Glycogen synthesis prevents metabolic imbalance and disruption of photosynthetic electron transport from photosystem II during transition to photomixotrophy in Synechocystis sp. PCC 6803.

Some cyanobacteria can grow photoautotrophically or photomixotrophically by using simultaneously CO2 and glucose. The switch between these trophic modes and the role of glycogen, their main carbon storage macromolecule, was investigated. We analysed the effect of glucose addition on the physiology, metabolic and photosynthetic state of Synechocystis sp. PCC 6803 and mutants lacking phosphoglucomutase and ADP-glucose pyrophosphorylase, with limitations in glycogen synthesis. Glycogen acted as a metabolic buffer: glucose addition increased growth and glycogen reserves in the wild-type (WT), but arrested growth in the glycogen synthesis mutants. Already 30 min after glucose addition, metabolites from the Calvin-Benson-Bassham cycle and the oxidative pentose phosphate shunt increased threefold more in the glycogen synthesis mutants than the WT. These alterations substantially affected the photosynthetic performance of the glycogen synthesis mutants, as O2 evolution and CO2 uptake were both impaired. We conclude that glycogen synthesis is essential during transitions to photomixotrophy to avoid metabolic imbalance that induces inhibition of electron transfer from PSII and subsequently accumulation of reactive oxygen species, loss of PSII core proteins, and cell death. Our study lays foundations for optimising photomixotrophy-based biotechnologies through understanding the coordination of the crosstalk between photosynthetic electron transport and metabolism.

Stress response requires an efficient connection between glycogen and central carbon metabolism by phosphoglucomutases in cyanobacteria.

Glycogen and starch are the main storage polysaccharides, acting as a source of carbon and energy when necessary. Interconversion of glucose-1-phosphate and glucose-6-phosphate by phosphoglucomutases connects the metabolism of these polysaccharides with central carbon metabolism. However, knowledge about how this connection affects the ability of cells to cope with environmental stresses is still scarce. The cyanobacterium Synechocystis sp. PCC 6803 has two enzymes with phosphoglucomutase activity, PGM (phosphoglucomutase) and PMM/PGM (phosphomannomutase/phosphoglucomutase). In this work, we generated a null mutant of PGM (∆PGM) that exhibits very reduced phosphoglucomutase activity (1% of wild type activity). Although this mutant accumulates moderate amounts of glycogen, its phenotype resembles that of glycogen-less mutants, including high light sensitivity and altered response to nitrogen deprivation. Using an on/off arsenite promoter, we demonstrate that PMM/PGM is essential for growth and responsible for the remaining phosphoglucomutase activity in the ∆PGM strain. Furthermore, overexpression of PMM/PGM in the ∆PGM strain is enough to revoke the phenotype of this mutant. These results emphasize the importance of an adequate flux between glycogen and central carbon metabolism to maintain cellular fitness and indicate that although PGM is the main phosphoglucomutase activity, the phosphoglucomutase activity of PMM/PGM can substitute it when expressed in sufficient amounts.

Back to the future: Transplanting the chloroplast TrxF-FBPase-SBPase redox system to cyanobacteria.

Fructose-1,6-bisphosphatase (FBPase) and sedoheptulose-1,7-bisphosphatase (SBPase) are two essential activities in the Calvin-Benson-Bassham cycle that catalyze two irreversible reactions and are key for proper regulation and functioning of the cycle. These two activities are codified by a single gene in all cyanobacteria, although some cyanobacteria contain an additional gene coding for a FBPase. Mutants lacking the gene coding for SBP/FBPase protein are not able to grow photoautotrophically and require glucose to survive. As this protein presents both activities, we have tried to elucidate which of the two are required for photoautrophic growth in Synechocystis sp PCC 6803. For this, the genes coding for plant FBPase and SBPase were introduced in a SBP/FBPase mutant strain, and the strains were tested for growth in the absence of glucose. Ectopic expression of only a plant SBPase gene did not allow growth in the absence of glucose although allowed mutation of both Synechocystis' FBPase genes. When both plant FBPase and SBPase genes were expressed, photoautrophic growth of the SBP/FBPase mutants was restored. This complementation was partial as the strain only grew in low light, but growth was impaired at higher light intensities. Redox regulation of the Calvin-Benson-Bassham cycle is essential to properly coordinate light reactions to carbon fixation in the chloroplast. Two of the best characterized proteins that are redox-regulated in the cycle are FBPase and SBPase. These two proteins are targets of the FTR-Trx redox system with Trx f being the main reductant in vivo. Introduction of the TrxF gene improves growth of the complemented strain, suggesting that the redox state of the proteins may be the cause of this phenotype. The redox state of the plant proteins was also checked in these strains, and it shows that the cyanobacterial redox system is able to reduce all of them (SBPase, FBPase, and TrxF) in a light-dependent manner. Thus, the TrxF-FBPase-SBPase plant chloroplast system is active in cyanobacteria despite that these organisms do not contain proteins related to them. Furthermore, our system opens the possibility to study specificity of the Trx system in vivo without the complication of the different isoforms present in plants.

