Minimized dark consumption of Calvin cycle intermediates facilitates the initiation of photosynthesis in Synechocystis sp. PCC 6803.

Cyanobacteria intricately regulate their metabolic pathways during the diurnal cycle to ensure survival and growth. Under dark conditions, the breakdown of glycogen, an energy reserve, in these organisms replenishes Calvin cycle intermediates, especially downstream glycolytic metabolites, which are necessary for photosynthesis initiation upon light irradiation. However, it remains unclear how the accumulation of these intermediates is maintained in the dark despite limited glycogen availability. Therefore, in this study, we investigated the regulation of downstream glycolytic metabolites of the Calvin cycle under dark and light treatment using Synechocystis sp. PCC 6803. Our results showed that during the dark period, low pyruvate kinase (Pyk) activity ensured metabolite accumulation, while endogenous Pyk overexpression significantly lowered the accumulation of glycolytic intermediates. Remarkably, wild type Synechocystis maintained oxygen evolution ability throughout dark treatment for over 2 d, while Pyk overexpression resulted in decreased oxygen evolution after 16 h of dark treatment. These results indicated that limiting Pyk activity via darkness treatment facilitates photosynthetic initiation by maintaining glycolytic intermediates. Similarly, phosphoenolpyruvate carboxylase (PepC) overexpression decreased oxygen evolution under dark treatment; however, its effect was lower than that of Pyk. Further, we noted that as PepC overexpression decreased the levels of glycolytic intermediates in the dark, sugar phosphates in the Calvin-Benson-Bassham (CBB) cycle showed high accumulation, suggesting that sugar phosphates play important roles in supporting photosynthesis initiation. Therefore, our study highlights the importance of controlling the metabolic pathways through which glycolytic and CBB cycle intermediates are consumed (defined as cataplerosis of CBB cycle) to ensure stable photosynthesis.

Dark accumulation of downstream glycolytic intermediates initiates robust photosynthesis in cyanobacteria.

Photosynthesis must maintain stability and robustness throughout fluctuating natural environments. In cyanobacteria, dark-to-light transition leads to drastic metabolic changes from dark respiratory metabolism to CO2 fixation through the Calvin-Benson-Bassham (CBB) cycle using energy and redox equivalents provided by photosynthetic electron transfer. Previous studies have shown that catabolic metabolism supports the smooth transition into CBB cycle metabolism. However, metabolic mechanisms for robust initiation of photosynthesis are poorly understood due to lack of dynamic metabolic characterizations of dark-to-light transitions. Here, we show rapid dynamic changes (on a time scale of seconds) in absolute metabolite concentrations and 13C tracer incorporation after strong or weak light irradiation in the cyanobacterium Synechocystis sp. PCC 6803. Integration of this data enabled estimation of time-resolved nonstationary metabolic flux underlying CBB cycle activation. This dynamic metabolic analysis indicated that downstream glycolytic intermediates, including phosphoglycerate and phosphoenolpyruvate, accumulate under dark conditions as major substrates for initial CO2 fixation. Compared with wild-type Synechocystis, significant decreases in the initial oxygen evolution rate were observed in 12 h dark preincubated mutants deficient in glycogen degradation or oxidative pentose phosphate pathways. Accordingly, the degree of decrease in the initial oxygen evolution rate was proportional to the accumulated pool size of glycolytic intermediates. These observations indicate that the accumulation of glycolytic intermediates is essential for efficient metabolism switching under fluctuating light environments.

Conserved Two-component Hik2-Rre1 Signaling Is Activated Under Temperature Upshift and Plastoquinone-reducing Conditions in the Cyanobacterium Synechococcus elongatus PCC 7942.

