Tocopherols protect Synechocystis sp. strain PCC 6803 from lipid peroxidation

Tocopherols (vitamin E) are lipid-soluble antioxidants synthesized only by photosynthetic eukaryotes and some cyanobacteria, and have been assumed to play important roles in protecting photosynthetic membranes from oxidative stress. To test this hypothesis, tocopherol-deficient mutants of Synechocystis sp. strain PCC 6803 (slr1736 and slr1737 mutants) were challenged with a series of reactive oxygen species-generating and lipid peroxidation-inducing chemicals in combination with high-light (HL) intensity stress. The tocopherol-deficient mutants and wild type were indistinguishable in their growth responses to HL in the presence and absence of superoxide and singlet oxygen-generating chemicals. However, the mutants showed enhanced sensitivity to linoleic or linolenic acid treatments in combination with HL, consistent with tocopherols playing a crucial role in protecting Synechocystis sp. strain PCC 6803 cells from lipid peroxidation. The tocopherol-deficient mutants were also more susceptible to HL treatment in the presence of sublethal levels of norflurazon, an inhibitor of carotenoid synthesis, suggesting carotenoids and tocopherols functionally interact or have complementary or overlapping roles in protecting Synechocystis sp. strain PCC 6803 from lipid peroxidation and HL stress.     

Isopentenyl diphosphate isomerase deficiency in Synechocystis sp. strain PCC6803

Isopentenyl diphosphate isomerase (IPP isomerase) in many organisms and in plastids is central to isoprenoid synthesis and involves the conversion between IPP and dimethylallyl diphosphate (DMAPP). It is shown that Synechocystis PCC6803 is deficient in IPP isomerase activity, consistent with the absence in its genome of an obvious homologue for the enzyme. Incorporation of [1-(14)C]IPP in cell extracts, primarily into C(20), occurs only upon priming with DMAPP in Synechocystis PCC6803 and in Synechococcus PCC7942. Isoprenoid synthesis in these cyanobacteria does not appear to involve interconversion of IPP and DMAPP, raising the possibility that they are not within the plastid evolutionary lineage.     

Isolation of salt-induced periplasmic proteins from Synechocystis sp. strain PCC 6803

Periplasmic proteins were obtained from control cells and salt-adapted cells of the cyanobacterium Synechocystis sp. PCC 6803 using the method of cold osmotic shock. Two of these proteins (PP 1, apparent mol. mass 27.6 kDa, and PP 3, apparent mol. mass 39.9 kDa) were accumulated in high amounts in the periplasm of salt-adapted cells, while the major periplasmic protein (PP 2, apparent mol. mass 36.0 kDa) was accumulated independently from salt. After isolation from gels and partial sequencing, the proteins could be assigned to proteins deduced from the complete genome sequence of Synechocystis. Neither salt-induced periplasmic proteins (PP 1, Slr0924 and PP 3, Slr1485) exhibited sequence similarity to proteins of known function from databases. The major protein (PP 2-Slr0513) showed significant sequence similarities to iron-binding proteins. All proteins included typical leader sequences at their N-terminus. Received: 21 September 1998 / Accepted: 17 December 1998     

Multiple light inputs control phototaxis in Synechocystis sp. strain PCC6803

The phototactic behavior of individual cells of the cyanobacterium Synechocystis sp. strain PCC6803 was studied with a glass slide-based phototaxis assay. Data from fluence rate-response curves and action spectra suggested that there were at least two light input pathways regulating phototaxis. We observed that positive phototaxis in wild-type cells was a low fluence response, with peak spectral sensitivity at 645 and 704 nm. This red-light-induced phototaxis was inhibited or photoreversible by infrared light (760 nm). Previous work demonstrated that a taxD1 mutant (Cyanobase accession no. sll0041; also called pisJ1) lacked positive but maintained negative phototaxis. Therefore, the TaxD1 protein, which has domains that are similar to sequences found in both bacteriophytochrome and the methyl-accepting chemoreceptor protein, is likely to be the photoreceptor that mediates positive phototaxis. Wild-type cells exhibited negative phototaxis under high-intensity broad-spectrum light. This phenomenon is predominantly blue light responsive, with a maximum sensitivity at approximately 470 nm. A weakly negative phototactic response was also observed in the spectral region between 600 and 700 nm. A deltataxD1 mutant, which exhibits negative phototaxis even under low-fluence light, has a similar action maximum in the blue region of the spectrum, with minor peaks from green to infrared (500 to 740 nm). These results suggest that while positive phototaxis is controlled by the red light photoreceptor TaxD1, negative phototaxis in Synechocystis sp. strain PCC6803 is mediated by one or more (as yet) unidentified blue light photoreceptors.     

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.     

Physical and genetic map of the chromosome of the unicellular cyanobacterium Synechocystis sp. strain PCC 6803.

A combined physical and genetic map of the cyanobacterium Synechocystis sp. strain PCC 6803 chromosome was constructed. An estimated genome size of 3.82 Mb was obtained by summing the sizes of 25 MluI or 40 NotI fragments seen by pulsed-field electrophoresis. The order of the restriction fragments was determined by using two independent experimental approaches: pulsed-field fragment hybridization and linking clone analysis. The relative positions of 30 known genes or gene clusters were localized.     

Type II isopentenyl diphosphate isomerase from Synechocystis sp. strain PCC 6803

Open reading frame sll1556 in the cyanobacterium Synechocystis sp. strain 6803 encodes a putative type II isopentenyl diphosphate (IPP) isomerase. The His(6)-tagged protein was produced in Escherichia coli and purified by Ni(2+) chromatography. The homotetrameric enzyme required NADPH, flavin mononucleotide, and Mg(2+) for activity; K(m)(IPP) was 52 microM, and k(cat)(IPP) was 0.23 s(-1).     

