Artificial neural networks prediction and optimization based on four light regions for light utilization from Synechocystis sp. PCC 6803.

Light is crucial in microalgae growth. However, dividing the microalgae growth region into light and dark regions has limitations. In this study, the light response of Synechocystis sp. PCC 6803 was investigated to define four light regions (FLRs): light compensation region, light limitation region, light saturation region, and photoinhibition region. The proportions of cells' residence time in the FLRs and the number of times cells (NTC) passed through the FLRs in photobioreactors were calculated by using MATLAB. Based on the FLRs and NTC passed through the FLRs, a growth model was established by using artificial neural network (ANN).The ANN model had a validation R2 value of 0.97, which was 76.36% higher than the model based on light-dark regions. The high accuracy of the ANN model was further verified through dynamic adjustment of light intensity experiments.This study confirmed the importance of the FLRs for studying microalgae growth dynamics.

Cryo-EM structures of the Synechocystis sp. PCC 6803 cytochrome b6f complex with and without the regulatory PetP subunit.

In oxygenic photosynthesis, the cytochrome b6f (cytb6f) complex links the linear electron transfer (LET) reactions occurring at photosystems I and II and generates a transmembrane proton gradient via the Q-cycle. In addition to this central role in LET, cytb6f also participates in a range of processes including cyclic electron transfer (CET), state transitions and photosynthetic control. Many of the regulatory roles of cytb6f are facilitated by auxiliary proteins that differ depending upon the species, yet because of their weak and transient nature the structural details of these interactions remain unknown. An apparent key player in the regulatory balance between LET and CET in cyanobacteria is PetP, a ∼10 kDa protein that is also found in red algae but not in green algae and plants. Here, we used cryogenic electron microscopy to determine the structure of the Synechocystis sp. PCC 6803 cytb6f complex in the presence and absence of PetP. Our structures show that PetP interacts with the cytoplasmic side of cytb6f, displacing the C-terminus of the PetG subunit and shielding the C-terminus of cytochrome b6, which binds the heme cn cofactor that is suggested to mediate CET. The structures also highlight key differences in the mode of plastoquinone binding between cyanobacterial and plant cytb6f complexes, which we suggest may reflect the unique combination of photosynthetic and respiratory electron transfer in cyanobacterial thylakoid membranes. The structure of cytb6f from a model cyanobacterial species amenable to genetic engineering will enhance future site-directed mutagenesis studies of structure-function relationships in this crucial ET complex.

Redesigning the photosynthetic light reactions to enhance photosynthesis - the PhotoRedesign consortium.

In this Perspective article, we describe the visions of the PhotoRedesign consortium funded by the European Research Council of how to enhance photosynthesis. The light reactions of photosynthesis in individual phototrophic species use only a fraction of the solar spectrum, and high light intensities can impair and even damage the process. In consequence, expanding the solar spectrum and enhancing the overall energy capacity of the process, while developing resilience to stresses imposed by high light intensities, could have a strong positive impact on food and energy production. So far, the complexity of the photosynthetic machinery has largely prevented improvements by conventional approaches. Therefore, there is an urgent need to develop concepts to redesign the light-harvesting and photochemical capacity of photosynthesis, as well as to establish new model systems and toolkits for the next generation of photosynthesis researchers. The overall objective of PhotoRedesign is to reconfigure the photosynthetic light reactions so they can harvest and safely convert energy from an expanded solar spectrum. To this end, a variety of synthetic biology approaches, including de novo design, will combine the attributes of photosystems from different photoautotrophic model organisms, namely the purple bacterium Rhodobacter sphaeroides, the cyanobacterium Synechocystis sp. PCC 6803 and the plant Arabidopsis thaliana. In parallel, adaptive laboratory evolution will be applied to improve the capacity of reimagined organisms to cope with enhanced input of solar energy, particularly in high and fluctuating light.

Thylakoid attachment to the plasma membrane in Synechocystis sp. PCC 6803 requires the AncM protein.

