Synechocystis Strain Research Articles

Regulation of biofilm formation and cellular buoyancy through modulating intracellular cyclic di-GMP levels in engineered cyanobacteria.

The second messenger cyclic dimeric (3'→5') GMP (cyclic di-GMP or c-di-GMP) has been implicated in the transition between motile and sessile lifestyles in bacteria. In this study, we demonstrate that biofilm formation, cellular aggregation or flocculation, and cellular buoyancy are under the control of c-di-GMP in Synechocystis sp. PCC 6803 (Synechocystis) and Fremyella diplosiphon. Synechocystis is a unicellular cyanobacterium and displays lower levels of c-di-GMP; F. diplosiphon is filamentous and displays higher intracellular c-di-GMP levels. We transformed Synechocystis and F. diplosiphon with a plasmid for constitutive expression of genes encoding diguanylate cylase (DGC) and phosphodiesterase (PDE) proteins from Vibrio cholerae or Escherichia coli, respectively. These engineered strains allowed us to modulate intracellular c-di-GMP levels. Biofilm formation and cellular deposition were induced in the DGC-expressing Synechocystis strain which exhibited high intracellular levels of c-di-GMP; whereas strains expressing PDE in Synechocystis and F. diplosiphon to drive low intracellular levels of c-di-GMP exhibited enhanced cellular buoyancy. In addition, the PDE-expressing F. diplosiphon strain showed elevated chlorophyll levels. These results imply roles for coordinating c-di-GMP homeostasis in regulating native cyanobacterial phenotypes. Engineering exogenous DGC or PDE proteins to regulate intracellular c-di-GMP levels represents an effective tool for uncovering cryptic phenotypes or modulating phenotypes in cyanobacteria for practical applications in biotechnology applicable in photobioreactors and in green biotechnologies, such as energy-efficient harvesting of cellular biomass or the treatment of metal-containing wastewaters.

Metabolomic basis of laboratory evolution of butanol tolerance in photosynthetic Synechocystis sp. PCC 6803.

BackgroundRecent efforts demonstrated the potential application of cyanobacteria as a “microbial cell factory” to produce butanol directly from CO2. However, cyanobacteria have very low tolerance to the toxic butanol, which limits the economic viability of this renewable system.ResultsThrough a long-term experimental evolution process, we achieved a 150% increase of the butanol tolerance in a model cyanobacterium Synechocystis sp. PCC 6803 after a continuous 94 passages for 395 days in BG11 media amended with gradually increased butanol concentration from 0.2% to 0.5% (v/v). To decipher the molecular mechanism responsible for the tolerance increase, we employed an integrated GC-MS and LC-MS approach to determine metabolomic profiles of the butanol-tolerant Synechocystis strains isolated from several stages of the evolution, and then applied PCA and WGCNA network analyses to identify the key metabolites and metabolic modules related to the increased tolerance. The results showed that unstable metabolites of 3-phosphoglyceric acid (3PG), D-fructose 6-phosphate (F6P), D-glucose 6-phosphate (G6P), NADPH, phosphoenolpyruvic acid (PEP), D-ribose 5-phosphate (R5P), and stable metabolites of glycerol, L-serine and stearic acid were differentially regulated during the evolution process, which could be related to tolerance increase to butanol in Synechocystis.ConclusionsThe study provided the first time-series description of the metabolomic changes related to the gradual increase of butanol tolerance, and revealed a metabolomic basis important for rational tolerance engineering in Synechocystis.Electronic supplementary materialThe online version of this article (doi:10.1186/s12934-014-0151-y) contains supplementary material, which is available to authorized users.

