Synechocystis Strain Research Articles

Mutational analysis of the inactivating factors, IF7 and IF17 from Synechocystis sp. PCC 6803: critical role of arginine amino acid residues for glutamine synthetase inactivation

The Synechocystis sp. PCC 6803 glutamine synthetase type I (GS) activity is controlled by a process that involves protein-protein interaction with two inactivating factors (IF7 and IF17). IF7 is a natively unfolded, 65-residue-long protein, homologous to the carboxy-terminal region of IF17. Both proteins have abundance of positively charged amino acid residues and a high isoelectric point. In this study, we analyse the IF amino acid residues involved in GS inactivation by a mutational approach, both in vitro and in vivo. The results clearly indicate that the GS-IF complex formation must be determined mainly by electrostatic interactions. We have identified three conserved arginine residues of IF7 and IF17 that are essential for the interaction of these proteins with GS. All these residues map in the homologous region of IFs. Furthermore, in vitro analysis of a truncated IF17 protein without the 82-residue-long amino-terminal part, together with the analysis of a Synechocystis strain expressing a chimeric protein, containing this amino-terminal part of IF17 fused to IF7, demonstrates that amino-terminal region of IF17 mostly confers a higher stability to this protein.

Functional analysis of promoter fragments of salt-tolerance related genes in <I>Spirulina</I>

In this work, the functions of promoter fragments of two potential salt-tolerance related genes of Spirulina (Spirulina platensis Geitl.) were studied using green fluorescent protein gene (gfp) as a reporter. The promoter structures of two salt-tolerance related genes of Spirulina were predicted using online promoter prediction software. pMD18-T and pUC18 vectors were used to clone the promoter sequences as well as the gfp gene and kanamycine resistance (kan) gene. The fragments containing pro-gfp-kanr were further cloned into pKW1188 vector and the resulting recombinant plasmids were then transformed into a host strain Synechocystis sp. (Synechocystis pevalekii Ercegovic) PCC6803. The resulting bacterial strains were grown under various concentrations of salinity for defining time intervals. The bacterial fluorescence was observed using laser confocal microscope. Our results showed that the transgenic bacteria grown at different concentrations of salinity for various periods produced varying fluorescence intensities. The bacteria treated with NaCl at the concentrations of 0.4mol/L to 0.6mol/L for 6 to 8 h showed the strongest fluorescent intensity. From the result of high salt induced expression of gfp, we predicted that the genes under control of these two promoters are likely to play important roles in the salt tolerance of Spirulina. Accordingly, we believed that a research platform for the studying functions of the promoters of the salt-tolerance related genes in Spirulina has been developed with the gfp as a reporter, the kanr gene as the selection marker, and Synechocystis. sp. PCC6803 as the expression host.

The role of reduction in iron uptake processes in a unicellular, planktonic cyanobacterium

In many aquatic environments the essential micronutrient iron is predominantly complexed by a heterogeneous pool of strong organic chelators. Research on iron uptake mechanisms of cyanobacteria inhabiting these environments has focused on endogenous siderophore production and internalization. However, as many cyanobacterial species do not produce siderophores, alternative Fe acquisition mechanisms must exist. Here we present a study of the iron uptake pathways in the unicellular, planktonic, non-siderophore producing strain Synechocystis sp. PCC 6803. By applying trace metal clean techniques and a chemically controlled growth medium we obtained reliable and reproducible short-term (radioactive assays) and long-term (growth experiments) iron uptake rates. We found that Synechocystis 6803 is capable of acquiring iron from exogenous ferrisiderophores (Ferrioxamine-B, FeAerobactin) and that unchelated, inorganic Fe is a highly available source of iron. Inhibition of iron uptake by the Fe(II)-specific ligand, ferrozine, indicated that reduction of both inorganic iron and ferrisiderophore complexes occurs before transport through the plasma membrane. Measurements of iron reduction rates and the inhibitory effect of ferrozine on growth supported this conclusion. The reduction-based uptake strategy is well suited for acquiring iron from multiple complexes in dilute aquatic environments and may play an important role in other cyanobacterial strains.