Thylakoids are the highly specialized internal membrane systems that harbor the photosynthetic electron transport machinery in cyanobacteria and in chloroplasts. In Synechocystis sp. PCC 6803, thylakoid membranes (TMs) are arranged in peripheral sheets that occasionally converge on the plasma membrane (PM) to form thylakoid convergence membranes (TCMs). TCMs connect several thylakoid sheets and form local contact sites called thylapses between the two membrane systems, at which the early steps of photosystem II (PSII) assembly occur. The protein CurT is one of the main drivers of TCM formation known so far. Here, we identify, by whole-genome sequencing of a curT- suppressor strain, the protein anchor of convergence membranes (AncM) as a factor required for the attachment of thylakoids to the PM at thylapses. An ancM- mutant is shown to have a photosynthetic phenotype characterized by reductions in oxygen-evolution rate, PSII accumulation, and PS assembly. Moreover, the ancM- strain exhibits an altered thylakoid ultrastructure with additional sheets and TCMs detached from the PM. By combining biochemical studies with fluorescence and correlative light-electron microscopy-based approaches, we show that AncM is an integral membrane protein located in biogenic TCMs that form thylapses. These data suggest an antagonistic function of AncM and CurT in shaping TM ultrastructure.

Phototaxis in Cyanobacteria: From Mutants to Models of Collective Behavior.

Cyanobacteria rely on photosynthesis, and thus have evolved complex responses to light. These include phototaxis, the ability of cells to sense light direction and move towards or away from it. Analysis of mutants has demonstrated that phototaxis requires the coordination of multiple photoreceptors and signal transduction networks. The output of these networks is relayed to type IV pili (T4P) that attach to and exert forces on surfaces or other neighboring cells to drive "twitching" or "gliding" motility. This, along with the extrusion of polysaccharides or "slime" by cells, facilitates the emergence of group behavior. We evaluate recent models that describe the emergence of collective colony-scale behavior from the responses of individual, interacting cells. We highlight the advantages of "active matter" approaches in the study of bacterial communities, discussing key differences between emergent behavior in cyanobacterial phototaxis and similar behavior in chemotaxis or quorum sensing.

Biosynthesis of a sulfated exopolysaccharide, synechan, and bloom formation in the model cyanobacterium Synechocystis sp. strain PCC 6803.

Extracellularpolysaccharides of bacteria contribute to biofilm formation, stress tolerance, and infectivity. Cyanobacteria, the oxygenic photoautotrophic bacteria, uniquely produce sulfated extracellular polysaccharides among bacteria to support phototrophic biofilms. In addition, sulfated polysaccharides of cyanobacteria and other organisms have been focused as beneficial biomaterial. However, very little is known about their biosynthesis machinery and function in cyanobacteria. Here, we found that the model cyanobacterium, Synechocystis sp. strain PCC 6803, formed bloom-like cell aggregates embedded in sulfated extracellular polysaccharides (designated as synechan) and identified whole set of genes responsible for synechan biosynthesis and its transcriptional regulation, thereby suggesting a model for the synechan biosynthesis apparatus. Because similar genes are found in many cyanobacterial genomes with wide variation, our findings may lead elucidation of various sulfated polysaccharides, their functions, and their potential application in biotechnology.

Bacteria are single-cell microorganisms that can form communities called biofilms, which stick to surfaces such as rocks, plants or animals. Biofilms confer protection to bacteria and allow them to colonize new environments. The physical scaffold of biofilms is a viscous matrix made of several molecules, the main one being polysaccharides, complex carbohydrates formed by many monosaccharides (single sugar molecules) joined together. Cyanobacteria, also known as blue-green algae, are a type of bacteria that produce oxygen and use sunlight as an energy source, just as plants and algae do. Cyanobacteria produce extracellular polysaccharides that contain sulfate groups. These sulfated polysaccharides are also produced by animals and algae but are not common in other bacteria or plants. One possible role of sulfated, extracellular polysaccharides in cyanobacteria is keeping cells together in the floating aggregates found in cyanobacterial blooms. These are visible discolorations of the water caused by an overgrowth of cyanobacteria that occur in lakes, estuaries and coastal waters. However, little is known about how these polysaccharides are synthesized in cyanobacteria and what their natural role is. Maeda et al. found a strain of cyanobacteria that formed bloom-like aggregates that were embedded in sulfated extracellular polysaccharides. Using genetic engineering techniques, the researchers identified a set of genes responsible for producing a sulfated extracellular polysaccharide and regulating its levels. They also found that cell aggregates of cyanobacteria can float without having intracellular gas vesicles, which was previously thought to enable blooms to float. The results of the present study could have applications for human health, since many sulfated polysaccharides have antiviral, antitumor or anti-inflammatory properties, and similar genes are found in many cyanobacteria. In addition, these findings could be useful for controlling toxic cyanobacterial blooms, which are becoming increasingly problematic for society.