The sRNA NsiR4 fine-tunes arginine synthesis in the cyanobacterium Synechocystis sp. PCC 6803 by post-transcriptional regulation of PirA.

As the only oxygenic phototrophs among prokaryotes, cyanobacteria employ intricate mechanisms to regulate common metabolic pathways. These mechanisms include small protein inhibitors exerting their function by protein-protein interaction with key metabolic enzymes and regulatory small RNAs (sRNAs). Here we show that the sRNA NsiR4, which is highly expressed under nitrogen limiting conditions, interacts with the mRNA of the recently described small protein PirA in the model strain Synechocystis sp. PCC 6803. In particular, NsiR4 targets the pirA 5'UTR close to the ribosome binding site. Heterologous reporter assays confirmed that this interaction interferes with pirA translation. PirA negatively impacts arginine synthesis under ammonium excess by competing with the central carbon/nitrogen regulator PII that binds to and thereby activates the key enzyme of arginine synthesis, N-acetyl-L-glutamate-kinase (NAGK). Consistently, ectopic nsiR4 expression in Synechocystis resulted in lowered PirA accumulation in response to ammonium upshifts, which also affected intracellular arginine pools. As NsiR4 and PirA are inversely regulated by the global nitrogen transcriptional regulator NtcA, this regulatory axis enables fine tuning of arginine synthesis and conveys additional metabolic flexibility under highly fluctuating nitrogen regimes. Pairs of small protein inhibitors and of sRNAs that control the abundance of these enzyme effectors at the post-transcriptional level appear as fundamental building blocks in the regulation of primary metabolism in cyanobacteria.

Exploring the Diversity of the Thioredoxin Systems in Cyanobacteria.

Cyanobacteria evolved the ability to perform oxygenic photosynthesis using light energy to reduce CO2 from electrons extracted from water and form nutrients. These organisms also developed light-dependent redox regulation through the Trx system, formed by thioredoxins (Trxs) and thioredoxin reductases (TRs). Trxs are thiol-disulfide oxidoreductases that serve as reducing substrates for target enzymes involved in numerous processes such as photosynthetic CO2 fixation and stress responses. We focus on the evolutionary diversity of Trx systems in cyanobacteria and discuss their phylogenetic relationships. The study shows that most cyanobacteria contain at least one copy of each identified Trx, and TrxA is the only one present in all genomes analyzed. Ferredoxin thioredoxin reductase (FTR) is present in all groups except Gloeobacter and Prochlorococcus, where there is a ferredoxin flavin-thioredoxin reductase (FFTR). Our data suggest that both TRs may have coexisted in ancestral cyanobacteria together with other evolutionarily related proteins such as NTRC or DDOR, probably used against oxidative stress. Phylogenetic studies indicate that they have different evolutionary histories. As cyanobacteria diversified to occupy new habitats, some of these proteins were gradually lost in some groups. Finally, we also review the physiological relevance of redox regulation in cyanobacteria through the study of target enzymes.

Extracellular vesicles as an alternative copper-secretion mechanism in bacteria.