Increased H 2 production in the cyanobacterium Synechocystis sp. strain PCC 6803 by redirecting the electron supply via genetic engineering of the nitrate assimilation pathway

The unicellular cyanobacterium Synechocystis sp. strain PCC 6803 contains a single bidirectional NiFe-Hox-hydrogenase, which evolves hydrogen under certain environmental conditions. The nitrate assimilation pathway is a potential competing pathway that may reduce the electron flow to the hydrogenase and thereby limit hydrogen production. To improve H 2 production, the nitrate assimilation pathway was disrupted by genetic engineering to redirect the electron flow towards the Hox-hydrogenase. Mutant strains disrupted in either nitrate reductase (Δ narB) or nitrite reductase (Δ nirA) or both nitrate reductase and nitrite reductase (Δ narB:Δ nirA) were constructed and tested for their ability to produce hydrogen. H 2 production and Hox-hydrogenase activities in all the mutant strains were higher than those in wild-type. Highest H 2 production was observed in the Δ narB:Δ nirA strain. Small changes were observed for Hox-hydrogenase enzyme activities and only minor changes in transcript levels of hoxH and hoxY were not correlated with H 2 production. The results suggest that the high rate of H 2 production observed in the Δ narB:Δ nirA strain of the cyanobacterium Synechocystis sp. strain PCC 6803 is the result of redirecting the electron supply from the nitrate assimilation pathway, through genetic engineering, towards the Hox-hydrogenase.

A synthetic DNA and fusion PCR approach to the ectopic expression of high levels of the D1 protein of photosystem II in Synechocystis sp. PCC 6803

A hybrid approach involving synthetic DNA, fusion PCR, and ectopic expression has been used to genetically manipulate the expression of the D1 protein of photosystem II (PSII) in the model cyanobacterium Synechocystis sp. PCC6803. Due to the toxicity of the full-length psbA gene in E. coli, a chimeric psbA2 gene locus was commercially synthesised and cloned in two halves. High-fidelity fusion PCR utilizing sequence overlap between the two synthetic gene halves allowed the production of a DNA fragment that was able to recombine the full-length psbA2 gene into the Synechocystis chromosome at an ectopic (non-native) location. This was accomplished by designing the synthetic DNA/fusion PCR product to have the psbA2 gene, with control sequences, interposed between chimeric sequences corresponding to an ectopic target chromosomal location. Additionally, a recipient strain of Synechocystis lacking all three psbA genes was produced by a combination of traditional marker replacement and markerless replacement techniques. Transformation of this multiple deletion strain by the synthetic DNA/fusion PCR product faithfully restored D1 expression in terms of its expression and PSII repair capacity. The advantages and potential issues for using this approach to rapidly introduce chimeric sequence characteristics as a general tool to produce novel genetic constructs are discussed.

The production of the sesquiterpene β-caryophyllene in a transgenic strain of the cyanobacterium Synechocystis

The plant secondary metabolite, β-caryophyllene, is a ubiquitous component of many plant resins that has traditionally been used in the cosmetics industry to provide a woody, spicy aroma to cosmetics and perfumes. Clinical studies have shown it to be potentially effective as an antibiotic, anesthetic, and anti-inflammatory agent. Additionally, there is significant interest in engineering phototrophic microorganisms with sesquiterpene synthase genes for the production of biofuels. Currently, the isolation of β-caryophyllene relies on purification methods from oleoresins extracted from large amounts of plant material. An engineered cyanobacterium platform that produces β-caryophyllene may provide a more sustainable and controllable means of production. To this end, the β-caryophyllene synthase gene ( QHS1) from Artemisia annua was stably inserted, via double homologous recombination, into the genome of the cyanobacterium Synechocystis sp. strain PCC6803. Gene insertion into Synechocystis was confirmed through PCR assays and sequencing reactions. Transcription and expression of QHS1 were confirmed using RT-PCR, and synthesis of β-caryophyllene was confirmed in the transgenic strain using GC-FID and GC–MS analysis.

