Genomic Structure of the Cyanobacterium Synechocystis sp. PCC 6803 Strain GT-S

Synechocystis sp. PCC 6803 is the most popular cyanobacterial strain, serving as a standard in the research fields of photosynthesis, stress response, metabolism and so on. A glucose-tolerant (GT) derivative of this strain was used for genome sequencing at Kazusa DNA Research Institute in 1996, which established a hallmark in the study of cyanobacteria. However, apparent differences in sequences deviating from the database have been noticed among different strain stocks. For this reason, we analysed the genomic sequence of another GT strain (GT-S) by 454 and partial Sanger sequencing. We found 22 putative single nucleotide polymorphisms (SNPs) in comparison to the published sequence of the Kazusa strain. However, Sanger sequencing of 36 direct PCR products of the Kazusa strains stored in small aliquots resulted in their identity with the GT-S sequence at 21 of the 22 sites, excluding the possibility of their being SNPs. In addition, we were able to combine five split open reading frames present in the database sequence, and to remove the C-terminus of an ORF. Aside from these, two of the Insertion Sequence elements were not present in the GT-S strain. We have thus become able to provide an accurate genomic sequence of Synechocystis sp. PCC 6803 for future studies on this important cyanobacterial strain.     

Precipitation of calcite induced by Synechocystis sp. PCC6803

Calcite with laminate structure was successfully prepared by culturing Synechocystis sp. PCC6803 with different concentrations of calcium chloride (CaCl₂) in BG11 media. S. PCC6803 was examined by scanning electron microscopy (SEM), transmission electron microscopy (TEM), laser confocal scanning microscope (LCSM) and energy dispersive spectroscopy (EDS). The effects of Ca²⁺ concentrations and pH values on calcification were investigated and the micro morphs of the CaCO₃ crystals were observed by means of SEM. These results showed that CaCO₃ crystals could be more easily formed with increasing the concentration of CaCl₂ in S. PCC6803 culture solution. S. PCC6803 could largely bind calcium ions, most of which were present in extracellular polymeric substances and on the cell wall. Inside the cells there were a lot of circular areas rich in calcium ions without the crystallization of calcium. Some cells produced a thicker gelatinous sheath outside of the translucent organic thin layer. And the cells inside also produced major changes that the original chloroplasts were almost transformed into starch grains whose sizes were from 0.5 to 1 μm with relatively uniform in sizes. At the same time the cell sizes significantly reduced to only about 8–9 μm almost changing to half of its original diameters. The calcite crystals with a highly preferred orientation induced by S. PCC6803 were observed with X-ray diffraction (XRD). A critical implication was that S. PCC6803 could induce bio-calcification and then mediate the further growth of CaCO₃ crystals in the biological system.     

A gene of Synechocystis sp. Strain PCC 6803 encoding a novel iron transporter

A mutant of Synechocystis sp. strain PCC 6803 disrupted for sll1878 exhibited greatly reduced Fe(3+) transport activity. The K(m) value of sll1878-dependent Fe(3+) transport in cells grown in iron-replete medium was 0.5 microM. Both the maximal rate and K(m) value were increased in iron-starved cells.     

Expression of holo-proteorhodopsin in Synechocystis sp. PCC 6803

Retinal-based photosynthesis may contribute to the free energy conversion needed for growth of an organism carrying out oxygenic photosynthesis, like a cyanobacterium. After optimization, this may even enhance the overall efficiency of phototrophic growth of such organisms in sustainability applications. As a first step towards this, we here report on functional expression of the archetype proteorhodopsin in Synechocystis sp. PCC 6803. Upon use of the moderate-strength psbA2 promoter, holo-proteorhodopsin is expressed in this cyanobacterium, at a level of up to 10⁵ molecules per cell, presumably in a hexameric quaternary structure, and with approximately equal distribution (on a protein-content basis) over the thylakoid and the cytoplasmic membrane fraction. These results also demonstrate that Synechocystis sp. PCC 6803 has the capacity to synthesize all-trans-retinal. Expressing a substantial amount of a heterologous opsin membrane protein causes a substantial growth retardation Synechocystis, as is clear from a strain expressing PROPS, a non-pumping mutant derivative of proteorhodopsin. Relative to this latter strain, proteorhodopsin expression, however, measurably stimulates its growth.     

