CP12 fine-tunes the Calvin-Benson cycle and carbohydrate metabolism in cyanobacteria.

The regulatory protein CP12 can bind glyceraldehyde 3-phosphate dehydrogenase (GapDH) and phosphoribulokinase (PRK) in oxygenic phototrophs, thereby switching on and off the flux through the Calvin-Benson cycle (CBC) under light and dark conditions, respectively. However, it can be assumed that CP12 is also regulating CBC flux under further conditions associated with redox changes. To prove this hypothesis, the mutant Δcp12 of the model cyanobacterium Synechocystis sp. PCC 6803 was compared to wild type and different complementation strains. Fluorescence microscopy showed for the first time the in vivo kinetics of assembly and disassembly of the CP12-GapDH-PRK complex, which was absent in the mutant Δcp12. Metabolome analysis revealed differences in the contents of ribulose 1,5-bisphosphate and dihydroxyacetone phosphate, the products of the CP12-regulated enzymes GapDH and PRK, between wild type and mutant Δcp12 under changing CO2 conditions. Growth of Δcp12 was not affected at constant light under different inorganic carbon conditions, however, the addition of glucose inhibited growth in darkness as well as under diurnal conditions. The growth defect in the presence of glucose is associated with the inability of Δcp12 to utilize external glucose. These phenotypes could be complemented by ectopic expression of the native CP12 protein, however, expression of CP12 variants with missing redox-sensitive cysteine pairs only partly restored the growth with glucose. These experiments indicated that the loss of GapDH-inhibition via CP12 is more critical than PRK association. Measurements of the NAD(P)H oxidation revealed an impairment of light intensity-dependent redox state regulation in Δcp12. Collectively, our results indicate that CP12-dependent regulation of the CBC is crucial for metabolic adjustment under conditions leading to redox changes such as diurnal conditions, glucose addition, and different CO2 conditions in cyanobacteria.

Advanced Image Analysis Methods for Automated Segmentation of Subnuclear Chromatin Domains.

The combination of ever-increasing microscopy resolution with cytogenetical tools allows for detailed analyses of nuclear functional partitioning. However, the need for reliable qualitative and quantitative methodologies to detect and interpret chromatin sub-nuclear organization dynamics is crucial to decipher the underlying molecular processes. Having access to properly automated tools for accurate and fast recognition of complex nuclear structures remains an important issue. Cognitive biases associated with human-based curation or decisions for object segmentation tend to introduce variability and noise into image analysis. Here, we report the development of two complementary segmentation methods, one semi-automated (iCRAQ) and one based on deep learning (Nucl.Eye.D), and their evaluation using a collection of A. thaliana nuclei with contrasted or poorly defined chromatin compartmentalization. Both methods allow for fast, robust and sensitive detection as well as for quantification of subtle nucleus features. Based on these developments, we highlight advantages of semi-automated and deep learning-based analyses applied to plant cytogenetics.

Evidence for Electron Transfer from the Bidirectional Hydrogenase to the Photosynthetic Complex I (NDH-1) in the Cyanobacterium Synechocystis sp. PCC 6803.

The cyanobacterial bidirectional [NiFe]-hydrogenase is a pentameric enzyme. Apart from the small and large hydrogenase subunits (HoxYH) it contains a diaphorase module (HoxEFU) that interacts with NAD(P)+ and ferredoxin. HoxEFU shows strong similarity to the outermost subunits (NuoEFG) of canonical respiratory complexes I. Photosynthetic complex I (NDH-1) lacks these three subunits. This led to the idea that HoxEFU might interact with NDH-1 instead. HoxEFUYH utilizes excited electrons from PSI for photohydrogen production and it catalyzes the reverse reaction and feeds electrons into the photosynthetic electron transport. We analyzed hydrogenase activity, photohydrogen evolution and hydrogen uptake, the respiration and photosynthetic electron transport of ΔhoxEFUYH, and a knock-out strain with dysfunctional NDH-1 (ΔndhD1/ΔndhD2) of the cyanobacterium Synechocystis sp. PCC 6803. Photohydrogen production was prolonged in ΔndhD1/ΔndhD2 due to diminished hydrogen uptake. Electrons from hydrogen oxidation must follow a different route into the photosynthetic electron transport in this mutant compared to wild type cells. Furthermore, respiration was reduced in ΔhoxEFUYH and the ΔndhD1/ΔndhD2 localization of the hydrogenase to the membrane was impaired. These data indicate that electron transfer from the hydrogenase to the NDH-1 complex is either direct, by the binding of the hydrogenase to the complex, or indirect, via an additional mediator.