Fatty acid composition of the heterotrophic nanoflagellate Paraphysomonas sp.: influence of diet and de novo biosynthesis

We compared the relative importance of dietary factors versus de novo synthesis in determin- ing the polyunsaturated fatty acid (PUFA) composi- tion of the heterotrophic nanoflagellate Paraphyso- monas sp. The flagellate was fed with different mutants of the picocyanobacterial strain Synechocystis PCC6803, which differ in their capability to synthe- size specific PUFAs. The desA, desB, and desD genes of Synechocystis PCC6803 encode lipid desaturases at the Δ12, Δ15, and Δ6 positions of 18C fatty acids (FAs), respectively. Thus, the use of desA - , desB - , desD - , and desA - /des D - mutants of Synechocystis PCC6803 as food sources permitted us to provide the heterotrophic flagellate with decreasing levels of unsaturated FAs. In each treatment, Paraphysomonas sp. exhibited the same FA composition pattern, i.e. high levels of 16:0 and 18:1, and significant amounts of 18C PUFAs and long-chain PUFAs such as 20:4(n-6), 20:5(n-3), and 22:6(n-3), which indicated that Paraphysomonas sp. is capable of synthesizing these PUFAs de novo. Results also showed that dietary 18C PUFAs seem to be preferentially accu- mulated in Paraphysomonas sp. lipids. This demon- strates that heterotrophic protists could play a key role in transferring essential compounds from pri- mary producers to metazoan consumers.

Spectroscopic and Functional Characterizations of Cyanobacterium Synechocystis PCC 6803 Mutants on and near the Heme Axial Ligand of Cytochrome b559 in Photosystem II

The functional role of cytochrome (cyt) b(559) in photosystem II (PSII) was investigated in H22K alpha and Y18S alpha cyt b(559) mutants of the cyanobacterium Synechocystis sp. PCC6803. H22K alpha and Y18S alpha cyt b(559) mutant carries one amino acid substitution on and near one of heme axial ligands of cyt b(559) in PSII, respectively. Both mutants grew photoautotrophically, assembled stable PSII, and exhibited the normal period-four oscillation in oxygen yield. However, both mutants showed several distinct chlorophyll a fluorescence properties and were more susceptible to photoinhibition than wild type. EPR results indicated the displacement of one of the two axial ligands to the heme of cyt b(559) in H22K alpha mutant reaction centers, at least in isolated reaction centers. The maximum absorption of cyt b(559) in Y18S alpha mutant PSII core complexes was shifted to 561 nm. Y18S alpha and H22K alpha mutant PSII core complexes contained predominately the low potential form of cyt b(559). The findings lend support to the concept that the redox properties of cyt b(559) are strongly influenced by the hydrophobicity and ligation environment of the heme. When the cyt b(559) mutations placed in a D1-D170A genetic background that prevents assembly of the manganese cluster, accumulation of PSII is almost completely abolished. Overall, our data support a functional role of cyt b(559) in protection of PSII under photoinhibition conditions in vivo.

Ferredoxin:NADP+ Oxidoreductase Association with Phycocyanin Modulates Its Properties