The highly conserved Hik2-Rre1 two-component system is a multi-stress responsive signal-transducing module that controls the expression of hsp and other genes in cyanobacteria. Previously, we found in Synechococcus elongatus PCC 7942 that the heat-inducible phosphorylation of Rre1 was alleviated in a hik34 mutant, suggesting that Hik34 positively regulates signaling. In this study, we examined the growth of the hik34 deletion mutant in detail, and newly identified suppressor mutations located in rre1 or sasA gene negating the phenotype. Subsequent analyses indicated that heat-inducible Rre1 phosphorylation is dependent on Hik2 and that Hik34 modulates this Hik2-dependent response. In the following part of this study, we focused on the mechanism to control the Hik2 activity. Other recent studies reported that Hik2 activity is regulated by the redox status of plastoquinone (PQ) through the 3Fe-4S cluster attached to the cyclic GMP, adenylyl cyclase, FhlA (GAF) domain. Consistent with this, Rre1 phosphorylation occurred after the addition of 2,5-dibromo-6-isopropyl-3-methyl-1,4-benzoquinone but not after the addition of 3-(3,4-dichlorophenyl)-1,1-dimethylurea to the culture medium, which corresponded to PQ-reducing or -oxidizing conditions, respectively, suggesting that the Hik2-to-Rre1 phosphotransfer was activated under PQ-reducing conditions. However, there was no correlation between the measured PQ redox status and Rre1 phosphorylation during the temperature upshift. Therefore, changes in the PQ redox status are not the direct reason for the heat-inducible Rre1 phosphorylation, while some redox regulation is likely involved as oxidation events dependent on 2,6-dichloro-1,4-benzoquinone prevented heat-inducible Rre1 phosphorylation. On the basis of these results, we propose a model for the control of Hik2-dependent Rre1 phosphorylation.

Coral symbionts evolved a functional polycistronic flavodiiron gene.

Photosynthesis in cyanobacteria, green algae, and basal land plants is protected against excess reducing pressure on the photosynthetic chain by flavodiiron proteins (FLV) that dissipate photosynthetic electrons by reducing O2. In these organisms, the genes encoding FLV are always conserved in the form of a pair of two-type isozymes (FLVA and FLVB) that are believed to function in O2 photo-reduction as a heterodimer. While coral symbionts (dinoflagellates of the family Symbiodiniaceae) are the only algae to harbor FLV in photosynthetic red plastid lineage, only one gene is found in transcriptomes and its role and activity remain unknown. Here, we characterized the FLV genes in Symbiodiniaceae and found that its coding region is composed of tandemly repeated FLV sequences. By measuring the O2-dependent electron flow and P700 oxidation, we suggest that this atypical FLV is active in vivo. Based on the amino-acid sequence alignment and the phylogenetic analysis, we conclude that in coral symbionts, the gene pair for FLVA and FLVB have been fused to construct one coding region for a hybrid enzyme, which presumably occurred when or after both genes were inherited from basal green algae to the dinoflagellate. Immunodetection suggested the FLV polypeptide to be cleaved by a post-translational mechanism, adding it to the rare cases of polycistronic genes in eukaryotes. Our results demonstrate that FLV are active in coral symbionts with genomic arrangement that is unique to these species. The implication of these unique features on their symbiotic living environment is discussed.

NADPH production in dark stages is critical for cyanobacterial photocurrent generation: a study using mutants deficient in oxidative pentose phosphate pathway.

Live cyanobacteria and algae integrated onto an extracellular electrode can generate a light-induced current (i.e., a photocurrent). Although the photocurrent is expected to be correlated with the redox environment of the photosynthetic cells, the relationship between the photocurrent and the cellular redox state is poorly understood. Here, we investigated the effect of the reduced nicotinamide adenine dinucleotide phosphate [NADP(H)] redox level of cyanobacterial cells (before light exposure) on the photocurrent using several mutants (Δzwf, Δgnd, and ΔglgP) deficient in the oxidative pentose phosphate (OPP) pathway, which is the metabolic pathway that produces NADPH in darkness. The NAD(P)H redox level and photocurrent in the cyanobacterium Synechocystis sp. PCC 6803 were measured noninvasively. Dysfunction of the OPP pathway led to oxidation of the photosynthetic NADPH pool in darkness. In addition, photocurrent induction was retarded and the current density was lower in Δzwf, Δgnd, and ΔglgP than in wild-type cells. Exogenously added glucose compensated the phenotype of ΔglgP and drove the OPP pathway in the mutant, resulting in an increase in the photocurrent. The results indicated that NADPH accumulated by the OPP pathway before illumination is a key factor for the generation of a photocurrent. In addition, measuring the photocurrent can be a non-invasive approach to estimate the cellular redox level related to NADP(H) pool in cyanobacteria.

