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.España, MINECO BIO2016-75634-PJunta de Andalucía P12-BIO-1119 , BIO-28

NADP(+)-isocitrate dehydrogenase from the cyanobacterium Anabaena sp. strain PCC 7120: purification and characterization of the enzyme and cloning, sequencing, and disruption of the icd gene

NADP(+)-isocitrate dehydrogenase (NADP(+)-IDH) from the dinitrogen-fixing filamentous cyanobacterium Anabaena sp. strain PCC 7120 was purified to homogeneity. The native enzyme is composed of two identical subunits (M(r), 57,000) and cross-reacts with antibodies obtained against the previously purified NADP(+)-IDH from the unicellular cyanobacterium Synechocystis sp. strain PCC 6803. Anabaena NADP(+)-IDH resembles in its physicochemical and kinetic parameters the typical dimeric IDHs from prokaryotes. The gene encoding Anabaena NADP(+)-IDH was cloned by complementation of an Escherichia coli icd mutant with an Anabaena genomic library. The complementing DNA was located on a 6-kb fragment. It encodes an NADP(+)-IDH that has the same mobility as that of Anabaena NADP(+)-IDH on nondenaturing polyacrylamide gels. The icd gene was subcloned and sequenced. Translation of the nucleotide sequence gave a polypeptide of 473 amino acids that showed high sequence similarity to the E. coli enzyme (59% identity) and with IDH1 and IDH2, the two subunits of the heteromultimeric NAD(+)-IDH from Saccharomyces cerevisiae (30 to 35% identity); however, a low level of similarity to NADP(+)-IDHs of eukaryotic origin was found (23% identity). Furthermore, Anabaena NADP(+)-IDH contains a 44-residue amino acid sequence in its central region that is absent in the other IDHs so far sequenced. Attempts to generate icd mutants by insertional mutagenesis were unsuccessful, suggesting an essential role of IDH in Anabaena sp. strain PCC 7120.España, Dirección General de Investigación Científica y Técnica (grant PB91-0127

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.Deutsche Forschungsgemeinschaft KL 3114/2-1Ministerio de Economía y Competitividad BIO2016-75634-PFEDER BIO2016-75634-

A new type of glutamine synthetase in cyanobacteria: The protein encoded by the glnN gene supports nitrogen assimilation in Synechocystis sp. strain PCC 6803

A new glutamine synthetase gene, glnN, which encodes a polypeptide of 724 amino acid residues (M(r), 79,416), has been identified in the unicellular cyanobacterium Synechocystis sp. strain PCC 6803; this is the second gene that encodes a glutamine synthetase (GS) in this cyanobacterium. The functionality of this gene was evidenced by its ability to complement an Escherichia coli glnA mutant and to support Synechocystis growth in a strain whose glnA gene was inactivated by insertional mutagenesis. In this mutant (strain SJCR3), as well as in the wild-type strain, the second GS activity was subject to regulation by the nitrogen source, being strongly enhanced in nitrogen-free medium. Transcriptional fusion of a chloramphenicol acetyltransferase (cat) gene with the 5'-upstream region of glnN suggested that synthesis of the second Synechocystis GS is regulated at the transcriptional level. Furthermore, the level of glnN mRNA, a transcript of about 2,300 bases, was found to be strongly increased in nitrogen-free medium. The glnN product is similar to the GS subunits of Bacteroides fragilis and Butyrivibrio fibrisolvens, two obligate anaerobic bacteria whose GSs are markedly different from other prokaryotic and eukaryotic GSs. However, significant similarity is evident in the five regions which are homologous in all of the GSs so far described. The new GS gene was also found in other cyanobacteria but not in N2-fixing filamentous species.Dirección General de Investigación Científica y Técnica PB91-012

A core of three amino acids at the carboxyl-terminal region of glutamine synthetase defines its regulation in cyanobacteria

Glutamine synthetase (GS) type I is a key enzyme in nitrogen metabolism, and its activity is finely controlled by cellular carbon/nitrogen balance. In cyanobacteria, a reversible process that involves protein-protein interaction with two proteins, the inactivating factors IF7 and IF17, regulates GS. Previously, we showed that three arginine residues of IFs are critical for binding and inhibition of GS. In this work, taking advantage of the specificity of GS/IFs interaction in the model cyanobacteria Synechocystis sp. PCC 6803 and Anabaena sp. PCC 7120, we have constructed a different chimeric GSs from these two cyanobacteria. Analysis of these proteins, together with a site-directed mutagenesis approach, indicates that a core of three residues (E419, N456 and R459) is essential for the inactivation process. The three residues belong to the last 56 amino acids of the C-terminus of SynechocystisGS. A protein-protein docking modeling of SynechocystisGS in complex with IF7 supports the role of the identified core for GS/IF interaction.España, MINECO BFU 2010 - 15708 and BFU 2013 - 41712Junta de Andalucía Bio - 28