Metal homeostasis is fundamental for optimal performance of cell metabolic pathways. Over the course of evolution, several systems emerged to warrant an intracellular metal equilibrium. When exposed to growth-challenging copper concentrations, Gram-negative bacteria quickly activate copper-detoxification mechanisms, dependent on transmembrane-protein complexes and metallochaperones that mediate metal efflux. Here, we show that vesiculation is also a common bacterial response mechanism to high copper concentrations, and that extracellular vesicles (EVs) play a role in transporting copper. We present evidence that bacteria from different ecological niches release copious amounts of EVs when exposed to copper. Along with the activation of the classical detoxification systems, we demonstrate that copper-stressed cells of the cyanobacterium Synechocystis sp. PCC6803 release EVs loaded with the copper-binding metallochaperone CopM. Under standard growth conditions, CopM-loaded EVs could also be isolated from a Synechocystis strain lacking a functional TolC-protein, which we characterize here as exhibiting a copper-sensitive phenotype. Analyses of Synechocystis tolC-mutant's EVs isolated from cells cultivated under standard conditions indicated the presence of copper therein, in significantly higher levels as compared to those from the wild-type. Altogether, these results suggest that release of EVs in bacteria represent a novel copper-secretion mechanism, shedding light into alternative mechanisms of bacterial metal resistance.

Depletion of m-type thioredoxin impairs photosynthesis, carbon fixation, and oxidative stress in cyanobacteria.

Thioredoxins (Trxs) are disulfide oxidoreductases that regulate many biological processes. The m-type thioredoxin (TrxA) is the only Trx present in all oxygenic photosynthetic organisms. Extensive biochemical and proteomic analyses have identified many TrxA target proteins in different photosynthetic organisms. However, the precise function of this essential protein in vivo is still poorly known. In this study, we generated a conditional Synechocystis sp. PCC 6803 mutant strain (STXA2) using an on-off promoter that is able to survive with only 2% of the TrxA level of the wild-type (WT) strain. STXA2 characterization revealed that TrxA depletion results in growth arrest and pronounced impairment of photosynthesis and the Calvin-Benson-Bassham (CBB) cycle. Analysis of the in vivo redox state of the bifunctional enzyme fructose-1,6-bisphosphatase/sedoheptulose-1,7-bisphosphatase showed higher levels of oxidation that affected enzyme activity in STXA2. This result implies that TrxA-mediated redox regulation of the CBB cycle is conserved in both cyanobacteria and chloroplasts, although the targets have different evolutionary origins. The STXA2 strain also accumulated more reactive oxygen species and was more sensitive to oxidative stress than the WT. Analysis of the in vivo redox state of 2-Cys peroxiredoxin revealed full oxidation, corresponding with TrxA depletion. Overall, these results indicate that depletion of TrxA in STXA2 greatly alters the cellular redox state, interfering with essential processes such as photosynthetic machinery operativity, carbon assimilation, and oxidative stress response. The TrxA regulatory role appears to be conserved along the evolution of oxygenic photosynthetic organisms.

The Novel PII-Interacting Protein PirA Controls Flux into the Cyanobacterial Ornithine-Ammonia Cycle.

Among prokaryotes, cyanobacteria have an exclusive position as they perform oxygenic photosynthesis. Cyanobacteria substantially differ from other bacteria in further aspects, e.g., they evolved a plethora of unique regulatory mechanisms to control primary metabolism. This is exemplified by the regulation of glutamine synthetase (GS) via small proteins termed inactivating factors (IFs). Here, we reveal another small protein, encoded by the ssr0692 gene in the model strain Synechocystis sp. PCC 6803, that regulates flux into the ornithine-ammonia cycle (OAC), the key hub of cyanobacterial nitrogen stockpiling and remobilization. This regulation is achieved by the interaction with the central carbon/nitrogen control protein PII, which commonly controls entry into the OAC by activating the key enzyme of arginine synthesis, N-acetyl-l-glutamate kinase (NAGK). In particular, the Ssr0692 protein competes with NAGK for PII binding and thereby prevents NAGK activation, which in turn lowers arginine synthesis. Accordingly, we termed it PII-interacting regulator of arginine synthesis (PirA). Similar to the GS IFs, PirA accumulates in response to ammonium upshift due to relief from repression by the global nitrogen control transcription factor NtcA. Consistent with this, the deletion of pirA affects the balance of metabolite pools of the OAC in response to ammonium shocks. Moreover, the PirA-PII interaction requires ADP and is prevented by PII mutations affecting the T-loop conformation, the major protein interaction surface of this signal processing protein. Thus, we propose that PirA is an integrator determining flux into N storage compounds not only depending on the N availability but also the energy state of the cell.IMPORTANCE Cyanobacteria contribute a significant portion to the annual oxygen yield and play important roles in biogeochemical cycles, e.g., as major primary producers. Due to their photosynthetic lifestyle, cyanobacteria also arouse interest as hosts for the sustainable production of fuel components and high-value chemicals. However, their broad application as microbial cell factories is hampered by limited knowledge about the regulation of metabolic fluxes in these organisms. Our research identified a novel regulatory protein that controls nitrogen flux, in particular arginine synthesis. Besides its role as a proteinogenic amino acid, arginine is a precursor for the cyanobacterial storage compound cyanophycin, which is of potential interest to biotechnology. Therefore, the obtained results will not only enhance our understanding of flux control in these organisms but also help to provide a scientific basis for targeted metabolic engineering and, hence, the design of photosynthesis-driven biotechnological applications.