Quantification of NAD(P)H in cyanobacterial cells by a phenol extraction method.

In photosynthetic organisms, it is recognized that the intracellular redox ratio of NADPH is regulated within an appropriate range for the cooperative function of a wide variety of physiological processes. However, despite its importance, there is large variability in the values of the NADPH fraction [NADPH/(NADPH + NADP+)] quantitatively estimated to date. In the present study, the light response of the NADPH fraction was investigated by applying a novel NADP(H) extraction method using phenol / chloroform / isoamyl alcohol (PCI) in the cyanobacterium Synechocystis sp. PCC 6803. The light response of NADP(H) observed using PCI extraction was qualitatively consistent with the NAD(P)H fluorescence time course measured in vivo. Moreover, the results obtained by PCI extraction and the fluorescence-based methods were also consistent in a mutant lacking the ability to oxidize NAD(P)H in the respiratory chain, and exhibiting a unique NADPH light response. These observations indicate that the PCI extraction method allowed quantitative determination of NADP(H) redox. Notably, the PCI extraction method showed that not all NADP(H) was oxidized or reduced by light-dark transition. Specifically, the fraction of NADPH was 42% in the dark-adapted cell, and saturated at 68% in light conditions.

Mediator-Microorganism Interaction in Microbial Solar Cell: a Fluo-Electrochemical Insight.

Microbial solar cells that mainly rely on the use of photosynthesic organisms are a promising alternative to photovoltaics for solar electricity production. In that way, we propose a new approach involving electrochemistry and fluorescence techniques. The coupled setup Electro-Pulse-Amplitude-Modulation ("e-PAM") enables the simultaneous recording of the produced photocurrent and fluorescence signals from the photosynthetic chain. This methodology was validated with a suspension of green alga Chlamydomonas reinhardtii in interaction with an exogenous redox mediator (2,6-dichlorobenzoquinone; DCBQ). The balance between photosynthetic chain events (PSII photochemical yield, quenching) and the extracted electricity can be monitored overtime. More particularly, the nonphotochemical quenching induced by DCBQ mirrors the photocurrent. This setup thus helps to distinguish the electron harvesting from some side effects due to quinones in real time. It therefore paves the way for future analyses devoted to the choice of the experimental conditions (redox mediator, photosynthetic organisms, and so on) to find the best electron extraction.

The endogenous redox rhythm is controlled by a central circadian oscillator in cyanobacterium Synechococcus elongatus PCC7942.

The intracellular redox and the circadian clock in photosynthetic organisms are two major regulators globally affecting various biological functions. Both of the global control systems have evolved as systems to adapt to regularly or irregularly changing light environments. Here, we report that the two global regulators mutually interact in cyanobacterium Synechococcus elongatus PCC7942, a model photosynthetic organism whose clock molecular mechanism is well known. Electrochemical assay using a transmembrane electron mediator revealed that intracellular redox of S. elongatus PCC7942 cell exhibited circadian rhythms under constant light conditions. The redox rhythm disappeared when transcription/translation of clock genes is defunctionalized, indicating that the transcription/translation controlled by a core KaiABC oscillator generates the circadian redox rhythm. Importantly, the amplitude of the redox rhythm at a constant light condition was large enough to affect the KaiABC oscillator. The findings indicated that the intracellular redox state is actively controlled to change in a 24-h cycle under constant light conditions by the circadian clock system.

Specific Interaction between Redox Phospholipid Polymers and Plastoquinone in Photosynthetic Electron Transport Chain.

Redox phospholipid polymers added in culture media are known to be capable of extracting electrons from living photosynthetic cells across bacterial cell membranes with high cytocompatibility. In the present study, we identify the intracellular redox species that transfers electrons to the polymers. The open-circuit electrochemical potential of an electrolyte containing the redox polymer and extracted thylakoid membranes shift to positive (or negative) under light irradiation, when an electron transport inhibitor specific to plastoquinone is added upstream (or downstream) in the photosynthetic electron transport chain. The same trend is also observed for a medium containing living photosynthetic cells of Synechococcus elongatus PCC7942. These results clearly indicate that the phospholipid redox polymers extract photosynthetic electrons mainly from plastoquinone.

Metabolomics-based development of bioproduction processes toward industrial-scale production.