Function and association of CyanoP in photosystem II of Synechocystis sp. PCC 6803

The CyanoP protein is a cyanobacterial homolog of the PsbP protein, which is an extrinsic subunit of photosystem II (PSII) in green plant species. The molecular function of CyanoP has been investigated in mutant strains of Synechocystis but inconsistent results have been reported by different laboratories. In this study, we generated and characterized a Synechocystis mutant in which entire region of the CyanoP gene was eliminated. After repeated subculture in CaCl2-depleted medium, growth retardation was clearly observed for a CyanoP knockout mutant of Synechocystis sp. PCC 6803 (∆P). The PSII-mediated oxygen-evolving activity of the ∆P cells was more susceptible to depletion of CaCl2 than that of wild-type cells. The 77 K fluorescence emission spectra indicated that energy coupling between phycobilisome and PSII was perturbed in both wild-type and ∆P cells under CaCl2-depleted conditions, and was more evident for the ∆P mutant. To examine the association of CyanoP with PSII complexes, we tested several detergents for solubilization of thylakoid membranes and showed that CyanoP was partly included in fractions containing large protein complexes in gel-filtration analysis. These results indicate that CyanoP constitutively stabilizes PSII functionality in vivo.

Broad-host-range vector system for synthetic biology and biotechnology in cyanobacteria.

Inspired by the developments of synthetic biology and the need for improved genetic tools to exploit cyanobacteria for the production of renewable bioproducts, we developed a versatile platform for the construction of broad-host-range vector systems. This platform includes the following features: (i) an efficient assembly strategy in which modules released from 3 to 4 donor plasmids or produced by polymerase chain reaction are assembled by isothermal assembly guided by short GC-rich overlap sequences. (ii) A growing library of molecular devices categorized in three major groups: (a) replication and chromosomal integration; (b) antibiotic resistance; (c) functional modules. These modules can be assembled in different combinations to construct a variety of autonomously replicating plasmids and suicide plasmids for gene knockout and knockin. (iii) A web service, the CYANO-VECTOR assembly portal, which was built to organize the various modules, facilitate the in silico construction of plasmids, and encourage the use of this system. This work also resulted in the construction of an improved broad-host-range replicon derived from RSF1010, which replicates in several phylogenetically distinct strains including a new experimental model strain Synechocystis sp. WHSyn, and the characterization of nine antibiotic cassettes, four reporter genes, four promoters, and a ribozyme-based insulator in several diverse cyanobacterial strains.

Chronic Response of Three Different Cyanobacterial Species on Growth, Pigment, and Metabolic Variations to the High Molecular Weight Polycyclic Aromatic Hydrocarbon – Pyrene

A study was carried out to investigate the chronic response of three different cyanobacterial species namely Anabaena fertilissima, Synechocystis sp., and Nostoc muscorum to Pyrene, a high molecular weight (HMW) polycyclic aromatic hydrocarbons (PAHs). Organisms were treated based upon LC50 (lethal concentration at which 50% reduction in growth) concentrations, i.e., 3.0 ppm for Anabaena fertilissima, 3.0 ppm for Synechocystis sp., and 0.5 ppm for Nostoc muscorum as well as finalized one lower and one higher dose to LC50. The influence of pyrene on growth, pigments, release of metabolites, such as carbohydrates, protein, amino acid, and phenols, was carried out. The test doses caused a concentration dependent decrease in carotenoids and phycobilliproteins and showed more sensitivity for pyrene. Depletion of carbohydrate by 47–98% and proteins by 39–81% was encountered with rise in pyrene concentrations after 16th day of exposure in case of high tolerant strain Synechocystis sp. However, phenols were found to rise by 64–93% with increased pyrene concentrations on the contrary, amino acids were reported to decline by 59–93%. The order for the sensitivity of the test species is as follows: Nostoc muscorum > Anabaena fertillissima > Synechocystis sp.

Engineering Synechocystis PCC6803 for Hydrogen Production: Influence on the Tolerance to Oxidative and Sugar Stresses

In the prospect of engineering cyanobacteria for the biological photoproduction of hydrogen, we have studied the hydrogen production machine in the model unicellular strain Synechocystis PCC6803 through gene deletion, and overexpression (constitutive or controlled by the growth temperature). We demonstrate that the hydrogenase-encoding hoxEFUYH operon is dispensable to standard photoautotrophic growth in absence of stress, and it operates in cell defense against oxidative (H2O2) and sugar (glucose and glycerol) stresses. Furthermore, we showed that the simultaneous over-production of the proteins HoxEFUYH and HypABCDE (assembly of hydrogenase), combined to an increase in nickel availability, led to an approximately 20-fold increase in the level of active hydrogenase. These novel results and mutants have major implications for those interested in hydrogenase, hydrogen production and redox metabolism, and their connections with environmental conditions.

