| 1. |
- Chen, Que, et al.
(författare)
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Combining retinal-based and chlorophyll-based (oxygenic) photosynthesis : Proteorhodopsin expression increases growth rate and fitness of a Delta PSI strain of Synechocystis sp. PCC6803
- 2019
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Ingår i: Metabolic engineering. - : Elsevier. - 1096-7176 .- 1096-7184. ; 52, s. 68-76
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Tidskriftsartikel (refereegranskat)abstract
- To fill the "green absorption gap", a green absorbing proteorhodopsin was expressed in a PSI-deletion strain (Delta PSI) of Synechocystis sp. PCC6803. Growth-rate measurements, competition experiments and physiological characterization of the proteorhodopsin-expressing strains, relative to the Delta PSI control strain, allow us to conclude that proteorhodopsin can enhance the rate of photoheterotrophic growth of Delta PSI Synechocystis strain. The physiological characterization included measurement of the amount of residual glucose in the spent medium and analysis of oxygen uptake- and production rates. To explore the use of solar radiation beyond the PAR region, a red-shifted variant Proteorhodopsin-D212N/F234S was expressed in a retinal-deficient PSI-deletion strain (Delta PSI/Delta SynACO). Via exogenous addition of retinal analogue an infrared absorbing pigment (maximally at 740 nm) was reconstituted in vivo. However, upon illumination with 746 nm light, it did not significantly stimulate the growth (rate) of this mutant. The inability of the proteorhodopsin-expressing Delta PSI strain to grow photoautotrophically is most likely due to a kinetic rather than a thermodynamic limitation of its NADPH-dehydrogenase in NADP(+)-reduction.
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| 2. |
- Chen, Que, et al.
(författare)
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Functional Expression of Gloeobacter Rhodopsin in PSI-Less Synechocystis sp. PCC6803
- 2019
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Ingår i: Frontiers in Bioengineering and Biotechnology. - : Frontiers Media S.A.. - 2296-4185. ; 7
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Tidskriftsartikel (refereegranskat)abstract
- The approach of providing an oxygenic photosynthetic organism with a cyclic electron transfer system, i.e., a far-red light-driven proton pump, is widely proposed to maximize photosynthetic efficiency via expanding the absorption spectrum of photosynthetically active radiation. As a first step in this approach, Gloeobacter rhodopsin was expressed in a PSI-deletion strain of Synechocystis sp. PCC6803. Functional expression of Gloeobacter rhodopsin, in contrast to Proteorhodopsin, did not stimulate the rate of photoheterotrophic growth of this Synechocystis strain, analyzed with growth rate measurements and competition experiments. Nevertheless, analysis of oxygen uptake and-production rates of the Gloeobacter rhodopsin-expressing strains, relative to the 1 PSI control strain, confirm that the proton-pumping Gloeobacter rhodopsin provides the cells with additional capacity to generate proton motive force. Significantly, expression of the Gloeobacter rhodopsin did modulate levels of pigment formation in the transgenic strain.
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| 3. |
- Cheregi, Otilia, et al.
(författare)
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Antibiotic Disc Assay for Measuring Cell Wall Function in Synechocystis sp. PCC6803
- 2016
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Ingår i: Bio-protocol. - 2331-8325. ; 6:24
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Tidskriftsartikel (refereegranskat)abstract
- This protocol describes how to investigate the integrity of the outer cell wall in thecyanobacterium Synechocystis sp. PCC6803 using antibiotics. It is adapted to the agar diffusion test(Bauer et al., 1966), in which filter paper discs impregnated with specified concentrations of antibioticswere placed on agar plates inoculated with bacteria. The antibiotics we tested, interfering with thebiosynthesis/function of bacterial cell walls, will diffuse into the agar and produce a zone ofcyanobacterial growth inhibition around the disc(s). The size of the inhibition zone reflects the sensitivityof the strain to the action of antibiotics, e.g., a mutation in a protein functioning within the cell wall or itsconstruction would render the mutant strain more sensitive to the respective antibiotic. The method hasproven to be useful for phenotyping a mutant of Synechocystis sp. PCC6803 lacking all three genesencoding Deg proteases. Deletion of these ATP-independent serine proteases was shown to haveimpact on the outer cell layers of Synechocystis cells.