In photosynthetic organisms, ferredoxin:NADP(+) oxidoreductase (FNR) is known to provide NADPH for CO(2) assimilation, but it also utilizes NADPH to provide reduced ferredoxin. The cyanobacterium Synechocystis sp. strain PCC6803 produces two FNR isoforms, a small one (FNR(S)) similar to the one found in plant plastids and a large one (FNR(L)) that is associated with the phycobilisome, a light-harvesting complex. Here we show that a mutant lacking FNR(L) exhibits a higher NADP(+)/NADPH ratio. We also purified to homogeneity a phycobilisome subcomplex comprising FNR(L,) named FNR(L)-PC. The enzymatic activities of FNR(L)-PC were compared with those of FNR(S). During NADPH oxidation, FNR(L)-PC exhibits a 30% decrease in the Michaelis constant K(m)((NADPH)), and a 70% increase in K(m)((ferredoxin)), which is in agreement with its predicted lower activity of ferredoxin reduction. During NADP(+) reduction, the FNR(L)-PC shows a 29/43% decrease in the rate of single electron transfer from reduced ferredoxin in the presence/absence of NADP(+). The increase in K(m)((ferredoxin)) and the rate decrease of single reduction are attributed to steric hindrance by the phycocyanin moiety of FNR(L)-PC. Both isoforms are capable of catalyzing the NADP(+) reduction under multiple turnover conditions. Furthermore, we obtained evidence that, under high ionic strength conditions, electron transfer from reduced ferredoxin is rate limiting during this process. The differences that we observe might not fully explain the in vivo properties of the Synechocystis mutants expressing only one of the isoforms. Therefore, we advocate that FNR localization and/or substrates availability are essential in vivo.

Carotenoid-triggered energy dissipation in phycobilisomes of Synechocystis sp. PCC 6803 diverts excitation away from reaction centers of both photosystems

Cyanobacteria are capable of using dissipation of phycobilisome-absorbed energy into heat as part of their photoprotective strategy. Non-photochemical quenching in cyanobacteria cells is triggered by absorption of blue-green light by the carotenoid-binding protein, and involves quenching of phycobilisome fluorescence. In this study, we find direct evidence that the quenching is accompanied by a considerable reduction of energy flow to the photosystems. We present light saturation curves of photosystems’ activity in quenched and non-quenched states in the cyanobacterium Synechocystis sp. PCC 6803. In the quenched state, the quantum efficiency of light absorbed by phycobilisomes drops by about 30–40% for both photoreactions—P700 photooxidation in the photosystem II-less strain and photosystem II fluorescence induction in the photosystem I-less strain of Synechocystis. A similar decrease of the excitation pressure on both photosystems leads us to believe that the core–membrane linker allophycocyanin APC-LCM is at or beyond the point of non-photochemical quenching. We analyze 77 K fluorescence spectra and suggest that the quenching center is formed at the level of the short-wavelength allophycocyanin trimers. It seems that both chlorophyll and APC-LCM may dissipate excess energy via uphill energy transfer at physiological temperatures, but neither of the two is at the heart of the carotenoid-binding protein-dependent non-photochemical quenching mechanism.

HETEROGENEITY OF THE CYANOBACTERIAL GENUS SYNECHOCYSTIS AND DESCRIPTION OF A NEW GENUS, GEMINOCYSTIS(1).

The study and revision of the unicellular cyanobacterial genus Synechocystis was based on the type species S. aquatilis Sauv. and strain PCC 6803, a reference strain for this species. Uniformity in rRNA gene sequence, morphology, and ultrastructure was observed in all available Synechocystis strains, with the exception of the strain PCC 6308, which has been considered by some to be a model strain for Synechocystis. This strain differs substantially from the typical Synechocystis cluster according to both molecular (<90% of similarity, differences in 16S-23S rRNA internal transcribed spacer [ITS] secondary structure) and phenotypic criteria (different ultrastructure of cells). This strain is herein classified into the new genus Geminocystis gen. nov., as a sister taxon to the genus Cyanobacterium. Geminocystis differs from Cyanobacterium by genetic position (<94.4% of similarity) and more importantly by its different type of cell division. Because strain PCC 6308 was designated as a reference strain of the Synechocystis cluster 1 in Bergey's Manual, the members of this genetic cluster have to be revised and reclassified into Geminocystis gen. nov. Only the members of the Synechocystis cluster 2 allied with PCC 6803 correspond both genetically and phenotypically to the type species of the genus Synechocystis (S. aquatilis).