Characterisation of acyltransferases from Synechocystis sp. PCC6803

As phylogenetic ancestors of plant chloroplasts cyanobacteria resemble plastids with respect to lipid and fatty acid composition. These membrane lipids show the typical prokaryotic fatty acid pattern in which the sn-2 position is exclusively esterified by C(16) acyl groups. In the course of de novo glycerolipid biosynthesis this prokaryotic fatty acid pattern is established by the sequential acylation of glycerol-3-phosphate with acyl-ACPs by the activity of different acyltransferases. In silico approaches allowed the identification of putative Synechocystis acyltransferases involved in glycerolipid metabolism. Functional expression studies in Escherichia coli showed that sll1848 codes for a lysophosphatidic acid acyltransferase with a high specificity for 16:0-ACP, whereas slr2060 encodes a lysophospholipid acyltransferase, with a broad acyl-ACP specificity but a strong preference for lysophosphatidyglycerol especially its sn-2 acyl isomer as acyl-acceptor. The generation and analysis of the corresponding Synechocystis knockout mutants revealed that lysophosphatidic acid acyltransferase unlike the lysophospholipid acyltransferase is essential for the vital functions of the cells.     

Reduction of Photoautotrophic Productivity in the Cyanobacterium Synechocystis sp. Strain PCC 6803 by Phycobilisome Antenna Truncation

Truncation of the algal light-harvesting antenna is expected to enhance photosynthetic productivity. The wild type and three mutant strains of Synechocystis sp. strain 6803 with a progressively smaller phycobilisome antenna were examined under different light and CO(2) conditions. Surprisingly, such antenna truncation resulted in decreased whole-culture productivity for this cyanobacterium.     

Effect of Glucose Utilization on Nitrite Excretion by the Unicellular Cyanobacterium Synechocystis sp. Strain PCC 6803

Up to 1 mM nitrite was excreted by Synechocystis strain 6803 cells growing under mixotrophic or photoheterotrophic conditions. This excretion is not due to a lower ratio of nitrite and nitrate reductase activities in the presence of glucose but seems to be related to a shortage of reduced ferredoxin, their electron donor, as a result of a decrease in noncyclic photosynthetic flow observed under these circumstances. Because about 60% of the reduced nitrate is excreted, the potential utilization of cyanobacteria for removal of nitrate from contaminated waters containing high concentrations of organic compounds is questioned.     

Ferredoxin and flavodoxin from the cyanobacterium Synechocystis sp PCC 6803.

The unicellular cyanobacterium Synechocystis sp PCC 6803 is capable of synthesizing two different Photosystem-I electron acceptors, ferredoxin and flavodoxin. Under normal growth conditions a [2Fe-2S] ferredoxin was recovered and purified to homogeneity. The complete amino-acid sequence of this protein was established. The isoelectric point (pI = 3.48), midpoint redox potential (Em = -0.412 V) and stability under denaturing conditions were also determined. This ferredoxin exhibits an unusual electrophoretic behavior, resulting in a very low apparent molecular mass between 2 and 3.5 kDa, even in the presence of high concentrations of urea. However, a molecular mass of 10,232 Da (apo-ferredoxin) is calculated from the sequence. Free thiol assays indicate the presence of a disulfide bridge in this protein. A small amount of ferredoxin was also found in another fraction during the purification procedure. The amino-acid sequence and properties of this minor ferredoxin were similar to those of the major ferredoxin. However, its solubility in ammonium sulfate and its reactivity with antibodies directed against spinach ferredoxin were different. Traces of flavodoxin were also recovered from the same fraction. The amount of flavodoxin was dramatically increased under iron-deficient growth conditions. An acidic isoelectric point was measured (pI = 3.76), close to that of ferredoxin. The midpoint redox potentials of flavodoxin are Em1 = -0.433 V and Em2 = -0.238 V at pH 7.8. Sequence comparison based on the 42 N-terminal amino acids indicates that Synechocystis 6803 flavodoxin most likely belongs to the long-chain class, despite an apparent molecular mass of 15 kDa determined by SDS-PAGE.     

Photoheterotrophic Fluxome in Synechocystis sp. Strain PCC 6803 and Its Implications for Cyanobacterial Bioenergetics