Design and analysis of LacI-repressed promoters and DNA-looping in a cyanobacterium

BackgroundCyanobacteria are solar-powered prokaryotes useful for sustainable production of valuable molecules, but orthogonal and regulated promoters are lacking. The Lac repressor (LacI) from Escherichia coli is a well-studied transcription factor that is orthogonal to cyanobacteria and represses transcription by binding a primary lac operator (lacO), blocking RNA-polymerase. Repression can be enhanced through DNA-looping, when a LacI-tetramer binds two spatially separated lacO and loops the DNA. Ptrc is a commonly used LacI-repressed promoter that is inefficiently repressed in the cyanobacterium Synechocystis PCC 6803. Ptrc2O, a version of Ptrc with two lacO, is more efficiently repressed, indicating DNA-looping. To investigate the inefficient repression of Ptrc and cyanobacterial DNA-looping, we designed a Ptrc-derived promoter library consisting of single lacO promoters, including a version of Ptrc with a stronger lacO (Ptrc1O-proximal), and dual lacO promoters with varying inter-lacO distances (the Ptrc2O-library).ResultsWe first characterized artificial constitutive promoters and used one for engineering a LacI-expressing strain of Synechocystis. Using this strain, we observed that Ptrc1O-proximal is similar to Ptrc in being inefficiently repressed. Further, the Ptrc2O-library displays a periodic repression pattern that remains for both non- and induced conditions and decreases with longer inter-lacO distances, in both E. coli and Synechocystis. Repression of Ptrc2O-library promoters with operators out of phase is less efficient in Synechocystis than in E. coli, whereas repression of promoters with lacO in phase is efficient even under induced conditions in Synechocystis. Two well-repressed Ptrc2O promoters were highly active when tested in absence of LacI in Synechocystis.ConclusionsThe artificial constitutive promoters herein characterized can be utilized for expression in cyanobacteria, as demonstrated for LacI. The inefficient repression of Ptrc and Ptrc1O-proximal in Synechocystis, as compared to E. coli, may be due to insufficient LacI expression, or differences in RNAP subunits. DNA-looping works as a transcriptional regulation mechanism similarly as in E. coli. DNA-looping contributes strongly to Ptrc2O-library repression in Synechocystis, even though they contain the weakly-repressed primary lacO of Ptrc1O-proximal and relatively low levels of LacI/cell. Hence, Synechocystis RNAP may be more sensitive to DNA-looping than E. coli RNAP, and/or the chromatin torsion resistance could be lower. Two strong and highly repressed Ptrc2O promoters could be used without induction, or together with an unstable LacI.

Crystal structure of the Psb28 accessory factor of Thermosynechococcus elongatus photosystem II at 2.3 Å

Members of the Psb28 family of proteins are accessory factors implicated in the assembly and repair of the photosystem II complex. We present here the crystal structure of the Psb28 protein (Tlr0493) found in the thermophilic cyanobacterium Thermosynechococcus elongatus at a resolution of 2.3Å. Overall the crystal structure of the Psb28 monomer is similar to the solution structures of C-terminally His-tagged Psb28-1 from Synechocystis sp. PCC 6803 obtained previously by nuclear magnetic resonance spectroscopy. One new aspect is that Escherichia coli-expressed T. elongatus Psb28 is able to form dimers in solution and packs as a dimer of dimers in the crystal. Analysis of wild type and mutant strains of Synechocystis 6803 by blue native-polyacrylamide gel electrophoresis suggests that Psb28-1, the closest homologue to T. elongatus Psb28 in this organism, also exists as an oligomer in vivo, most likely a dimer. In line with the prediction based on the crystal structure of T. elongatus Psb28, the addition of a 3× Flag-tag to the C-terminus of Synechocystis 6803 Psb28-1 interferes with the accumulation of the Psb28-1 oligomer in vivo. In contrast, the more distantly related Psb28-2 protein found in Synechocystis 6803 lacks the residues that stabilize dimer formation in the T. elongatus Psb28 crystal and is detected as a monomer in vivo. Overall our data suggest that the dimer interface in the Psb28 crystal might be physiologically relevant.

