Evolution of Thylakoid Structural Diversity.

Oxygenic photosynthesis evolved billions of years ago, becoming Earth's main source of biologically available carbon and atmospheric oxygen. Since then, phototrophic organisms have diversified from prokaryotic cyanobacteria into several distinct clades of eukaryotic algae and plants through endosymbiosis events. This diversity can be seen in the thylakoid membranes, complex networks of lipids, proteins, and pigments that perform the light-dependent reactions of photosynthesis. In this review, we highlight the structural diversity of thylakoids, following the evolutionary history of phototrophic species. We begin with a molecular inventory of different thylakoid components and then illustrate how these building blocks are integrated to form membrane networks with diverse architectures. We conclude with an outlook on understanding how thylakoids remodel their architecture and molecular organization during dynamic processes such as biogenesis, repair, and environmental adaptation.

Dual Functions of a Rubisco Activase in Metabolic Repair and Recruitment to Carboxysomes.

Rubisco, the key enzyme of CO2 fixation in photosynthesis, is prone to inactivation by inhibitory sugar phosphates. Inhibited Rubisco undergoes conformational repair by the hexameric AAA+ chaperone Rubisco activase (Rca) in a process that is not well understood. Here, we performed a structural and mechanistic analysis of cyanobacterial Rca, a close homolog of plant Rca. In the Rca:Rubisco complex, Rca is positioned over the Rubisco catalytic site under repair and pulls the N-terminal tail of the large Rubisco subunit (RbcL) into the hexamer pore. Simultaneous displacement of the C terminus of the adjacent RbcL opens the catalytic site for inhibitor release. An alternative interaction of Rca with Rubisco is mediated by C-terminal domains that resemble the small Rubisco subunit. These domains, together with the N-terminal AAA+ hexamer, ensure that Rca is packaged with Rubisco into carboxysomes. The cyanobacterial Rca is a dual-purpose protein with functions in Rubisco repair and carboxysome organization.

Structure and Function of a Bacterial Gap Junction Analog.

Multicellular lifestyle requires cell-cell connections. In multicellular cyanobacteria, septal junctions enable molecular exchange between sister cells and are required for cellular differentiation. The structure of septal junctions is poorly understood, and it is unknown whether they are capable of controlling intercellular communication. Here, we resolved the in situ architecture of septal junctions by electron cryotomography of cryo-focused ion beam-milled cyanobacterial filaments. Septal junctions consisted of a tube traversing the septal peptidoglycan. Each tube end comprised a FraD-containing plug, which was covered by a cytoplasmic cap. Fluorescence recovery after photobleaching showed that intercellular communication was blocked upon stress. Gating was accompanied by a reversible conformational change of the septal junction cap. We provide the mechanistic framework for a cell junction that predates eukaryotic gap junctions by a billion years. The conservation of a gated dynamic mechanism across different domains of life emphasizes the importance of controlling molecular exchange in multicellular organisms.

Structure and energy transfer in photosystems of oxygenic photosynthesis.

Oxygenic photosynthesis is the principal converter of sunlight into chemical energy on Earth. Cyanobacteria and plants provide the oxygen, food, fuel, fibers, and platform chemicals for life on Earth. The conversion of solar energy into chemical energy is catalyzed by two multisubunit membrane protein complexes, photosystem I (PSI) and photosystem II (PSII). Light is absorbed by the pigment cofactors, and excitation energy is transferred among the antennae pigments and converted into chemical energy at very high efficiency. Oxygenic photosynthesis has existed for more than three billion years, during which its molecular machinery was perfected to minimize wasteful reactions. Light excitation transfer and singlet trapping won over fluorescence, radiation-less decay, and triplet formation. Photosynthetic reaction centers operate in organisms ranging from bacteria to higher plants. They are all evolutionarily linked. The crystal structure determination of photosynthetic protein complexes sheds light on the various partial reactions and explains how they are protected against wasteful pathways and why their function is robust. This review discusses the efficiency of photosynthetic solar energy conversion.

ATP synthase.