Regulation of glutamine synthetase activity in the unicellular cyanobacterium Synechocystis sp. strain PCC 6803 by the nitrogen source: Effect of ammonium

Glutamine synthetase activity from Synechocystis sp. strain PCC 6803 is regulated as a function of the nitrogen source available in the medium. Addition of 0.25 mM NH4Cl to nitrate-grown cells promotes a clear short-term inactivation of glutamine synthetase, whose enzyme activity decreases to 5 to 10% of the initial value in 25 min. The intracellular levels of glutamine, determined under various conditions, taken together with the results obtained with azaserine (an inhibitor of transamidases), rule out the possibility that glutamine per se is responsible for glutamine synthetase inactivation. Nitrogen starvation attenuates the ammonium-mediated glutamine synthetase inactivation, indicating that glutamine synthetase regulation is modulated through the internal balance between carbon-nitrogen compounds and carbon compounds. The parallelism observed between the glutamine synthetase activity and the internal concentration of α-ketoglutarate suggests that this metabolite could play a role as a positive effector of glutamine synthetase activity in Synechocystis sp. Despite the similarities of this physiological system to that described for enterobacteria, the lack of in vivo 32P labeling of glutamine synthetase during the inactivation process excludes the existence of an adenylylation-deadenylylation system in this cyanobacterium.Dirección General de Investigación Científica y Técnica PB 88-002

CopM is a novel copper-binding protein involved in copper resistance in Synechocystis sp. PCC 6803

Copper resistance system in the cyanobacterium Synechocystis sp. PCC 6803 comprises two operons, copMRS and copBAC, which are expressed in response to copper in the media. copBAC codes for a heavy-metal efflux–resistance nodulation and division (HME-RND) system, while copMRS codes for a protein of unknown function, CopM, and a two-component system CopRS, which controls the expression of these two operons. Here, we report that CopM is a periplasmic protein able to bind Cu(I) with high affinity (KD ∼3 × 10−16). Mutants lacking copM showed a sensitive copper phenotype similar to mutants affected in copB, but lower than mutants of the two-component system CopRS, suggesting that CopBAC and CopM constitute two independent resistance mechanisms. Moreover, constitutive expression of copM is able to partially suppress the copper sensitivity of the copR mutant strain, pointing out that CopM per se is able to confer copper resistance. Furthermore, constitutive expression of copM was able to reduce total cellular copper content of the copR mutant to the levels determined in the wild-type (WT) strain. Finally, CopM was localized not only in the periplasm but also in the extracellular space, suggesting that CopM can also prevent copper accumulation probably by direct copper binding outside the cell.España, Ministerio de Economía y Competitividad BFU2010–15708España, Ministerio de Economía y Competitividad BFU2013–41712-

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.Ministerio de Economía y Competitividad CTQ 2015-64445-R, BFU2013- 41712-P, BIO2016-75634PJunta de Andalucía BIO-284Generalitat Valenciana Prometeo 018/201

Overoxidation of 2-cys peroxiredoxin in prokaryotes: Cyanobacterial 2-cys peroxiredoxins sensitive to oxidative stress

In eukaryotic organisms, hydrogen peroxide has a dual effect; it is potentially toxic for the cell but also has an important signaling activity. According to the previously proposed floodgate hypothesis, the signaling activity of hydrogen peroxide in eukaryotes requires a transient increase in its concentration, which is due to the inactivation by overoxidation of 2-Cys peroxiredoxin (2-Cys Prx). Sensitivity to overoxidation depends on the structural GGLG and YF motifs present in eukaryotic 2-Cys Prxs and is believed to be absent from prokaryotic enzymes, thus representing a paradoxical gain of function exclusive to eukaryotic organisms. Here we show that 2-Cys Prxs from several prokaryotic organisms, including cyanobacteria, contain the GG(L/ V/I)G and YF motifs characteristic of sensitive enzymes. In search of the existence of overoxidation-sensitive 2-Cys Prxs in prokaryotes, we have analyzed the sensitivity to overoxidation of 2-Cys Prxs from two cyanobacterial strains, Anabaena sp. PCC7120 and Synechocystis sp. PCC6803. In vitro analysis of wild type and mutant variants of the Anabaena 2-Cys Prx showed that this enzyme is overoxidized at the peroxidatic cysteine residue, thus constituting an exception among prokaryotes. Moreover, the 2-Cys Prx from Anabaena is readily and reversibly overoxidized in vivo in response to high light and hydrogen peroxide, showing higher sensitivity to overoxidation than the Synechocystis enzyme. These cyanobacterial strains have different strategies to cope with hydrogen peroxide. While Synechocystis has low content of less sensitive 2-Cys Prx and high catalase activity, Anabaena contains abundant and sensitive 2-Cys Prx, but low catalase activity, which is remarkably similar to the chloroplast system.Ministerio de Educación y Ciencia BIO2007-60644, BFU2007-60300Junta de Andalucía P06-CVI01578, BIO-182, BIO-28