Synechocystis sp. PCC 6803 Requires the Bidirectional Hydrogenase to Metabolize Glucose and Arginine Under Oxic Conditions.

The cyanobacterium Synechocystis sp.PCC 6803 possesses a bidirectional NiFe-hydrogenase, HoxEFUYH. It functions to produce hydrogen under dark, fermentative conditions and photoproduces hydrogen when dark-adapted cells are illuminated. Unexpectedly, we found that the deletion of the large subunit of the hydrogenase (HoxH) in Synechocystis leads to an inability to grow on arginine and glucose under continuous light in the presence of oxygen. This is surprising, as the hydrogenase is an oxygen-sensitive enzyme. In wild-type (WT) cells, thylakoid membranes largely disappeared, cyanophycin accumulated, and the plastoquinone (PQ) pool was highly reduced, whereas ΔhoxH cells entered a dormant-like state and neither consumed glucose nor arginine at comparable rates to the WT. Hydrogen production was not traceable in the WT under these conditions. We tested and could show that the hydrogenase does not work as an oxidase on arginine and glucose but has an impact on the redox states of photosynthetic complexes in the presence of oxygen. It acts as an electron valve as an immediate response to the supply of arginine and glucose but supports the input of electrons from arginine and glucose oxidation into the photosynthetic electron chain in the long run, possibly via the NDH-1 complex. Despite the data presented in this study, the latter scenario requires further proof. The exact role of the hydrogenase in the presence of arginine and glucose remains unresolved. In addition, a unique feature of the hydrogenase is its ability to shift electrons between NAD(H), NADP(H), ferredoxin, and flavodoxin, which was recently shown in vitro and might be required for fine-tuning. Taken together, our data show that Synechocystis depends on the hydrogenase to metabolize organic carbon and nitrogen in the presence of oxygen, which might be an explanation for its prevalence in aerobic cyanobacteria.

In-vivo quantification of electron flow through photosystem I - Cyclic electron transport makes up about 35% in a cyanobacterium.

Photosynthetic electron flow, driven by photosystem I and II, provides chemical energy for carbon fixation. In addition to a linear mode a second cyclic route exists, which only involves photosystem I. The exact contributions of linear and cyclic transport are still a matter of debate. Here, we describe the development of a method that allows quantification of electron flow in absolute terms through photosystem I in a photosynthetic organism for the first time. Specific in-vivo protocols allowed to discern the redox states of plastocyanin, P700 and the FeS-clusters including ferredoxin at the acceptor site of PSI in the cyanobacterium Synechocystis sp. PCC 6803 with the near-infrared spectrometer Dual-KLAS/NIR. P700 absorbance changes determined with the Dual-KLAS/NIR correlated linearly with direct determinations of PSI concentrations using EPR. Dark-interval relaxation kinetics measurements (DIRKPSI) were applied to determine electron flow through PSI. Counting electrons from hydrogen oxidation as electron donor to photosystem I in parallel to DIRKPSI measurements confirmed the validity of the method. Electron flow determination by classical PSI yield measurements overestimates electron flow at low light intensities and saturates earlier compared to DIRKPSI. Combination of DIRKPSI with oxygen evolution measurements yielded a proportion of 35% of surplus electrons passing PSI compared to PSII. We attribute these electrons to cyclic electron transport, which is twice as high as assumed for plants. Counting electrons flowing through the photosystems allowed determination of the number of quanta required for photosynthesis to 11 per oxygen produced, which is close to published values.

