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

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

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

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

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

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

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

Protein NirP1 regulates nitrite reductase and nitrite excretion in cyanobacteria.

When the supply of inorganic carbon is limiting, photosynthetic cyanobacteria excrete nitrite, a toxic intermediate in the ammonia assimilation pathway from nitrate. It has been hypothesized that the excreted nitrite represents excess nitrogen that cannot be further assimilated due to the missing carbon, but the underlying molecular mechanisms are unclear. Here, we identified a protein that interacts with nitrite reductase, regulates nitrogen metabolism and promotes nitrite excretion. The protein, which we named NirP1, is encoded by an unannotated gene that is upregulated under low carbon conditions and controlled by transcription factor NtcA, a central regulator of nitrogen homeostasis. Ectopic overexpression of nirP1 in Synechocystis sp. PCC 6803 resulted in a chlorotic phenotype, delayed growth, severe changes in amino acid pools, and nitrite excretion. Coimmunoprecipitation experiments indicated that NirP1 interacts with nitrite reductase, a central enzyme in the assimilation of ammonia from nitrate/nitrite. Our results reveal that NirP1 is widely conserved in cyanobacteria and plays a crucial role in the coordination of C/N primary metabolism by targeting nitrite reductase.

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

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

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

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

Machine learning predicts system-wide metabolic flux control in cyanobacteria.

Metabolic fluxes and their control mechanisms are fundamental in cellular metabolism, offering insights for the study of biological systems and biotechnological applications. However, quantitative and predictive understanding of controlling biochemical reactions in microbial cell factories, especially at the system level, is limited. In this work, we present ARCTICA, a computational framework that integrates constraint-based modelling with machine learning tools to address this challenge. Using the model cyanobacterium Synechocystis sp. PCC 6803 as chassis, we demonstrate that ARCTICA effectively simulates global-scale metabolic flux control. Key findings are that (i) the photosynthetic bioproduction is mainly governed by enzymes within the Calvin-Benson-Bassham (CBB) cycle, rather than by those involve in the biosynthesis of the end-product, (ii) the catalytic capacity of the CBB cycle limits the photosynthetic activity and downstream pathways and (iii) ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO) is a major, but not the most, limiting step within the CBB cycle. Predicted metabolic reactions qualitatively align with prior experimental observations, validating our modelling approach. ARCTICA serves as a valuable pipeline for understanding cellular physiology and predicting rate-limiting steps in genome-scale metabolic networks, and thus provides guidance for bioengineering of cyanobacteria.

ApcE plays an important role in light-induced excitation energy dissipation in the Synechocystis PCC6803 phycobilisomes.

Phycobilisomes (PBs) play an important role in cyanobacterial photosynthesis. They capture light and transfer excitation energy to the photosynthetic reaction centres. PBs are also central to some photoprotective and photoregulatory mechanisms that help sustain photosynthesis under non-optimal conditions. Amongst the mechanisms involved in excitation energy dissipation that are activated in response to excessive illumination is a recently discovered light-induced mechanism that is intrinsic to PBs and has been the least studied. Here, we used single-molecule spectroscopy and developed robust data analysis methods to explore the role of a terminal emitter subunit, ApcE, in this intrinsic, light-induced mechanism. We isolated the PBs from WT Synechocystis PCC 6803 as well as from the ApcE-C190S mutant of this strain and compared the dynamics of their fluorescence emission. PBs isolated from the mutant (i.e., ApcE-C190S-PBs), despite not binding some of the red-shifted pigments in the complex, showed similar global emission dynamics to WT-PBs. However, a detailed analysis of dynamics in the core revealed that the ApcE-C190S-PBs are less likely than WT-PBs to enter quenched states under illumination but still fully capable of doing so. This result points to an important but not exclusive role of the ApcE pigments in the light-induced intrinsic excitation energy dissipation mechanism in PBs.

Metabolic engineering of Synechocystis sp. PCC 6803 for the improved production of phenylpropanoids.