This study investigated metabolic responses in Synechocystis sp. strain PCC 6803 to photosynthetic impairment. We used 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU; a photosystem II inhibitor) to block O₂ evolution and ATP/NADPH generation by linear electron flow. Based on ¹³C-metabolic flux analysis (¹³C-MFA) and RNA sequencing, we have found that Synechocystis sp. PCC 6803 employs a unique photoheterotrophic metabolism. First, glucose catabolism forms a cyclic route that includes the oxidative pentose phosphate (OPP) pathway and the glucose-6-phosphate isomerase (PGI) reaction. Glucose-6-phosphate is extensively degraded by the OPP pathway for NADPH production and is replenished by the reversed PGI reaction. Second, the Calvin cycle is not fully functional, but RubisCO continues to fix CO₂ and synthesize 3-phosphoglycerate. Third, the relative flux through the complete tricarboxylic acid (TCA) cycle and succinate dehydrogenase is small under heterotrophic conditions, indicating that the newly discovered cyanobacterial TCA cycle (via the γ-aminobutyric acid pathway or α-ketoglutarate decarboxylase/succinic semialdehyde dehydrogenase) plays a minimal role in energy metabolism. Fourth, NAD(P)H oxidation and the cyclic electron flow (CEF) around photosystem I are the two main ATP sources, and the CEF accounts for at least 40% of total ATP generation from photoheterotrophic metabolism (without considering maintenance loss). This study not only demonstrates a new topology for carbohydrate oxidation but also provides quantitative insights into metabolic bioenergetics in cyanobacteria.     

Accumulation of poly-beta-hydroxybutyrate in cyanobacterium Synechocystis sp. PCC6803

Accumulation of poly-beta-hydroxybutyrate (PHB) by photoautotrophic microorganisms makes it possible to reduce the production cost of PHB. The Synechocystis sp. PCC6803 cells grown in BG11 medium under balanced, nitrogen-starved or phosphorus-starved conditions were observed by transmission electron microscope. Many electron-transparent granules in the nitrogen-starved cells had a diameter up to 0.8 micron. In contrast, the number of granules in the normally cultured cells decreased obviously and only zero to three much smaller granules were in each cell. These granules were similar to those in bacteria capable of synthesizing PHB. They were proved to be PHB by gas chromatography after subjecting the cells to methanolysis. Effects of glucose as carbon source and light intensity on PHB accumulation in Synechocystis sp. PCC6803 under nitrogen-starved cultivation were further studied. Glucose and illumination promoted cell growth but did not favor PHB synthesis. After 7 days of growth under nitrogen-starved photoautotrophic conditions, the intracellular level of PHB was up to 4.1% of cellular dry weight and the PHB concentration in the culture broth was 27 mg/l.     

Optimum conditions for transformation of Synechocystis sp. PCC 6803

This study was conducted to determine the optimal conditions for introduction of exogenous DNA into Synechocystis sp. PCC 6803. Of the three transformation techniques studied, electroporation, ultrasonic transformation and natural transformation, natural transformation showed the highest efficiency. Additionally, this study demonstrated that the higher plasmid concentration and longer homologous recombining fragments resulted in a greater number of transformants. For successful transformation, the lowest concentration of plasmid was 0.02 microg/ml, and the shortest homologous recombining fragment was 0.2 kb. Use of Synechocystis sp. PCC 6803 in the logarithmic growth phase resulted in two-fold higher transformation rate than that of the same organism when cells in the latent phase or the plateau phase were used for transformation. Pretreatment of the host strain, Synechocystis sp. PCC 6803, with EDTA (2 mM) for two days prior to transformation increased the transformation efficiency by 23%. Additionally, incubation of the cells and DNA for 5 h under light conditions increased the transformation efficiency by two orders of magnitude. Moreover, recovery treatment of the cells before they were plated onto antibiotic medium also increased the transformation efficiency.     

The ggpS Gene from Synechocystis sp. Strain PCC 6803 Encoding Glucosyl-Glycerol-Phosphate Synthase Is Involved in Osmolyte Synthesis

A salt-sensitive mutant of Synechocystis sp. strain PCC 6803 defective in the synthesis of the compatible solute glucosylglycerol (GG) was used to search for the gene encoding GG-phosphate synthase (GGPS), the key enzyme in GG synthesis. Cloning and sequencing of the mutated region and the corresponding wild-type region revealed that a deletion of about 13 kb occurred in the genome of mutant 11. This deletion affected at least 10 open reading frames, among them regions coding for proteins showing similarities to trehalose (otsA homolog)- and glycerol-3-phosphate-synthesizing enzymes. After construction and characterization of mutants defective in these genes, it became obvious that an otsA homolog (sll1566) (T. Kaneko et al., DNA Res. 3:109–136, 1996) encodes GGPS, since only the mutant affected in sll1566 showed salt sensitivity combined with a complete absence of GG accumulation. Furthermore, the overexpression of sll1566 in Escherichia coli led to the appearance of GGPS activity in the heterologous host. The overexpressed protein did not show the salt dependence that is characteristic for the GGPS in crude protein extracts of Synechocystis.     