English

Two cyanobacterial strains Synechocystis “AHZ-HB-MK” and “AHZ-HB-P2A” were isolated from tannery effluent form Lahore, Pakistan and their tolerance against K2CrO4was examined. Although growth was completely inhibited at 10 µg mL-1 in AHZ-HB-P2Astrain, cell growth of AHZ-HB-MK strain was observed even at a concentration of 100 µg mL-1. To show reasons for different tolerance between the two strains, we compared changes in nonprotein thiols, glutathione (GSH and GSSG), cysteine and cystine, after K2CrO4 treatment in both strains. After K2CrO4 treatment GSH completely disappeared in both strains, suggesting that GSH was involved in detoxification. GSSG content in former strain remarkably increased almost three and six times after 7-days treatment with 10 and 100 µg mL-1 K2CrO4, respectively while it decreased in the later strain. Cysteine content in the former strain increased almost three and eleven times after 1 day and 7 days treatment with 100 µg mL-1 K2CrO4, respectively, such increase in cysteine content was not observed in later strain. These data revealed that former strain could induce glutathione and cysteine but later strain could not. The difference in the ability to induce the nonprotein thiols between two strains must be responsible for differential tolerance in K2CrO4 observed in the growth inhibition.   Key words: Chromium, cysteine, cyanobacteria, glutathione, nonprotein thiol,Synechocystis.

Exopolysaccharides protect Synechocystis against the deleterious effects of Titanium dioxide nanoparticles in natural and artificial waters

We have studied the effect of TiO2 nanoparticles (NPs) on the model cyanobacteria Synechocystis PCC6803. We used well-characterized NPs suspensions in artificial and natural (Seine River, France) waters. We report that NPs trigger direct (cell killing) and indirect (cell sedimentation precluding the capture of light, which is crucial to photosynthesis) deleterious effects. Both toxic effects increase with NPs concentration and are exacerbated by the presence of UVAs that increase the production of Reactive Oxygen Species (hydroxyl and superoxide radicals) by TiO2 NPs. Furthermore, we compared the responses of the wild-type strain of Synechocystis, which possesses abundant exopolysaccharides surrounding the cells, to that of an EPS-depleted mutant. We show, for the first time, that the exopolysaccharides play a crucial role in Synechocystis protection against cell killing caused by TiO2 NPs.

Influence of exopolysaccharides on the electrophoretic properties of the model cyanobacterium Synechocystis

The influence of extracellular polymeric substances (EPS) on cell electrokinetics was investigated in the model cyanobacterium Synechocystis, in wild-type strains and in ten EPS-depleted mutants. The charge density and the softness of the EPS polyelectrolyte layer were calculated from the dependence of the electrophoretic mobility values of the cells with the ionic strength of the surrounding fluid. Electrophoretic mobility data showed that the eleven Synechocystis strains investigated behave as soft particles and cannot be adequately described by classical electrokinetic models of rigid particles. EPS surrounding the cells, especially those released in the growth medium, significantly increased the softness of the cell surface. Furthermore, the anionic nature of EPS resulted in negative surface charge densities, which appeared to be strongly dependent on the composition of the suspending fluid, as documented by a strong reduction of their absolute values in the presence of calcium cations. These finding stresses the importance of the physicochemical properties of EPS and cell surfaces of cyanobacteria, for both cell-to-medium and cell-to-cell communications. In turn, these results emphasize that, whenever possible, natural waters should be used for meaningful ecotoxicological analyses of potential toxics.

Cyanobacterial defense mechanisms against foreign DNA transfer and their impact on genetic engineering

