Reduction of photosystem I reaction center by recombinant DrgA protein in isolated thylakoid membranes of the cyanobacterium <Emphasis Type="Italic">Synechocystis</Emphasis> sp. PCC 6803

To study the function of soluble NAD(P)H:quinone oxidoreductase of the cyanobacterium Synechocystis sp. PCC 6803 encoded by drgA gene, recombinant DrgA protein carrying 12 histidine residues on the C-terminal end was expressed in Escherichia coli and purified. Recombinant DrgA is a flavoprotein that exhibits quinone reductase and nitroreductase activities with NAD(P)H as the electron donor. Using EPR spectroscopy, it was demonstrated that addition of recombinant DrgA protein and NADPH to DCMU-treated isolated thylakoid membranes of the cyanobacterium increased the dark rereduction rate of the photosystem I reaction center (P700⁺). Thus, DrgA can participate in electron transfer from NADPH to the electron transport chain of the Synechocystis sp. PCC 6803 thylakoid membrane.     

Ferredoxin:NADP+ Oxidoreductase Association with Phycocyanin Modulates Its Properties

In photosynthetic organisms, ferredoxin:NADP⁺ oxidoreductase (FNR) is known to provide NADPH for CO₂ assimilation, but it also utilizes NADPH to provide reduced ferredoxin. The cyanobacterium Synechocystis sp. strain PCC6803 produces two FNR isoforms, a small one (FNR_S) similar to the one found in plant plastids and a large one (FNR_L) that is associated with the phycobilisome, a light-harvesting complex. Here we show that a mutant lacking FNR_L exhibits a higher NADP⁺/NADPH ratio. We also purified to homogeneity a phycobilisome subcomplex comprising FNR_L, named FNR_L-PC. The enzymatic activities of FNR_L-PC were compared with those of FNR_S. During NADPH oxidation, FNR_L-PC exhibits a 30% decrease in the Michaelis constant K_m(NADPH), and a 70% increase in K_{m(ferredoxin)}, which is in agreement with its predicted lower activity of ferredoxin reduction. During NADP⁺ reduction, the FNR_L-PC shows a 29/43% decrease in the rate of single electron transfer from reduced ferredoxin in the presence/absence of NADP⁺. The increase in K_{m(ferredoxin)} and the rate decrease of single reduction are attributed to steric hindrance by the phycocyanin moiety of FNR_L-PC. Both isoforms are capable of catalyzing the NADP⁺ reduction under multiple turnover conditions. Furthermore, we obtained evidence that, under high ionic strength conditions, electron transfer from reduced ferredoxin is rate limiting during this process. The differences that we observe might not fully explain the in vivo properties of the Synechocystis mutants expressing only one of the isoforms. Therefore, we advocate that FNR localization and/or substrates availability are essential in vivo.     

EPR study of electron transport in the cyanobacterium Synechocystis sp. PCC 6803: Oxygen-dependent interrelations between photosynthetic and respiratory electron transport chains

In this work, we investigated electron transport processes in the cyanobacterium Synechocystis sp. PCC 6803, with a special emphasis focused on oxygen-dependent interrelations between photosynthetic and respiratory electron transport chains. Redox transients of the photosystem I primary donor P700 and oxygen exchange processes were measured by the EPR method under the same experimental conditions. To discriminate between the factors controlling electron flow through photosynthetic and respiratory electron transport chains, we compared the P700 redox transients and oxygen exchange processes in wild type cells and mutants with impaired photosystem II and terminal oxidases (CtaI, CydAB, CtaDEII). It was shown that the rates of electron flow through both photosynthetic and respiratory electron transport chains strongly depended on the transmembrane proton gradient and oxygen concentration in cell suspension. Electron transport through photosystem I was controlled by two main mechanisms: (i) oxygen-dependent acceleration of electron transfer from photosystem I to NADP⁺, and (ii) slowing down of electron flow between photosystem II and photosystem I governed by the intrathylakoid pH. Inhibitor analysis of P700 redox transients led us to the conclusion that electron fluxes from dehydrogenases and from cyclic electron transport pathway comprise 20-30% of the total electron flux from the intersystem electron transport chain to P700⁺.     

