Is the A-1 Pigment in Photosystem I Part of P700? A (P700+-P700) FTIR Difference Spectroscopy Study of A-1 Mutants.

The involvement of the second pair of chlorophylls, termed A-1A and A-1B, in light-induced electron transfer in photosystem I (PSI) is currently debated. Asparagines at PsaA600 and PsaB582 are involved in coordinating the A-1B and A-1A pigments, respectively. Here we have mutated these asparagine residues to methionine in two single mutants and a double mutant in PSI from Synechocystis sp. PCC 6803, which we term NA600M, NB582M, and NA600M/NB582M mutants. (P700+-P700) FTIR difference spectra (DS) at 293 K were obtained for the wild-type and the three mutant PSI samples. The wild-type and mutant FTIR DS differ considerably. This difference indicates that the observed changes in the (P700+-P700) FTIR DS cannot be due to only the PA and PB pigments of P700. Comparison of the wild-type and mutant FTIR DS allows the assignment of different features to both A-1 pigments in the FTIR DS for wild-type PSI and assesses how these features shift upon cation formation and upon mutation. While the exact role the A-1 pigments play in the species we call P700 is unclear, we demonstrate that the vibrational modes of the A-1A and A-1B pigments are modified upon P700+ formation. Previously, we showed that the A-1 pigments contribute to P700 in green algae. In this manuscript, we demonstrate that this is also the case in cyanobacterial PSI. The nature of the mutation-induced changes in algal and cyanobacterial PSI is similar and can be considered within the same framework, suggesting a universality in the nature of P700 in different photosynthetic organisms.

Functional consequences of modification of the photosystem I/photosystem II ratio in the cyanobacterium Synechocystis sp. PCC 6803.

The stoichiometry of photosystem II (PSII) and photosystem I (PSI) varies between photoautotrophic organisms. The cyanobacterium Synechocystis sp. PCC 6803 maintains two- to fivefold more PSI than PSII reaction center complexes, and we sought to modify this stoichiometry by changing the promoter region of the psaAB operon. We thus generated mutants with varied psaAB expression, ranging from ~3% to almost 200% of the wild-type transcript level, but all showing a reduction in PSI levels, relative to wild type, suggesting a role of the psaAB promoter region in translational regulation. Mutants with 25%-70% of wild-type PSI levels were photoautotrophic, with whole-chain oxygen evolution rates on a per-cell basis comparable to that of wild type. In contrast, mutant strains with <10% of the wild-type level of PSI were obligate photoheterotrophs. Variable fluorescence yields of all mutants were much higher than those of wild type, indicating that the PSI content is localized differently than in wild type, with less transfer of PSII-absorbed energy to PSI. Strains with less PSI saturate at a higher light intensity, enhancing productivity at higher light intensities. This is similar to what is found in mutants with reduced antennae. With 3-(3,4-dichlorophenyl)-1,1-dimethylurea present, P700+ re-reduction kinetics in the mutants were slower than in wild type, consistent with the notion that there is less cyclic electron transport if less PSI is present. Overall, strains with a reduction in PSI content displayed surprisingly vigorous growth and linear electron transport. IMPORTANCE: Consequences of reduction in photosystem I content were investigated in the cyanobacterium Synechocystis sp. PCC 6803 where photosystem I far exceeds the number of photosystem II complexes. Strains with less photosystem I displayed less cyclic electron transport, grew more slowly at lower light intensity and needed more light for saturation but were surprisingly normal in their whole-chain electron transport rates, implying that a significant fraction of photosystem I is dispensable for linear electron transport in cyanobacteria. These strains with reduced photosystem I levels may have biotechnological relevance as they grow well at higher light intensities.

Structure and genome editing of type I-B CRISPR-Cas.

Type I CRISPR-Cas systems employ multi-subunit effector Cascade and helicase-nuclease Cas3 to target and degrade foreign nucleic acids, representing the most abundant RNA-guided adaptive immune systems in prokaryotes. Their ability to cause long fragment deletions have led to increasing interests in eukaryotic genome editing. While the Cascade structures of all other six type I systems have been determined, the structure of the most evolutionarily conserved type I-B Cascade is still missing. Here, we present two cryo-EM structures of the Synechocystis sp. PCC 6714 (Syn) type I-B Cascade, revealing the molecular mechanisms that underlie RNA-directed Cascade assembly, target DNA recognition, and local conformational changes of the effector complex upon R-loop formation. Remarkably, a loop of Cas5 directly intercalated into the major groove of the PAM and facilitated PAM recognition. We further characterized the genome editing profiles of this I-B Cascade-Cas3 in human CD3+ T cells using mRNA-mediated delivery, which led to unidirectional 4.5 kb deletion in TRAC locus and achieved an editing efficiency up to 41.2%. Our study provides the structural basis for understanding target DNA recognition by type I-B Cascade and lays foundation for harnessing this system for long range genome editing in human T cells.