Cyanobacteria display a large diversity of cellular forms ranging from unicellular to complex multicellular filaments or aggregates. Species in the group present a wide range of metabolic characteristics including the fixation of atmospheric nitrogen, resistance to extreme environments, production of hydrogen, secondary metabolites and exopolysaccharides. These characteristics led to the growing interest in cyanobacteria across the fields of ecology, evolution, cell biology and biotechnology. The number of available cyanobacterial genome sequences has increased considerably in recent years, with more than 140 fully sequenced genomes to date. Genetic engineering of cyanobacteria is widely applied to the model unicellular strains Synechocystis sp. PCC 6803 and Synechococcus elongatus PCC 7942. However the establishment of transformation protocols in many other cyanobacterial strains is challenging. One obstacle to the development of these novel model organisms is that many species have doubling times of 48 h or more, much longer than the bacterial models E. coli or B. subtilis. Furthermore, cyanobacterial defense mechanisms against foreign DNA pose a physical and biochemical barrier to DNA insertion in most strains. Here we review the various barriers to DNA uptake in the context of lateral gene transfer among microbes and the various mechanisms for DNA acquisition within the prokaryotic domain. Understanding the cyanobacterial defense mechanisms is expected to assist in the development and establishment of novel transformation protocols that are specifically suitable for this group.

Complete Sequence of the First Chimera Genome Constructed by Cloning the Whole Genome of Synechocystis Strain PCC6803 into the Bacillus subtilis 168 Genome

Genome synthesis of existing or designed genomes is made feasible by the first successful cloning of a cyanobacterium, Synechocystis PCC6803, in Gram-positive, endospore-forming Bacillus subtilis. Whole-genome sequence analysis of the isolate and parental B. subtilis strains provides clues for identifying single nucleotide polymorphisms (SNPs) in the 2 complete bacterial genomes in one cell.

Metabolic phenotyping of the cyanobacterium Synechocystis 6803 engineered for production of alkanes and free fatty acids

We demonstrate a simple high-throughput single-cell approach that exploits the ultrahigh brightness and non-invasive nature of synchrotron infrared beam to characterize strains of the cyanobacterium Synechocystis 6803 (S. 6803) constructed with altered metabolic traits affecting the acyl-CoA pool. Their metabolic responses to the modified traits were phenotyped by single-cell synchrotron radiation Fourier transform infrared (SR-FTIR) spectromicroscopy and multivariate analysis. SR-FTIR difference spectra and cluster vector plots segregated the strains as phenotypic populations based on signals in the hydrocarbon and biomolecular fingerprint regions, although each population incorporated a stochastic distribution of cells with different metabolic properties. All engineered strains exhibited an increase in FTIR features attributed to functional groups in hydrocarbon, fatty acid (FA), and/or FA ester chains, and a decrease in polysaccharide features. The metabolic signatures obtained by SR-FTIR were consistent with detailed qualitative and quantitative metabolic information provided in GC/MS/NMR data. A strain with extra copies of the FAR and FAD genes, encoding, respectively, the fatty acyl-ACP reductase and fatty aldehyde decarbonylase enzymes in the alkane biosynthesis pathway, showed up to a fivefold increase in the intracellular levels of heptadecane, a threefold increase in 9-heptadecene, and a significant increase in secreted 16:0 and 18:0 free FAs (FFAs). Inactivation of the AAS gene, encoding acyl-ACP synthetase, prevented re-thioesterification of FFAs generated from membrane lipid recycling and led to elevated levels and of intracellular FFAs of an altered composition, and a decrease in heptadecane and secreted FFAs. Introduction of a FatB gene, encoding a thioesterase (TE), which catalyzes the liberation of FFAs from acyl-ACP, yielded little effect in itself. However, the activity of the TE enzyme was clearly manifested in combination with AAS inactivation; A TE-containing train lacking AAS showed a dramatic (30-fold) increase in intracellular FFAs (with the majority being 16:0) and increases in heptadecane and secreted FFAs.

Subunit Organization of a Synechocystis Hetero-Oligomeric Thylakoid FtsH Complex Involved in Photosystem II Repair

