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The assimilation of nitrate, a most important soil nitrogen source, is tightly regulated in microorganisms and plants. In Aspergillus nidulans, during the transcriptional activation process of nitrate assimilatory genes, the interaction between the pathway-specific transcription factor NirA and the exportin KapK/CRM1 is disrupted, and this leads to rapid nuclear accumulation and transcriptional activity of NirA. In this work by mass spectrometry, we found that in the absence of nitrate, when NirA is inactive and predominantly cytosolic, methionine 169 in the nuclear export sequence (NES) is oxidized to methionine sulfoxide (Metox169). This oxidation depends on FmoB, a flavin-containing monooxygenase which in vitro uses methionine and cysteine, but not glutathione, as oxidation substrates. The function of FmoB cannot be replaced by alternative Fmo proteins present in A. nidulans. Exposure of A. nidulans cells to nitrate led to rapid reduction of NirA-Metox169 to Met169; this reduction being independent from thioredoxin and classical methionine sulfoxide reductases. Replacement of Met169 by isoleucine, a sterically similar but not oxidizable residue, led to partial loss of NirA activity and insensitivity to FmoB-mediated nuclear export. In contrast, replacement of Met169 by alanine transformed the protein into a permanently nuclear and active transcription factor. Co-immunoprecipitation analysis of NirA-KapK interactions and subcellular localization studies of NirA mutants lacking different parts of the protein provided evidence that Met169 oxidation leads to a change in NirA conformation. Based on these results we propose that in the presence of nitrate the activation domain is exposed, but the NES is masked by a central portion of the protein (termed nitrate responsive domain, NiRD), thus restricting active NirA molecules to the nucleus. In the absence of nitrate, Met169 in the NES is oxidized by an FmoB-dependent process leading to loss of protection by the NiRD, NES exposure, and relocation of the inactive NirA to the cytosol.

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 (β-α-β-β-β-α) at its N-terminal region (residues 1–100), whereas the C terminus (residues 100–197) 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.

The assimilation of nitrate, a most important soil nitrogen source, is tightly regulated in microorganisms and plants. In Aspergillus nidulans, during the transcriptional activation process of nitrate assimilatory genes, the interaction between the pathway-specific transcription factor NirA and the exportin KapK/CRM1 is disrupted, and this leads to rapid nuclear accumulation and transcriptional activity of NirA. In this work by mass spectrometry, we found that in the absence of nitrate, when NirA is inactive and predominantly cytosolic, methionine 169 in the nuclear export sequence (NES) is oxidized to methionine sulfoxide (Metox169). This oxidation depends on FmoB, a flavin-containing monooxygenase which in vitro uses methionine and cysteine, but not glutathione, as oxidation substrates. The function of FmoB cannot be replaced by alternative Fmo proteins present in A. nidulans. Exposure of A. nidulans cells to nitrate led to rapid reduction of NirA-Metox169 to Met169; this reduction being independent from thioredoxin and classical methionine sulfoxide reductases. Replacement of Met169 by isoleucine, a sterically similar but not oxidizable residue, led to partial loss of NirA activity and insensitivity to FmoB-mediated nuclear export. In contrast, replacement of Met169 by alanine transformed the protein into a permanently nuclear and active transcription factor. Co-immunoprecipitation analysis of NirA-KapK interactions and subcellular localization studies of NirA mutants lacking different parts of the protein provided evidence that Met169 oxidation leads to a change in NirA conformation. Based on these results we propose that in the presence of nitrate the activation domain is exposed, but the NES is masked by a central portion of the protein (termed nitrate responsive domain, NiRD), thus restricting active NirA molecules to the nucleus. In the absence of nitrate, Met169 in the NES is oxidized by an FmoB-dependent process leading to loss of protection by the NiRD, NES exposure, and relocation of the inactive NirA to the cytosol.

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.

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.

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.

A light-repressed transcript encodes the LrtA protein in cyanobacteria. We show that half-life of lrtA transcript from Synechocystis sp. PCC 6803 is higher in dark-treated cells as compared to light-grown cells, suggesting post-transcriptional control of lrtA expression. The lrtA 5´ untranslated leader region is involved in that darkness-dependent regulation. We also found that Synechocystis sp. PCC 6803 LrtA is a ribosome-associated protein present in both 30S and 70S ribosomal particles. In order to investigate the function of this protein we have constructed a deletion mutant of the lrtA gene. Cells lacking LrtA (∆lrtA) had significantly lower amount of 70S particles and a greater amount of 30S and 50S particles, suggesting a role of LrtA in stabilizing 70S particles. Synechocystis strains with different amounts of LrtA protein: wild-type, ∆lrtA, and LrtAS (overexpressing lrtA) showed no differences in their growth rate under standard laboratory conditions. However, a clear LrtA dose-dependent effect was observed in the presence of the antibiotic tylosin, being the LrtAS strains the most sensitive. Similar results were obtained under hyperosmotic stress caused by sorbitol. Conversely, after prolonged periods of starvation, ∆lrtA strains were delayed in their growth with respect to the wild-type and the LrtAS strains. A positive role of LrtA protein in post-stress survival is proposed.

Background Cyanobacteria, as phototrophic organisms with low nutritional requirements and great metabolic versatility, are attractive for the sustainable production of value-added chemicals from CO 2 and sunlight. One limitation of these strategies is that carbon is partitioned towards biomass synthesis rather than product synthesis. An alternative to conventional metabolic engineering approaches involves controlling regulatory circuits to enhance the flow of carbon towards the synthesis of desired compounds. The carbon-flow-regulator A (CfrA) is pivotal in redirecting carbon flux during nitrogen deficiency in cyanobacteria, promoting glycogen accumulation by inhibiting 2,3-phosphoglycerate mutase enzyme. The moderately halotolerant cyanobacterium Synechocystis sp. PCC 6803 accumulates sucrose and glucosylglycerol (GG) as compatible solutes under salt stress. Sucrose is a valuable carbon source for heterotrophic organisms, whether they are cultivated independently or in co-cultures. In this context, we explored the potential biotechnological relevance of CfrA in redirecting carbon flow towards sucrose production. Results A strain that overexpresses cfrA , independently of nitrogen growth conditions, and carries a plasmid that expresses sucrose-phosphate synthase (SPS) from Synechocystis sp. PCC 6803 and the heterologous sucrose permease CscB inducibly (P ars -cfrA /suc strain) was constructed and analysed. In this strain, cfrA expression increased sucrose production by 40% compared to non-induced levels. The fixed carbon was partially redirected towards sucrose production at the expense of glycogen accumulation and biomass generation. Furthermore, an improvement in the photosynthetic activity of this strain was observed due to the presence of this carbon sink. The effect of eliminating GG synthesis (Δ ggpS /P ars -cfrA /suc strain) on sucrose production was also analyzed. Under high salinity conditions (400 mM NaCl), this strain exhibited a maximum sucrose accumulation of 2.72 g/L. Encapsulation of the P ars -cfrA /suc strain has also been studied. Conclusions Our results indicate that modulating carbon flow through CfrA overexpression can substantially boost sucrose production. Glycogen accumulation, mediated by CfrA, enhances sucrose production, which is partly derived from the use of stored glycogen. Furthermore, immobilising Synechocystis cells in alginate improves sucrose production and facilitates its utilisation. Given the widespread occurrence of the cfrA gene in cyanobacteria, its potential as a target in various biotechnological strategies that require the redirection of carbon flow should be considered.