A protease-mediated mechanism regulates the cytochrome c6/plastocyanin switch in Synechocystis sp. PCC 6803.

After the Great Oxidation Event (GOE), iron availability was greatly decreased, and photosynthetic organisms evolved several alternative proteins and mechanisms. One of these proteins, plastocyanin, is a type I blue-copper protein that can replace cytochrome c6 as a soluble electron carrier between cytochrome b6f and photosystem I. In most cyanobacteria, expression of these two alternative proteins is regulated by copper availability, but the regulatory system remains unknown. Herein, we provide evidence that the regulatory system is composed of a BlaI/CopY-family transcription factor (PetR) and a BlaR-membrane protease (PetP). PetR represses petE (plastocyanin) expression and activates petJ (cytochrome c6), while PetP controls PetR levels in vivo. Using whole-cell extracts, we demonstrated that PetR degradation requires both PetP and copper. Transcriptomic analysis revealed that the PetRP system regulates only four genes (petE, petJ, slr0601, and slr0602), highlighting its specificity. Furthermore, the presence of petE and petRP in early branching cyanobacteria indicates that acquisition of these genes could represent an early adaptation to decreased iron bioavailability following the GOE.

CfrA, a Novel Carbon Flow Regulator, Adapts Carbon Metabolism to Nitrogen Deficiency in Cyanobacteria.

Cyanobacteria unable to fix atmospheric nitrogen have evolved sophisticated adaptations to survive to long periods of nitrogen starvation. These genetic programs are still largely unknown-as evidenced by the many proteins whose expression is regulated in response to nitrogen availability, but which belong to unknown or hypothetical categories. In Synechocystis sp. PCC 6803, the global nitrogen regulator NtcA activates the expression of the sll0944 gene upon nitrogen deprivation. This gene encodes a protein that is highly conserved in cyanobacteria, but of unknown function. Based on the results described herein, we named the product of sll0944 carbon flow regulator A (CfrA). We analyzed the phenotypes of strains containing different levels of CfrA, including a knock-out strain (ΔcfrA), and two strains overexpressing CfrA from either the constitutive P trc promoter (Ptrc-cfrA) or the arsenite-inducible promoter P arsB (Pars-cfrA). Our results show that the amount of CfrA determines the accumulation of glycogen, and affects the synthesis of protein and photosynthetic pigments as well as amino acid pools. Strains with high levels of CfrA present high levels of glycogen and a decrease in photosynthetic pigments and protein content when nitrogen is available. Possible interactions between CfrA and the pyruvate dehydrogenase complex or PII protein have been revealed. The phenotype associated with CfrA overexpression is also observed in PII-deficient strains; however, it is lethal in this genetic background. Taken together, our results indicate a role for CfrA in the adaptation of carbon flux during acclimation to nitrogen deficiency.

Dynamics of the intrinsically disordered inhibitor IF7 of glutamine synthetase in isolation and in complex with its partner.

