Incorporation of the chlorophyll d-binding light-harvesting protein from Acaryochloris marina and its localization within the photosynthetic apparatus of Synechocystis sp. PCC6803

The gene encoding a chlorophyll d-binding light-harvesting protein, pcbA from Acaryochloris marina (now called as accessory Chlorophyll Binding Protein CBPII) marked with a His-tag was transformed into the genome of Synechocystis PCC6803. Protein gel electrophoresis and western blotting confirmed that this foreign chlorophyll d-binding protein CBPII was expressed and integrated into the thylakoid membrane and bound with chlorophyll a, the only type of chlorophyll present in Synechocystis PCC 6803. Native electrophoresis suggested that CBPII interacts with photosystem II of Synechocystis PCC 6803. Surprisingly, spectral analyses showed that the phycobiliproteins were suppressed in the transformed Synechocystis pcbA⁺, with a lower ratio of phycobilins to chlorophyll a. These results suggest that there are competitive interactions between the external antenna system of phycobiliproteins and the integral antenna system of chlorophyll-bound protein complexes.     

Inactivation of the geranylgeranyl reductase (ChlP) gene in the cyanobacterium Synechocystis sp. PCC 6803

Geranylgeranyl reductase catalyses the reduction of geranylgeranyl pyrophosphate to phytyl pyrophosphate required for synthesis of chlorophylls, phylloquinone and tocopherols. The gene chlP (ORF sll1091) encoding the enzyme has been inactivated in the cyanobacterium Synechocystis sp. PCC 6803. The resulting ΔchlP mutant accumulates exclusively geranylgeranylated chlorophyll a instead of its phytylated analogue as well as low amounts of α-tocotrienol instead of α-tocopherol. Whereas the contents of chlorophyll and total carotenoids are decreased, abundance of phycobilisomes is increased in ΔchlP cells. The mutant assembles functional photosystems I and II as judged from 77 K fluorescence and electron transport measurements. However, the mutant is unable to grow photoautotrophically due to instability and rapid degradation of the photosystems in the absence of added glucose. We suggest that instability of the photosystems in ΔchlP is directly related to accumulation of geranylgeranylated chlorophyll a. Increased rigidity of the chlorophyll isoprenoid tail moiety due to three additional CC bonds is the likely cause of photooxidative stress and reduced stability of photosynthetic pigment-protein complexes assembled with geranylgeranylated chlorophyll a in the ΔchlP mutant.     

Temperature-dependent hyper-activation of monoglucosyldiacylglycerol synthase is post-translationally regulated in Synechocystis sp. PCC 6803

The mechanism of monoglucosyldiacylglycerol (MGlcDG) increase following heat shock in Synechocystis sp. PCC 6803 was examined by measuring MGlcDG synthase (Sll1377) activity. Temperature-dependent activation of Sll1377 was observed in the membrane fraction of Synechocystis sp. PCC 6803, whereas the Sll1377 protein level remained unchanged, suggesting that the activity is post-translationally regulated without covalent modification of Sll1377 by soluble enzymes. Four individual mutations introduced into recombinant Sll1377 (D147, D200, R329, and R331) significantly reduced the activity and blocked temperature-dependent activation, suggesting that these amino acid residues are essential for Sll1377 activity at both normal growth temperature and the higher temperature.     

Remodeling of phosphatidylglycerol in Synechocystis PCC6803

The phosphatidylglycerol deficient ΔpgsA mutant of Synechocystis PCC6803 provided a unique experimental system for investigating in vivo retailoring of exogenously added dioleoylphosphatidylglycerol in phosphatidylglycerol-depleted cells. Gas chromatographic analysis of fatty acid composition suggested that diacyl-phosphatidylglycerols were synthesized from the artificial synthetic precursor. The formation of new, retailored lipid species was confirmed by negative-ion electrospray ionization–Fourier-transform ion cyclotron resonance and ion trap tandem mass spectrometry. Various isomeric diacyl-phosphatidylglycerols were identified indicating transesterification of the exogenously added dioleoylphosphatidyl-glycerol at the sn-1 or sn-2 positions. Polyunsaturated fatty acids were incorporated selectively into the sn-1 position. Our experiments with Synechocystis PCC6803/ΔpgsA mutant cells demonstrated lipid remodeling in a prokaryotic photosynthetic bacterium. Our data suggest that the remodeling of diacylphosphatidylglycerol likely involves reactions catalyzed by phospholipase A₁ and A₂ or acyl-hydrolase, lysophosphatidylglycerol acyltransferase and acyl-lipid desaturases.     