Ethylene Synthesis and Regulated Expression of Recombinant Protein in Synechocystis sp. PCC 6803

The ethylene-forming enzyme (EFE) from Pseudomonas syringae catalyzes the synthesis of ethylene which can be easily detected in the headspace of closed cultures. A synthetic codon-optimized gene encoding N-terminal His-tagged EFE (EFEh) was expressed in Synechocystis sp. PCC 6803 (Synechocystis) and Escherichia coli (E. coli) under the control of diverse promoters in a self-replicating broad host-range plasmid. Ethylene synthesis was stably maintained in both organisms in contrast to earlier work in Synechococcus elongatus PCC 7942. The rate of ethylene accumulation was used as a reporter for protein expression in order to assess promoter strength and inducibility with the different expression systems. Several metal-inducible cyanobacterial promoters did not function in E. coli but were well-regulated in cyanobacteria, albeit at a low level of expression. The E. coli promoter P_trc resulted in constitutive expression in cyanobacteria regardless of whether IPTG was added or not. In contrast, a Lac promoter variant, P_A1lacO-1, induced EFE-expression in Synechocystis at a level of expression as high as the Trc promoter and allowed a fine level of IPTG-dependent regulation of protein-expression. The regulation was tight at low cell density and became more relaxed in more dense cultures. A synthetic quorum-sensing promoter system was also constructed and shown to function well in E. coli, however, only a very low level of EFE-activity was observed in Synechocystis, independent of cell density.     

Premethylation of Foreign DNA Improves Integrative Transformation Efficiency in Synechocystis sp. Strain PCC 6803

Restriction digestion of foreign DNA is one of the key biological barriers against genetic transformation in microorganisms. To establish a high-efficiency transformation protocol in the model cyanobacterium, Synechocystis sp. strain PCC 6803 (Synechocystis 6803), we investigated the effects of premethylation of foreign DNA on the integrative transformation of this strain. In this study, two type II methyltransferase-encoding genes, i.e., sll0729 (gene M) and slr0214 (gene C), were cloned from the chromosome of Synechocystis 6803 and expressed in Escherichia coli harboring an integration plasmid. After premethylation treatment in E. coli, the integration plasmid was extracted and used for transformation of Synechocystis 6803. The results showed that although expression of methyltransferase M had little impact on the transformation of Synechocystis 6803, expression of methyltransferase C resulted in 11- to 161-fold-higher efficiency in the subsequent integrative transformation of Synechocystis 6803. Effective expression of methyltransferase C, which could be achieved by optimizing the 5′ untranslated region, was critical to efficient premethylation of the donor DNA and thus high transformation efficiency in Synechocystis 6803. Since premethylating foreign DNA prior to transforming Synechocystis avoids changing the host genetic background, the study thus provides an improved method for high-efficiency integrative transformation of Synechocystis 6803.     

Proteomic analysis of heterotrophy in Synechocystis sp. PCC 6803

To provide an insight into the heterotrophic metabolism of cyanobacteria, a proteomic approach has been employed with the model organism Synechocystis sp. PCC 6803. The soluble proteins from Synechocystis grown under photoautotrophic and light-activated heterotrophic conditions were separated by 2-DE and identified by MALDI-MS or LC-MS/MS analysis. 2-DE gels made using narrow- and micro-range IPG strips allowed quantitative comparison of more than 900 spots. Out of 67 abundant protein spots identified, 13 spots were increased and 9 decreased under heterotrophy, representing all the major fold changes. Proteomic alterations and activity levels of selected enzymes indicate a shift in the central carbon metabolism in response to trophic change. The significant reduction in light-saturated rate of photosynthesis as well as in the expression levels of rubisco and CO(2)-concentrating mechanism proteins under heterotrophy indicates the down-regulation of the photosynthetic machinery. Alterations in the expression level of proteins involved in carbon utilization pathways refer to enhanced glycolysis, oxidative pentose phosphate pathway as well as tricarboxylic acid cycle under heterotrophy. Proteomic evidences also suggest an enhanced biosynthesis of amino acids such as histidine and serine during heterotrophic growth.     

A Physical Map of the Genome of a Unicellular Cyanobacterium Synechocystis sp. Strain PCC6803

An accurate physical map of the genome of a cyanobacterium,Synechocystis sp. strain PCC6803, was constructed on the basisof restriction and linking clone analysis. The genome contained6 recognition sites for AscI, 25 sites for MluI, and 31 sitesfor SplI, and the entire genome size was estimated to be 3.6Mb. Sixteen genes or gene clusters, including those involvedin the photosynthetic systems, were localized on the physicalmapof the genome by hybridization. In the course of the above analysis,two extra chromosomal units with approximate sizes of 110 kband 125 kb were identified.