Oxygenic photosynthesis is the principal converter of sunlight into chemical energy. Cyanobacteria and plants provide aerobic life with oxygen, food, fuel, fibers, and platform chemicals. Four multisubunit membrane proteins are involved: photosystem I (PSI), photosystem II (PSII), cytochrome b6f (cyt b6f), and ATP synthase (FOF1). ATP synthase is likewise a key enzyme of cell respiration. Over three billion years, the basic machinery of oxygenic photosynthesis and respiration has been perfected to minimize wasteful reactions. The proton-driven ATP synthase is embedded in a proton tight-coupling membrane. It is composed of two rotary motors/generators, FO and F1, which do not slip against each other. The proton-driven FO and the ATP-synthesizing F1 are coupled via elastic torque transmission. Elastic transmission decouples the two motors in kinetic detail but keeps them perfectly coupled in thermodynamic equilibrium and (time-averaged) under steady turnover. Elastic transmission enables operation with different gear ratios in different organisms.

Metabolic compensation and circadian resilience in prokaryotic cyanobacteria.

For a biological oscillator to function as a circadian pacemaker that confers a fitness advantage, its timing functions must be stable in response to environmental and metabolic fluctuations. One such stability enhancer, temperature compensation, has long been a defining characteristic of these timekeepers. However, an accurate biological timekeeper must also resist changes in metabolism, and this review suggests that temperature compensation is actually a subset of a larger phenomenon, namely metabolic compensation, which maintains the frequency of circadian oscillators in response to a host of factors that impinge on metabolism and would otherwise destabilize these clocks. The circadian system of prokaryotic cyanobacteria is an illustrative model because it is composed of transcriptional and nontranscriptional oscillators that are coupled to promote resilience. Moreover, the cyanobacterial circadian program regulates gene activity and metabolic pathways, and it can be manipulated to improve the expression of bioproducts that have practical value.

The inner workings of an ancient biological clock.

Circadian clocks evolved in diverse organisms as an adaptation to the daily swings in ambient light and temperature that derive from Earth's rotation. These timing systems, based on intracellular molecular oscillations, synchronize organisms' behavior and physiology with the 24-h environmental rhythm. The cyanobacterial clock serves as a special model for understanding circadian rhythms because it can be fully reconstituted in vitro. This review summarizes recent advances that leverage new biochemical, biophysical, and mathematical approaches to shed light on the molecular mechanisms of cyanobacterial Kai proteins that support the clock, and their homologues in other bacteria. Many questions remain in circadian biology, and the tools developed for the Kai system will bring us closer to the answers.

Orange carotenoid proteins: structural understanding of evolution and function.

Cyanobacteria uniquely contain a primitive water-soluble carotenoprotein, the orange carotenoid protein (OCP). Nearly all extant cyanobacterial genomes contain genes for the OCP or its homologs, implying an evolutionary constraint for cyanobacteria to conserve its function. Genes encoding the OCP and its two constituent structural domains, the N-terminal domain, helical carotenoid proteins (HCPs), and its C-terminal domain, are found in the most basal lineages of extant cyanobacteria. These three carotenoproteins exemplify the importance of the protein for carotenoid properties, including protein dynamics, in response to environmental changes in facilitating a photoresponse and energy quenching. Here, we review new structural insights for these carotenoproteins and situate the role of the protein in what is currently understood about their functions.

A systematic exploration of bacterial form I rubisco maximal carboxylation rates.

Autotrophy is the basis for complex life on Earth. Central to this process is rubisco-the enzyme that catalyzes almost all carbon fixation on the planet. Yet, with only a small fraction of rubisco diversity kinetically characterized so far, the underlying biological factors driving the evolution of fast rubiscos in nature remain unclear. We conducted a high-throughput kinetic characterization of over 100 bacterial form I rubiscos, the most ubiquitous group of rubisco sequences in nature, to uncover the determinants of rubisco's carboxylation velocity. We show that the presence of a carboxysome CO2 concentrating mechanism correlates with faster rubiscos with a median fivefold higher rate. In contrast to prior studies, we find that rubiscos originating from α-cyanobacteria exhibit the highest carboxylation rates among form I enzymes (≈10 s-1 median versus <7 s-1 in other groups). Our study systematically reveals biological and environmental properties associated with kinetic variation across rubiscos from nature.

The Microbiology of Biological Soil Crusts.