Current trends in high throughput proteomics in cyanobacteria

Advancements in genome sequencing and high throughput proteomics of cyanobacterial strains led to 13 published reports, from a small number of laboratories. These successful studies focused on Synechocystis, Nostoc and Anabaena strains, prochlorococcus, and halotolerant Euhalothece. The implications of emerging quantitative aspects developed and applied in these large-scale studies are assessed in the wake of advanced cyanobacterial research. Furthermore, contributions from traditional and early high throughput analysis of cyanobacterial proteomics are compared and summarised. Finally, opinions are provided to link both the trends and the future challenges. This review aims to push the synergy between proteomics and cyanobacterial research to improve both the technical and biological significance.

Photosynthetic Regulation of the Cyanobacterium Synechocystis sp. PCC 6803 Thioredoxin System and Functional Analysis of TrxB (Trx x) and TrxQ (Trx y) Thioredoxins

The expression of the genes encoding the ferredoxin-thioredoxin system including the ferredoxin-thioredoxin reductase (FTR) genes ftrC and ftrV and the four different thioredoxin genes trxA (m-type; slr0623), trxB (x-type; slr1139), trxC (sll1057) and trxQ (y-type; slr0233) of the cyanobacterium Synechocystis sp. PCC 6803 has been studied according to changes in the photosynthetic conditions. Experiments of light-dark transition indicate that the expression of all these genes except trxQ decreases in the dark in the absence of glucose in the growth medium. The use of two electron transport inhibitors, 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU) and 2,5-dibromo-3-methyl-6-isopropyl-p-benzoquinone (DBMIB), reveals a differential effect on thioredoxin genes expression being trxC and trxQ almost unaffected, whereas trxA, trxB, and the ftr genes are down-regulated. In the presence of glucose, DCMU does not affect gene expression but DBMIB still does. Analysis of the single TrxB or TrxQ and the double TrxB TrxQ Synechocystis mutant strains reveal different functions for each of these thioredoxins under different growth conditions. Finally, a Synechocystis strain was generated containing a mutated version of TrxB (TrxBC34S), which was used to identify the potential in-vivo targets of this thioredoxin by a proteomic analysis.

The thioredoxin reductase‐glutaredoxins‐ferredoxin crossroad pathway for selenate tolerance in Synechocystis PCC6803

Most organisms use two systems to maintain the redox homeostasis of cellular thiols. In the thioredoxin (Trx) system, NADPH sequentially reduces thioredoxin reductases (NTR), Trxs and protein disulfides. In the glutaredoxin (Grx) system, NADPH reduces the glutathione reductase enzyme occurring in most organisms, glutathione, Grxs, and protein disulfides or glutathione-protein mixed disulfides. As little is known concerning these enzymes in cyanobacteria, we have undertaken their analysis in the model strain Synechocystis PCC6803. We found that Grx1 and Grx2 are active, and that Grx2 but not Grx1 is crucial to tolerance to hydrogen peroxide and selenate. We also found that Synechocystis has no genuine glutathione reductase and uses NTR as a Grx electron donor, in a novel integrative pathway NADPH-NTR-Grx1-Grx2-Fed7 (ferredoxin 7), which operates in protection against selenate, the predominant form of selenium in the environment. This is the first report on the occurrence of a physical interaction between a Grx and a Fed, and of an electron transfer between two Grxs. These findings are discussed in terms of the (i) selectivity of Grxs and Feds (Synechocystis possesses nine Feds), (ii) crucial importance of NTR for cell fitness and (iii) resistance to selenate, in absence of a Thauera selenatis-like selenate reductase.

Transformation and gene replacement in the facultatively chemoheterotrophic, unicellular cyanobacterium Synechocystis sp. PCC6714 by electroporation

The unicellular cyanobacterium Synechocystis sp. PCC6714 can grow not only under photoautotrophic conditions, but also under chemoheterotrophic conditions if glucose is added to the medium. This makes it useful for the study of many aspects of bioenergetic mechanisms. In contrast to its closely related strain Synechocystis sp. PCC6803, which cannot grow chemoheterotrophically, Synechocystis PCC6714 is not naturally transformable. To enable gene transfer in this strain, we established a method for the introduction of self-replicating IncQ plasmids and for gene replacement using electroporation.