Glutamine synthetase (GS) catalyzes the ATP-dependent formation of glutamine from glutamate and ammonia. The activity of Synechocystis sp. PCC 6803 GS is regulated, among other mechanisms, by protein-protein interactions with a 65-residue-long, intrinsically disordered protein (IDP), named IF7. IDPs explore diverse conformations in their free states and, in some cases, in their molecular complexes. We used both nuclear magnetic resonance (NMR) at 11.7 T and small angle X-ray scattering (SAXS) to study the size and the dynamics in the picoseconds-to-nanosecond (ps-ns) timescale of: (i) isolated IF7; and (ii) the IF7/GS complex. Our SAXS findings, together with MD results, show: (i) some of the possible IF7 structures in solution; and, (ii) that the presence of IF7 affected the structure of GS in solution. The joint use of SAXS and NMR shows that movements of each amino acid of IF7 were uncorrelated with those of its neighbors. Residues of IF7 with the largest values of the relaxation rates (R1, R2 and ηxy), in the free and bound species, were mainly clustered around: (i) the C terminus of the protein; and (ii) Ala30. These residues, together with Arg8 (which is a hot-spot residue in the interaction with GS), had a restricted mobility in the presence of GS. The C-terminal region, which appeared more compact in our MD simulations of isolated IF7, seemed to be involved in non-native contacts with GS that help in the binding between the two macromolecules.

Lethality caused by ADP-glucose accumulation is suppressed by salt-induced carbon flux redirection in cyanobacteria.

Cyanobacteria are widely distributed photosynthetic organisms. During the day they store carbon, mainly as glycogen, to provide the energy and carbon source they require for maintenance during the night. Here, we generate a mutant strain of the freshwater cyanobacterium Synechocystis sp. PCC 6803 lacking both glycogen synthases. This mutant has a lethal phenotype due to massive accumulation of ADP-glucose, the substrate of glycogen synthases. This accumulation leads to alterations in its photosynthetic capacity and a dramatic decrease in the adenylate energy charge of the cell to values as low as 0.1. Lack of ADP-glucose pyrophosphorylase, the enzyme responsible for ADP-glucose synthesis, or reintroduction of any of the glycogen synthases abolishes the lethal phenotype. Viability of the glycogen synthase mutant is also fully recovered in NaCl-supplemented medium, which redirects the surplus of ADP-glucose to synthesize the osmolite glucosylglycerol. This alternative metabolic sink also suppresses phenotypes associated with the defective response to nitrogen deprivation characteristic of glycogen-less mutants, restoring the capacity to degrade phycobiliproteins. Thus, our system is an excellent example of how inadequate management of the adenine nucleotide pools results in a lethal phenotype, and the influence of metabolic carbon flux in cell viability and fitness.

Redox interference in nitrogen status via oxidative stress is mediated by 2-oxoglutarate in cyanobacteria.

Reactive oxygen species (ROS) are generated naturally in photosynthetic organisms by respiration and photosynthesis. Therefore, detoxification of these compounds, avoiding oxidative stress, is essential for proper cell function. In cyanobacteria, some observations point to a crosstalk between ROS homeostasis, in particular hydrogen peroxide, and nitrogen metabolism by a mechanism independent of known redox regulators. Using glutamine synthetase (GS), a finely regulated enzyme essential for nitrogen assimilation, as a tool, we were able to monitor nitrogen metabolism in relation to oxidative stress. We show that hydrogen peroxide clearly alters the expression of different genes related to nitrogen metabolism, both in the wild-type strain of the cyanobacterium Synechocystis sp. PCC 6803 and in a mutant strain lacking the catalase-peroxidase encoded by the katG gene and therefore highly sensitive to oxidative stress. As cyanobacteria perceive nitrogen status by sensing intracellular 2-oxoglutarate (2-OG) concentrations, the hydrogen peroxide effect was analysed under different nitrogen conditions in the wild-type, the ∆katG strain and in a strain able to transport 2-OG. The results obtained demonstrate that hydrogen peroxide interferes with signalling of cellular carbon : nitrogen status by decreasing the intracellular concentrations of 2-OG and hence altering the function of the 2-OG-sensing global nitrogen regulator NtcA.

Characterization of TrxC, an Atypical Thioredoxin Exclusively Present in Cyanobacteria.