Biological soil crusts are thin, inconspicuous communities along the soil atmosphere ecotone that, until recently, were unrecognized by ecologists and even more so by microbiologists. In its broadest meaning, the term biological soil crust (or biocrust) encompasses a variety of communities that develop on soil surfaces and are powered by photosynthetic primary producers other than higher plants: cyanobacteria, microalgae, and cryptogams like lichens and mosses. Arid land biocrusts are the most studied, but biocrusts also exist in other settings where plant development is constrained. The minimal requirement is that light impinge directly on the soil; this is impeded by the accumulation of plant litter where plants abound. Since scientists started paying attention, much has been learned about their microbial communities, their composition, ecological extent, and biogeochemical roles, about how they alter the physical behavior of soils, and even how they inform an understanding of early life on land. This has opened new avenues for ecological restoration and agriculture.

HEPN-MNT Toxin-Antitoxin System: The HEPN Ribonuclease Is Neutralized by OligoAMPylation.

Prokaryotic toxin-antitoxin (TA) systems are composed of a toxin capable of interfering with key cellular processes and its neutralizing antidote, the antitoxin. Here, we focus on the HEPN-MNT TA system encoded in the vicinity of a subtype I-D CRISPR-Cas system in the cyanobacterium Aphanizomenon flos-aquae. We show that HEPN acts as a toxic RNase, which cleaves off 4 nt from the 3' end in a subset of tRNAs, thereby interfering with translation. Surprisingly, we find that the MNT (minimal nucleotidyltransferase) antitoxin inhibits HEPN RNase through covalent di-AMPylation (diadenylylation) of a conserved tyrosine residue, Y109, in the active site loop. Furthermore, we present crystallographic snapshots of the di-AMPylation reaction at different stages that explain the mechanism of HEPN RNase inactivation. Finally, we propose that the HEPN-MNT system functions as a cellular ATP sensor that monitors ATP homeostasis and, at low ATP levels, releases active HEPN toxin.

Structure and function of the Si3 insertion integrated into the trigger loop/helix of cyanobacterial RNA polymerase.

Cyanobacteria and evolutionarily related chloroplasts of algae and plants possess unique RNA polymerases (RNAPs) with characteristics that distinguish them from canonical bacterial RNAPs. The largest subunit of cyanobacterial RNAP (cyRNAP) is divided into two polypeptides, ß'1 and ß'2, and contains the largest known lineage-specific insertion domain, Si3, located in the middle of the trigger loop and spanning approximately half of the ß'2 subunit. In this study, we present the X-ray crystal structure of Si3 and the cryo-EM structures of the cyRNAP transcription elongation complex plus the NusG factor with and without incoming nucleoside triphosphate (iNTP) bound at the active site. Si3 has a well-ordered and elongated shape that exceeds the length of the main body of cyRNAP, fits into cavities of cyRNAP in the absence of iNTP bound at the active site and shields the binding site of secondary channel-binding proteins such as Gre and DksA. A small transition from the trigger loop to the trigger helix upon iNTP binding results in a large swing motion of Si3; however, this transition does not affect the catalytic activity of cyRNAP due to its minimal contact with cyRNAP, NusG, or DNA. This study provides a structural framework for understanding the evolutionary significance of these features unique to cyRNAP and chloroplast RNAP and may provide insights into the molecular mechanism of transcription in specific environment of photosynthetic organisms and organelle.

Clocking out and letting go to unleash green biotech applications in a photosynthetic host.

Cyanobacteria are photosynthetic bacteria whose gene expression patterns are globally regulated by their circadian (daily) clocks. Due to their ability to use sunlight as their energy source, they are also attractive hosts for "green" production of pharmaceuticals, renewable fuels, and chemicals. However, despite the application of traditional genetic tools such as the identification of strong promoters to enhance the expression of heterologous genes, cyanobacteria have lagged behind other microorganisms such as Escherichia coli and yeast as economically efficient cell factories. The previous approaches have ignored large-scale constraints within cyanobacterial metabolic networks on transcription, predominantly the pervasive control of gene expression by the circadian (daily) clock. Here, we show that reprogramming gene expression by releasing circadian repressor elements in the transcriptional regulatory pathways coupled with inactivation of the central oscillating mechanism enables a dramatic enhancement of expression in cyanobacteria of heterologous genes encoding both catalytically active enzymes and polypeptides of biomedical significance.

Machine learning reveals the transcriptional regulatory network and circadian dynamics of Synechococcus elongatus PCC 7942.