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| 4. |
- Cheregi, Otilia, et al.
(författare)
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Inactivation of the Deg protease family in the cyanobacterium Synechocystis sp. PCC 6803 has impact on the outer cell layers
- 2015
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Ingår i: Journal of Photochemistry and Photobiology. B. - : Elsevier. - 1011-1344 .- 1873-2682. ; , s. 383-394
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Tidskriftsartikel (refereegranskat)abstract
- The serine type Deg/HtrA proteases are distributed in a wide range of organisms from Escherichia coli to humans. The cyanobacterium Synechocystis sp. PCC 6803 possesses three Deg protease orthologues: HtrA, HhoA and HhoB. Previously we compared Synechocystis 6803 wild type cells exposed to mild or severe stress conditions with a mutant lacking all three Deg proteases and demonstrated that stress had strong impact on the proteomes and metabolomes [1]. To identify the biochemical processes, which this protease family is involved in, here we compared Synechocystis sp. PCC 6803 wild type cells with a mutant lacking all three Deg proteases grown under normal growth conditions (30 °C and 40 μmol photons m−2 s−1). Deletion of the Deg proteases lead to the down-regulation of proteins related to the biosynthesis of outer cell layers (e.g. the GDP mannose 4,6-dehydratase) and affected protein secretion. During the late growth phase of the culture Deg proteases were found to be secreted to the extracellular medium of the Synechocystis sp. PCC 6803 wild type strain. While cyanobacterial Deg proteases seem to act mainly in the periplasmic space, deletion of the three proteases influences the proteome and metabolome of the whole cell. Impairments in the outer cell layers of the triple mutant might explain the higher sensitivity toward light and oxidative stress, which was observed earlier by Barker and coworkers [2].
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| 5. |
- Cheregi, Otilia, et al.
(författare)
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Insights into the Cyanobacterial Deg/HtrA Proteases
- 2016
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Ingår i: Frontiers in Plant Science. - : Frontiers Media S.A. - 1664-462X. ; 7
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Tidskriftsartikel (refereegranskat)abstract
- Proteins are the main machinery for all living processes in a cell; they provide structural elements, regulate biochemical reactions as enzymes, and are the interface to the outside as receptors and transporters. Like any other machinery proteins have to be assembled correctly and need maintenance after damage, e.g., caused by changes in environmental conditions, genetic mutations, and limitations in the availability of cofactors. Proteases and chaperones help in repair, assembly, and folding of damaged and misfolded protein complexes cost-effective, with low energy investment compared with neo-synthesis. Despite their importance for viability, the specific biological role of most proteases in vivo is largely unknown. Deg/HtrA proteases, a family of serinetype ATP-independent proteases, have been shown in higher plants to be involved in the degradation of the Photosystem II reaction center protein D1. The objective of this review is to highlight the structure and function of their cyanobacterial orthologs. Homology modeling was used to find specific features of the SynDeg/HtrA proteases of Synechocystis sp. PCC 6803. Based on the available data concerning their location and their physiological substrates we conclude that these Deg proteases not only have important housekeeping and chaperone functions within the cell, but also are needed for remodeling the cell exterior.
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| 6. |
- Cheregi, Otilia, et al.