Pyruvate kinase 2 from Synechocystis sp. PCC 6803 increased substrate affinity via glucose-6-phosphate and ribose-5-phosphate for phosphoenolpyruvate consumption.

Pyruvate kinase (Pyk, EC 2.7.1.40) is a glycolytic enzyme that generates pyruvate and adenosine triphosphate (ATP) from phosphoenolpyruvate (PEP) and adenosine diphosphate (ADP), respectively. Pyk couples pyruvate and tricarboxylic acid metabolisms. Synechocystis sp. PCC 6803 possesses two pyk genes (encoded pyk1, sll0587 and pyk2, sll1275). A previous study suggested that pyk2 and not pyk1 is essential for cell viability; however, its biochemical analysis is yet to be performed. Herein, we biochemically analyzed Synechocystis Pyk2 (hereafter, SyPyk2). The optimum pH and temperature of SyPyk2 were 7.0 and 55 °C, respectively, and the Km values for PEP and ADP under optimal conditions were 1.5 and 0.053 mM, respectively. SyPyk2 is activated in the presence of glucose-6-phosphate (G6P) and ribose-5-phosphate (R5P); however, it remains unaltered in the presence of adenosine monophosphate (AMP) or fructose-1,6-bisphosphate. These results indicate that SyPyk2 is classified as PykA type rather than PykF, stimulated by sugar monophosphates, such as G6P and R5P, but not by AMP. SyPyk2, considering substrate affinity and effectors, can play pivotal roles in sugar catabolism under nonphotosynthetic conditions.

Phosphorus deficiency alleviates iron limitation in Synechocystis cyanobacteria through direct PhoB-mediated gene regulation.

Iron and phosphorus are essential nutrients that exist at low concentrations in surface waters and may be co-limiting resources for phytoplankton growth. Here, we show that phosphorus deficiency increases the growth of iron-limited cyanobacteria (Synechocystis sp. PCC 6803) through a PhoB-mediated regulatory network. We find that PhoB, in addition to its well-recognized role in controlling phosphate homeostasis, also regulates key metabolic processes crucial for iron-limited cyanobacteria, including ROS detoxification and iron uptake. Transcript abundances of PhoB-targeted genes are enriched in samples from phosphorus-depleted seawater, and a conserved PhoB-binding site is widely present in the promoters of the target genes, suggesting that the PhoB-mediated regulation may be highly conserved. Our findings provide molecular insights into the responses of cyanobacteria to simultaneous iron/phosphorus nutrient limitation.

The redox-sensitive R-loop of the carbon control protein SbtB contributes to the regulation of the cyanobacterial CCM.

SbtB is a PII-like protein that regulates the carbon-concentrating mechanism (CCM) in cyanobacteria. SbtB proteins can bind many adenyl nucleotides and possess a characteristic C-terminal redox sensitive loop (R-loop) that forms a disulfide bridge in response to the diurnal state of the cell. SbtBs also possess an ATPase/ADPase activity that is modulated by the redox-state of the R-loop. To investigate the R-loop in the cyanobacterium Synechocystis sp. PCC 6803, site-specific mutants, unable to form the hairpin and permanently in the reduced state, and a R-loop truncation mutant, were characterized under different inorganic carbon (Ci) and light regimes. Growth under diurnal rhythm showed a role of the R-loop as sensor for acclimation to changing light conditions. The redox-state of the R-loop was found to impact the binding of the adenyl-nucleotides to SbtB, its membrane association and thereby the CCM regulation, while these phenotypes disappeared after truncation of the R-loop. Collectively, our data imply that the redox-sensitive R-loop provides an additional regulatory layer to SbtB, linking the CO2-related signaling activity of SbtB with the redox state of cells, mainly reporting the actual light conditions. This regulation not only coordinates CCM activity in the diurnal rhythm but also affects the primary carbon metabolism.