Novel Cyanobacterial Biosensor for Detection of Herbicides

The aim of this work was to generate a cyanobacterial biosensor that could be used to detect herbicides and other environmental pollutants. A representative freshwater cyanobacterium, Synechocystis sp. strain PCC6803, was chromosomally marked with the luciferase gene luc (from the firefly Photinus pyralis) to create a novel bioluminescent cyanobacterial strain. Successful expression of the luc gene during growth of Synechocystis sp. strain PCC6803 cultures was characterized by measuring optical density and bioluminescence. Bioluminescence was optimized with regard to uptake of the luciferase substrate, luciferin, and the physiology of the cyanobacterium. Bioassays demonstrated that a novel luminescent cyanobacterial biosensor has been developed which responded to a range of compounds including different herbicide types and other toxins. This biosensor is expected to provide new opportunities for the rapid screening of environmental samples or for the investigation of potential environmental damage.     

NADPH fluorescence in the cyanobacterium Synechocystis sp. PCC 6803: A versatile probe for in vivo measurements of rates,yields and pools

We measured the kinetics of light-induced NADPH formation and subsequent dark consumption by monitoring in vivo its fluorescence in the cyanobacterium Synechocystis PCC 6803. Spectral data allowed the signal changes to be attributed to NAD(P)H and signal linearity vs the chlorophyll concentration was shown to be recoverable after appropriate correction. Parameters associated to reduction of NADP⁺ to NADPH by ferredoxin–NADP⁺-oxidoreductase were determined: After single excitation of photosystem I, half of the signal rise is observed in 8 ms; Evidence for a kinetic limitation which is attributed to an enzyme bottleneck is provided; After two closely separated saturating flashes eliciting two photosystem I turnovers in less than 2 ms, more than 50% of the cytoplasmic photoreductants (reduced ferredoxin and photosystem I acceptors) are diverted from NADPH formation by competing processes. Signal quantitation in absolute NADPH concentrations was performed by adding exogenous NADPH to the cell suspensions and by estimating the enhancement factor of in vivo fluorescence (between 2 and 4). The size of the visible (light-dependent) NADP (NADP⁺ + NADPH) pool was measured to be between 1.4 and 4 times the photosystem I concentration. A quantitative discrepancy is found between net oxygen evolution and NADPH consumption by the light-activated Calvin–Benson cycle. The present study shows that NADPH fluorescence is an efficient probe for studying in vivo the energetic metabolism of cyanobacteria which can be used for assessing multiple phenomena occurring over different time scales.     

The rpaC gene product regulates phycobilisome-photosystem II interaction in cyanobacteria

State transitions in cyanobacteria are a physiological adaptation mechanism that changes the interaction of the phycobilisomes with the Photosystem I and Photosystem II core complexes. A random mutagenesis study in the cyanobacterium Synechocystis sp. PCC6803 identified a gene named rpaC which appeared to be specifically required for state transitions. rpaC is a conserved cyanobacterial gene which was tentatively suggested to code for a novel signal transduction factor. The predicted gene product is a 9-kDa integral membrane protein. We have further examined the role of rpaC by overexpressing the gene in Synechocystis 6803 and by inactivating the ortholog in a second cyanobacterium, Synechococcus sp. PCC7942. Unlike the Synechocystis 6803 null mutant, the Synechococcus 7942 null mutant is unable to segregate, indicating that the gene is essential for cell viability in this cyanobacterium. The Synechocystis 6803 overexpressor is also unable to segregate, indicating that the cells can only tolerate a limited gene copy number. The non-segregated Synechococcus 7942 mutant can perform state transitions but shows a perturbed phycobilisome-Photosystem II interaction. Based on these results, we propose that the rpaC gene product controls the stability of the phycobilisome-Photosystem II supercomplex, and is probably a structural component of the complex.     

