The cyanobacterial type I NAD(P)H dehydrogenase (NDH-1) complexes play a crucial role in a variety of bioenergetic reactions such as respiration, CO2 uptake, and cyclic electron transport around ...photosystem I. Two types of NDH-1 complexes, NDH-1MS and NDH-1MS', are involved in the CO2 uptake system. However, the composition and function of the complexes still remain largely unknown. Here, we found that deletion of ndhM caused inactivation of NDH-1-dependent cyclic electron transport around photosystem I and abolishment of CO2 uptake, resulting in a lethal phenotype under air CO2 condition. The mutation of NdhM abolished the accumulation of the hydrophilic subunits of the NDH-1, such as NdhH, NdhI, NdhJ, and NdhK, in the thylakoid membrane, resulting in disassembly of NDH-1MS and NDH-1MS' as well as NDH-1L. In contrast, the accumulation of the hydrophobic subunits was not affected in the absence of NdhM. In the cytoplasm, the NDH-1 subcomplex assembly intermediates including NdhH and NdhK were seriously affected in the Delta ndhM mutant but not in the NdhI-deleted mutant Delta ndhI. In vitro protein interaction analysis demonstrated that NdhM interacts with NdhK, NdhH, NdhI, and NdhJ but not with other hydrophilic subunits of the NDH-1 complex. These results suggest that NdhM localizes in the hydrophilic subcomplex of NDH-1 complexes as a core subunit and is essential for the function of NDH-1MS and NDH-1MS' involved in CO2 uptake in Synechocystis sp. strain PCC 6803.
Main conclusion : The Ycf46 mutant of Synechocystis showed growth inhibition under low dissolved CO sub( 2 ) conditions, suggesting a role for the Ycf46 protein in the process of photosynthetic CO ...sub( 2 ) uptake and utilization. Abstract: Hypothetical chloroplast open reading frame Ycf46 proteins are highly conserved in all cyanobacterial lineages and most algal chloroplast genomes, but their exact function is still unknown. In the cyanobacterium Synechocystis sp. PCC 6803, the Ycf46 encoding gene slr0374 is part of an operon (with slr0373 and slr0376) and responds to many environmental stresses. Transcript levels of the slr0373, slr0374 and slr0376 genes were increased under a low concentration of dissolved inorganic carbon (C sub(i)). Compared with the wild type, the mutant lacking slr0374 showed growth arrest under C sub(i)-deficient conditions but not under iron-deficient or low-light conditions. In addition, the mutant grew more slowly than the wild type under pH 6.0 conditions in which CO sub(2) was the dominant C sub(i) source, indicating the mutant cells had weak CO sub(2) uptake and/or utilization ability. Supplying a high concentration of CO sub(2) (5 %, v/v) to the mutant restored its phenotype to the wild type level. The photosynthetic activity of the mutant was inhibited to a lesser extent by a carbonic anhydrase inhibitor than that of the wild type, which specifically blocked CO sub(2) uptake. Inactivation of slr0374 decreased expression of the ecaB gene and reduced carbonic anhydrase activity. A subcellular localization assay indicated that the Ycf46 protein was soluble. By co-immunoprecipitation assay using Slr0374 as a bait-protein, potential interacting proteins in the size range of 30 kDa were identified. These results suggest that the Ycf46 protein plays a role in the regulation of photosynthesis in cyanobacteria, especially in CO sub(2) uptake and utilization.
PHB (poly-hydroxy-butyrate) represents a promising bioplastic alternative with good biodegradation properties. Furthermore, PHB can be produced in a completely carbon-neutral fashion in the natural ...producer cyanobacterium Synechocystis sp. PCC 6803. This strain has been used as model system in past attempts to boost the intracellular production of PHB above ~ 15% per cell-dry-weight (CDW).
We have created a new strain that lacks the regulatory protein PirC (product of sll0944), which exhibits a higher activity of the phosphoglycerate mutase resulting in increased PHB pools under nutrient limiting conditions. To further improve the intracellular PHB content, two genes involved in PHB metabolism, phaA and phaB, from the known producer strain Cupriavidus necator, were introduced under the control of the strong promotor PpsbA2. The resulting strain, termed PPT1 (ΔpirC-REphaAB), produced high amounts of PHB under continuous light as well under a day-night regime. When grown in nitrogen and phosphorus depleted medium, the cells produced up to 63% per CDW. Upon the addition of acetate, the content was further increased to 81% per CDW. The produced polymer consists of pure PHB, which is highly isotactic.
The amounts of PHB achieved with PPT1 are the highest ever reported in any known cyanobacterium and demonstrate the potential of cyanobacteria for a sustainable, industrial production of PHB.