FtsH metalloproteases are key components of the photosystem II (PSII) repair cycle, which operates to maintain photosynthetic activity in the light. Despite their physiological importance, the structure and subunit composition of thylakoid FtsH complexes remain uncertain. Mutagenesis has previously revealed that the four FtsH homologs encoded by the cyanobacterium Synechocystis sp PCC 6803 are functionally different: FtsH1 and FtsH3 are required for cell viability, whereas FtsH2 and FtsH4 are dispensable. To gain insights into FtsH2, which is involved in selective D1 protein degradation during PSII repair, we used a strain of Synechocystis 6803 expressing a glutathione S-transferase (GST)-tagged derivative (FtsH2-GST) to isolate FtsH2-containing complexes. Biochemical analysis revealed that FtsH2-GST forms a hetero-oligomeric complex with FtsH3. FtsH2 also interacts with FtsH3 in the wild-type strain, and a mutant depleted in FtsH3, like ftsH2(-) mutants, displays impaired D1 degradation. FtsH3 also forms a separate heterocomplex with FtsH1, thus explaining why FtsH3 is more important than FtsH2 for cell viability. We investigated the structure of the isolated FtsH2-GST/FtsH3 complex using transmission electron microscopy and single-particle analysis. The three-dimensional structural model obtained at a resolution of 26 Å revealed that the complex is hexameric and consists of alternating FtsH2/FtsH3 subunits.

The Antisense RNA As1_flv4 in the Cyanobacterium Synechocystis sp. PCC 6803 Prevents Premature Expression of the flv4-2 Operon upon Shift in Inorganic Carbon Supply

The functional relevance of natural cis-antisense transcripts is mostly unknown. Here we have characterized the association of three antisense RNAs and one intergenically encoded noncoding RNA with an operon that plays a crucial role in photoprotection of photosystem II under low carbon conditions in the cyanobacterium Synechocystis sp. PCC 6803. Cyanobacteria show strong gene expression dynamics in response to a shift of cells from high carbon to low levels of inorganic carbon (C(i)), but the regulatory mechanisms are poorly understood. Among the most up-regulated genes in Synechocystis are flv4, sll0218, and flv2, which are organized in the flv4-2 operon. The flavodiiron proteins encoded by this operon open up an alternative electron transfer route, likely starting from the Q(B) site in photosystem II, under photooxidative stress conditions. Our expression analysis of cells shifted from high carbon to low carbon demonstrated an inversely correlated transcript accumulation of the flv4-2 operon mRNA and one antisense RNA to flv4, designated as As1_flv4. Overexpression of As1_flv4 led to a decrease in flv4-2 mRNA. The promoter activity of as1_flv4 was transiently stimulated by C(i) limitation and negatively regulated by the AbrB-like transcription regulator Sll0822, whereas the flv4-2 operon was positively regulated by the transcription factor NdhR. The results indicate that the tightly regulated antisense RNA As1_flv4 establishes a transient threshold for flv4-2 expression in the early phase after a change in C(i) conditions. Thus, it prevents unfavorable synthesis of the proteins from the flv4-2 operon.

Thermorecovery of cyanobacterial fatty acids at elevated temperatures

We have developed a genetic system we call “thermorecovery” that allows us to lyse cyanobacterial cultures and hydrolyze membrane lipids to release free fatty acids (FFAs), a biofuel precursor. The system uses thermostable lipases encoded by genes from thermophilic organisms that have been transferred into the cyanobacterial genome and can be synthesized by turning off CO2 availability and subsequently activated by increasing the concentrated culture temperature. When synthesized in FFA-producing strains, the lipase Fnl from Fervidobacterium nodosum Rt17-B1 released the most FFA. Of the seven candidate lipases investigated, Fnl-synthesizing strains yielded 42.7±1.5mg/l FFA at 47°C. We also determined that the optimal production conditions for SD338, the Synechocystis strain synthesizing Fnl, was to keep the cell concentrates at 46°C for two days after a one-day CO2 limitation pretreatment of the culture. A 4-l continuous semi-batch production experiment with SD338 showed that daily harvested cultures (1l) released an average of 43.9±6.6mg fatty acid and this productivity lasted for at least 20 days without significant decline. This improved thermorecovery process can be used in conjunction with other means to genetically engineer cyanobacteria to produce biofuels or biofuel precursors as the final step in recovery of membrane lipids.