Synthesis of fatty acids de novo is required for photosynthetic acclimation of Synechocystis sp. PCC 6803 to high temperature

The role of fatty acid synthesis in the acclimation of the photosynthetic machinery to high temperature was investigated in a mutant of the cyanobacterium Synechocystis sp. PCC 6803 that had a lower than wild-type level of enoyl-(acyl-carrier-protein) reductase FabI, a key component of the type-II fatty acid synthase system. The mutant exhibited marked impairment in the tolerance and acclimation of cells to high temperature: photoautotrophic growth of the mutant was severely inhibited at 40 °C. Moreover, mutant cells were unable to achieve wild-type enhancement of the thermal stability of photosystem II (PSII) when the growth temperature was raised from 25 °C to 38 °C. Enhancement of the thermal stability of PSII was abolished when wild-type cells were treated with triclosan, a specific inhibitor of FabI, and the enhancement of thermal stability was also blocked in darkness and in the presence of chloramphenicol. Analysis of fatty acids in thylakoid membranes revealed that levels of unsaturated fatty acids did not differ between mutant and wild-type cells, indicating that the saturation of fatty acids in membrane lipids might not be responsible for the enhancement of thermal stability at elevated temperatures. Our observations suggest that the synthesis de novo of fatty acids, as well as proteins, is required for the enhancement of the thermal stability of PSII during the acclimation of Synechocystis cells to high temperature.     

Computational protein structure modeling and analysis of UV-B stress protein in Synechocystis PCC 6803

This study focuses on Ultra Violet stress (UVS) gene product which is a UV stress induced protein from cyanobacteria, Synechocystis PCC 6803. Three dimensional structural modeling of target UVS protein was carried out by homology modeling method. 3F2I pdb from Nostoc sp. PCC 7120 was selected as a suitable template protein structure. Ultimately, the detection of active binding regions was carried out for characterization of functional sites in modeled UV-B stress protein. The top five probable ligand binding sites were predicted and the common binding residues between target and template protein was analyzed. It has been validated for the first time that modeled UVS protein structure from Synechocystis PCC 6803 was structurally and functionally similar to well characterized UVS protein of another cyanobacterial species, Nostoc sp PCC 7120 because of having same structural motif and fold with similar protein topology and function. Investigations revealed that UVS protein from Synechocystis sp. might play significant role during ultraviolet resistance. Thus, it could be a potential biological source for remediation for UV induced stress.     

Light-dependent expression of superoxide dismutase from cyanobacterium<Emphasis Type="Italic"> Synechocystis</Emphasis> sp. strain PCC 6803

The oxygenic phototrophic cyanobacterium Synechocystis sp. strain PCC 6803 inevitably evolves superoxide during photosynthesis. Synechocystis 6803 contains only one type of superoxide dismutase, designated as SodB; therefore, this protein plays an important role in preventing oxidative damages caused by light. Because there was no direct evidence that SodB in Synechocystis 6803 could be regulated by light, the relationship between SodB and light was investigated in the present study. The activity of SodB from the cells grown in continuous light culture was about 3.5-fold higher than that from the cells cultivated in continuous dark. Illumination maximally activated SodB within 12 h. The level of sodB mRNA increased 12-fold by light, and that of SodB protein proportionally. Therefore, the expression and activity of SodB from Synechocystis 6803 were dependent on the light.     