Growth arrest of Synechocystis sp. PCC6803 by superoxide generated from heterologously expressed Rhodobacter sphaeroides chlorophyllide a reductase

The photosynthetic growth of Synechocystis sp. PCC6803 ceased upon expression of Rhodobacter sphaeroides chlorophyllide a reductase (COR). However, an increase in cytosolic superoxide dismutase level in the recombinant Synechocystis sp. PCC6803 completely reversed the growth cessation. This demonstrates that COR generates superoxide in Synechocystis sp. PCC6803. Considering the dissolved oxygen (DO) level suitable for COR, the intracellular DO of this oxygenic photosynthetic cell appears to be low enough to support COR-mediated superoxide generation. The growth arrest of Synechocystis sp. PCC6803 by COR may give an insight into the evolutionary path from bacteriochlorophyll a biosynthetic pathway to chlorophyll a, which bypasses COR reaction.     

Photoheterotrophic Fluxome in Synechocystis sp. Strain PCC 6803 and Its Implications for Cyanobacterial Bioenergetics

This study investigated metabolic responses in Synechocystis sp. strain PCC 6803 to photosynthetic impairment. We used 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU; a photosystem II inhibitor) to block O₂ evolution and ATP/NADPH generation by linear electron flow. Based on ¹³C-metabolic flux analysis (¹³C-MFA) and RNA sequencing, we have found that Synechocystis sp. PCC 6803 employs a unique photoheterotrophic metabolism. First, glucose catabolism forms a cyclic route that includes the oxidative pentose phosphate (OPP) pathway and the glucose-6-phosphate isomerase (PGI) reaction. Glucose-6-phosphate is extensively degraded by the OPP pathway for NADPH production and is replenished by the reversed PGI reaction. Second, the Calvin cycle is not fully functional, but RubisCO continues to fix CO₂ and synthesize 3-phosphoglycerate. Third, the relative flux through the complete tricarboxylic acid (TCA) cycle and succinate dehydrogenase is small under heterotrophic conditions, indicating that the newly discovered cyanobacterial TCA cycle (via the γ-aminobutyric acid pathway or α-ketoglutarate decarboxylase/succinic semialdehyde dehydrogenase) plays a minimal role in energy metabolism. Fourth, NAD(P)H oxidation and the cyclic electron flow (CEF) around photosystem I are the two main ATP sources, and the CEF accounts for at least 40% of total ATP generation from photoheterotrophic metabolism (without considering maintenance loss). This study not only demonstrates a new topology for carbohydrate oxidation but also provides quantitative insights into metabolic bioenergetics in cyanobacteria.     

PHOTOTACTIC MOTILITY OF SYNECHOCYSTIS SP. UNIWG (CYANOBACTERIA) FROM BRACKISH ENVIRONMENT1

Although type IV pilus has been implicated in the phototactic motility of some unicellular cyanobacteria, its regulatory mechanism and the effect of environmental factors on motility are still unknown. Equally important is the ability of cyanobacterial cells to anchor themselves to an environment that is conducive for survival. We compared the motility of a newly isolated unicellular brackish cyanobacterium, Synechocystis sp. UNIWG, with the morphologically and phylogenetically similar freshwater cyanobacterium Synechocystis sp. PCC6803 under different environmental conditions. The phototactic motility of Synechocystis sp. UNIWG on semisolid BG‐11 medium with various concentrations of nitrogen source was significantly faster than that of Synechocystis PCC6803. Interestingly, the cell surface of Synechocystis sp. UNIWG showed the presence of rigid spicules when grown in liquid BG‐11, a phenomenon that was absent in Synechocystis PCC6803. Negative staining of Synechocystis sp. UNIWG revealed the presence of two distinct pilus morphotypes, which resembled type IV pili and thin pili of Synechocystis PCC6803. This finding suggested a similar pattern of phototactic motility in both strains. However, the rigid spicules on Synechocystis sp. UNIWG seem to be more of a hindrance during type IV motility. It was determined that the spicules were degraded when the cells moved, such as under prolonged darkness and/or depletion of nitrogen source, indicating that the function of the spicules is to attach the cell to an environment that is conducive for its survival. Thus, Synechocystis sp. UNIWG shows phototaxis regulation that is more complex than Synechocystis PCC6803.     