The AbrB2 Autorepressor, Expressed from an Atypical Promoter, Represses the Hydrogenase Operon To Regulate Hydrogen Production in Synechocystis Strain PCC6803

We have thoroughly investigated the abrB2 gene (sll0822) encoding an AbrB-like regulator in the wild-type strain of the model cyanobacterium Synechocystis strain PCC6803. We report that abrB2 is expressed from an active but atypical promoter that possesses an extended -10 element (TGTAATAT) that compensates for the absence of a -35 box. Strengthening the biological significance of these data, we found that the occurrence of an extended -10 promoter box and the absence of a -35 element are two well-conserved features in abrB2 genes from other cyanobacteria. We also show that AbrB2 is an autorepressor that is dispensable to cell growth under standard laboratory conditions. Furthermore, we demonstrate that AbrB2 also represses the hox operon, which encodes the Ni-Fe hydrogenase of biotechnological interest, and that the hox operon is weakly expressed even though it possesses the two sequences resembling canonical -10 and -35 promoter boxes. In both the AbrB2-repressed promoters of the abrB2 gene and the hox operon, we found a repeated DNA motif [TT-(N(5))-AAC], which could be involved in AbrB2 repression. Supporting this hypothesis, we found that a TT-to-GG mutation of one of these elements increased the activity of the abrB2 promoter. We think that our abrB2-deleted mutant with increased expression of the hox operon and hydrogenase activity, together with the reporter plasmids we constructed to analyze the abrB2 gene and the hox operon, will serve as useful tools to decipher the function and the regulation of hydrogen production in Synechocystis.

A Cyanobacterium <i>Synechocystis</i> sp. PCC 6803 Glutaredoxin Gene (<i>slr1562</i>) Protects <i>Escherichia coli</i> against Abiotic Stresses

Problem statement: Glutaredoxins (GRXs) are ubiquitous small heat stable glutathione-dependent oxidoreductase enzymes that play a crucial role in plant development and response to oxidative stress. Approach: Cyanobacterium Synechocystis strain PCC 6803 contains two genes (slr1562 and ssr2061) encoding glutaredoxins. In the present investigation the slr1562 gene (grxC) was isolated and characterized. Results: The results revealed that the amino acid sequence deduced from GrxC protein share high identity with those of GRXs from other organisms and contain the consensus GRX family domain with a CPFC active site. Northern blotting analysis revealed that the expression of slr1562 gene could be induced by oxidative and salt stresses. Moreover, the protein GrxC was successfully overexpressed as a soluble fraction in Escherichia coli JM109. The over-expression of GrxC in Escherichia coli cells significantly increased resistance of cells to oxidative, drought and salt stresses. Conclusion/Recommendations: These results suggest that the slr1562 gene could play an important role in regulating abiotic tolerance against oxidative, drought and salt stresses in different organisms.

High performance analysis of the cyanobacterial metabolism via liquid chromatography coupled to a LTQ-Orbitrap mass spectrometer: evidence that glucose reprograms the whole carbon metabolism and triggers oxidative stress

Cyanobacteria are environmentally important photosynthetic microorganisms attracting a growing attention in various areas of basic and applied researches. To better understand their metabolism, we presently report on the development of a robust and simple protocol for facile extraction and high throughput analysis of the metabolites of the widely-used strain Synechocystis PCC6803 through liquid chromatography coupled to high resolution mass spectrometry (LC/MS). Our analytical method was developed and tested with 102 reference compounds representative of the chemical diversity of polar cell metabolites, and Synechocystis cell extracts spiked with 37 reference compounds. These samples were analyzed with two chromatographic systems, each coupled to a LTQ-Orbitrap mass spectrometer: a liquid chromatographic system equipped with a pentafluorophenylpropyl column (the PFPP-LC/MS system), and an ultra-high performance liquid chromatographic system with a C18-reversed phase column (the C18-UHPLC/MS system). We showed that the PFPP-LC/MS method performs better than the C18-UHPLC/MS method in terms of retention, separation and detection of metabolites. Consequently, we applied the PFPP-LC/MS method to analyze the metabolome of Synechocystis growing under various conditions of light and glucose, which strongly influence cell growth. We found that glucose increases glucose storage (synthesis of glycogen-like polysaccharide) and catabolism (oxidative pentose phosphate pathway and glycolysis), while it decreases the Calvin–Benson cycle that consumes photosynthetic electrons for CO2 assimilation. Depending on light and glucose availabilities, this global metabolic reprogramming can generate an oxidative stress, likely through the recombination of the glucose-spared electrons with the photosynthetic oxygen thereby producing toxic reactive oxygen species.