Exploring the photosynthetic production capacity of sucrose by cyanobacteria

Because cyanobacteria are photosynthetic, fast-growing microorganisms that can accumulate sucrose under salt stress, they have a potential application as a sugar source for the biomass-derived production of renewable fuels and chemicals. In the present study, the production of sucrose by the cyanobacteria Synechocystis sp. PCC6803, Synechococcus elongatus PCC7942, and Anabaena sp. PCC7120 was examined. The three species displayed different growth curves and intracellular sucrose accumulation rates in response to NaCl. Synechocystis sp. PCC6803 was used to examine the impact of modifying the metabolic pathway on the levels of sucrose production. The co-overexpression of sps (slr0045), spp (slr0953), and ugp (slr0207) lead to a 2-fold increase in intracellular sucrose accumulation, whereas knockout of ggpS (sll1566) resulted in a 1.5-fold increase in the production of this sugar. When combined, these genetic modifications resulted in a fourfold increase in intracellular sucrose accumulation. To explore methods for optimizing the transport of the intracellular sucrose to the growth medium, the acid-wash technique and the CscB (sucrose permease)-dependent export method were evaluated using Synechocystis sp. PCC6803. Whereas the acid-wash technique proved to be effective, the CscB-dependent export method was not effective. Taken together, these results suggest that using genetic engineering, photosynthetic cyanobacteria can be optimized for efficient sucrose production.     

Effects of fatty acid activation on photosynthetic production of fatty acid-based biofuels in Synechocystis sp. PCC6803

Background

Direct conversion of solar energy and carbon dioxide to drop in fuel molecules in a single biological system can be achieved from fatty acid-based biofuels such as fatty alcohols and alkanes. These molecules have similar properties to fossil fuels but can be produced by photosynthetic cyanobacteria.

Results

Synechocystis sp. PCC6803 mutant strains containing either overexpression or deletion of the slr1609 gene, which encodes an acyl-ACP synthetase (AAS), have been constructed. The complete segregation and deletion in all mutant strains was confirmed by PCR analysis. Blocking fatty acid activation by deleting slr1609 gene in wild-type Synechocystis sp. PCC6803 led to a doubling of the amount of free fatty acids and a decrease of alkane production by up to 90 percent. Overexpression of slr1609 gene in the wild-type Synechocystis sp. PCC6803 had no effect on the production of either free fatty acids or alkanes. Overexpression or deletion of slr1609 gene in the Synechocystis sp. PCC6803 mutant strain with the capability of making fatty alcohols by genetically introducing fatty acyl-CoA reductase respectively enhanced or reduced fatty alcohol production by 60 percent.

Conclusions

Fatty acid activation functionalized by the slr1609 gene is metabolically crucial for biosynthesis of fatty acid derivatives in Synechocystis sp. PCC6803. It is necessary but not sufficient for efficient production of alkanes. Fatty alcohol production can be significantly improved by the overexpression of slr1609 gene.     

Using photosystem I as a reporter protein for C analysis in a coculture containing cyanobacterium and a heterotrophic bacterium

¹³C metabolism analysis of a microbial community is often hindered by the time-consuming and complicated separation procedure for a single species. However, a “reporter protein,” produced uniquely by one cell type, retains ¹³C fingerprint information in microbial consortia. This study describes the use of photosystem I (PSI), a multi-subunit protein complex universally found in oxygenic phototrophs, as a reliable reporter protein to probe microalgal metabolism (i.e., cyanobacterium Synechocystis sp. PCC 6803) in a mixed culture with heterotrophic bacteria (i.e., Escherichia coli). We demonstrate that efficient purification of PSI and subsequent ¹³C-based amino acid analyses may decipher photomixotrophic metabolism of Synechocystis 6803 in the coculture. This study also indicates that a supplement of NaHCO₃ at high concentration could significantly improve the robustness of cyanobacterial growth against bacterial contamination.     

The Slr0924 protein of Synechocystis sp. strain PCC 6803 resembles a subunit of the chloroplast protein import complex and is mainly localized in the thylakoid lumen

An isolated 25 kDa protein of Synechocystis sp. PCC 6803 was N-terminally sequenced and assigned to a protein encoded by the ORF slr0924. This ORF shows a certain degree of sequence similarity to a subunit from the protein Translocon at the Inner envelope of pea Chloroplasts (Tic22). The deduced amino acid sequence of Slr0924 has a N-terminal extension, that contains two possible translational start points and two possible cleavage sites for leader peptidases. Immunostaining with an antibody raised to the over-produced protein revealed two cross-reacting forms, which probably correspond to a larger intermediate and the mature protein. Immunogold labelling of thin sections showed that the protein is located mainly in the thylakoid region. This result was verified by thylakoid membrane fractionation indicating that Slr0924 is a lumenal protein. The slr0924 gene product is essential for the viability of Synechocystis sp. PCC 6803 as shown by interposon mutagenesis. The merodiploid strain showed reduced photosynthetic activity compared to the wild-type. Furthermore, growth of the merodiploid strain was found to be completely inhibited after cultivation with glucose. Accordingly, the amount of the slr0924 gene product was regulated by glucose and light intensities in wild-type cells. The potential function of the protein in Synechocystis sp. PCC 6803 will be discussed.     