Microbial biomanufacturing offers a promising, environment-friendly platform for next-generation chemical production. However, its limited industrial implementation is attributed to the slow production rates of target compounds and the time-intensive engineering of high-yield strains. This review highlights how metabolomics expedites bioproduction development, as demonstrated through case studies of its integration into microbial strain engineering, culture optimization, and model construction. The Design-Build-Test-Learn (DBTL) cycle serves as a standard workflow for strain engineering. Process development, including the optimization of culture conditions and scale-up, is crucial for industrial production. In silico models facilitate the development of strains and processes. Metabolomics is a powerful driver of the DBTL framework, process development, and model construction.

Recent research advances on non-linear phenomena in various biosystems.

All biological phenomena can be classified as open, dissipative and non-linear. Moreover, the most typical phenomena are associated with non-linearity, dissipation and openness in biological systems. In this review article, four research topics on non-linear biosystems are described to show the examples from various biological systems. First, membrane dynamics of a lipid bilayer for the cell membrane is described. Since the cell membrane separates the inside of the cell from the outside, self-organizing systems that form spatial patterns on membranes often depend on non-linear dynamics. Second, various data banks based on recent genomics analysis supply the data including vast functional proteins from many organisms and their variable species. Since the proteins existing in nature are only a very small part of the space represented by amino acid sequence, success of mutagenesis-based molecular evolution approach crucially depends on preparing a library with high enrichment of functional proteins. Third, photosynthetic organisms depend on ambient light, the regular and irregular changes of which have a significant impact on photosynthetic processes. The light-driven process proceeds through many redox couples in the cyanobacteria constituting chain of redox reactions. The fourth topic focuses on a vertebrate model, the zebrafish, which can help to understand, predict and control the chaos of complex biological systems. In particular, during early developmental stages, developmental differentiation occurs dynamically from a fertilized egg to divided and mature cells. These exciting fields of complexity, chaos, and non-linear science have experienced impressive growth in recent decades. Finally, future directions for non-linear biosystems are presented.

Order-of-magnitude enhancement in photocurrent generation of Synechocystis sp. PCC 6803 by outer membrane deprivation.

Biophotovoltaics (BPV) generates electricity from reducing equivalent(s) produced by photosynthetic organisms by exploiting a phenomenon called extracellular electron transfer (EET), where reducing equivalent(s) is transferred to external electron acceptors. Although cyanobacteria have been extensively studied for BPV because of their high photosynthetic activity and ease of handling, their low EET activity poses a limitation. Here, we show an order-of-magnitude enhancement in photocurrent generation of the cyanobacterium Synechocystis sp. PCC 6803 by deprivation of the outer membrane, where electrons are suggested to stem from pathway(s) downstream of photosystem I. A marked enhancement of EET activity itself is verified by rapid reduction of exogenous electron acceptor, ferricyanide. The extracellular organic substances, including reducing equivalent(s), produced by this cyanobacterium serve as respiratory substrates for other heterotrophic bacteria. These findings demonstrate that the outer membrane is a barrier that limits EET. Therefore, depriving this membrane is an effective approach to exploit the cyanobacterial reducing equivalent(s).

Ferrihydrite Reduction by Photosynthetic Synechocystis sp. PCC 6803 and Its Correlation With Electricity Generation.

Microbial extracellular electron transfer (EET) to solid-state electron acceptors such as anodes and metal oxides, which was originally identified in dissimilatory metal-reducing bacteria, is a key process in microbial electricity generation and the biogeochemical cycling of metals. Although it is now known that photosynthetic microorganisms can also generate (photo)currents via EET, which has attracted much interest in the field of biophotovoltaics, little is known about the reduction of metal (hydr)oxides via photosynthetic microbial EET. The present work quantitatively assessed the reduction of ferrihydrite in conjunction with the EET of the photosynthetic microbe Synechocystis sp. PCC 6803. Microbial reduction of ferrihydrite was found to be initiated in response to light but proceeded at higher rates when exogenous glucose was added, even under dark conditions. These results indicate that current generation from Synechocystis cells does not always need light irradiation. The qualitative trends exhibited by the ferrihydrite reduction rates under various conditions showed significant correlation with those of the microbial currents. Notably, the maximum concentration of Fe(II) generated by the cyanobacterial cells under dark conditions in the presence of glucose was comparable to the levels observed in the photic layers of Fe-rich microbial mats.