Synechococcus elongatus is an important cyanobacterium that serves as a versatile and robust model for studying circadian biology and photosynthetic metabolism. Its transcriptional regulatory network (TRN) is of fundamental interest, as it orchestrates the cell's adaptation to the environment, including its response to sunlight. Despite the previous characterization of constituent parts of the S. elongatus TRN, a comprehensive layout of its topology remains to be established. Here, we decomposed a compendium of 300 high-quality RNA sequencing datasets of the model strain PCC 7942 using independent component analysis. We obtained 57 independently modulated gene sets, or iModulons, that explain 67% of the variance in the transcriptional response and 1) accurately reflect the activity of known transcriptional regulations, 2) capture functional components of photosynthesis, 3) provide hypotheses for regulon structures and functional annotations of poorly characterized genes, and 4) describe the transcriptional shifts under dynamic light conditions. This transcriptome-wide analysis of S. elongatus provides a quantitative reconstruction of the TRN and presents a knowledge base that can guide future investigations. Our systems-level analysis also provides a global TRN structure for S. elongatus PCC 7942.

Recognition of cyanobacteria promoters via Siamese network-based contrastive learning under novel non-promoter generation.

It is a vital step to recognize cyanobacteria promoters on a genome-wide scale. Computational methods are promising to assist in difficult biological identification. When building recognition models, these methods rely on non-promoter generation to cope with the lack of real non-promoters. Nevertheless, the factitious significant difference between promoters and non-promoters causes over-optimistic prediction. Moreover, designed for E. coli or B. subtilis, existing methods cannot uncover novel, distinct motifs among cyanobacterial promoters. To address these issues, this work first proposes a novel non-promoter generation strategy called phantom sampling, which can eliminate the factitious difference between promoters and generated non-promoters. Furthermore, it elaborates a novel promoter prediction model based on the Siamese network (SiamProm), which can amplify the hidden difference between promoters and non-promoters through a joint characterization of global associations, upstream and downstream contexts, and neighboring associations w.r.t. k-mer tokens. The comparison with state-of-the-art methods demonstrates the superiority of our phantom sampling and SiamProm. Both comprehensive ablation studies and feature space illustrations also validate the effectiveness of the Siamese network and its components. More importantly, SiamProm, upon our phantom sampling, finds a novel cyanobacterial promoter motif ('GCGATCGC'), which is palindrome-patterned, content-conserved, but position-shifted.

Carbon isotope fractionation by an ancestral rubisco suggests that biological proxies for CO2 through geologic time should be reevaluated.

The history of Earth's carbon cycle reflects trends in atmospheric composition convolved with the evolution of photosynthesis. Fortunately, key parts of the carbon cycle have been recorded in the carbon isotope ratios of sedimentary rocks. The dominant model used to interpret this record as a proxy for ancient atmospheric CO2 is based on carbon isotope fractionations of modern photoautotrophs, and longstanding questions remain about how their evolution might have impacted the record. Therefore, we measured both biomass (εp) and enzymatic (εRubisco) carbon isotope fractionations of a cyanobacterial strain (Synechococcus elongatus PCC 7942) solely expressing a putative ancestral Form 1B rubisco dating to ≫1 Ga. This strain, nicknamed ANC, grows in ambient pCO2 and displays larger εp values than WT, despite having a much smaller εRubisco (17.23 ± 0.61‰ vs. 25.18 ± 0.31‰, respectively). Surprisingly, ANC εp exceeded ANC εRubisco in all conditions tested, contradicting prevailing models of cyanobacterial carbon isotope fractionation. Such models can be rectified by introducing additional isotopic fractionation associated with powered inorganic carbon uptake mechanisms present in Cyanobacteria, but this amendment hinders the ability to accurately estimate historical pCO2 from geological data. Understanding the evolution of rubisco and the CO2 concentrating mechanism is therefore critical for interpreting the carbon isotope record, and fluctuations in the record may reflect the evolving efficiency of carbon fixing metabolisms in addition to changes in atmospheric CO2.

Synchronization of the circadian clock to the environment tracked in real time.