Minor pilins are involved in motility and natural competence in the cyanobacterium Synechocystis sp. PCC 6803.

Cyanobacteria synthesize type IV pili, which are known to be essential for motility, adhesion and natural competence. They consist of long flexible fibers that are primarily composed of the major pilin PilA1 in Synechocystis sp. PCC 6803. In addition, Synechocystis encodes less abundant pilin-like proteins, which are known as minor pilins. In this study, we show that the minor pilin PilA5 is essential for natural transformation but is dispensable for motility and flocculation. In contrast, a set of minor pilins encoded by the pilA9-slr2019 transcriptional unit are necessary for motility but are dispensable for natural transformation. Neither pilA5-pilA6 nor pilA9-slr2019 are essential for pilus assembly as mutant strains showed type IV pili on the cell surface. Three further gene products with similarity to PilX-like minor pilins have a function in flocculation of Synechocystis. The results of our study indicate that different minor pilins facilitate distinct pilus functions. Further, our microarray analysis demonstrated that the transcription levels of the minor pilin genes change in response to surface contact. A total of 122 genes were determined to have altered transcription between planktonic and surface growth, including several plasmid genes which are involved exopolysaccharide synthesis and the formation of bloom-like aggregates.

Enhancing photosynthesis at high light levels by adaptive laboratory evolution.

Photosynthesis is readily impaired by high light (HL) levels. Photosynthetic organisms have therefore evolved various mechanisms to cope with the problem. Here, we have dramatically enhanced the light tolerance of the cyanobacterium Synechocystis by adaptive laboratory evolution (ALE). By combining repeated mutagenesis and exposure to increasing light intensities, we generated strains that grow under extremely HL intensities. HL tolerance was associated with more than 100 mutations in proteins involved in various cellular functions, including gene expression, photosynthesis and metabolism. Co-evolved mutations were grouped into five haplotypes, and putative epistatic interactions were identified. Two representative mutations, introduced into non-adapted cells, each confer enhanced HL tolerance, but they affect photosynthesis and respiration in different ways. Mutations identified by ALE that allow photosynthetic microorganisms to cope with altered light conditions could be employed in assisted evolution approaches and could strengthen the robustness of photosynthesis in crop plants.

The NAD Kinase Slr0400 Functions as a Growth Repressor in Synechocystis sp. PCC 6803.

NADP+, the phosphorylated form of nicotinamide adenine dinucleotide (NAD), plays an essential role in many cellular processes. NAD kinase (NADK), which is conserved in all living organisms, catalyzes the phosphorylation of NAD+ to NADP+. However, the physiological role of phosphorylation of NAD+ to NADP+ in the cyanobacterium Synechocystis remains unclear. In this study, we report that slr0400, an NADK-encoding gene in Synechocystis, functions as a growth repressor under light-activated heterotrophic growth conditions and light and dark cycle conditions in the presence of glucose. We show, via characterization of NAD(P)(H) content and enzyme activity, that NAD+ accumulation in slr0400-deficient mutant results in the unsuppressed activity of glycolysis and tricarboxylic acid (TCA) cycle enzymes. In determining whether Slr0400 functions as a typical NADK, we found that constitutive expression of slr0400 in an Arabidopsis nadk2-mutant background complements the pale-green phenotype. Moreover, to determine the physiological background behind the growth advantage of mutants lacking slr04000, we investigated the photobleaching phenotype of slr0400-deficient mutant under high-light conditions. Photosynthetic analysis found in the slr0400-deficient mutant resulted from malfunctions in the Photosystem II (PSII) photosynthetic machinery. Overall, our results suggest that NADP(H)/NAD(H) maintenance by slr0400 plays a significant role in modulating glycolysis and the TCA cycle to repress the growth rate and maintain the photosynthetic capacity.