Time-of-day-dependent responses of cyanobacterial cellular viability against oxidative stress.

As an adaptation to periodic fluctuations of environmental light, photosynthetic organisms have evolved a circadian clock. Control by the circadian clock of many cellular physiological functions, including antioxidant enzymes, metabolism and the cell cycle, has attracted attention in the context of oxidative stress tolerance. However, since each physiological function works in an integrated manner to deal with oxidative stress, whether or not cell responses to oxidative stress are under circadian control remains an open question. In fact, circadian rhythms of oxidative stress tolerance have not yet been experimentally demonstrated. In the present work, we applied an assay using methyl viologen (MV), which generates reactive oxygen species (ROS) under light irradiation, and experimentally verified the circadian rhythms of oxidative stress tolerance in photosynthetic cells of the cyanobacterium Synechococcus elongatus PCC 7942, a standard model species for investigation of the circadian clock. Here, we report that ROS generated by MV treatment causes damage to stroma components and not to the photosynthetic electron transportation chain, leading to reduced cell viability. The degree of decrease in cell viability was dependent on the subjective time at which oxidative stress was applied. Thus, oxidative stress tolerance was shown to exhibit circadian rhythms. In addition, the rhythmic pattern of oxidative stress tolerance disappeared in mutant cells lacking the essential clock genes. Notably, ROS levels changed periodically, independent of the MV treatment. Thus, we demonstrate for the first time that in cyanobacterial cells, oxidative stress tolerance shows circadian oscillation.

Extracellular electron transfer mediated by a cytocompatible redox polymer to study the crosstalk among the mammalian circadian clock, cellular metabolism, and cellular redox state.

The circadian clock is an endogenous biological timekeeping system that controls various physiological and cellular processes with a 24 h rhythm. The crosstalk among the circadian clock, cellular metabolism, and cellular redox state has attracted much attention. To elucidate this crosstalk, chemical compounds have been used to perturb cellular metabolism and the redox state. However, an electron mediator that facilitates extracellular electron transfer (EET) has not been used to study the mammalian circadian clock due to potential cytotoxic effects of the mediator. Here, we report evidence that a cytocompatible redox polymer pMFc (2-methacryloyloxyethyl phosphorylcholine-co-vinyl ferrocene) can be used as the mediator to study the mammalian circadian clock. EET mediated by oxidized pMFc (ox-pMFc) extracted intracellular electrons from human U2OS cells, resulting in a longer circadian period. Analyses of the metabolome and intracellular redox species imply that ox-pMFc receives an electron from glutathione, thereby inducing pentose phosphate pathway activation. These results suggest novel crosstalk among the circadian clock, metabolism, and redox state. We anticipate that EET mediated by a redox cytocompatible polymer will provide new insights into the mammalian circadian clock system, which may lead to the development of new treatments for circadian clock disorders.

Machine-Learning Analysis to Predict the Exciton Valley Polarization Landscape of 2D Semiconductors.

We demonstrate the applicability of employing machine-learning-based analysis to predict the low-temperature exciton valley polarization landscape of monolayer tungsten diselenide (1L-WSe2) using position-dependent information extracted from its photoluminescence (PL) spectra at room temperature. We performed low- and room-temperature polarization-resolved PL mapping and used the obtained experimental data to create regression models for the prediction using the Random Forest machine-learning algorithm. The local information extracted from the room-temperature PL spectra and the low-temperature exciton valley polarization was used as the input and output data for the machine-learning process, respectively. The spatial distribution of the exciton valley polarization in a 1L-WSe2 sample that was not used for the learning of the decision trees was successfully predicted. Furthermore, we numerically obtained the degree of importance for each input variable and demonstrated that this parameter provides helpful information for examining the physics that shape the spatially heterogeneous valley polarization landscape of 1L-WSe2.

Planar Perovskite Solar Cells with High Efficiency and Fill Factor Obtained Using Two-Step Growth Process.