Two novel heteropolymer-forming proteins maintain the multicellular shape of the cyanobacterium Anabaena sp. PCC 7120.

Polymerizing and filament-forming proteins are instrumental for numerous cellular processes such as cell division and growth. Their function in stabilization and localization of protein complexes and replicons is achieved by a filamentous structure. Known filamentous proteins assemble into homopolymers consisting of single subunits - for example, MreB and FtsZ in bacteria - or heteropolymers that are composed of two subunits, for example, keratin and α/ß tubulin in eukaryotes. Here, we describe two novel coiled-coil-rich proteins (CCRPs) in the filament-forming cyanobacterium Anabaena sp. PCC 7120 (hereafter Anabaena) that assemble into a heteropolymer and function in the maintenance of the Anabaena multicellular shape (termed trichome). The two CCRPs - Alr4504 and Alr4505 (named ZicK and ZacK) - are strictly interdependent for the assembly of protein filaments in vivo and polymerize nucleotide independently in vitro, similar to known intermediate filament (IF) proteins. A ΔzicKΔzacK double mutant is characterized by a zigzagged cell arrangement and hence a loss of the typical linear Anabaena trichome shape. ZicK and ZacK interact with themselves, with each other, with the elongasome protein MreB, the septal junction protein SepJ and the divisome associate septal protein SepI. Our results suggest that ZicK and ZacK function in cooperation with SepJ and MreB to stabilize the Anabaena trichome and are likely essential for the manifestation of the multicellular shape in Anabaena. Our study reveals the presence of filament-forming IF-like proteins whose function is achieved through the formation of heteropolymers in cyanobacteria.

Phylogenetic Analyses and GAGA-Motif Binding Studies of BBR/BPC Proteins Lend to Clues in GAGA-Motif Recognition and a Regulatory Role in Brassinosteroid Signaling.

Plant GAGA-motif binding factors are encoded by the BARLEY B RECOMBINANT / BASIC PENTACYSTEINE (BBR/BPC) family, which fulfill indispensable functions in growth and development. BBR/BPC proteins control flower development, size of the stem cell niche and seed development through transcriptional regulation of homeotic transcription factor genes. They are responsible for the context dependent recruitment of Polycomb repressive complexes (PRC) or other repressive proteins to GAGA-motifs, which are contained in Polycomb repressive DNA-elements (PREs). Hallmark of the protein family is the highly conserved BPC domain, which is required for DNA binding. Here we study the evolution and diversification of the BBR/BPC family and its DNA-binding domain. Our analyses supports a further division of the family into four main groups (I-IV) and several subgroups, to resolve a strict monophyletic descent of the BPC domain. We prove a polyphyletic origin for group III proteins, which evolved from group I and II members through extensive loss of domains in the N-terminus. Conserved motif searches lend to the identification of a WAR/KHGTN consensus and a TIR/K motif at the very C-terminus of the BPC-domain. We could show by DPI-ELISA that this signature is required for DNA-binding in AtBPC1. Additional binding studies with AtBPC1, AtBPC6 and mutated oligonucleotides consolidated the binding to GAGA tetramers. To validate these findings, we used previously published ChIP-seq data from GFP-BPC6. We uncovered that many genes of the brassinosteroid signaling pathway are targeted by AtBPC6. Consistently, bpc6, bpc4 bpc6, and lhp1 bpc4 bpc4 mutants display brassinosteroid-dependent root growth phenotypes. Both, a function in brassinosteroid signaling and our phylogenetic data supports a link between BBR/BPC diversification in the land plant lineage and the complexity of flower and seed plant evolution.