Reconstruction of the absorption spectrum of Synechocystis sp. PCC 6803 optical mutants from the in vivo signature of individual pigments.

In this work, we reconstructed the absorption spectrum of different Synechocystis sp. PCC 6803 optical strains by summing the computed signature of all pigments present in this organism. To do so, modifications to in vitro pigment spectra were first required: namely wavelength shift, curve smoothing, and the package effect calculation derived from high pigment densities were applied. As a result, we outlined a plausible shape for the in vivo absorption spectrum of each chromophore. These are flatter and slightly broader in physiological conditions yet the mean weight-specific absorption coefficient remains identical to the in vitro conditions. Moreover, we give an estimate of all pigment concentrations without applying spectrophotometric correlations, which are often prone to error. The computed cell spectrum reproduces in an accurate manner the experimental spectrum for all the studied wavelengths in the wild-type, Olive, and PAL strain. The gathered pigment concentrations are in agreement with reported values in literature. Moreover, different illumination set-ups were evaluated to calculate the mean absorption cross-section of each chromophore. Finally, a qualitative estimate of light-limited cellular growth at each wavelength is given. This investigation describes a novel way to approach the cell absorption spectrum and shows all its inherent potential for photosynthesis research.

In vivo localization and oligomerization of PixD and PixE for controlling phototaxis in the cyanobacterium Synechocystis sp. PCC 6803.

Phototaxis is a phenomenon where cyanobacteria move toward a light source. Previous studies have shown that the blue-light-using-flavin (BLUF)-type photoreceptor PixD and the response regulator-like protein PixE control the phototaxis in the cyanobacterium Synechocystis sp. PCC 6803. The pixD-null mutant moves away from light, whereas WT, pixE mutant, and pixD pixE double mutant move toward the light. This indicates that PixE functions downstream of PixD and influences the direction of movement. However, it is still unclear how the light signal received by PixD is transmitted to PixE, and then subsequently transmitted to the type IV pili motor mechanism. Here, we investigated intracellular localization and oligomerization of PixD and PixE to elucidate mechanisms of phototaxis regulation. Blue-native PAGE analysis, coupled with western blotting, indicated that most PixD exist as a dimer in soluble fractions, whereas PixE localized in ~250 kDa and ~450 kDa protein complexes in membrane fractions. When blue-native PAGE was performed after illuminating the membrane fractions with blue light, PixE levels in the ~250 kDa and ~450 kDa complexes were reduced and increased, respectively. These results suggest that PixE, localized in the ~450 kDa complex, controls activity of the motor ATPase PilB1 to regulate pilus motility.

Integrated Photonic System for Early Warning of Cyanobacterial Blooms in Aquaponics.

Cyanobacterial blooms produce hazardous toxins, deplete oxygen, and secrete compounds that confer undesirable organoleptic properties to water. To prevent bloom appearance, the World Health Organization has established an alert level between 500 and 2000 cells·mL-1, beyond the capabilities of most optical sensors detecting the cyanobacteria fluorescent pigments. Flow cytometry, cell culturing, and microscopy may reach these detection limits, but they involve both bulky and expensive laboratory equipment or long and tedious protocols. Thus, no current technology allows fast, sensitive, and in situ detection of cyanobacteria. Here, we present a simple, user-friendly, low-cost, and portable photonic system for in situ detection of low cyanobacterial concentrations in water samples. The system integrates high-performance preconcentration elements and optical components for fluorescence measurement of specific cyanobacterial pigments, that is, phycocyanin. Phycocyanin has demonstrated to be more selective to cyanobacteria than other pigments, such as chlorophyll-a, and to present an excellent linear correlation with bacterial concentration from 102 to 104 cell·mL-1 (R2 = 0.99). Additionally, the high performance of the preconcentration system leads to detection limits below 435 cells·mL-1 after 10 min in aquaponic water samples. Due to its simplicity, compactness, and sensitivity, we envision the current technology as a powerful tool for early warning and detection of low pathogen concentrations in water samples.

Hik36-Hik43 and Rre6 act as a two-component regulatory system to control cell aggregation in Synechocystis sp. PCC6803.