BACKGROUND: Phenylpropanoids are a large group of plant secondary metabolites with various biological functions, derived from aromatic amino acids. Cyanobacteria are promising host organisms for sustainable production of plant phenylpropanoids. We have previously engineered Synechocystis sp. PCC 6803 to produce trans-cinnamic acid (tCA) and p-coumaric acid (pCou), the first intermediates of phenylpropanoid pathway, by overexpression of phenylalanine- and tyrosine ammonia lyases. In this study, we aimed to enhance the production of the target compounds tCA and pCou in Synechocystis. RESULTS: We eliminated the 4-hydroxyphenylpyruvate dioxygenase (HPPD) activity, which is a competing pathway consuming tyrosine and, possibly, phenylalanine for tocopherol synthesis. Moreover, several genes of the terminal steps of the shikimate pathway were overexpressed alone or in operons, such as aromatic transaminases, feedback insensitive cyclohexadienyl dehydrogenase (TyrC) from Zymomonas mobilis and the chorismate mutase (CM) domain of the fused chorismate mutase/prephenate dehydratase enzyme from Escherichia coli. The obtained engineered strains demonstrated nearly 1.5 times enhanced tCA and pCou production when HPPD was knocked out compared to the parental production strains, accumulating 138 ± 3.5 mg L-1 of tCA and 72.3 ± 10.3 mg L-1 of pCou after seven days of photoautotrophic growth. However, there was no further improvement when any of the pathway genes were overexpressed. Finally, we used previously obtained AtPRM8 and TsPRM8 Synechocystis strains with deregulated shikimate pathway as a background for the overexpression of synthetic constructs with ppd knockout. CONCLUSIONS: HPPD elimination enhances the tCA and pCou productivity to a similar extent. The use of PRM8 based strains as a background for overexpression of synthetic constructs, however, did not promote tCA and pCou titers, which indicates a tight regulation of the terminal steps of phenylalanine and tyrosine synthesis. This work contributes to establishing cyanobacteria as hosts for phenylpropanoid production.

Mutagenesis of Ile184 in the cd-loop of the photosystem II D1 protein modifies acceptor-side function via spontaneous mutation of D1-His252 in Synechocystis sp. PCC 6803.

The Photosystem II water-plastoquinone oxidoreductase is a multi-subunit complex which catalyses the light-driven oxidation of water to molecular oxygen in oxygenic photosynthesis. The D1 reaction centre protein exists in multiple forms in cyanobacteria, including D1FR which is expressed under far-red light. We investigated the role of Phe184 that is found in the lumenal cd-loop of D1FR but is typically an isoleucine in other D1 isoforms. The I184F mutant in Synechocystis sp. PCC 6803 was similar to the control strain but accumulated a spontaneous mutation that introduced a Gln residue in place of His252 located on the opposite side of the thylakoid membrane. His252 participates in the protonation of the secondary plastoquinone electron acceptor QB. The I184F:H252Q double mutant exhibited reduced high-light-induced photodamage and an altered QB-binding site that impaired herbicide binding. Additionally, the H252Q mutant had a large increase in the variable fluorescence yield although the number of photochemically active PS II centres was unchanged. In the I184F:H252Q mutant the extent of the increased fluorescence yield decreased. Our data indicates substitution of Ile184 to Phe modulates PS II-specific variable fluorescence in cells with the His252 to Gln substitution by modifying the QB-binding site.

Inhibition of Polyphosphate Degradation in Synechocystis sp. PCC6803 through Inactivation of the phoU Gene.

Phosphorus is an essential but non-renewable nutrient resource critical for agriculture. Luxury phosphorus uptake allows microalgae to synthesize polyphosphate and accumulate phosphorus, but, depending on the strain of algae, polyphosphate may be degraded within 4 hours of accumulation. We studied the recovery of phosphorus from wastewater through luxury uptake by an engineered strain of Synechocystis sp. with inhibited polyphosphate degradation and the effect of this engineered Synechocystis biomass on lettuce growth. First, a strain (ΔphoU) lacking the phoU gene, which encodes a negative regulator of environmental phosphate concentrations, was generated to inhibit polyphosphate degradation in cells. Polyphosphate concentrations in the phoU knock-out strain were maintained for 24 h and then decreased slowly. In contrast, polyphosphate concentrations in the wild-type strain increased up to 4 h and then decreased rapidly. In addition, polyphosphate concentration in the phoU knockout strain cultured in semi-permeable membrane bioreactors with artificial wastewater medium was 2.5 times higher than that in the wild type and decreased to only 16% after 48 h. The biomass of lettuce treated with the phoU knockout strain (0.157 mg P/m2) was 38% higher than that of the lettuce treated with the control group. These results indicate that treating lettuce with this microalgal biomass can be beneficial to crop growth. These results suggest that the use of polyphosphate-accumulating microalgae as biofertilizers may alleviate the effects of a diminishing phosphorous supply. These findings can be used as a basis for additional genetic engineering to increase intracellular polyphosphate levels.