The circadian system of the cyanobacterium Synechococcus elongatus PCC 7942 relies on a three-protein nanomachine (KaiA, KaiB, and KaiC) that undergoes an oscillatory phosphorylation cycle with a period of ~24 h. This core oscillator can be reconstituted in vitro and is used to study the molecular mechanisms of circadian timekeeping and entrainment. Previous studies showed that two key metabolic changes that occur in cells during the transition into darkness, changes in the ATP/ADP ratio and redox status of the quinone pool, are cues that entrain the circadian clock. By changing the ATP/ADP ratio or adding oxidized quinone, one can shift the phase of the phosphorylation cycle of the core oscillator in vitro. However, the in vitro oscillator cannot explain gene expression patterns because the simple mixture lacks the output components that connect the clock to genes. Recently, a high-throughput in vitro system termed the in vitro clock (IVC) that contains both the core oscillator and the output components was developed. Here, we used IVC reactions and performed massively parallel experiments to study entrainment, the synchronization of the clock with the environment, in the presence of output components. Our results indicate that the IVC better explains the in vivo clock-resetting phenotypes of wild-type and mutant strains and that the output components are deeply engaged with the core oscillator, affecting the way input signals entrain the core pacemaker. These findings blur the line between input and output pathways and support our previous demonstration that key output components are fundamental parts of the clock.

A c-di-GMP binding effector controls cell size in a cyanobacterium.

Cyclic-di-GMP (c-di-GMP) is a ubiquitous bacterial signaling molecule. It is also a critical player in the regulation of cell size and cell behaviors such as cell aggregation and phototaxis in cyanobacteria, which constitute an important group of prokaryotes for their roles in the ecology and evolution of the Earth. However, c-di-GMP receptors have never been revealed in cyanobacteria. Here, we report the identification of a c-di-GMP receptor, CdgR, from the filamentous cyanobacterium Anabaena PCC 7120. Crystal structural analysis and genetic studies demonstrate that CdgR binds c-di-GMP at the dimer interface and this binding is required for the control of cell size in a c-di-GMP-dependent manner. Different functions of CdgR, in ligand binding and signal transmission, could be separated genetically, allowing us to dissect its molecular signaling functions. The presence of the apo-form of CdgR triggers cell size reduction, consistent with the similar effects observed with a decrease of c-di-GMP levels in cells. Furthermore, we found that CdgR exerts its function by interacting with a global transcription factor DevH, and this interaction was inhibited by c-di-GMP. The lethal effect triggered by conditional depletion of DevH or by the production of several point-mutant proteins of CdgR in cells indicates that this signaling pathway plays critical functions in Anabaena. Our studies revealed a mechanism of c-di-GMP signaling in the control of cell size, an important and complex trait for bacteria. CdgR is highly conserved in cyanobacteria, which will greatly expand our understanding of the roles of c-di-GMP signaling in these organisms.

An SI3-σ arch stabilizes cyanobacteria transcription initiation complex.

Multisubunit RNA polymerases (RNAPs) associate with initiation factors (σ in bacteria) to start transcription. The σ factors are responsible for recognizing and unwinding promoter DNA in all bacterial RNAPs. Here, we report two cryo-EM structures of cyanobacterial transcription initiation complexes at near-atomic resolutions. The structures show that cyanobacterial RNAP forms an "SI3-σ" arch interaction between domain 2 of σA (σ2) and sequence insertion 3 (SI3) in the mobile catalytic domain Trigger Loop (TL). The "SI3-σ" arch facilitates transcription initiation from promoters of different classes through sealing the main cleft and thereby stabilizing the RNAP-promoter DNA open complex. Disruption of the "SI3-σ" arch disturbs cyanobacteria growth and stress response. Our study reports the structure of cyanobacterial RNAP and a unique mechanism for its transcription initiation. Our data suggest functional plasticity of SI3 and provide the foundation for further research into cyanobacterial and chloroplast transcription.

Making a living off the rainbow's edge: How phycobilisomes adapt structurally to absorb far-red light.

Cyanobacteria harvest light by using architecturally complex, soluble, light-harvesting complexes known as phycobilisomes (PBSs). PBS diversity includes specialized subunit paralogs that are tuned to specific regions of the light spectrum; some cyanobacterial lineages can even absorb far-red light. In a recent issue of the Journal of Biological Chemistry, Gisriel et al. reported the cryo-electron microscopic structure of a far-red PBS core, showing how bilin binding in the α-subunits of allophycocyanin paralogs can modify the bilin-binding site to red shift the absorbance spectrum. This work helps explain how cyanobacteria can grow in environments where most of the visible light has been filtered out.