(författare)
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Marine microalgae for outdoor biomass production - a laboratory study simulating seasonal light and temperature for the west coast of Sweden
- 2021
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Ingår i: Physiologia plantarum. - : Wiley. - 1399-3054 .- 0031-9317. ; 173:2, s. 543-554
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Tidskriftsartikel (refereegranskat)abstract
- At Nordic latitudes, year-round outdoor cultivation of microalgae is debatable due to seasonal variations in productivity. Shall the same species/strains be used throughout the year, or shall seasonal-adapted ones be used? To elucidate this, a laboratory study was performed where two out of 167 marine microalgal strains were selected for intended cultivation at the west coast of Sweden. The two local strains belong to Nannochloropsis granulata (Ng) and Skeletonema marinoi (Sm142). They were cultivated in photobioreactors and compared in conditions simulating variations in light and temperature of a year divided into three growth seasons (spring, summer and winter). The strains grew similarly well in summer (and also in spring), but Ng produced more biomass (0.225 versus 0.066 g DW L-1 d-1 ) which was more energy rich (25.0 versus 16.6 MJ kg-1 DW). In winter, Sm142 grew faster and produced more biomass (0.017 versus 0.007 g DW L-1 d-1 ), having similar energy to the other seasons. The higher energy of the Ng biomass is attributed to a higher lipid content (40 versus 16% in summer). The biomass of both strains was richest in proteins (65%) in spring. In all seasons, Sm142 was more effective in removing phosphorus from the cultivation medium (6.58 versus 4.14 mg L-1 d-1 in summer), whereas Ng was more effective in removing nitrogen only in summer (55.0 versus 30.8 mg L-1 d-1 ). Our results suggesting that, depending on the purpose, either the same or different local species can be cultivated are relevant when designing outdoor pilot studies. This article is protected by copyright. All rights reserved.
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| 7. |
- Cheregi, Otilia, et al.
(författare)
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Microalgae biotechnology in Nordic countries - the potential of local strains.
- 2019
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Ingår i: Physiologia plantarum. - : Wiley. - 1399-3054 .- 0031-9317. ; 166:1, s. 438-450
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Forskningsöversikt (refereegranskat)abstract
- Climate change, energy use and food security are the main challenges that our society is facing nowadays. Biofuels and feedstock from microalgae can be part of the solution if high and continuous production is to be ensured. This could be attained in year-round, low cost, outdoor cultivation systems using strains that are not only champion producers of desired compounds but also have robust growth in a dynamic climate. Using microalgae strains adapted to the local conditions may be advantageous particularly in Nordic countries. Here, we review the current status of laboratory and outdoor-scale cultivation in Nordic conditions of local strains for biofuel, high-value compounds and water remediation. Strains suitable for biotechnological purposes were identified from the large and diverse pool represented by saline (NE Atlantic Ocean), brackish (Baltic Sea) and fresh water (lakes and rivers) sources. Energy-efficient annual rotation for cultivation of strains well adapted to Nordic climate has the potential to provide high biomass yields for biotechnological purposes.
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| 8. |
- Cheregi, Otilia, et al.
(författare)
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Presence of state transitions in the cryptophyte alga Guillardia theta
- 2015
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Ingår i: Journal of Experimental Botany. - : Oxford University Press. - 0022-0957 .- 1460-2431. ; 66:20, s. 6461-6470
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Tidskriftsartikel (refereegranskat)abstract
- Plants and algae have developed various regulatory mechanisms for optimal delivery of excitation energy to the photosystems even during fluctuating light conditions; these include state transitions as well as non-photochemical quenching. The former process maintains the balance by redistributing antennae excitation between the photosys-tems, meanwhile the latter by dissipating excessive excitation inside the antennae. In the present study, these mecha-nisms have been analysed in the cryptophyte alga Guillardia theta. Photoprotective non-photochemical quenching was observed in cultures only after they had entered the stationary growth phase. These cells displayed a diminished overall photosynthetic efficiency, measured as CO2 assimilation rate and electron transport rate. However, in the logarithmic growth phase G. theta cells redistributed excitation energy via a mechanism similar to state transitions. These state transitions were triggered by blue light absorbed by the membrane integrated chlorophyll a/c antennae, and green light absorbed by the lumenal biliproteins was ineffective. It is proposed that state transitions in G. thetaare induced by small re-arrangements of the intrinsic antennae proteins, resulting in their coupling/uncoupling to the photosystems in state 1 or state 2, respectively. G. theta therefore represents a chromalveolate algae able to perform state transitions.
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| 9. |
- Cheregi, Otilia, et al.
(författare)
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Regulation of the scp Genes in the Cyanobacterium Synechocystis sp PCC 6803-What is New?