Phycobilisome protein ApcG interacts with PSII and regulates energy transfer in Synechocystis.

Photosynthetic organisms harvest light using pigment-protein complexes. In cyanobacteria, these are water-soluble antennae known as phycobilisomes (PBSs). The light absorbed by PBS is transferred to the photosystems in the thylakoid membrane to drive photosynthesis. The energy transfer between these complexes implies that protein-protein interactions allow the association of PBS with the photosystems. However, the specific proteins involved in the interaction of PBS with the photosystems are not fully characterized. Here, we show in Synechocystis sp. PCC 6803 that the recently discovered PBS linker protein ApcG (sll1873) interacts specifically with PSII through its N-terminal region. Growth of cyanobacteria is impaired in apcG deletion strains under light-limiting conditions. Furthermore, complementation of these strains using a phospho-mimicking version of ApcG causes reduced growth under normal growth conditions. Interestingly, the interaction of ApcG with PSII is affected when a phospho-mimicking version of ApcG is used, targeting the positively charged residues interacting with the thylakoid membrane, suggesting a regulatory role mediated by phosphorylation of ApcG. Low-temperature fluorescence measurements showed decreased PSI fluorescence in apcG deletion and complementation strains. The PSI fluorescence was the lowest in the phospho-mimicking complementation strain, while the pull-down experiment showed no interaction of ApcG with PSI under any tested condition. Our results highlight the importance of ApcG for selectively directing energy harvested by the PBS and imply that the phosphorylation status of ApcG plays a role in regulating energy transfer from PSII to PSI.

Improved capacity for the repair of photosystem II via reinforcement of the translational and antioxidation systems in Synechocystis sp. PCC 6803.

In the cyanobacterium Synechocystis sp. PCC 6803, translation factor EF-Tu is inactivated by reactive oxygen species (ROS) via oxidation of Cys82 and the oxidation of EF-Tu enhances the inhibition of the repair of photosystem II (PSII) by suppressing protein synthesis. In our present study, we generated transformants of Synechocystis that overexpressed a mutated form of EF-Tu, designated EF-Tu (C82S), in which Cys82 had been replaced by a Ser residue, and ROS-scavenging enzymes individually or together. Expression of EF-Tu (C82S) alone in Synechocystis enhanced the repair of PSII under strong light, with the resultant mitigation of PSII photoinhibition, but it stimulated the production of ROS. However, overexpression of superoxide dismutase and catalase, together with the expression of EF-Tu (C82S), lowered intracellular levels of ROS and enhanced the repair of PSII more significantly under strong light, via facilitation of the synthesis de novo of the D1 protein. By contrast, the activity of photosystem I was hardly affected in wild-type cells and in all the lines of transformed cells under the same strong-light conditions. Furthermore, transformed cells that overexpressed EF-Tu (C82S), superoxide dismutase, and catalase were able to survive longer under stronger light than wild-type cells. Thus, the reinforced capacity for both protein synthesis and ROS scavenging allowed both photosynthesis and cell proliferation to tolerate strong light.

Quantifying the Energy Spillover between Photosystems II and I in Cyanobacterial Thylakoid Membranes and Cells.

The spatial separation of photosystems I and II (PSI and PSII) is thought to be essential for efficient photosynthesis by maintaining a balanced flow of excitation energy between them. Unlike the thylakoid membranes of plant chloroplasts, cyanobacterial thylakoids do not form tightly appressed grana stacks that enforce strict lateral separation. The coexistence of the two photosystems provides a ground for spillover-excitation energy transfer from PSII to PSI. Spillover has been considered as a pathway of energy transfer from the phycobilisomes to PSI and may also play a role in state transitions as means to avoid overexcitation of PSII. Here, we demonstrate a significant degree of energy spillover from PSII to PSI in reconstituted membranes and isolated thylakoid membranes of Thermosynechococcus (Thermostichus) vulcanus and Synechocystis sp. PCC 6803 by steady-state and time-resolved fluorescence spectroscopy. The quantum yield of spillover in these systems was determined to be up to 40%. Spillover was also found in intact cells but to a considerably lower degree (20%) than in isolated thylakoid membranes. The findings support a model of coexistence of laterally separated microdomains of PSI and PSII in the cyanobacterial cells as well as domains where the two photosystems are energetically connected. The methodology presented here can be applied to probe spillover in other photosynthetic organisms.