Cyanobacteria form a diverse group of oxygenic photosynthetic prokaryotes considered to be the antecessor of plant chloroplast. They contain four different thioredoxins isoforms, three of them corresponding to m, x and y type present in plant chloroplast, while the fourth one (named TrxC) is exclusively found in cyanobacteria. TrxC has a modified active site (WCGLC) instead of the canonical (WCGPC) present in most thioredoxins. We have purified it and assayed its activity but surprisingly TrxC lacked all the classical activities, such as insulin precipitation or activation of the fructose-1,6-bisphosphatase. Mutants lacking trxC or over-expressing it were generated in the model cyanobacterium Synechocystis sp. PCC 6803 and their phenotypes have been analyzed. The ΔtrxC mutant grew at similar rates to WT in all conditions tested although it showed an increased carotenoid content especially under low carbon conditions. Overexpression strains showed reduced growth under the same conditions and accumulated lower amounts of carotenoids. They also showed lower oxygen evolution rates at high light but higher Fv'/Fm' and Non-photochemical-quenching (NPQ) in dark adapted cells, suggesting a more oxidized plastoquinone pool. All these data suggest that TrxC might have a role in regulating photosynthetic adaptation to low carbon and/or high light conditions.

The Distinctive Regulation of Cyanobacterial Glutamine Synthetase.

Glutamine synthetase (GS) features prominently in bacterial nitrogen assimilation as it catalyzes the entry of bioavailable nitrogen in form of ammonium into cellular metabolism. The classic example, the comprehensively characterized GS of enterobacteria, is subject to exquisite regulation at multiple levels, among them gene expression regulation to control GS abundance, as well as feedback inhibition and covalent modifications to control enzyme activity. Intriguingly, the GS of the ecologically important clade of cyanobacteria features fundamentally different regulatory systems to those of most prokaryotes. These include the interaction with small proteins, the so-called inactivating factors (IFs) that inhibit GS linearly with their abundance. In addition to this protein interaction-based regulation of GS activity, cyanobacteria use alternative elements to control the synthesis of GS and IFs at the transcriptional level. Moreover, cyanobacteria evolved unique RNA-based regulatory mechanisms such as glutamine riboswitches to tightly tune IF abundance. In this review, we aim to outline the current knowledge on the distinctive features of the cyanobacterial GS encompassing the overall control of its activity, sensing the nitrogen status, transcriptional and post-transcriptional regulation, as well as strain-specific differences.

The Cyanobacterial Ribosomal-Associated Protein LrtA from Synechocystis sp. PCC 6803 Is an Oligomeric Protein in Solution with Chameleonic Sequence Properties.

The LrtA protein of Synechocystis sp. PCC 6803 intervenes in cyanobacterial post-stress survival and in stabilizing 70S ribosomal particles. It belongs to the hibernating promoting factor (HPF) family of proteins, involved in protein synthesis. In this work, we studied the conformational preferences and stability of isolated LrtA in solution. At physiological conditions, as shown by hydrodynamic techniques, LrtA was involved in a self-association equilibrium. As indicated by Nuclear Magnetic Resonance (NMR), circular dichroism (CD) and fluorescence, the protein acquired a folded, native-like conformation between pH 6.0 and 9.0. However, that conformation was not very stable, as suggested by thermal and chemical denaturations followed by CD and fluorescence. Theoretical studies of its highly-charged sequence suggest that LrtA had a Janus sequence, with a context-dependent fold. Our modelling and molecular dynamics (MD) simulations indicate that the protein adopted the same fold observed in other members of the HPF family (ß-α-ß-ß-ß-α) at its N-terminal region (residues 1­100), whereas the C terminus (residues 100­197) appeared disordered and collapsed, supporting the overall percentage of overall secondary structure obtained by CD deconvolution. Then, LrtA has a chameleonic sequence and it is the first member of the HPF family involved in a self-association equilibrium, when isolated in solution.

Unprecedented pathway of reducing equivalents in a diflavin-linked disulfide oxidoreductase.

Flavoproteins participate in a wide variety of physiologically relevant processes that typically involve redox reactions. Within this protein superfamily, there exists a group that is able to transfer reducing equivalents from FAD to a redox-active disulfide bridge, which further reduces disulfide bridges in target proteins to regulate their structure and function. We have identified a previously undescribed type of flavin enzyme that is exclusive to oxygenic photosynthetic prokaryotes and that is based on the primary sequence that had been assigned as an NADPH-dependent thioredoxin reductase (NTR). However, our experimental data show that the protein does not transfer reducing equivalents from flavins to disulfides as in NTRs but functions in the opposite direction. High-resolution structures of the protein from Gloeobacter violaceus and Synechocystis sp. PCC6803 obtained by X-ray crystallography showed two juxtaposed FAD molecules per monomer in redox communication with an active disulfide bridge in a variant of the fold adopted by NTRs. We have tentatively named the flavoprotein "DDOR" (diflavin-linked disulfide oxidoreductase) and propose that its activity is linked to a thiol-based transfer of reducing equivalents in bacterial membranes. These findings expand the structural and mechanistic repertoire of flavoenzymes with oxidoreductase activity and pave the way to explore new protein engineering approaches aimed at designing redox-active proteins for diverse biotechnological applications.

