) in oxygenic phototrophs, thereby switching on and off the flux through the Calvin-Benson cycle (CBC) under light and dark conditions, respectively. However, it can be assumed that CP12 is also regulating CBC flux under further conditions associated with redox changes. To prove this hypothesis, the mutant Δcp12 of the model cyanobacterium Synechocystis sp. PCC 6803 was compared to wild type and different complementation strains. Fluorescence microscopy showed for the first time the in vivo kinetics of assembly and disassembly of the CP12-GapDH-PRK complex, which was absent in the mutant Δcp12. Metabolome analysis revealed differences in the contents of ribulose 1,5-bisphosphate and dihydroxyacetone phosphate, the products of the CP12-regulated enzymes GapDH and PRK, between wild type and mutant Δcp12 under changing CO2 conditions. Growth of Δcp12 was not affected at constant light under different inorganic carbon conditions, however, the addition of glucose inhibited growth in darkness as well as under diurnal conditions. The growth defect in the presence of glucose is associated with the inability of Δcp12 to utilize external glucose. These phenotypes could be complemented by ectopic expression of the native CP12 protein, however, expression of CP12 variants with missing redox-sensitive cysteine pairs only partly restored the growth with glucose. These experiments indicated that the loss of GapDH-inhibition via CP12 is more critical than PRK association. Measurements of the NAD(P)H oxidation revealed an impairment of light intensity-dependent redox state regulation in Δcp12. Collectively, our results indicate that CP12-dependent regulation of the CBC is crucial for metabolic adjustment under conditions leading to redox changes such as diurnal conditions, glucose addition, and different CO2 conditions in cyanobacteria.

Advances and challenges in photosynthetic hydrogen production.

The vision to replace coal with hydrogen goes back to Jules Verne in 1874. However, sustainable hydrogen production remains challenging. The most elegant approach is to utilize photosynthesis for water splitting and to subsequently save solar energy as hydrogen. Cyanobacteria and green algae are unicellular photosynthetic organisms that contain hydrogenases and thereby possess the enzymatic equipment for photosynthetic hydrogen production. These features of cyanobacteria and algae have inspired artificial and semi-artificial in vitro techniques, that connect photoexcited materials or enzymes with hydrogenases or mimics of these for hydrogen production. These in vitro methods have on their part been models for the fusion of cyanobacterial and algal hydrogenases to photosynthetic photosystem I (PSI) in vivo, which recently succeeded as proofs of principle.

Synechocystis sp. PCC 6803 Requires the Bidirectional Hydrogenase to Metabolize Glucose and Arginine Under Oxic Conditions.

The cyanobacterium Synechocystis sp.PCC 6803 possesses a bidirectional NiFe-hydrogenase, HoxEFUYH. It functions to produce hydrogen under dark, fermentative conditions and photoproduces hydrogen when dark-adapted cells are illuminated. Unexpectedly, we found that the deletion of the large subunit of the hydrogenase (HoxH) in Synechocystis leads to an inability to grow on arginine and glucose under continuous light in the presence of oxygen. This is surprising, as the hydrogenase is an oxygen-sensitive enzyme. In wild-type (WT) cells, thylakoid membranes largely disappeared, cyanophycin accumulated, and the plastoquinone (PQ) pool was highly reduced, whereas ΔhoxH cells entered a dormant-like state and neither consumed glucose nor arginine at comparable rates to the WT. Hydrogen production was not traceable in the WT under these conditions. We tested and could show that the hydrogenase does not work as an oxidase on arginine and glucose but has an impact on the redox states of photosynthetic complexes in the presence of oxygen. It acts as an electron valve as an immediate response to the supply of arginine and glucose but supports the input of electrons from arginine and glucose oxidation into the photosynthetic electron chain in the long run, possibly via the NDH-1 complex. Despite the data presented in this study, the latter scenario requires further proof. The exact role of the hydrogenase in the presence of arginine and glucose remains unresolved. In addition, a unique feature of the hydrogenase is its ability to shift electrons between NAD(H), NADP(H), ferredoxin, and flavodoxin, which was recently shown in vitro and might be required for fine-tuning. Taken together, our data show that Synechocystis depends on the hydrogenase to metabolize organic carbon and nitrogen in the presence of oxygen, which might be an explanation for its prevalence in aerobic cyanobacteria.

Pyruvate:ferredoxin oxidoreductase and low abundant ferredoxins support aerobic photomixotrophic growth in cyanobacteria.

The decarboxylation of pyruvate is a central reaction in the carbon metabolism of all organisms. It is catalyzed by the pyruvate:ferredoxin oxidoreductase (PFOR) and the pyruvate dehydrogenase (PDH) complex. Whereas PFOR reduces ferredoxin, the PDH complex utilizes NAD+. Anaerobes rely on PFOR, which was replaced during evolution by the PDH complex found in aerobes. Cyanobacteria possess both enzyme systems. Our data challenge the view that PFOR is exclusively utilized for fermentation. Instead, we show, that the cyanobacterial PFOR is stable in the presence of oxygen in vitro and is required for optimal photomixotrophic growth under aerobic and highly reducing conditions while the PDH complex is inactivated. We found that cells rely on a general shift from utilizing NAD(H)- to ferredoxin-dependent enzymes under these conditions. The utilization of ferredoxins instead of NAD(H) saves a greater share of the Gibbs-free energy, instead of wasting it as heat. This obviously simultaneously decelerates metabolic reactions as they operate closer to their thermodynamic equilibrium. It is common thought that during evolution, ferredoxins were replaced by NAD(P)H due to their higher stability in an oxidizing atmosphere. However, the utilization of NAD(P)H could also have been favored due to a higher competitiveness because of an accelerated metabolism.