We report here the characterization of a novel orphan response regulator Slr1588 directly involved in the synthesis and transport of compatible solutes against salt stress. In the Delta ...slr1588mutant, salt tolerance was found to be decreased by 2-3-fold. Using a high performance Q-EXACTIVE hybrid quadrupole-Orbitrap mass spectrometer, we found that proteins involved in the synthesis and transport of glucosylglycerol, a key compatible solute, were up-regulated in the Delta slr1588mutant grown in 4.0% NaCl, suggesting that Slr1588 might function as a repressor for glucosylglycerol metabolism. The functional assignment was further confirmed using an electrophoretic mobility shift assay (EMSA) showing that the purified his-tagged Slr1588 could bind in vitrodirectly to the upstream regions of sll1566(ggpS) genes required for glucosylglycerol biosynthesis. In addition, quantitative proteomic analysis showed that the biosynthesis of another key compatible solute in Synechocystis, sucrose, was also up-regulated in the Delta slr1588mutant under 4.0% NaCl, and EMSA showed that the purified his-tagged Slr1588 bound in vitrodirectly to the upstream regions of sll0045(spsA) gene required for sucrose biosynthesis. Moreover, proteomic analysis showed that 113 and 127 unique proteins were and up- and down-regulated in the Delta slr1588mutant grown under 4.0% NaCl, respectively. Notably, a dozen transporter genes were down-regulated in the Delta slr1588mutant under salt stress. The study revealed a novel salt-tolerant regulatory mechanism mediated by Slr1588, and also provided a proteomic description of the possible Slr1588 regulon in Synechocystis.
The fluorescence emission of the phycobilisome (PBS) core-membrane linker protein (L sub(CM)) can be directly quenched by photoactivated orange carotenoid protein (OCP) at room temperature both in ...vitro and in vivo, which suggests the crucial role of the OCP-L sub(CM) interaction in non-photochemical quenching (NPQ.) of cyanobacteria. This implication was further supported (i) by low-temperature (77 K) fluorescence emission and excitation measurements which showed a specific quenching of the corresponding long-wavelength fluorescence bands which belong to the PBS terminal emitters in the presence of photo-activated OCP, (ii) by systematic investigation of the fluorescence quenching and recovery in wild type and L sub(CM)-less cells of the model cyanobacterium Synechocystis sp. PCC 6803, and (iii) by the impact of dephosphorylation of isolated PBS on the quenching. The OCP binding site within the PBS and the most probable geometrical arrangement of the OCP-allophycocyanin (APC) complex was determined in silico using the crystal structures of OCP and APC. Geometrically modeled attachment of OCP to the PBS core is not at variance with the OCP-L sub(CM) interaction. It was concluded that besides being a very central element in the PBS to reaction center excitation energy transfer and PBS assembly, L sub(CM) also has an essential role in the photoprotective light adaptation processes of cyanobacteria.
Culturing cyanobacteria in a highly alkaline environment is a possible strategy for controlling contamination by other organisms. Synechocystis PCC 6803 cells were grown in continuous cultures to ...assess their growth performance at different pH values. Light conversion efficiency linearly decreased with the increase in pH and ranged between 12.5 % (PAR) at pH 7.5 (optimal) and decreased to 8.9 % at pH 11.0. Photosynthetic activity, assessed by measuring both chlorophyll fluorescence and photosynthesis rate, was not much affected going from pH 7.5 to 11.0, while productivity, growth yield, and biomass yield on light energy declined by 32, 28, and 26 % respectively at pH 11.0. Biochemical composition of the biomass did not change much within pH 7 and 10, while when grown at pH 11.0, carbohydrate content increased by 33 % while lipid content decreased by about the same amount. Protein content remained almost constant (average 65.8 % of dry weight). Cultures maintained at pH above 11.0 could grow free of contaminants (protozoa and other competing microalgae belonging to the species of Poterioochromonas).