A hemoprotein with a zinc-mirror heme site ties heme availability to carbon metabolism in cyanobacteria.

Heme has a critical role in the chemical framework of the cell as an essential protein cofactor and signaling molecule that controls diverse processes and molecular interactions. Using a phylogenomics-based approach and complementary structural techniques, we identify a family of dimeric hemoproteins comprising a domain of unknown function DUF2470. The heme iron is axially coordinated by two zinc-bound histidine residues, forming a distinct two-fold symmetric zinc-histidine-iron-histidine-zinc site. Together with structure-guided in vitro and in vivo experiments, we further demonstrate the existence of a functional link between heme binding by Dri1 (Domain related to iron 1, formerly ssr1698) and post-translational regulation of succinate dehydrogenase in the cyanobacterium Synechocystis, suggesting an iron-dependent regulatory link between photosynthesis and respiration. Given the ubiquity of proteins containing homologous domains and connections to heme metabolism across eukaryotes and prokaryotes, we propose that DRI (Domain Related to Iron; formerly DUF2470) functions at the molecular level as a heme-dependent regulatory domain.

Impact of Carbon Fixation, Distribution and Storage on the Production of Farnesene and Limonene in Synechocystis PCC 6803 and Synechococcus PCC 7002.

Terpenes are high-value chemicals which can be produced by engineered cyanobacteria from sustainable resources, solar energy, water and CO2. We previously reported that the euryhaline unicellular cyanobacteria Synechocystis sp. PCC 6803 (S.6803) and Synechococcus sp. PCC 7002 (S.7002) produce farnesene and limonene, respectively, more efficiently than other terpenes. In the present study, we attempted to enhance farnesene production in S.6803 and limonene production in S.7002. Practically, we tested the influence of key cyanobacterial enzymes acting in carbon fixation (RubisCO, PRK, CcmK3 and CcmK4), utilization (CrtE, CrtR and CruF) and storage (PhaA and PhaB) on terpene production in S.6803, and we compared some of the findings with the data obtained in S.7002. We report that the overproduction of RubisCO from S.7002 and PRK from Cyanothece sp. PCC 7425 increased farnesene production in S.6803, but not limonene production in S.7002. The overexpression of the crtE genes (synthesis of terpene precursors) from S.6803 or S.7002 did not increase farnesene production in S.6803. In contrast, the overexpression of the crtE gene from S.6803, but not S.7002, increased farnesene production in S.7002, emphasizing the physiological difference between these two model cyanobacteria. Furthermore, the deletion of the crtR and cruF genes (carotenoid synthesis) and phaAB genes (carbon storage) did not increase the production of farnesene in S.6803. Finally, as a containment strategy of genetically modified strains of S.6803, we report that the deletion of the ccmK3K4 genes (carboxysome for CO2 fixation) did not affect the production of limonene, but decreased the production of farnesene in S.6803.

Multi-biomarker response of cyanobacteria Synechocystis salina and Microcystis aeruginosa to diclofenac.

The cyanobacterial response to pharmaceuticals is less frequently investigated compared to green algae. Pharmaceuticals can influence not only the growth rate of cyanobacteria culture, but can also cause changes at the cellular level. The effect of diclofenac (DCF) as one of the for cyanobacteria has been rarely tested, and DCF has never been applied with cellular biomarkers. The aim of this work was to test the response of two unicellular cyanobacteria (Synechocystis salina and Microcystis aeruginosa) toward DCF (100 mg L-1) under photoautotrophic growth conditions. Such endpoints were analyzed as cells number, DCF uptake, the change in concentrations of photosynthetic pigments, the production of toxins, and chlorophyll a in vivo fluorescence. It was noted that during a 96 h exposure, cell proliferation was not impacted. Nevertheless, a biochemical response was observed. The increased production of microcystin was noted for M. aeruginosa. Due to the negligible absorption of DCF into cells, it is possible that the biochemical changes are induced by an external signal. The application of non-standard biomarkers demonstrates the effect of DCF on microorganism metabolism without a corresponding effect on biomass. The high resistance of cyanobacteria to DCF and the stimulating effect of DCF on the secretion of toxins raise concerns for environment biodiversity.

Recombinant protein synthesis and isolation of human interferon alpha-2 in cyanobacteria.