Production of optically pure d-lactate from CO2 by blocking the PHB and acetate pathways and expressing d-lactate dehydrogenase in cyanobacterium Synechocystis sp. PCC 6803

Lactate is an important industrial material with numerous potential applications, and its production from carbon dioxide is very attractive. d-Lactate is an essential monomer for production of thermostable polylactide. The photoautotrophic prokaryote cyanobacterium Synechocystis sp. PCC 6803 represents a promising host for biosynthesis of d-lactate from CO₂ as it only contains d-lactate dehydrogenase. The production of d-lactate from CO₂ by an engineered strain of Synechocystis sp. PCC 6803 with overexpressing d-lactate dehydrogenase and a soluble transhydrogenase has been reported recently. Here, we report an alternative engineering strategy to produce d-lactate from CO₂. This strategy involves blocking two competitive pathways, the native poly-3-hydroxybutyrate and acetate pathways from the acetyl-CoA node, and introducing a more efficient d-lactate dehydrogenase into Synechocystis sp. PCC 6803. The engineered strain of Synechocystis sp. PCC 6803 was capable of producing 1.06 g/L of d-lactate from CO₂. This alternative strategy for the production of optically pure d-lactate could also be used to produce other acetyl-CoA-derived chemicals from CO₂ by using engineered cyanobacteria.     

Acclimation to high-light conditions in cyanobacteria: from gene expression to physiological responses

Photosynthetic organisms have evolved various acclimatory responses to high-light (HL) conditions to maintain a balance between energy supply (light harvesting and electron transport) and consumption (cellular metabolism) and to protect the photosynthetic apparatus from photodamage. The molecular mechanism of HL acclimation has been extensively studied in the unicellular cyanobacterium Synechocystis sp. PCC 6803. Whole genome DNA microarray analyses have revealed that the change in gene expression profile under HL is closely correlated with subsequent acclimatory responses such as (1) acceleration in the rate of photosystem II turnover, (2) downregulation of light harvesting capacity, (3) development of a protection mechanism for the photosystems against excess light energy, (4) upregulation of general protection mechanism components, and (5) regulation of carbon and nitrogen assimilation. In this review article, we survey recent progress in the understanding of the molecular mechanisms of these acclimatory responses in Synechocystis sp. PCC 6803. We also briefly describe attempts to understand HL acclimation in various cyanobacterial species in their natural environments.     

Remodeling of photosynthetic electron transport in Synechocystis sp. PCC 6803 for future hydrogen production from water

Photosynthetic microorganisms such as the cyanobacterium Synechocystis sp. PCC 6803 (Synechocystis) can be exploited for the light-driven synthesis of valuable compounds. Thermodynamically, it is most beneficial to branch-off photosynthetic electrons at ferredoxin (Fd), which provides electrons for a variety of fundamental metabolic pathways in the cell, with the ferredoxin-NADP⁺ Oxido-Reductase (FNR, PetH) being the main target. In order to re-direct electrons from Fd to another consumer, the high electron transport rate between Fd and FNR has to be reduced. Based on our previous in vitro experiments, corresponding FNR-mutants at position FNR_K190 (Wiegand, K., et al.: “Rational redesign of the ferredoxin-NADP-oxido-reductase/ferredoxin-interaction for photosynthesis-dependent H₂-production”. Biochim Biophys Acta, 2018) have been generated in Synechocystis cells to study their impact on the cellular metabolism and their potential for a future hydrogen-producing design cell. Out of two promising candidates, mutation FNR_K190D proved to be lethal due to oxidative stress, while FNR_K190A was successfully generated and characterized: The light induced NADPH formation is clearly impaired in this mutant and it shows also major metabolic adaptations like a higher glucose metabolism as evidenced by quantitative mass spectrometric analysis. These results indicate a high potential for the future use of photosynthetic electrons in engineered design cells – for instance for hydrogen production. They also show substantial differences of interacting proteins in an in vitro environment vs. physiological conditions in whole cells.     