The Entner-Doudoroff Pathway Contributes to Glycogen Breakdown During High to Low CO2 Shifts in the Cyanobacterium Synechocystis sp. PCC 6803.

Cyanobacteria perform plant-like oxygenic photosynthesis to convert inorganic carbon into organic compounds and can also use internal carbohydrate reserves under specific conditions. A mutant collection with defects in different routes for sugar catabolism was studied to analyze which of them is preferentially used to degrade glycogen reserves in light-exposed cells of Synechocystis sp. PCC 6803 shifted from high to low CO2 conditions. Mutants defective in the glycolytic Embden-Meyerhof-Parnas pathway or in the oxidative pentose-phosphate (OPP) pathway showed glycogen levels similar to wild type under high CO2 (HC) conditions and were able to degrade it similarly after shifts to low CO2 (LC) conditions. In contrast, the mutant Δeda, which is defective in the glycolytic Entner-Doudoroff (ED) pathway, accumulated elevated glycogen levels under HC that were more slowly consumed during the LC shift. In consequence, the mutant Δeda showed a lowered ability to respond to the inorganic carbon shifts, displayed a pronounced lack in the reactivation of growth when brought back to HC, and differed significantly in its metabolite composition. Particularly, Δeda accumulated enhanced levels of proline, which is a well-known metabolite to maintain redox balances via NADPH levels in many organisms under stress conditions. We suggest that deletion of eda might promote the utilization of the OPP shunt that dramatically enhance NADPH levels. Collectively, the results point at a major regulatory contribution of the ED pathway for the mobilization of glycogen reserves during rapid acclimation to fluctuating CO2 conditions.

In-vivo quantification of electron flow through photosystem I - Cyclic electron transport makes up about 35% in a cyanobacterium.

Photosynthetic electron flow, driven by photosystem I and II, provides chemical energy for carbon fixation. In addition to a linear mode a second cyclic route exists, which only involves photosystem I. The exact contributions of linear and cyclic transport are still a matter of debate. Here, we describe the development of a method that allows quantification of electron flow in absolute terms through photosystem I in a photosynthetic organism for the first time. Specific in-vivo protocols allowed to discern the redox states of plastocyanin, P700 and the FeS-clusters including ferredoxin at the acceptor site of PSI in the cyanobacterium Synechocystis sp. PCC 6803 with the near-infrared spectrometer Dual-KLAS/NIR. P700 absorbance changes determined with the Dual-KLAS/NIR correlated linearly with direct determinations of PSI concentrations using EPR. Dark-interval relaxation kinetics measurements (DIRKPSI) were applied to determine electron flow through PSI. Counting electrons from hydrogen oxidation as electron donor to photosystem I in parallel to DIRKPSI measurements confirmed the validity of the method. Electron flow determination by classical PSI yield measurements overestimates electron flow at low light intensities and saturates earlier compared to DIRKPSI. Combination of DIRKPSI with oxygen evolution measurements yielded a proportion of 35% of surplus electrons passing PSI compared to PSII. We attribute these electrons to cyclic electron transport, which is twice as high as assumed for plants. Counting electrons flowing through the photosystems allowed determination of the number of quanta required for photosynthesis to 11 per oxygen produced, which is close to published values.

Glycolytic Shunts Replenish the Calvin-Benson-Bassham Cycle as Anaplerotic Reactions in Cyanobacteria.

The recent discovery of the Entner-Doudoroff (ED) pathway as a third glycolytic route beside Embden-Meyerhof-Parnas (EMP) and oxidative pentose phosphate (OPP) pathway in oxygenic photoautotrophs requires a revision of their central carbohydrate metabolism. In this study, unexpectedly, we observed that deletion of the ED pathway alone, and even more pronounced in combination with other glycolytic routes, diminished photoautotrophic growth in continuous light in the cyanobacterium Synechocystis sp. PCC 6803. Furthermore, we found that the ED pathway is required for optimal glycogen catabolism in parallel to an operating Calvin-Benson-Bassham (CBB) cycle. It is counter-intuitive that glycolytic routes, which are a reverse to the CBB cycle and do not provide any additional biosynthetic intermediates, are important under photoautotrophic conditions. However, observations on the ability to reactivate an arrested CBB cycle revealed that they form glycolytic shunts that tap the cellular carbohydrate reservoir to replenish the cycle. Taken together, our results suggest that the classical view of the CBB cycle as an autocatalytic, completely autonomous cycle that exclusively relies on its own enzymes and CO2 fixation to regenerate ribulose-1,5-bisphosphate for Rubisco is an oversimplification. We propose that in common with other known autocatalytic cycles, the CBB cycle likewise relies on anaplerotic reactions to compensate for the depletion of intermediates, particularly in transition states and under fluctuating light conditions that are common in nature.

In-vivo turnover frequency of the cyanobacterial NiFe-hydrogenase during photohydrogen production outperforms in-vitro systems.

Cyanobacteria provide all components for sunlight driven biohydrogen production. Their bidirectional NiFe-hydrogenase is resistant against low levels of oxygen with a preference for hydrogen evolution. However, until now it was unclear if its catalytic efficiency can keep pace with the photosynthetic electron transfer rate. We identified NikKLMQO (sll0381-sll0385) as a nickel transporter, which is required for hydrogen production. ICP-MS measurements were used to quantify hydrogenase molecules per cell. We found 400 to 2000 hydrogenase molecules per cell depending on the conditions. In-vivo turnover frequencies of the enzyme ranged from 62 H2/s in the wild type to 120 H2/s in a mutant during photohydrogen production. These frequencies are above maximum in-vivo photosynthetic electron transfer rates of 47 e-/s (equivalent to 24 H2/s). They are also above those of existing in-vitro systems working with unlimited electron supply and show that in-vivo photohydrogen production is limited by electron delivery to the enzyme.