Synechocystis sp. PCC 6803 (Synechocystis) is a unicellular photosynthetic microorganism that has been used as a model for photo-biochemical research. It comprises a potential cell factory for the generation of valuable bioactive compounds, therapeutic proteins, and possibly biofuels. Fusion constructs of recombinant proteins with the CpcA α-subunit or CpcB ß-subunit of phycocyanin in Synechocystis have enabled true over-expression of several isoprenoid pathway enzymes and biopharmaceutical proteins to levels of 10-20 % of the total cellular protein. The present work employed the human interferon α-2 protein, as a study case of over-expression and downstream processing. It advanced the state of the art in the fusion constructs for protein overexpression technology by developing the bioresource for target protein separation from the fusion construct and isolation in substantially enriched or pure form. The work brings the cyanobacterial cell factory concept closer to meaningful commercial application for the photosynthetic production of useful recombinant proteins.

Erythromycin mediates co-flocculation between cyanobacterium Synechocystis sp. PCC 6803 and filamentous fungi in liquid cultivation without organic compounds.

Photoautotrophic cyanobacteria assimilate the greenhouse gas carbon dioxide as their sole carbon source for producing useful bioproducts. However, harvesting the cells from their liquid media is a major bottleneck in the process. Thus, an easy-to-harvest method, such as auto-flocculation, is desirable. Here, we found that cyanobacterium Synechocystis sp. PCC 6803 co-flocculated with a natural fungal contamination in the presence of the antibiotic erythromycin (EM) but not without EM. The fungi in the co-flocculated biomass were isolated and found to consist of five species with the filamentous Purpureocillium lilacinum and Aspergillus protuberus making up 71% of the overall fungal population. The optimal co-cultivation for flocculation was an initial 5 mg (fresh weight) of fungi, an initial cell density of Synechocystis of 0.2 OD730, 10 µM EM, and 14 days of cultivation in 100 mL of BG11 medium with no organic compound. This yielded 248 ± 28 mg/L of the Synechocystis-fungi flocculated biomass from 560 ± 35 mg/L of total biomass, a 44 ± 2% biomass flocculation efficiency. Furthermore, the EM treated Synechocystis cells in the Synechocystis-fungi flocculate had a normal cell color and morphology, while those in the axenic suspension exhibited strong chlorosis. Thus, the occurrence of the Synechocystis-fungi flocculation was mediated by EM, and the co-flocculation with the fungi protected Synechocystis against the development of chlorosis. Transcriptomic analysis suggested that the EM-mediated co-flocculation was a result of down-regulation of the minor pilin genes and up-regulation of several genes including the chaperone gene for pilin regulation, the S-layer protein genes, the exopolysaccharide-polymerization gene, and the genes for signaling proteins involved in cell attachment and abiotic-stress responses. The CuSO4 stress can also mediate Synechocystis-fungi flocculation but at a lower flocculation efficiency than that caused by EM. The EM treatment may be applied in the co-culture between other cyanobacteria and fungi to mediate cell bio-flocculation.

The role of the 5' sensing function of ribonuclease E in cyanobacteria.

RNA degradation is critical for synchronising gene expression with changing conditions in prokaryotic and eukaryotic organisms. In bacteria, the preference of the central ribonucleases RNase E, RNase J and RNase Y for 5'-monophosphorylated RNAs is considered important for RNA degradation. For RNase E, the underlying mechanism is termed 5' sensing, contrasting to the alternative 'direct entry' mode, which is independent of monophosphorylated 5' ends. Cyanobacteria, such as Synechocystis sp. PCC 6803 (Synechocystis), encode RNase E and RNase J homologues. Here, we constructed a Synechocystis strain lacking the 5' sensing function of RNase E and mapped on a transcriptome-wide level 283 5'-sensing-dependent cleavage sites. These included so far unknown targets such as mRNAs encoding proteins related to energy metabolism and carbon fixation. The 5' sensing function of cyanobacterial RNase E is important for the maturation of rRNA and several tRNAs, including tRNAGluUUC. This tRNA activates glutamate for tetrapyrrole biosynthesis in plant chloroplasts and in most prokaryotes. Furthermore, we found that increased RNase activities lead to a higher copy number of the major Synechocystis plasmids pSYSA and pSYSM. These results provide a first step towards understanding the importance of the different target mechanisms of RNase E outside Escherichia coli.