Structural framework for the understanding spectroscopic and functional signatures of the cyanobacterial Orange Carotenoid Protein families.

The Orange Carotenoid Protein (OCP) is a unique photoreceptor crucial for cyanobacterial photoprotection. Best studied Synechocystis sp. PCC 6803 OCP belongs to the large OCP1 family. Downregulated by the Fluorescence Recovery Protein (FRP) in low-light, high-light-activated OCP1 binds to the phycobilisomes and performs non-photochemical quenching. Recently discovered families OCP2 and OCP3 remain structurally and functionally underexplored, and no systematic comparative studies have ever been conducted. Here we present two first crystal structures of OCP2 from morphoecophysiologically different cyanobacteria and provide their comprehensive structural, spectroscopic and functional comparison with OCP1, the recently described OCP3 and all-OCP ancestor. Structures enable correlation of spectroscopic signatures with the effective number of hydrogen and discovered here chalcogen bonds anchoring the ketocarotenoid in OCP, as well as with the rotation of the echinenone's ß-ionone ring in the CTD. Structural data also helped rationalize the observed differences in OCP/FRP and OCP/phycobilisome functional interactions. These data are expected to foster OCP research and applications in optogenetics, targeted carotenoid delivery and cyanobacterial biomass engineering.

Regulation of L-aspartate oxidase contributes to NADP+ biosynthesis in Synechocystis sp. PCC 6803.

Cyanobacteria have been promoted as a biomass resource that can contribute to carbon neutrality. Synechocystis sp. PCC 6803 is a model cyanobacterium that is widely used in various studies. NADP+ and NAD+ are electron receptors involved in energy metabolism. The NADP+/NAD+ ratio in Synechocystis sp. PCC 6803 is markedly higher than that in the heterotrophic bacterium Escherichia coli. In Synechocystis sp. PCC 6803, NADP+ primarily functions as an electron receptor during the light reaction of photosynthesis, and NADP+ biosynthesis is essential for photoautotrophic growth. Generally, the regulatory enzyme of NADP+ biosynthesis is NAD kinase, which catalyzes the phosphorylation of NAD+. However, a previous study suggested that the regulation of another enzyme contributes to NADP+ biosynthesis in Synechocystis sp. PCC 6803 under photoautotrophic conditions. L-Aspartate oxidase is the first enzyme in NAD(P)+ biosynthesis. In this study, we biochemically characterized Synechocystis sp. PCC 6803 L-aspartate oxidase and determined the phenotype of a Synechocystis sp. PCC 6803 mutant overexpressing L-aspartate oxidase. The catalytic efficiency of L-aspartate oxidase from Synechocystis sp. PCC 6803 was lower than that of L-aspartate oxidases and NAD kinases from other organisms. L-Aspartate oxidase activity was affected by different metabolites such as NADP+ and ATP. The L-aspartate oxidase-overexpressing strain grew faster than the wild-type strain under photoautotrophic conditions. The L-aspartate oxidase-overexpressing strain accumulated NADP+ under photoautotrophic conditions. These results indicate that the regulation of L-aspartate oxidase contributes to NADP+ biosynthesis in Synechocystis sp. PCC 6803 under photoautotrophic conditions. These findings provide insight into the regulatory mechanism of cyanobacterial NADP+ biosynthesis.

Transmembrane and PAS domains of the histidine kinase Hik33 as regulators of cold and light responses in the cyanobacterium Synechocystis sp. PCC 6803.

The PAS (Per-ARNT-Sim) domain is a sensory protein regulatory module found in archaea, prokaryotes, and eukaryotes. Histidine and serine/threonine protein kinases, chemo- and photoreceptors, circadian rhythm regulators, ion channels, phosphodiesterases, and other cellular response regulators are among these proteins. Hik33 is a multifunctional sensory histidine kinase that is implicated in cyanobacterial responses to cold, salt, hyperosmotic, and oxidative stressors. The functional roles of individual Hik33 domains in signal transduction were investigated in this study. Synechocystis Hik33 deletion variants were developed, in which either both or a portion of the transmembrane domains and/or the PAS domain were deleted. Cold stress was applied to the mutant strains either under illumination or in the dark. The findings show that the transmembrane domains govern temperature responses, whereas PAS domain may be involved in regulation of downstream gene expression in light-dependent manner.