- 2015
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Ingår i: Molecules. - : MDPI AG. - 1431-5157 .- 1420-3049. ; 20:8, s. 14621-14637
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Tidskriftsartikel (refereegranskat)abstract
- In the cyanobacterium Synechocystis sp. PCC 6803 there are five genes encoding small CAB-like (SCP) proteins, which have been shown to be up-regulated under stress. Analyses of the promoter sequences of the scp genes revealed the existence of an NtcA binding motif in two scp genes, scpB and scpE. Binding of NtcA, the key transcriptional regulator during nitrogen stress, to the promoter regions was shown by electrophoretic mobility shift assay. The metabolite 2-oxoglutarate did not increase the affinity of NtcA for binding to the promoters of scpB and scpE. A second motif, the HIP1 palindrome 5' GGCGATCGCC 3', was detected in the upstream regions of scpB and scpC. The transcription factor encoded by sll1130 has been suggested to recognize this motif to regulate heat-responsive genes. Our data suggest that HIP1 is not a regulatory element within the scp genes. Further, the presence of the high light regulatory (HLR1) motif was confirmed in scpB-E, in accordance to their induced transcriptions in cells exposed to high light. The HLR1 motif was newly discovered in eight additional genes.
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| 10. |
- Cheregi, Otilia, et al.
(författare)
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The search for new chlorophyll-binding proteins in the cyanobacterium Synechocystis sp. PCC 6803
- 2012
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Ingår i: Journal of Biotechnology. - : Elsevier. - 0168-1656 .- 1873-4863. ; 162:1, s. 124-133
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Tidskriftsartikel (refereegranskat)abstract
- Light harvesting provides a major challenge in the production of biofuels from microorganisms; while sunlight provides the energy necessary for biomass/biofuel production, at the same time it damages the cells. The genome of Synechocystis sp. PCC 6803 was searched for open reading frames that might code for yet unidentified chlorophyll-binding proteins with low molecular mass that could be involved in stress-adaptation. Amongst 9,167 hypothetical ORFs corresponding to potential polypeptides of 100 amino acids or less, two were identified that had the potential to be pigment-binding, because they i) encoded a potential transmembrane region, ii) showed sequence similarity with known chlorophyll-binding domains, iii) were conserved in other cyanobacterial species, and iv) their codon adaptation index indicated significant translation probability. The two ORFs were located complementary (antisense) and internal to the ferrochelatase (hemH) and the pyruvate dehydrogenase (pdh) genes and therefore were named a-fch and a-pdh, respectively. Transcription of both genes was confirmed; however, no translated proteins could be detected immunologically. Whereas mutations within a-pdh or a-fch did not lead to any obvious phenotype, it is clear that transcripts and proteins over and above the currently known set may play a role in defining the physiology of cyanobacteria and other organisms.
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| 11. |
- Cheregi, Otilia, et al.
(författare)
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Transcriptome analysis reveals insights into adaptive responses of two marine microalgae species to Nordic seasons
- 2023
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Ingår i: Algal Research. - 2211-9264. ; 74
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Tidskriftsartikel (refereegranskat)abstract
- There is an increasing interest in algae-based biomass produced outdoors in natural and industrial settings for biotechnological applications. To predict the yield and biochemical composition of the biomass, it is important to understand how the transcriptome of species and strains of interest is affected by seasonal changes. Here we studied the effects of Nordic winter and summer on the transcriptome of two phytoplankton species, namely the diatom Skeletonema marinoi (Sm) and the eustigmatophyte Nannochloropsis granulata (Ng), recently identified as potentially important for biomass production on the west coast of Sweden. Cultures were grown in photobioreactors in simulated Nordic summer and winter, and the gene expression in two phases was quantified by Illumina RNA-sequencing. Five paired comparisons were made among the four conditions. Sm was overall more responsive to seasons since 70 % of the total transcriptome (14,783 genes) showed differential expression in at least one comparison as compared to 1.6 % (1403 genes) for Ng. For both species, we observed larger differences between the seasons than between the phases of the same season. In summer phase 1, Sm cells focused on photosynthesis and polysaccharide biosynthesis. Nitrate assimilation and recycling of intracellular nitrogen for protein biosynthesis were more active in summer phase 2 and throughout winter. Lipid catabolism was upregulated in winter relative to summer to supply carbon for respiration. Ng favored lipid accumulation in summer, while in winter activated different lipid remodeling pathways as compared to Sm. To cope with winter, Ng upregulated breakdown and transport of carbohydrates for energy production. Taken together, our transcriptome data reveal insights into adaptive seasonal responses of Sm and Ng important for biotechnological applications on the west coast of Sweden, but more work is required to decipher the molecular mechanisms behind these responses.