Identification of the direct regulon of NtcA during early acclimation to nitrogen starvation in the cyanobacterium Synechocystis sp. PCC 6803.

In cyanobacteria, nitrogen homeostasis is maintained by an intricate regulatory network around transcription factor NtcA. Although mechanisms controlling NtcA activity appear to be well understood, its regulon remains poorly defined. To determine the NtcA regulon during the early stages of nitrogen starvation for the model cyanobacterium Synechocystis sp. PCC 6803, we performed chromatin immunoprecipitation, followed by sequencing (ChIP-seq), in parallel with transcriptome analysis (RNA-seq). Through combining these methods, we determined 51 genes activated and 28 repressed directly by NtcA. In addition to genes associated with nitrogen and carbon metabolism, a considerable number of genes without current functional annotation were among direct targets providing a rich reservoir for further studies. The NtcA regulon also included eight non-coding RNAs, of which Ncr1071, Syr6 and NsiR7 were experimentally validated, and their putative targets were computationally predicted. Surprisingly, we found substantial NtcA binding associated with delayed expression changes indicating that NtcA can reside in a poised state controlled by other factors. Indeed, a role of PipX as modulating factor in nitrogen regulation was confirmed for selected NtcA-targets. We suggest that the indicated poised state of NtcA enables a more differentiated response to nitrogen limitation and can be advantageous in native habitats of Synechocystis.

The isolated, twenty-three-residue-long, N-terminal region of the glutamine synthetase inactivating factor binds to its target.

Glutamine synthetase (GS) catalyzes the ATP-dependent formation of glutamine from glutamate and ammonia. The activity of Synechocystis sp. PCC 6803 GS type I is regulated by protein-protein interactions with a 65-residue-long protein (IF7). IF7 binds initially to GS through residues at its N terminus. In this work, we studied the conformational preferences of the N-terminal region of IF7 (IF7pep, residues Ala7-Ala29), its binding to GS and its functional properties. Isolated IF7pep populated a nascent helix in aqueous solution. IF7pep was bound to GS with an affinity constant of 0.4µM, and a 1:1 stoichiometry. IF7pep did not inactivate GS, suggesting that there were other IF7 regions important to carry out the inactivating function. Binding of IF7pep to GS was electrostatically-driven and it did not follow a kinetic two-state model.

Intrinsically disordered inhibitor of glutamine synthetase is a functional protein with random-coil-like pKa values.

The sequential action of glutamine synthetase (GS) and glutamate synthase (GOGAT) in cyanobacteria allows the incorporation of ammonium into carbon skeletons. In the cyanobacterium Synechocystis sp. PCC 6803, the activity of GS is modulated by the interaction with proteins, which include a 65-residue-long intrinsically disordered protein (IDP), the inactivating factor IF7. This interaction is regulated by the presence of charged residues in both IF7 and GS. To understand how charged amino acids can affect the binding of an IDP with its target and to provide clues on electrostatic interactions in disordered states of proteins, we measured the pKa values of all IF7 acidic groups (Glu32, Glu36, Glu38, Asp40, Asp58, and Ser65, the backbone C-terminus) at 100 mM NaCl concentration, by using NMR spectroscopy. We also obtained solution structures of IF7 through molecular dynamics simulation, validated them on the basis of previous experiments, and used them to obtain theoretical estimates of the pKa values. Titration values for the two Asp and three Glu residues of IF7 were similar to those reported for random-coil models, suggesting the lack of electrostatic interactions around these residues. Furthermore, our results suggest the presence of helical structure at the N-terminus of the protein and of conformational changes at acidic pH values. The overall experimental and in silico findings suggest that local interactions and conformational equilibria do not play a role in determining the electrostatic features of the acidic residues of IF7.