Inactivation of sll0136 gene in Synechocystis sp. PCC 6803 results in the disturbance in protein biogenesis of photosynthetic complexes

The genome of cyanobacterium Synechocystis sp. PCC 6803 contains the sll0136 (pepP) gene encoding the putative homolog of proline aminopeptidase PII (AMPPII) of the heterotrophic bacterium Escherichia coli. AMPPII is known to cleave the N-terminal amino acid residue of peptides and proteins only in the case of a penultimate proline position. The Synechocystis sp. PCC 6803 insertion mutant with inactivated pepP gene is characterized by the reduced content of phycobiliproteins and also proteins of photosystem II, which may be related to the reduced synthesis or stability of corresponding proteins. A possible involvement of PepP in biogenesis of proteins of the photosynthetic apparatus is discussed.     

Dissection of respiratory and cyclic electron transport in Synechocystis sp. PCC 6803

Cyclic electron transport (CET) is an attractive hypothesis for regulating photosynthetic electron transport and producing the additional ATP in oxygenic phototrophs. The concept of CET has been established in the last decades, and it is proposed to function in the progenitor of oxygenic photosynthesis, cyanobacteria. The in vivo activity of CET is frequently evaluated either from the redox state of the reaction center chlorophyll in photosystem (PS) I, P700, in the absence of PSII activity or by comparing PSI and PSII activities through the P700 redox state and chlorophyll fluorescence, respectively. The evaluation of CET activity, however, is complicated especially in cyanobacteria, where CET shares the intersystem chain, including plastoquinone, cytochrome b₆/f complex, plastocyanin, and cytochrome c₆, with photosynthetic linear electron transport (LET) and respiratory electron transport (RET). Here we sought to distinguish the in vivo electron transport rates in RET and CET in the cyanobacterium Synechocystis sp. PCC 6803. The reduction rate of oxidized P700 (P700⁺) decreased to less than 10% when PSII was inhibited, indicating that PSII is the dominant electron source to PSI but P700⁺ is also reduced by electrons derived from other sources. The oxidative pentose phosphate (OPP) pathway functions as the dominant electron source for RET, which was found to be inhibited by glycolaldehyde (GA). In the condition where the OPP pathway and respiratory terminal oxidases were inhibited by GA and KCN, the P700⁺ reduction rate was less than 1% of that without any inhibitors. This study indicate that the electron transport to PSI when PSII is inhibited is dominantly derived from the OPP pathway in Synechocystis sp. PCC 6803.

     

Respiratory terminal oxidases alleviate photo-oxidative damage in photosystem I during repetitive short-pulse illumination in <Emphasis Type="Italic">Synechocystis</Emphasis> sp. PCC 6803

Oxygenic phototrophs are vulnerable to damage by reactive oxygen species (ROS) that are produced in photosystem I (PSI) by excess photon energy over the demand of photosynthetic CO₂ assimilation. In plant leaves, repetitive short-pulse (rSP) illumination produces ROS to inactivate PSI. The production of ROS is alleviated by oxidation of the reaction center chlorophyll in PSI, P700, during the illumination with the short-pulse light, which is supported by flavodiiron protein (FLV). In this study, we found that in the cyanobacterium Synechocystis sp. PCC 6803 P700 was oxidized and PSI was not inactivated during rSP illumination even in the absence of FLV. Conversely, the mutant deficient in respiratory terminal oxidases was impaired in P700 oxidation during the illumination with the short-pulse light to suffer from photo-oxidative damage in PSI. Interestingly, the other cyanobacterium Synechococcus sp. PCC 7002 could not oxidize P700 without FLV during rSP illumination. These data indicate that respiratory terminal oxidases are critical to protect PSI from ROS damage during rSP illumination in Synechocystis sp. PCC 6803 but not Synechococcus sp. PCC 7002.     