Arginine inhibits the arginine biosynthesis rate-limiting enzyme and leads to the accumulation of intracellular aspartate in Synechocystis sp. PCC 6803.

Cyanobacteria are oxygen-evolving photosynthetic prokaryotes that affect the global carbon and nitrogen turnover. Synechocystis sp. PCC 6803 (Synechocystis 6803) is a model cyanobacterium that has been widely studied and can utilize and uptake various nitrogen sources and amino acids from the outer environment and media. l-arginine is a nitrogen-rich amino acid used as a nitrogen reservoir in Synechocystis 6803, and its biosynthesis is strictly regulated by feedback inhibition. Argininosuccinate synthetase (ArgG; EC 6.3.4.5) is the rate-limiting enzyme in arginine biosynthesis and catalyzes the condensation of citrulline and aspartate using ATP to produce argininosuccinate, which is converted to l-arginine and fumarate through argininosuccinate lyase (ArgH). We performed a biochemical analysis of Synechocystis 6803 ArgG (SyArgG) and obtained a Synechocystis 6803 mutant overexpressing SyArgG and ArgH of Synechocystis 6803 (SyArgH). The specific activity of SyArgG was lower than that of other arginine biosynthesis enzymes and SyArgG was inhibited by arginine, especially among amino acids and organic acids. Both arginine biosynthesis enzyme-overexpressing strains grew faster than the wild-type Synechocystis 6803. Based on previous reports and our results, we suggest that SyArgG is the rate-limiting enzyme in the arginine biosynthesis pathway in cyanobacteria and that arginine biosynthesis enzymes are similarly regulated by arginine in this cyanobacterium. Our results contribute to elucidating the regulation of arginine biosynthesis during nitrogen metabolism.

KEY MESSAGE: This study revealed the catalytic efficiency and inhibition of cyanobacterial argininosuccinate synthetase by arginine and demonstrated that a strain overexpressing this enzyme grew faster than the wild-type strain.

Design, synthesis and activity evaluation of pseudilin analogs against cyanobacteria as IspD inhibitors.

The discovery of safe, effective, and selective chemical algicides is the stringent need for the algicides development, and it is also one of the effective routes to control cyanobacteria harmful algal blooms and to meet the higher requirements of environmental and ecological. In this work, a series of novel bromo-N-phenyl-5-o-hydroxyphenylpyrazole-3-carboxyamides were rationally designed as pseudilin analogs by bioisosteric replacement and molecular hybridization strategies, in which the pyrrole unit of pseudilin was replaced with pyrazole and further combined with the dominant structural fragments of algicide diuron. The synthesis was carried out by a facile four-step routeincluding cyclization, amidation, transanulation, and halogenation. The biological activity evaluation on AtIspD, EcIspD, Synechocystis sp. PCC6803 and Microcystis aeruginosa FACHB905 revealed that most compounds had good EcIspD and excellent cyanobacteria inhibitory activity. In particular, compound 6bb exhibited potent algicidal activity against PCC6803 and FACHB905 with EC50 = 1.28 µM and 0.37 µM, respectively, 1.4-fold and 4.0-fold enhancement compared to copper sulfate (EC50 = 1.79 and 1.49 µM, respectively), and it also showed the best inhibitory activity of EcIspD. The binding of 6bb to EcIspD was explored by molecular docking, and it was confirmed that 6bb could bind to the EcIspD active site. Compound 6bb was proven to be a potential structure for the further development of novel algicides that targets IspD in the MEP pathway.

High-throughput profiling of metabolic responses to exogenous nutrients in Synechocystis sp. PCC 6803.