The Psb27 Protein Facilitates Manganese Cluster Assembly in Photosystem II

Photosystem II (PSII) is a large membrane protein complex that uses light energy to convert water to molecular oxygen. This enzyme undergoes an intricate assembly process to ensure accurate and efficient positioning of its many components. It has been proposed that the Psb27 protein, a lumenal extrinsic subunit, serves as a PSII assembly factor. Using a psb27 genetic deletion strain (Deltapsb27) of the cyanobacterium Synechocystis sp. PCC 6803, we have defined the role of the Psb27 protein in PSII biogenesis. While the Psb27 protein was not essential for photosynthetic activity, various PSII assembly assays revealed that the Deltapsb27 mutant was defective in integration of the Mn(4)Ca(1)Cl(x) cluster, the catalytic core of the oxygen-evolving machinery within the PSII complex. The other lumenal extrinsic proteins (PsbO, PsbU, PsbV, and PsbQ) are key components of the fully assembled PSII complex and are important for the water oxidation reaction, but we propose that the Psb27 protein has a distinct function separate from these subunits. We show that the Psb27 protein facilitates Mn(4)Ca(1)Cl(x) cluster assembly in PSII at least in part by preventing the premature association of the other extrinsic proteins. Thus, we propose an exchange of lumenal subunits and cofactors during PSII assembly, in that the Psb27 protein is replaced by the other extrinsic proteins upon assembly of the Mn(4)Ca(1)Cl(x) cluster. Furthermore, we show that the Psb27 protein provides a selective advantage for cyanobacterial cells under conditions such as nutrient deprivation where Mn(4)Ca(1)Cl(x) cluster assembly efficiency is critical for survival.

An AbrB-Like protein regulates the expression of the bidirectional hydrogenase in Synechocystis sp. strain PCC 6803.

In the unicellular cyanobacterium Synechocystis sp. strain PCC 6803, the pentameric bidirectional Ni-Fe hydrogenase (HoxEFUYH) is the sole enzyme involved in hydrogen metabolism. Recent investigations implicated the transcription factor LexA in the regulation of the hox genes in this cyanobacterium, suggesting the factor to work as an activator. In this work, we show evidence that LexA cannot account exclusively for the regulation of the hox genes in this cyanobacterium. Therefore, we investigated which additional transcription factors interact in and may regulate the expression of the hox genes in Synechocystis sp. strain PCC 6803. By using DNA affinity assays, a transcription factor with similarity to the transition state regulator AbrB from Bacillus subtilis was isolated. Electrophoretic mobility shift assays showed that the AbrB-like protein specifically interacts with the promoter region of the hox genes as well as with its own promoter region. In addition, results obtained with two genetically modified strains of Synechocystis sp. strain PCC 6803, one with a not fully segregated inactivation mutation of the abrB-like gene and the other overexpressing the same abrB-like gene, suggest that this transcription factor functions as a regulator of hox gene expression.

Photoassembly of the Manganese Cluster in Mutants Perturbed in the High Affinity Mn-Binding Site of the H2O-Oxidation Complex of Photosystem II

