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The PII superfamily consists of widespread signal transduction proteins found in all domains of life. In addition to canonical PII proteins involved in C/N sensing, structurally similar PII-like proteins evolved to fulfill diverse, yet poorly understood cellular functions. In cyanobacteria, the bicarbonate transporter SbtA is co-transcribed with the conserved PII-like protein, SbtB, to augment intracellular inorganic carbon levels for efficient CO 2 fixation. We identified SbtB as a sensor of various adenine nucleotides including the second messenger nucleotides cyclic AMP (cAMP) and c-di-AMP. Moreover, many SbtB proteins possess a C-terminal extension with a disulfide bridge of potential redox-regulatory function, which we call R-loop. Here, we reveal an unusual ATP/ADP apyrase (diphosphohydrolase) activity of SbtB that is controlled by the R-loop. We followed the sequence of hydrolysis reactions from ATP over ADP to AMP in crystallographic snapshots and unravel the structural mechanism by which changes of the R-loop redox state modulate apyrase activity. We further gathered evidence that this redox state is controlled by thioredoxin, suggesting that it is generally linked to cellular metabolism, which is supported by physiological alterations in site-specific mutants of the SbtB protein. Finally, we present a refined model of how SbtB regulates SbtA activity, in which both the apyrase activity and its redox regulation play a central role. This highlights SbtB as a central switch point in cyanobacterial cell physiology, integrating not only signals from the energy state (adenyl-nucleotide binding) and the carbon supply via cAMP binding but also from the day/night status reported by the C-terminal redox switch.

Nitrogen limitation imposes a major transition in the lifestyle of nondiazotrophic cyanobacteria that is controlled by a complex interplay of regulatory factors involving the pervasive signal processor P II Immediately upon nitrogen limitation, newly fixed carbon is redirected toward glycogen synthesis. How the metabolic switch for diverting fixed carbon toward the synthesis of glycogen or of cellular building blocks is operated was so far poorly understood. Here, using the nondiazotrophic cyanobacterium Synechocystis sp. PCC 6803 as model system, we identified a novel P II interactor, the product of the sll0944 gene, which we named PirC. We show that PirC binds to and inhibits the activity of 2,3-phosphoglycerate-independent phosphoglycerate mutase (PGAM), the enzyme that deviates newly fixed CO 2 toward lower glycolysis. The binding of PirC to either P II or PGAM is tuned by the metabolite 2-oxoglutarate (2-OG), which accumulates upon nitrogen starvation. In these conditions, the high levels of 2-OG dissociate the PirC-P II complex to promote PirC binding to and inhibition of PGAM. Accordingly, a PirC-deficient mutant showed strongly reduced glycogen levels upon nitrogen deprivation, whereas polyhydroxybutyrate granules were overaccumulated compared to wild-type. Metabolome analysis revealed an imbalance in 3-phosphoglycerate to pyruvate levels in the pirC mutant, confirming that PirC controls the carbon flux in cyanobacteria via mutually exclusive interaction with either P II or PGAM., Competing Interests: The authors declare no competing interest., (Copyright © 2021 the Author(s). Published by PNAS.)

Cyanobacteria are phototrophic prokaryotes that evolved oxygenic photosynthesis ∼2.7 billion y ago and are presently responsible for ∼10% of total global photosynthetic production. To cope with the evolutionary pressure of dropping ambient CO 2 concentrations, they evolved a CO 2 -concentrating mechanism (CCM) to augment intracellular inorganic carbon (C i ) levels for efficient CO 2 fixation. However, how cyanobacteria sense the fluctuation in C i is poorly understood. Here we present biochemical, structural, and physiological insights into SbtB, a unique P II -like signaling protein, which provides new insights into C i sensing. SbtB is highly conserved in cyanobacteria and is coexpressed with CCM genes. The SbtB protein from the cyanobacterium Synechocystis sp. PCC 6803 bound a variety of adenosine nucleotides, including the second messenger cAMP. Cocrystal structures unraveled the individual binding modes of trimeric SbtB with AMP and cAMP. The nucleotide-binding pocket is located between the subunit clefts of SbtB, perfectly matching the structure of canonical P II proteins. This clearly indicates that proteins of the P II superfamily arose from a common ancestor, whose structurally conserved nucleotide-binding pocket has evolved to sense different adenyl nucleotides for various signaling functions. Moreover, we provide physiological and biochemical evidence for the involvement of SbtB in C i acclimation. Collectively, our results suggest that SbtB acts as a C i sensor protein via cAMP binding, highlighting an evolutionarily conserved role for cAMP in signaling the cellular carbon status., Competing Interests: The authors declare no conflict of interest.