Characterisation of CO2 and HCO3 − uptake in the cyanobacterium Synechocystis sp. PCC6803

The availability of a complete genome database for the cyanobacterium Synechocystissp. PCC6803 (glucose-tolerant strain) has raised expectations that this organism would become a reference strain for work aimed at understanding the CO₂-concentrating mechanism (CCM) in cyanobacteria. However, the amount of physiological data available has been relatively limited. In this report we provide data on the relative contributions of net HCO₃ ⁻ uptake and CO₂ uptake under steady state photosynthetic conditions. Cells were compared after growth at high CO₂ (2% v/v in air) or limiting CO₂ conditions (20 ppm CO₂). Synechocystishas a very high dependence on net HCO₃ ⁻ uptake at low to medium concentrations of inorganic carbon (Ci). At high Ci concentrations net CO₂ uptake became more important but did not contribute more than 40% to the rate of photosynthetic O₂ evolution. The data also confirm that high Ci cells of Synechocystissp. PCC6803 possess a strong capacity for net HCO₃ ⁻ uptake under steady state photosynthetic conditions. Time course experiments show that induction of maximal Ci uptake capacity on a shift from high CO₂ to low CO₂ conditions was near completion by four hours. By contrast, relaxation of the induced state on return of cells to high CO₂, takes in excess of 230 h. Experiments were conducted to determine if Synechocystissp. PCC6803 is able to exhibit a `fast induction' response under severe Ci limitation and whether glucose was capable of causing a rapid inactivation in Ci uptake capacity. Clear evidence for either response was not found. This revised version was published online in August 2006 with corrections to the Cover Date.     

Role of the GlgX protein in glycogen metabolism of the cyanobacterium, Synechococcus elongatus PCC 7942

The putative glgX gene encoding isoamylase-type debranching enzyme was isolated from the cyanobacterium, Synechococcus elongatus PCC 7942. The deduced amino acid sequence indicated that the residues essential to the catalytic activity and substrate binding in bacterial and plant isoamylases and GlgX proteins were all conserved in the GlgX protein of S. elongatus PCC 7942. The role of GlgX in the cyanobacterium was examined by insertional inactivation of the gene. Disruption of the glgX gene resulted in the enhanced fluctuation of glycogen content in the cells during light–dark cycles of the culture, although the effect was marginal. The glycogen of the glgX mutant was enriched with very short chains with degree of polymerization 2 to 4. When the mutant was transformed with putative glgX genes of Synechocystis sp. PCC 6803, the short chains were decreased as compared to the parental mutant strain. The result indicated that GlgX protein contributes to form the branching pattern of polysaccharide in S. elongatus PCC 7942.     

Evaluation of central metabolism based on a genomic database of<Emphasis Type="Italic">Synechocystis</Emphasis> PCC6803

Cyanobacteria produce industrially important secondary metabolites such as lipopeptide, oligosaccharide, fatty acid (esp. sulfolipid),etc. Among them,Synechocystis PCC6803 is the first strain with a publicly available full genome sequence, as of 1996, and is one of the most extensively studied photosynthetic microorganisms. Using this genomic information, the central metabolism ofSynechocystis PCC6803 was reconstructed, including photosynthesis, oxidative phosphorylation, glycolysis, pyruvate metabolism, TCA cycle, carbon fixation, and transport system. Each biochemical reaction was carefully incorporated into the model, taking into consideration the metabolite formula, stoichiometry, charge balance, and thermodynamic properties using information from genomic and metabolic databases as well as biochemical literature. The metabolic flux of the model was calculated using flux balance analysis according to its cultivation with various carbon sources. The results of simulation were in accordance with experimental data, which suggests that the central metabolism model can properly estimate the behavior ofSynechocystis PCC6803. This model would aid in the understanding of the whole cell metabolism ofSynechocystis PCC6803, the first effort of its kind for photosynthetic bacteria.     