Expression of the CMV‐CP Gene in Synechocystis 6803 Affects Cyanobacterial Photosynthesis

Previous work has shown that the presence of excess coat protein (CP) of cucumber mosaic virus (CMV) in the chloroplasts was related with mosaic symptoms. However, whether these mosaic symptoms are directly induced by the interaction between CP and chloroplasts is unknown. To directly demonstrate the interaction between CP and the chloroplast, Synechocystis sp. PCC 6803 was used as the chloroplast model. The cDNA encoding the CMV‐CP was cloned in a cyanobacterial shuttle vector (pKT‐CP) and transferred to Synechocystis sp. PCC 6803. The CP was expressed in the cyanobacterium with the psbA promoter. The expression of CMV‐CP hindered the growth of transgenic cyanobacterium cells and decreased its photosynthetic rate and the PS II activity. The transgenic cells showed increased fluorescence (F) from the phycobilisome terminal emitters and increased fluorescence (F) from PS II. The absorption spectra at room temperature showed the Chl and the phycocyanin absorption peak of the mutant strain significantly decreased. These results showed that CP may directly affect the cyanobacterium cells and decreased its photosynthesis, especially the PS II activity. These data might provide new evidence for mosaic symptoms being directly induced by the interaction between CP and chloroplasts.     

Increase in the rate of photosynthetic linear electron transport in cyanobacteruim <Emphasis Type="Italic">Synechocystis</Emphasis> sp. PCC 6803 lacking phycobilisomes

Compensating changes in the pigment apparatus of photosynthesis that resulted from a complete loss of phycobilisomes (PBS) were investigated in the cells of a PAL mutant of cyanobacterium Synechocystis sp. PCC 6803. The ratio PBS/chlorophyll calculated on the basis of the intensity of bands in the action spectra of photosynthetic activity of two photosystems in the wild strain was 1: 70 for PSII and 1: 300 for PSI. Taking into consideration the number of chlorophyll molecules per reaction center in each photosystem, these ratios could be interpreted as association of PBS with dimers of PSII and trimers of PSI as well as greater dependence of PSII as compared with PSI on light absorption by PBS. The ratio PSI/PSII determined by photochemical cross-section of the reactions of two photosystems was 3.5: 1.0 for wild strain of Synechocystis sp. PCC 6803 and 0.7: 1.0 for the PAL mutant. A fivefold increase in the relative content of PSII in pigment apparatus corresponds to a 5-fold increase in the intensity of bands at 685 and 695 nm as related to the band of PSI at 726 nm recorded in low-temperature fluorescence spectrum of the PAL mutant. Inhibition of PSII with diuron resulted in a pronounced stimulation of chlorophyll fluorescence in the PAL mutant as compared to the wild strain of Synechocystis sp. PCC 6803; these data suggested an activation of electron transfer between PSII and PSI in the mutant cells. Thus, the lack of PBS in the mutant strain of Synechocystis sp. PCC 6803 was compensated for by the higher relative content of PSII in the pigment apparatus of photosynthesis and by a rise in the rate of linear electron transport.     

Nitric oxide represses photosystem II and NDH-1 in the cyanobacterium Synechocystis sp. PCC 6803

Photosynthetic electron transfer comprises a series of light-induced redox reactions catalysed by multiprotein machinery in the thylakoid. These protein complexes possess cofactors susceptible to redox modifications by reactive small molecules. The gaseous radical nitric oxide (NO), a key signalling molecule in green algae and plants, has earlier been shown to bind to Photosystem (PS) II and obstruct electron transfer in plants. The effects of NO on cyanobacterial bioenergetics however, have long remained obscure. In this study, we exposed the model cyanobacterium Synechocystis sp. PCC 6803 to NO under anoxic conditions and followed changes in whole-cell fluorescence and oxidoreduction of P700 in vivo. Our results demonstrate that NO blocks photosynthetic electron transfer in cells by repressing PSII, PSI, and likely the NDH dehydrogenase-like complex 1 (NDH-1). We propose that iron?sulfur clusters of NDH-1 complex may be affected by NO to such an extent that ferredoxin-derived electron injection to the plastoquinone pool, and thus cyclic electron transfer, may be inhibited. These findings reveal the profound effects of NO on Synechocystis cells and demonstrate the importance of controlled NO homeostasis in cyanobacteria.