The oceanic N 2 -fixing cyanobacterium Trichodesmium spp. form extensive surface blooms and contribute significantly to marine carbon and nitrogen cycles in the oligotrophic subtropical and tropical oceans. Trichodesmium grows in salinities from 27 to 43 parts per thousand (ppt), yet its salt acclimation strategy remains enigmatic because the genome of Trichodesmium erythraeum strain IMS101 lacks all genes for the biosynthesis of any known compatible solute. Using NMR and liquid chromatography coupled to mass spectroscopy, we identified the main compatible solute in T. erythraeum strain IMS101 as the quaternary ammonium compound N,N,N-trimethyl homoserine (or homoserine betaine) and elucidated its biosynthetic pathway. The identification of this compatible solute explains how Trichodesmium spp. can thrive in the marine system at varying salinities and provides further insight into the diversity of microbial salt acclimation., Competing Interests: The authors declare no conflict of interest.

Glutamine synthetase (GS), a key enzyme in biological nitrogen assimilation, is regulated in multiple ways in response to varying nitrogen sources and levels. Here we show a small regulatory RNA, NsiR4 (nitrogen stress-induced RNA 4), which plays an important role in the regulation of GS in cyanobacteria. NsiR4 expression in the unicellular Synechocystis sp. PCC 6803 and in the filamentous, nitrogen-fixing Anabaena sp. PCC 7120 is stimulated through nitrogen limitation via NtcA, the global transcriptional regulator of genes involved in nitrogen metabolism. NsiR4 is widely conserved throughout the cyanobacterial phylum, suggesting a conserved function. In silico target prediction, transcriptome profiling on pulse overexpression, and site-directed mutagenesis experiments using a heterologous reporter system showed that NsiR4 interacts with the 5'UTR of gifA mRNA, which encodes glutamine synthetase inactivating factor (IF)7. In Synechocystis, we observed an inverse relationship between the levels of NsiR4 and the accumulation of IF7 in vivo. This NsiR4-dependent modulation of gifA (IF7) mRNA accumulation influenced the glutamine pool and thus [Formula: see text] assimilation via GS. As a second target, we identified ssr1528, a hitherto uncharacterized nitrogen-regulated gene. Competition experiments between WT and an ΔnsiR4 KO mutant showed that the lack of NsiR4 led to decreased acclimation capabilities of Synechocystis toward oscillating nitrogen levels. These results suggest a role for NsiR4 in the regulation of nitrogen metabolism in cyanobacteria, especially for the adaptation to rapid changes in available nitrogen sources and concentrations. NsiR4 is, to our knowledge, the first identified bacterial sRNA regulating the primary assimilation of a macronutrient.

Photorespiratory 2-phosphoglycolate (2PG) metabolism is essential for photosynthesis in higher plants but thought to be superfluous in cyanobacteria because of their ability to concentrate CO(2) internally and thereby inhibit photorespiration. Here, we show that 3 routes for 2PG metabolism are present in the model cyanobacterium Synechocystis sp. strain PCC 6803. In addition to the photorespiratory C2 cycle characterized in plants, this cyanobacterium also possesses the bacterial glycerate pathway and is able to completely decarboxylate glyoxylate via oxalate. A triple mutant with defects in all 3 routes of 2PG metabolism exhibited a high-CO(2)-requiring (HCR) phenotype. All these catabolic routes start with glyoxylate, which can be synthesized by 2 different forms of glycolate dehydrogenase (GlcD). Mutants defective in one or both GlcD proteins accumulated glycolate under high CO(2) level and the double mutant DeltaglcD1/DeltaglcD2 was unable to grow under low CO(2). The HCR phenotype of both the double and the triple mutant could not be attributed to a significantly reduced affinity to CO(2), such as in other cyanobacterial HCR mutants defective in the CO(2)-concentrating mechanism (CCM). These unexpected findings of an HCR phenotype in the presence of an active CCM indicate that 2PG metabolism is essential for the viability of all organisms that perform oxygenic photosynthesis, including cyanobacteria and C3 plants, at ambient CO(2) conditions. These data and phylogenetic analyses suggest cyanobacteria as the evolutionary origin not only of oxygenic photosynthesis but also of an ancient photorespiratory 2PG metabolism.

In plants and microorganisms, salt stress regulates the expression of large numbers of genes. However, the machinery that senses salt stress remains to be characterized. In this study we identified sensory histidine kinases that are involved in the perception of salt stress in the cyanobacterium Synechocystis sp. strain PCC 6803. A library of strains with mutations in all 43 histidine kinases was screened by DNA microarray analysis of genomewide gene expression under salt stress. The results suggested that four histidine kinases, namely, Hik16, Hik33, Hik34, and Hik41, perceived and transduced salt signals. However, Hik33, Hik34, and Hik16 acting with Hik41 regulated the expression of different sets of genes. These histidine kinases regulated the expression of approximately 20% of the salt-inducible genes, whereas the induction of the remaining salt-inducible genes was unaffected by mutations in any of the histidine kinases, suggesting that additional sensory mechanisms might operate in the perception of salt stress. We also used DNA microarrays to investigate the effect of various salts on gene expression. Our results indicate that Hik33 responds to sodium salts and not to KCl, whereas the Hik16/Hik41 system responds only to NaCl.