Cyanobacteria fix carbon dioxide and release carbon-containing compounds into the wider ecosystem, yet they are sensitive to small metabolites that may impact their growth and physiology. Several cyanobacteria can grow mixotrophically, but we currently lack a molecular understanding of how specific nutrients may alter the compounds they release, limiting our knowledge of how environmental factors might impact primary producers and the ecosystems they support. In this study, we develop a high-throughput phytoplankton culturing platform and identify how the model cyanobacterium Synechocystis sp. PCC 6803 responds to nutrient supplementation. We assess growth responses to 32 nutrients at two concentrations, identifying 15 that are utilized mixotrophically. Seven nutrient sources significantly enhance growth, while 19 elicit negative growth responses at one or both concentrations. High-throughput exometabolomics indicates that oxidative stress limits Synechocystis' growth but may be alleviated by antioxidant metabolites. Furthermore, glucose and valine induce strong changes in metabolite exudation in a possible effort to correct pathway imbalances or maintain intracellular elemental ratios. This study sheds light on the flexibility and limits of cyanobacterial physiology and metabolism, as well as how primary production and trophic food webs may be modulated by exogenous nutrients.IMPORTANCECyanobacteria capture and release carbon compounds to fuel microbial food webs, yet we lack a comprehensive understanding of how external nutrients modify their behavior and what they produce. We developed a high throughput culturing platform to evaluate how the model cyanobacterium Synechocystis sp. PCC 6803 responds to a broad panel of externally supplied nutrients. We found that growth may be enhanced by metabolites that protect against oxidative stress, and growth and exudate profiles are altered by metabolites that interfere with central carbon metabolism and elemental ratios. This work contributes a holistic perspective of the versatile response of Synechocystis to externally supplied nutrients, which may alter carbon flux into the wider ecosystem.

Indirect interactions involving the PsbM or PsbT subunits and the PsbO, PsbU and PsbV proteins stabilize assembly and activity of Photosystem II in Synechocystis sp. PCC 6803.

The low-molecular-weight PsbM and PsbT proteins of Photosystem II (PS II) are both located at the monomer-monomer interface of the mature PS II dimer. Since the extrinsic proteins are associated with the final step of assembly of an active PS II monomer and, in the case of PsbO, are known to impact the stability of the PS II dimer, we have investigated the potential cooperativity between the PsbM and PsbT subunits and the PsbO, PsbU and PsbV extrinsic proteins. Blue-native polyacrylamide electrophoresis and western blotting detected stable PS II monomers in the ∆PsbM:∆PsbO and ∆PsbT:∆PsbO mutants that retained sufficient oxygen-evolving activity to support reduced photoautotrophic growth. In contrast, the ∆PsbM:∆PsbU and ∆PsbT:∆PsbU mutants assembled dimeric PS II at levels comparable to wild type and supported photoautotrophic growth at rates similar to those obtained with the corresponding ∆PsbM and ∆PsbT cells. Removal of PsbV was more detrimental than removal of PsbO. Only limited levels of dimeric PS II were observed in the ∆PsbM:∆PsbV mutant and the overall reduced level of assembled PS II in this mutant resulted in diminished rates of photoautotrophic growth and PS II activity below those obtained in the ∆PsbM:∆PsbO and ∆PsbT:∆PsbO strains. In addition, the ∆PsbT:∆PsbV mutant did not assemble active PS II centers although inactive monomers could be detected. The inability of the ∆PsbT:∆PsbV mutant to grow photoautotrophically, or to evolve oxygen, suggested a stable oxygen-evolving complex could not assemble in this mutant.

Whole-cell biophotovoltaic systems for renewable energy generation: A systematic analysis of existing knowledge.

The development of carbon-neutral fuel sources is an essential step in addressing the global fossil energy crisis. Whole-cell biophotovoltaic systems (BPVs) are a renewable, non-polluting energy-generating device that utilizes oxygenic photosynthetic microbes (OPMs) to split water molecules and generate bioelectricity under the driving of light energy. Since 2006, BPVs have been widely studied, with the order magnitudes of power density increasing from 10-4 mW/m2 to 103 mW/m2. This review examines the extracellular electron transfer (EET) mechanisms and regulation techniques of BPVs from biofilm to external environment. It is found that the EET of OPMs is mainly mediated by membrane proteins, with terminal oxidase limiting the power output. Synechocystis sp. PCC6803 and Chlorella vulgaris are two species that produce high power density in BPVs. The use of metal nanoparticles mixing, 3D pillar array electrodes, microfluidic technology, and transient-state operation models can significantly enhance power density. Challenges and potential research directions are discussed, including a deeper analysis of EET mechanisms and dynamics, the development of modular devices, integration of multiple regulatory components, and the exploration of novel BPV technologies.

Solution Structures of Two Different FRP-OCP Complexes as Revealed via SEC-SANS.