Hybrid organic-inorganic perovskite solar cells (PSCs) have been regarded as the most promising next-generation photovoltaics (PVs) because of their potential for low-cost fabrication and advances in their development. Superior quality of the photoactive perovskite layer is a main factor for further increasing the PV performance of the organic-inorganic perovskite solar cells (PSCs). Herein, we successfully obtained perovskite with a high crystallinity and large grain size by utilizing excess PbI2 and SSGP technique and demonstrated a superior PV performance of normal-architecture planar PSCs. The SSGP PSCs with the highest fill factor (FF) reported thus far (83.4%) to our knowledge were obtained without sacrificing other PV parameters. Moreover, a high efficiency of 21.3% (21.6%) with a high FF of 80.0% (81.2%) in forward (reverse) scan was achieved. The unencapsulated SSGP PSCs showed robust continuous light-soaking and thermal stability under harsh characterization conditions. Additionally, we achieved a high efficiency of 20.1% with a negligible hysteresis on the large active area SSGP PSCs (∼1 cm2). The optical properties, efficient carrier extraction, and reduction of recombination loss of the SSGP perovskite significantly contribute to the high PV performance and robust stability of SSGP PSCs.

Extracellular Electron Transfer via Outer Membrane Cytochromes in a Methanotrophic Bacterium Methylococcus capsulatus (Bath).

Electron exchange reactions between microbial cells and solid materials, referred to as extracellular electron transfer (EET), have attracted attention in the fields of microbial physiology, microbial ecology, and biotechnology. Studies of model species of iron-reducing, or equivalently, current-generating bacteria such as Geobacter spp. and Shewanella spp. have revealed that redox-active proteins, especially outer membrane c-type cytochromes (OMCs), play a pivotal role in the EET process. Recent (meta)genomic analyses have revealed that diverse microorganisms that have not been demonstrated to have EET ability also harbor OMC-like proteins, indicating that EET via OMCs could be more widely preserved in microorganisms than originally thought. A methanotrophic bacterium Methylococcus capsulatus (Bath) was reported to harbor multiple OMC genes whose expression is elevated by Cu starvation. However, the physiological role of these genes is unknown. Therefore, in this study, we explored whether M. capsulatus (Bath) displays EET abilities via OMCs. In electrochemical analysis, M. capsulatus (Bath) generated anodic current only when electron donors such as formate were available, and could reduce insoluble iron oxides in the presence of electron donor compounds. Furthermore, the current-generating and iron-reducing activities of M. capsulatus (Bath) cells that were cultured in a Cu-deficient medium, which promotes high levels of OMC expression, were higher than those cultured in a Cu-supplemented medium. Anodic current production by the Cu-deficient cells was significantly suppressed by disruption of MCA0421, a highly expressed OMC gene, and by treatment with carbon monoxide (CO) gas (an inhibitor of c-type cytochromes). Our results provide evidence of EET in M. capsulatus (Bath) and demonstrate the pivotal role of OMCs in this process. This study raises the possibility that EET to solid compounds is a novel survival strategy of methanotrophic bacteria.

Real-time monitoring of intracellular redox changes in Methylococcus capsulatus (Bath) for efficient bioconversion of methane to methanol.

This study aimed to develop a novel method for real-time monitoring of the intracellular redox states in a methanotroph Methylococcus capsulatus, using Peredox as a genetically encoded fluorescent sensor of the NADH:NAD+ ratio. As expected, the fluorescence derived from the Peredox-expressing M. capsulatus transformant increased by supplementation of electron donor compounds (methane and formate), while it decreased by specifically inhibiting the methanol oxidation reaction. Electrochemical measurements confirmed that the Peredox fluorescence reliably represents the intracellular redox changes. This study is the first to construct a reliable redox-monitoring method for methanotrophs, which will facilitate to develop more efficient methane-to-methanol bioconversion processes.

Draft genome sequences of three Holospora species (Holospora obtusa, Holospora undulata, and Holospora elegans), endonuclear symbiotic bacteria of the ciliate Paramecium caudatum.

We present draft genome sequences of three Holospora species, hosted by the ciliate Paramecium caudatum; that is, the macronucleus-specific H. obtusa and the micronucleus-specific H. undulata and H. elegans. We investigate functions of orthologous core genes conserved across the three Holospora species, which may be essential for the infection and survival in the host nucleus.