In response to environmental stress the model cyanobacterium, Synechocystis sp. PCC6803 can switch from a planktonic state to autoaggregation and biofilm formation. The precise mechanism of this transition remains unknown. Here we investigated the role of a candidate two-component regulatory system (TCS) in controlling morphological changes, as a way to understand the intermediate molecular steps that are part of the signaling pathway. A bacterial two-hybrid assay showed that the response regulator Rre6 formed a TCS together with a split histidine kinase consisting of Hik36 and Hik43. Individual disruption mutants displayed autoaggregation in a static culture. In contrast, unlike in the wild type, high salinity did not induce biofilm formation in Δhik36, Δhik43 and Δrre6. The expression levels of exopolysaccharide (EPS) production genes were higher in Δhik36 and Δhik43, compared with the wild type, but lower in Δrre6, suggesting that the TCS regulated EPS production in Synechocystis. Rre6 interacted physically with the motor protein PilT2, that is a component of the type IV pilus system. This interaction was enhanced in a phosphomimic version of Rre6. Taken together, Hik36-Hik43-Rre6 function as an upstream component of the pili-related signal transduction cascade and control the prevention of cell adhesion and biofilm formation.

The role of transcriptional repressor activity of LexA in salt-stress responses of the cyanobacterium Synechocystis sp. PCC 6803.

Different from typical LexA repressors in heterotrophic bacteria exerting SOS response by auto-cleavage, cyanobacterial LexAs, especially that of Synechocystis sp. PCC 6803 (S.6803), have been suggested be involved in regulation of a number of genes related to various cellular processes, rather than the typical SOS regulon. When and how cyanobacterial LexAs are triggered to regulate its target genes have remained unknown. In this study, we found the profound repressing effect of LexA on salt-stress inducible genes in S.6803. The repressing activity of LexA was likely to persist during salt stress and the salt response of these genes was mainly achieved by other regulators than LexA, suggesting that the physiological role of LexA is fine-tuning of gene expression in response to environmental changes. Although the amount and oligomeric state of LexA were unchanged upon salt stress, two-dimensional electrophoresis and liquid chromatography-tandem mass spectrometry analyses detected a change in posttranslational modification in a small fraction of LexA molecules, possibly dephosphorylation of Ser173, after 30 min upon the upshift in salt concentration. Activity of LexA in S.6803 may be under gradual control by posttranslational modification to fine-tune gene expression, which is contrasted with the digital switching-off regulation by auto-cleavage in heterotrophic bacteria.

Long-Chain Saturated Fatty Acids, Palmitic and Stearic Acids, Enhance the Repair of Photosystem II.

Free fatty acids (FFA) generated in cyanobacterial cells can be utilized for the biodiesel that is required for our sustainable future. The combination of FFA and strong light induces severe photoinhibition of photosystem II (PSII), which suppresses the production of FFA in cyanobacterial cells. In the present study, we examined the effects of exogenously added FFA on the photoinhibition of PSII in Synechocystis sp. PCC 6803. The addition of lauric acid (12:0) to cells accelerated the photoinhibition of PSII by inhibiting the repair of PSII and the de novo synthesis of D1. α-Linolenic acid (18:3) affected both the repair of and photodamage to PSII. Surprisingly, palmitic (16:0) and stearic acids (18:0) enhanced the repair of PSII by accelerating the de novo synthesis of D1 with the mitigation of the photoinhibition of PSII. Our results show chemical potential of FFA in the regulation of PSII without genetic manipulation.

Model quantification of the light-induced thylakoid membrane processes in Synechocystis sp. PCC 6803 in vivo and after exposure to radioactive irradiation.