The cyanobacterial FtsH4 protease controls accumulation of protein factors involved in the biogenesis of photosystem I.

Membrane-bound FtsH proteases are universally present in prokaryotes and in mitochondria and chloroplasts of eukaryotic cells. These metalloproteases are often critical for viability and play both protease and chaperone roles to maintain cellular homeostasis. In contrast to most bacteria bearing a single ftsH gene, cyanobacteria typically possess four FtsH proteases (FtsH1-4) forming heteromeric (FtsH1/3 and FtsH2/3) and homomeric (FtsH4) complexes. The functions and substrate repertoire of each complex are however poorly understood. To identify substrates of the FtsH4 protease complex we established a trapping assay in the cyanobacterium Synechocystis PCC 6803 utilizing a proteolytically inactivated trapFtsH4-His. Around 40 proteins were specifically enriched in trapFtsH4 pulldown when compared with the active FtsH4. As the list of putative FtsH4 substrates contained Ycf4 and Ycf37 assembly factors of Photosystem I (PSI), its core PsaB subunit and the IsiA chlorophyll-binding protein that associates with PSI during iron stress, we focused on these PSI-related proteins. Therefore, we analysed their degradation by FtsH4 in vivo in Synechocystis mutants and in vitro using purified substrates. The data confirmed that FtsH4 degrades Ycf4, Ycf37, IsiA, and also the individual PsaA and PsaB subunits in the unassembled state but not when assembled within the PSI complexes. A possible role of FtsH4 in the PSI life-cycle is discussed.

Conformational changes in a Photosystem II hydrogen bond network stabilize the oxygen-evolving complex.

The Mn4CaO5 oxygen-evolving complex (OEC) in Photosystem II (PSII) is assembled in situ and catalyzes water oxidation. After OEC assembly, the PsbO extrinsic subunit docks to the lumenal face of PSII and both stabilizes the OEC and facilitates efficient proton transfer to the lumen. D1 residue R334 is part of a hydrogen bond network involved in proton release during catalysis and interacts directly with PsbO. D1-R334 has recently been observed in different conformations in apo- and holo-OEC PSII structures. We generated a D1-R334G point mutant in Synechocystis sp. PCC 6803 to better understand this residue's function. D1-R334G PSII is active under continuous light, but the OEC is unstable in darkness. Isolated D1-R334G core complexes have little bound PsbO and less manganese as the wild type control. The S2 intermediate is stabilized in D1-R334G indicating that the local environment around the OEC has been altered. These results suggest that the hydrogen bond network that includes D1-R334 exists in a different functional conformation during PSII biogenesis in the absence of PsbO.

Self-Assembly of Nanofilaments in Cyanobacteria for Protein Co-localization.

Cyanobacteria offer great potential as alternative biotechnological hosts due to their photoautotrophic capacities. However, in comparison to established heterotrophic hosts, several key aspects, such as product titers, are still lagging behind. Nanobiotechnology is an emerging field with great potential to improve existing hosts, but so far, it has barely been explored in microbial photosynthetic systems. Here, we report the establishment of large proteinaceous nanofilaments in the unicellular model cyanobacterium Synechocystis sp. PCC 6803 and the fast-growing cyanobacterial strain Synechococcus elongatus UTEX 2973. Transmission electron microscopy and electron tomography demonstrated that expression of pduA*, encoding a modified bacterial microcompartment shell protein, led to the generation of bundles of longitudinally aligned nanofilaments in S. elongatus UTEX 2973 and shorter filamentous structures in Synechocystis sp. PCC 6803. Comparative proteomics showed that PduA* was at least 50 times more abundant than the second most abundant protein in the cell and that nanofilament assembly had only a minor impact on cellular metabolism. Finally, as a proof-of-concept for co-localization with the filaments, we targeted a fluorescent reporter protein, mCitrine, to PduA* by fusion with an encapsulation peptide that natively interacts with PduA. The establishment of nanofilaments in cyanobacterial cells is an important step toward cellular organization of heterologous pathways and the establishment of cyanobacteria as next-generation hosts.