Photosynthetic organisms have established photoprotective mechanisms in order to dissipate excess light energy into heat, which is commonly known as non-photochemical quenching. Cyanobacteria utilize the orange carotenoid protein (OCP) as a high-light sensor and quencher to regulate the energy flow in the photosynthetic apparatus. Triggered by strong light, OCP undergoes conformational changes to form the active red state (OCPR). In many cyanobacteria, the back conversion of OCP to the dark-adapted state is assisted by the fluorescence recovery protein (FRP). However, the exact molecular events involving OCP and its interaction with FRP remain largely unraveled so far due to their metastability. Here, we use small-angle neutron scattering combined with size exclusion chromatography (SEC-SANS) to unravel the solution structures of FRP-OCP complexes using a compact mutant of OCP lacking the N-terminal extension (∆NTEOCPO) and wild-type FRP. The results are consistent with the simultaneous presence of stable 2:2 and 2:1 FRP-∆NTEOCPO complexes in solution, where the former complex type is observed for the first time. For both complex types, we provide ab initio low-resolution shape reconstructions and compare them to homology models based on available crystal structures. It is likely that both complexes represent intermediate states of the back conversion of OCP to its dark-adapted state in the presence of FRP, which are of transient nature in the photocycle of wild-type OCP. This study demonstrates the large potential of SEC-SANS in revealing the solution structures of protein complexes in polydisperse solutions that would otherwise be averaged, leading to unspecific results.

Interactome Analysis of ClpX Reveals Its Regulatory Role in Metabolism and Photosynthesis in Cyanobacteria.

Protein homeostasis is essential for cyanobacteria to maintain proper cellular function under adverse and fluctuating conditions. The AAA+ superfamily of proteolytic complexes in cyanobacteria plays a critical role in this process, including ClpXP, which comprises a hexameric ATPase ClpX and a tetradecameric peptidase ClpP. Despite the physiological effects of ClpX on growth and photosynthesis, its potential substrates and underlying mechanisms in cyanobacteria remain unknown. In this study, we employed a streptavidin-biotin affinity pull-down assay coupled with label-free proteome quantitation to analyze the interactome of ClpX in the model cyanobacterium Synechocystis sp. PCC 6803 (hereafter Synechocystis). We identified 503 proteins as potential ClpX-binding targets, many of which had novel interactions. These ClpX-binding targets were found to be involved in various biological processes, with particular enrichment in metabolic processes and photosynthesis. Using protein-protein docking, GST pull-down, and biolayer interferometry assays, we confirmed the direct association of ClpX with the photosynthetic proteins, ferredoxin-NADP+ oxidoreductase (FNR) and phycocyanin subunit (CpcA). Subsequent functional investigations revealed that ClpX participates in the maintenance of FNR homeostasis and functionality in Synechocystis grown under different light conditions. Overall, our study provides a comprehensive understanding of the extensive functions regulated by ClpX in cyanobacteria to maintain protein homeostasis and adapt to environmental challenges.

High Myristic Acid in Glycerolipids Enhances the Repair of Photodamaged Photosystem II under Strong Light.

Cyanobacteria inhabit areas with a broad range of light, temperature and nutrient conditions. The robustness of cyanobacterial cells, which can survive under different conditions, may depend on the resilience of photosynthetic activity. Cyanothece sp. PCC 8801 (Cyanothece), a freshwater cyanobacterium isolated from a Taiwanese rice field, had a higher repair activity of photodamaged photosystem II (PSII) under intense light than Synechocystis sp. PCC 6803 (Synechocystis), another freshwater cyanobacterium. Cyanothece contains myristic acid (14:0) as the major fatty acid at the sn-2 position of the glycerolipids. To investigate the role of 14:0 in the repair of photodamaged PSII, we used a Synechocystis transformant expressing a T-1274 encoding a lysophosphatidic acid acyltransferase (LPAAT) from Cyanothece. The wild-type and transformant cells contained 0.2 and 20.1 mol% of 14:0 in glycerolipids, respectively. The higher content of 14:0 in the transformants increased the fluidity of the thylakoid membrane. In the transformants, PSII repair was accelerated due to an enhancement in the de novo synthesis of D1 protein, and the production of singlet oxygen (1O2), which inhibited protein synthesis, was suppressed. The high content of 14:0 increased transfer of light energy received by phycobilisomes to PSI and CP47 in PSII and the content of carotenoids. These results indicated that an increase in 14:0 reduced 1O2 formation and enhanced PSII repair. The higher content of 14:0 in the glycerolipids may be required as a survival strategy for Cyanothece inhabiting a rice field under direct sunlight.