Measurements of OJIP-SMT patterns of fluorescence induction (FI) in Synechocystis sp. PCC 6803 (Synechocystis) cells on a time scale up to several minutes were mathematically treated within the framework of thylakoid membrane (T-M) model (Belyaeva et al., Photosynth Res 140:1-19, 2019) that was renewed to account for the state transitions effects. Principles of describing electron transfer in reaction centers of photosystems II and I (PSII and PSI) and cytochrome b6f complex remained unchanged, whereas parameters for dissipative reactions of non-radiative charge recombination were altered depending on the oxidation state of QB-site (neutral, reduced by one electron, empty, reduced by two electrons). According to our calculations, the initial content of plastoquinol (PQH2) in the total quinone pool of Synechocystis cells adapted to darkness for 10 min ranged between 20 and 40%. The results imply that the PQ pool mediates photosynthetic and respiratory charge flows. The redistribution of PBS antenna units responsible for the increase of Chl fluorescence in cyanobacteria (qT2 → 1) upon state 2 → 1 transition or the fluorescence lowering (qT1 → 2) due to state 1 → 2 transition were described in the model by exponential functions. Parameters of dynamically changed effective cross section were found by means of simulations of OJIP-SMT patterns observed on Synechocystis cells upon strong (3000 µmol photons m-2s-1) and moderate (1000 µmol photons m-2s-1) actinic light intensities. The corresponding light constant values kLΣAnt = 1.2 ms-1 and 0.4 ms-1 define the excitation of total antenna pool dynamically redistributed between PSII and PSI reaction centers. Although the OCP-induced quenching of antenna excitation is not involved in the model, the main features of the induction signals have been satisfactorily explained. In the case of strong illumination, the effective cross section decreases by approximately 33% for irradiated Synechocystis cells as compared to untreated cells. Under moderate light, the irradiated Synechocystis cells showed in simulations the same cross section as the untreated cells. The thylakoid model renewed with state transitions description allowed simulation of fluorescence induction OJIP-SMT curves detected on time scale from microseconds to minutes.

A multi-parametric screening platform for photosynthetic trait characterization of microalgae and cyanobacteria under inorganic carbon limitation.

Microalgae and cyanobacteria are considered as important model organisms to investigate the biology of photosynthesis; moreover, they are valuable sources of biomolecules for several biotechnological applications. Understanding the species-specific traits of photosynthetic electron transport is extremely important, because it contributes to the regulation of ATP/NADPH ratio, which has direct/indirect links to carbon fixation and other metabolic pathways and thus overall growth and biomass production. In the present work, a cuvette-based setup is developed, in which a combination of measurements of dissolved oxygen, pH, chlorophyll fluorescence and NADPH kinetics can be performed without disturbing the physiological status of the sample. The suitability of the system is demonstrated using a model cyanobacterium Synechocystis sp. PCC6803, as well as biofuel-candidate microalgae species, such as Chlorella sorokiniana, Dunaliella salina and Nannochloropsis limnetica undergoing inorganic carbon (Ci) limitation. Inorganic carbon limitation, induced by photosynthetic Ci uptake under continuous illumination, caused a decrease in the effective quantum yield of PSII (Y(II)) and loss of oxygen-evolving capacity in all species investigated here; these effects were largely recovered by the addition of NaHCO3. Detailed analysis of the dark-light and light-dark transitions of NADPH production/uptake and changes in chlorophyll fluorescence kinetics revealed species- and condition-specific responses. These responses indicate that the impact of decreased Calvin-Benson cycle activity on photosynthetic electron transport pathways involving several sections of the electron transport chain (such as electron transfer via the QA-QB-plastoquinone pool, the redox state of the plastoquinone pool) can be analyzed with high sensitivity in a comparative manner. Therefore, the integrated system presented here can be applied for screening for specific traits in several significant species at different stages of inorganic carbon limitation, a condition that strongly impacts primary productivity.

iTRAQ-based proteomic analysis of the hepatopancreas from Litopenaeus vannamei after trans-vp28 gene Synechocystis sp. PCC6803 immunization.

Litopenaeus vannamei (Pacific white shrimp) is one of the most commercially important varieties of shrimp cultivated in the world. Shrimp farming is a high-risk, capital-intensive industry that is susceptible to periodic outbreaks of diseases caused by viral and bacterial pathogens. Thus, there is a need to develop economically viable methods of disease control. The hepatopancreas of crustaceans are known to have an important role in their innate immune response. In this study, we have explored the immune response of the hepatopancreas from L. vannamei fed with trans-vp28 gene Synechocystis sp. PCC6803 using iTRAQ-based proteomics. A total of 214 differentially expressed proteins (DEPs) were identified, of which 143 were up-regulated and 71 were down-regulated. These proteins have diverse roles in the cell cytoskeleton and cell phagocytosis, antioxidant defense process and the response of immune related proteins. Among these proteins, the immunity associated with the functional annotation of L. vannamei was further analysed. In addition, 4 DEPs (act1, N/A, H and C7M84_013542) were analysed using parallel reaction monitoring (PRM). This is the first report of proteomics in the hepatopancreas of L. vannamei immunized with trans-vp28 gene Synechocystis sp. PCC6803.