The light-driven, oxidative assembly of Mn2+ ions into the H2O-oxidation complex (WOC) of the photosystem II (PSII) reaction center is termed photoactivation and culminates in the formation of the oxygen-evolving (Mn4-Ca) center of the WOC. Initial binding and photooxidation of Mn2+ to the apoprotein is critically dependent upon aspartate 170 of the D1 protein (D1-D170) of the high affinity Mn site [Nixon and Diner (1992) Biochemistry 31, 942-948]. Three O2-evolving mutant strains of Synechocystis, D1-D170E, D1-D170H, and D1-D170V, were studied in terms of the kinetics of photoactivation under both continuous and flashing light. Photoactivation using single turnover flashes revealed D1-D170H and D1-D170V, but not D1-D170E, were prone to form substantial amounts ( approximately 40-50%) of inactive centers ascribed to photoligation of aberrant nonfunctional Mn based upon the reversibility of the inactivation and similarity to previous in vitro results [Chen, C., Kazimir, J., and Cheniae, G. M. (1995) Biochemistry 34, 13511-13526]. On the other hand, D1-D170E lowers the quantum efficiency of photoactivation compared to the wild-type by the largest amount (80% decrease) versus D1-D170H and D1-D170V, which do not produce measurable decreases in quantum efficiency. The low quantum efficiency of photoactivation in D1-D170E is due to the destabilization of photoactivation intermediates. Numerical analysis indicates that the PSII centers in D1-D170E are heterogeneous with respect to photoactivation kinetics and that the majority of centers are characterized by intermediates that decay approximately 10-fold more rapidly than the wild-type control. Additionally, the kinetics of O2 release during the S3-S0 transition was markedly retarded in D1-D170E, in contrast to D1-D170H and D1-D170V, which did not exhibit a discernible slow-down compared to the wild-type.

Toxicity assessment of crude and partially purified extracts of marine Synechocystis and Synechococcus cyanobacterial strains in marine invertebrates

Among the Cyanoprokaryota, the genera Synechocystis and Synechococcus have rarely been studied with respect to potential toxicity. This is particularly true with marine environments where studies about the toxicity of cyanobacteria are restricted to filamentous forms at the warmer temperate and tropical regions and also to filamentous forms at cold seas such as the Baltic Sea. In this study, we describe the effects of cyanobacterial strains of the Synechocystis and Synechococcus genera isolated from the marine coast of Portugal, on marine invertebrates. Crude and partially purified extracts at a concentration of 100 mg/ml of freeze-dried material of the marine strains were tested for acute toxicity in nauplii of the brine shrimp Artemia salina, in the rotifer Brachionus plicatillis and in embryos of the sea urchin Paracentrotus lividus and the mussel Mytilus galloprovincialis. The cyanobacterial extracts, especially the crude extract, had an impact on A. salina nauplii. No significant toxic effects were registered against the rotifer. A negative impact of all strains was recorded on the embryonic development of the sea urchin, with toxic effects resulting in an inhibition of embryogenesis or development of smaller larvae. To the mussel embryos, the effects of cyanobacterial extracts resulted in a complete inhibition of embryogenesis. The results of all assays indicate that Synechocystis and Synechococcus marine strains contained toxic compounds to marine invertebrates.

The ammonium‐inactivated cyanobacterial glutamine synthetase I is reactivated in vivo by a mechanism involving proteolytic removal of its inactivating factors

The Synechocystis sp. PCC 6803 glutamine synthetase type I (GS) activity is controlled by a process that involves protein-protein interaction with two inactivating factors (IF7 and IF17). Following addition of ammonium, the genes encoding these proteins, gifA and gifB, respectively, are derepressed, leading to the synthesis of IF7 and IF17 and consequently GS is inactivated. Upon ammonium removal, the GS activity rapidly returns to the initial level within 20 min. In this study, we analyse the mechanism underlying GS reactivation and find that this process involves IF7 and IF17 degradation. We show that the presence of ammonium as nitrogen source enhances IF17 but not IF7 stability independently of gif gene transcription. Studies with Synechocystis crude extracts under different conditions revealed that IF7 and IF17 display different stabilities in vitro. We found that IF7 is degraded in vitro by the activity of metalloproteases. Furthermore, the involvement of soluble processing metallopeptidases in IF7 degradation has also been demonstrated in vivo, by analysing Synechocystis mutant strains devoid of genes of the prp family. Finally, using a Synechocystis strain lacking GS type I, we establish the crucial role of the target protein GS for in vivo IF7 and IF17 stability.