Genes essential to iron transport in the cyanobacterium Synechocystis sp. strain PCC 6803

Genes encoding polypeptides of an ATP binding cassette (ABC)-type ferric iron transporter that plays a major role in iron acquisition in Synechocystis sp. strain PCC 6803 were identified. These genes are slr1295, slr0513, slr0327, and recently reported sll1878 (Katoh et al., J. Bacteriol. 182:6523-6524, 2000) and were designated futA1, futA2, futB, and futC, respectively, for their involvement in ferric iron uptake. Inactivation of these genes individually or futA1 and futA2 together greatly reduced the activity of ferric iron uptake in cells grown in complete medium or iron-deprived medium. All the fut genes are expressed in cells grown in complete medium, and expression was enhanced by iron starvation. The futA1 and futA2 genes appear to encode periplasmic proteins that play a redundant role in iron binding. The deduced products of futB and futC genes contain nucleotide-binding motifs and belong to the ABC transporter family of inner-membrane-bound and membrane-associated proteins, respectively. These results and sequence similarities among the four genes suggest that the Fut system is related to the Sfu/Fbp family of iron transporters. Inactivation of slr1392, a homologue of feoB in Escherichia coli, greatly reduced the activity of ferrous iron transport. This system is induced by intracellular low iron concentrations that are achieved in cells exposed to iron-free medium or in the fut-less mutants grown in complete medium.     

Identification of secreted proteins of the cyanobacterium Synechocystis sp. strain PCC 6803

We investigated the spectrum of secreted proteins in the cyanobacterium Synechocystis, and identified these proteins by amino-terminal sequencing. In total, seven sequences have been determined that corresponded to the proteins Sll0044, Sll1694, Sll1891, Slr0924, Slr0841, Slr0168, and Slr1855. The protein Sll1694 of 18 kDa that formed one of two major bands on SDS-PAGE was identified as cyanobacterial pilin, PilA. The amino-terminal sequence of another protein that formed a second major band was blocked. The analysis of the data revealed that five of seven proteins had distinct putative leader sequences for secretion.     

Purification and properties of glutamine synthetases from the cyanobacteria Synechocystis sp. strain PCC 6803 and Calothrix sp. strain PCC 7601.

Glutamine synthetases (GSs) from two cyanobacteria, one unicellular (Synechocystis sp. strain PCC 6803) and the other filamentous (Calothrix sp. strain PCC 7601 [Fremyella diplosiphon]), were purified to homogeneity. The biosynthetic activities of both enzymes were strongly inhibited by ADP, indicating that the energy charge of the cell might regulate the GS activity. Both cyanobacteria exhibited an ammonium-mediated repression of GS synthesis. In addition, the Synechocystis sp. showed an inactivation of GS promoted by ammonium that had not been demonstrated previously in cyanobacteria.     

ADP-ribosylation of glutamine synthetase in the cyanobacterium Synechocystis sp. strain PCC 6803.

Glutamine synthetase (GS) inactivation was observed in crude cell extracts and in the high-speed supernatant fraction from the cyanobacterium Synechocystis sp. strain PCC 6803 following the addition of ammonium ions, glutamine, or glutamate. Dialysis of the high-speed supernatant resulted in loss of inactivation activity, but this could be restored by the addition of NADH, NADPH, or NADP+ and, to a lesser extent, NAD+, suggesting that inactivation of GS involved ADP-ribosylation. This form of modification was confirmed both by labelling experiments using [32P]NAD+ and by chemical analysis of the hydrolyzed enzyme. Three different forms of GS, exhibiting no activity, biosynthetic activity only, or transferase activity only, could be resolved by chromatography, and the differences in activity were correlated with the extent of the modification. Both biosynthetic and transferase activities were restored to the completely inactive form of GS by treatment with phosphodiesterase.     

Metabolic engineering of Synechocystis sp. PCC 6803 for enhanced ethanol production based on flux balance analysis

Synechocystis sp. PCC 6803 is an attractive host for bio-ethanol production due to its ability to directly convert atmospheric carbon dioxide into ethanol using photosystems. To enhance ethanol production in Synechocystis sp. PCC 6803, metabolic engineering was performed based on in silico simulations, using the genome-scale metabolic model. Comprehensive reaction knockout simulations by flux balance analysis predicted that the knockout of NAD(P)H dehydrogenase enhanced ethanol production under photoautotrophic conditions, where ammonium is the nitrogen source. This deletion inhibits the re-oxidation of NAD(P)H, which is generated by ferredoxin-NADP⁺ reductase and imposes re-oxidation in the ethanol synthesis pathway. The effect of deleting the ndhF1 gene, which encodes NADH dehydrogenase subunit 5, on ethanol production was experimentally evaluated using ethanol-producing strains of Synechocystis sp. PCC 6803. The ethanol titer of the ethanol-producing ∆ndhF1 strain increased by 145%, compared with that of the control strain.

     

Active NDH-1 complexes from the cyanobacterium Synechocystis sp. strain PCC 6803

We identified eight bands by staining native gels for NADPH-nitrobluetetrazolium oxidoreductase activity after electrophoresis ofn-dodecyl-ß-D-maltoside-treated membranes of Synechocystissp. strain PCC 6803. Among them, bands A, C, D and E were attributedto the activity of NADPH dehydrogenase (NDH-1). Band A is ahighly active supercomplex of NDH-1 (about 1,000 kDa) that wasabsent in the     

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.