The active site of photosynthetic water oxidation by Photosystem II (PSII) is a manganese-calcium cluster (Mn sub(4)CaO sub(5)). A postulated catalytic base is assumed to be crucial. CP43-Arg357, ...which is a candidate for the identity of this base, is a second-sphere ligand of the Mn sub(4)-Ca cluster and is located near a putative proton exit pathway, which begins with residue D1-D61. Transient absorption spectroscopy and time-resolved O sub(2) polarography reveal that in the D1-D61N mutant, the transfer of an electron from the Mn sub(4)CaO sub(5) cluster to Y sub(Z) super(OX) and O sub(2) release during the final step of the catalytic cycle, the S sub(3)-S sub(0) transition, proceed simultaneously but are more dramatically decelerated than previously thought (t sub(1/2) of up to 50 ms vs a t sub(1/2) of 1.5 ms in the wild type). Using a bare platinum electrode to record the flash-dependent yields of O sub(2) from mutant and wild-type PSII has allowed the observation of the kinetics of release of O sub(2) from extracted thylakoid membranes at various pH values and in the presence of deuterated water. In the mutant, it was possible to resolve a clear lag phase prior to the appearance of O sub(2), indicating formation of an intermediate before the onset of O sub(2) formation. The lag phase and the photochemical miss factor were more sensitive to isotope substitution in the mutant, indicating that proton efflux in the mutant proceeds via an alternative pathway. The results are discussed in comparison with earlier results obtained from the substitution of CP43-Arg357 with lysine and in regard to hypotheses concerning the nature of the final steps in photosynthetic water oxidation. These considerations led to the conclusion that proton expulsion during the initial phase of the S sub(3)-S sub(0) transition starts with the deprotonation of the primary catalytic base, probably CP43-Arg357, followed by efficient proton egress involving the carboxyl group of D1-D61 in a process that constitutes the lag phase immediately prior to O sub(2) formation chemistry.
•Native biosynthetic genes for PHB production overexpressed in Synechocystis.•Overexpression of phaA and phaB resulted in the highest level of PHB.•Metabolic engineering leads to increased production ...of the valuable polymer PHB.
Synechocystis sp. PCC 6803 strains overexpressing pha genes were constructed and characterized for poly-3-hydroxybutyrate (PHB) production. These pha overexpressing strains showed slightly reduced growth rates. Under N-deprived condition, the strains overexpressing (OE) phaAB, phaEC and phaABEC showed significantly higher PHB contents than the wild type. The maximum PHB content, a 2.6-fold increase producing 26% PHB (dcw), was observed in OE phaAB cells grown for 9days in N-deprived medium. Under this condition, these OE phaAB cells increased PHB production to 35% PHB (dcw) upon addition of 0.4% (w/v) acetate. Higher PHB granules in OE phaAB cells were clearly visualized by both Nile red staining and TEM imaging. All OE strains under N-deficient condition had increased glgX transcript levels. Overall results demonstrate an enhanced PHB production in Synechocystis cells overexpressing pha genes, particularly phaA and phaB, when grown in N-deprived medium containing 0.4% (w/v) acetate.
Intense sunlight is dangerous for photosynthetic organisms. Cyanobacteria, like plants, protect themselves from light-induced stress by dissipating excess absorbed energy as heat. Recently, it was ...discovered that a soluble orange carotenoid protein, the OCP, is essential for this photoprotective mechanism. Here we show that the OCP is also a member of the family of photoactive proteins; it is a unique example of a photoactive protein containing a carotenoid as the photoresponsive chromophore. Upon illumination with blue-green light, the OCP undergoes a reversible transformation from its dark stable orange form to a red "active" form. The red form is essential for the induction of the photoprotective mechanism. The illumination induces structural changes affecting both the carotenoid and the protein. Thus, the OCP is a photoactive protein that senses light intensity and triggers photoprotection.
Of the two natural metabolic pathways for making terpenoids, biotechnological utilization of the mevalonate (MVA) pathway has enabled commercial production of valuable compounds, while the more ...recently discovered but stoichiometrically more efficient methylerythritol phosphate (MEP) pathway is underdeveloped. We conducted a study on the overexpression of each enzyme in the MEP pathway in the unicellular cyanobacterium Synechocystis sp. PCC 6803, to identify potential targets for increasing flux towards terpenoid production, using isoprene as a reporter molecule. Results showed that the enzymes Ipi, Dxs and IspD had the biggest impact on isoprene production. By combining and creating operons out of those genes, isoprene production was increased 2-fold compared to the base strain. A genome-scale model was used to identify targets upstream of the MEP pathway that could redirect flux towards terpenoids. A total of ten reactions from the Calvin-Benson-Bassham cycle, lower glycolysis and co-factor synthesis pathways were probed for their effect on isoprene synthesis by co-expressing them with the MEP enzymes, resulting in a 60% increase in production from the best strain. Lastly, we studied two isoprene synthases with the highest reported catalytic rates. Only by expressing them together with Dxs and Ipi could we get stable strains that produced 2.8 mg/g isoprene per dry cell weight, a 40-fold improvement compared to the initial strain.
•The effect of overexpressing every MEP enzymes was examined in Synechocystis.•Genetic insulators were systematically tested to enhance expression levels.•A metabolic model was used to find targets that increased isoprene production.•Several bottlenecks were identified for efficient terpenoid production.
PDF