Cyanobacterial monogalactosyldiacylglycerol-synthesis pathway is involved in normal unsaturation of galactolipids and low-temperature adaptation of Synechocystis sp. PCC 6803

We characterized certain physiological functions of cyanobacterial monoglucosyldiacylglycerol using a Synechocystis sp. PCC 6803 mutant in which the gene for monoglucosyldiacylglycerol synthase had been disrupted and its function complemented by inclusion of an Arabidopsis monogalactosyldiacylglycerol synthase gene. By using this method, we prepared the first viable monoglucosyldiacylglycerol-deficient mutant of cyanobacterium and found that monoglucosyldiacylglycerol is not essential for its growth and photosynthesis under a set of “normal growth conditions” when monogalactosyldiacylglycerol is adequately supplied by the Arabidopsis monogalactosyldiacylglycerol synthase. The mutant had healthy thylakoid membranes and normal pigment content. The membrane lipid composition of the mutant was similar with that of WT except lack of monoglucosyldiacylglycerol and a slight increase in the level of phosphatidylglycerol at both normal and low temperatures. However, the ratio of unsaturated fatty acids in monogalactosyldiacylglycerol and digalactosyldiacylglycerol was reduced in the mutant compared with WT. Although the growth of the mutant was indistinguishable with that of WT at normal growth temperature, it was markedly retarded at low temperature compared with that of WT. Our data indicated the possibility that cyanobacterial monogalactosyldiacylglycerol-synthesis pathway might be required for the adequate unsaturation level of fatty acids in galactolipids and affect the low-temperature sensitivity.     

A role for mrgA, a DPS family protein, in the internal transport of Fe in the cyanobacterium Synechocystis sp. PCC6803

The mrgA protein of the cyanobacterium Synechocystis sp. PCC6803 is a member of the DPS Fe storage protein family. The physiological role of this protein was studied using a disruption mutant in the mrgA gene (slr1894) and by measuring intracellular Fe quotas, 77K chlorophyll fluorescence and growth rates. It was found that the deletion of the mrgA gene did not impair the Fe storage capacity, as the intracellular Fe quotas of the ΔmrgA cells were comparable to those of the wild type. Furthermore, the cellular response to decreasing external Fe concentrations, as detected by the emergence of the CP43′ 77K fluorescence band, was similar in wild type and mutant cultures. On the other hand, a considerable slow down in the growth rate of ΔmrgA cultures was observed upon transfer from Fe replete to Fe depleted medium, indicating impeded utilization of the plentiful intracellular Fe. Based on these results, we suggest that mrgA plays an important role in the transport of intracellular Fe from storage (within bacterioferritins) to biosynthesis of metal cofactors throughout the cell's growth.     

Synechocystis sp. PCC 6803 — a useful tool in the study of the genetics of cyanobacteria

The cyanobacterium Synechocystis sp. PCC 6803 was the first phototrophic organism to be fully sequenced. The genomic sequence has revealed the structure of the genome and its gene constituents (3167 genes), as well as the relative map positions of each gene. The functions of nearly half of the genes has been deduced using similarity searches. The genome sequence has also allowed for the implementation of systematic strategies to study gene function and the mechanisms of gene regulation on a genome-wide level. Two genome databases, CyanoBase and CyanoMutants, have been established and act as a central repository for information on gene structure and gene function, respectively. As a result of the genome sequencing and the establishment of these databases, Synechocystis sp. PCC 6803 provides an extremely versatile and easy model to study the genetic systems of photosynthetic organisms. This revised version was published online in June 2006 with corrections to the Cover Date.     

Lack of digalactosyldiacylglycerol increases the sensitivity of Synechocystis sp. PCC 6803 to high light stress

The physiological role of digalactosyldiacylglycerol (DGDG) in photosynthesis was examined using a dgdA mutant of Synechocystis sp. PCC 6803 that is defective in the biosynthesis of DGDG. The dgdA mutant cells showed normal growth under low light (LL) conditions. However, their growth was retarded under high light (HL) conditions and under Ca²⁺- and/or Cl⁻-limited conditions compared to wild-type cells. The retardation in growth of the mutant cells was recovered by exogenous supply of DGDG in the growth medium. The dgdA mutant showed increased sensitivity to photoinhibition. Although both photodamage and repair processes of photosynthesis were affected, the repair process was more severely affected than the photodamage process, suggesting that DGDG plays an important role in the photosynthetic repair cycle.