Phosphoenolpyruvate carboxylase from the cyanobacterium Synechocystis sp. PCC 6803 is under global metabolic control by PII signaling.

Phosphoenolpyruvate carboxylase (PEPC) is the second major carbon-fixing enzyme in photoautotrophic organisms. PEPC is required for the synthesis of amino acids of the glutamate and aspartate family by replenishing the TCA cycle. Furthermore, in cyanobacteria, PEPC, together with malate dehydrogenase and malic enzyme, forms a metabolic shunt for the synthesis of pyruvate from PEP. During this process, CO2 is first fixed and later released again. Due to its central metabolic position, it is crucial to fully understand the regulation of PEPC. Here, we identify PEPC from the cyanobacterium Synechocystis sp. PCC 6803 (PEPC) as a novel interaction partner for the global signal transduction protein PII . In addition to an extensive characterization of PEPC, we demonstrate specific PII -PEPC complex formation and its enzymatic consequences. PEPC activity is tuned by the metabolite-sensing properties of PII : Whereas in the absence of PII, PEPC is subjected to ATP inhibition, it is activated beyond its basal activity in the presence of PII . Furthermore, PII -PEPC complex formation is inhibited by ADP and PEPC activation by PII -ATP is mitigated in the presence of 2-OG, linking PEPC regulation to the cell's global carbon/nitrogen status. Finally, physiological relevance of the in vitro measurements was proven by metabolomic analyses of Synechocystis wild-type and PII -deficient cells.

Transcriptome Analysis Reveals IsiA-Regulatory Mechanisms Underlying Iron Depletion and Oxidative-Stress Acclimation in Synechocystis sp. Strain PCC 6803.

Microorganisms in nature are commonly exposed to various stresses in parallel. The isiA gene encodes an iron stress-induced chlorophyll-binding protein which is significantly induced under iron starvation and oxidative stress. Acclimation of oxidative stress and iron deficiency was investigated using a regulatory mutant of the Synechocystis sp. strain PCC 6803. In this study, the ΔisiA mutant grew more slowly in oxidative-stress and iron depletion conditions compared to the wild-type (WT) counterpart under the same conditions. Thus, we performed transcriptome sequencing (RNA-seq) analysis of the WT strain and the ΔisiA mutant under double-stress conditions to obtain a comprehensive view of isiA-regulated genes. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses showed significant differences between the WT strain and ΔisiA mutant, mainly related to photosynthesis and the iron-sulfur cluster. The deletion of isiA affects the expression of various genes that are involved in cellular processes and structures, such as photosynthesis, phycobilisome, and the proton-transporting ATPase complex. Weighted gene coexpression network analysis (WGCNA) demonstrated three functional modules in which the turquoise module was negatively correlated with superoxide dismutase (SOD) activity. Coexpression network analysis identified several hub genes of each module. Cotranscriptional PCR and reads coverage using the Integrative Genomics Viewer demonstrated that isiA, isiB, isiC, ssl0461, and dfp belonged to the isi operon. Three sRNAs related to oxidative stress were identified. This study enriches our knowledge of IsiA-regulatory mechanisms under iron deficiency and oxidative stress.IMPORTANCE This study analyzed the impact of isiA deletion on the transcriptomic profile of Synechocystis The isiA gene encodes an iron stress-induced chlorophyll-binding protein, which is significantly induced under iron starvation. The deletion of isiA affects the expression of various genes that are involved in photosynthesis and ABC transporters. WGCNA revealed three functional modules in which the blue module was correlated with oxidative stress. We further demonstrated that the isi operon contained the following five genes: isiA, isiB, isiC, ssl0461, and dfp by cotranscriptional PCR. Three sRNAs were identified that were related to oxidative stress. This study enhances our knowledge of IsiA-regulatory mechanisms under iron deficiency and oxidative stress.