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| 12. |
- Dukic, Emilija, et al.
(författare)
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K+ and Cl- channels/transporters independently fine-tune photosynthesis in plants.
- 2019
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Ingår i: Scientific reports. - : Springer Science and Business Media LLC. - 2045-2322. ; 9:1
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Tidskriftsartikel (refereegranskat)abstract
- In variable light environments, plants adjust light use in photosynthetic electron transport and photoprotective dissipation in the thylakoid membrane. In this respect, roles of the K+/H+ antiporter KEA3, the Cl- channel/transporter CLCe and the voltage-dependent Cl- channel VCCN1 have been unraveled in Arabidopsis thaliana. Here we report that they independently adjust photosynthesis on the basis of analyses using single and higher order loss-of-function mutants. In short experiments of photosynthetic response on transition from dark to low light, we reveal a sequential functioning of VCCN1 and CLCe in the activation of photoprotection and of KEA3 in its downregulation to a low steady state while adjusting the electron transport. On transition from low to high light, VCCN1 accelerates the activation of photoprotection, whereas KEA3 slows it down on transition from high to low light. Based on parallel electrochromic band shift measurements, the mechanism behind is that VCCN1 builds up a pH gradient across the thylakoid membrane, whereas KEA3 dissipates this gradient, which affects photoprotection. CLCe regulates photosynthesis by a pH-independent mechanism likely involving Cl- homeostasis. Nevertheless, all genotypes grow well in alternating high and low light. Taken together, the three studied ion channels/transporters function independently in adjusting photosynthesis to the light environment.
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| 13. |
- Ekendahl, Susanne, 1965, et al.
(författare)
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Energieffektiv odling av marina mikroalger vid den svenska västkusten - potentiella applikationer : RISE Rapport 2020: 72
- 2020
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Rapport (övrigt vetenskapligt/konstnärligt)abstract
- An energy-efficient cultivation method for microalgae has been further developed for the Nordic climate, specifically for the Swedish west coast. The principle for the cultivation process has earlier been developed by RISE for freshwater microalgae. In this project, we have used marine species, and thereby also seawater, in the cultivation. Utilization of natural seawater lowers the environmental impact since, during some circumstances, freshwater may become limiting even in Sweden. The aim of the study was to investigate if selected marine species could be rotated during the year to achieve a longer cultivation season and increased production of energy-rich algal biomass. Winter, spring and summer at the Swedish west coast were simulated in cultivation experiments in laboratory bioreactors with the species Skeletonema marinoi, usually thriving and blooming on the west coast in winter and spring, and Nannochloropsis granulata, which blooms in summer and produces high amounts of lipids. Nannochloropsis was shown to be more suitable for cultivation during spring and summer, when it comes to production of biomass and energy, while Skeletonema grew better during winter and displayed a higher uptake of phosphate than Nannochloropsis. The productivity of Nannochloropsis was in the same range as that of a previously studied freshwater microalgal species, Tetradesmus obliquus. We have shown the principle for a rotational cultivation strategy and that species adapted for a cold climate can give higher productivity during the cold season. Nevertheless, the selected species (Skeletonema) generally showed too low productivity when cultivated during winter to be energy efficient. More species need to be studied to identify those with a high productivity during winter conditions. Even if cold-adapted species are less productive than the summer-adapted ones, considering the available sunlight, they could still be used to prolong the growth season. The cultivation process of Nannochloropsis could also be optimized in an outdoor environment to prolong the season in different ways, since it was tolerant to cold conditions. In practice, the work-related consequences of a rotational cultivation should be weighed against its benefits for each application. Possible applications mainly include cleaning of air and seawater, production of energy, biomass and biomaterials for the industry.
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| 14. |
- Kieselbach, Thomas, et al.
(författare)
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Proteomic analysis of the phycobiliprotein antenna of the cryptophyte alga Guillardia theta cultured under different light intensities
- 2018
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Ingår i: Photosynthesis Research. - : Springer. - 0166-8595 .- 1573-5079. ; 135:1–3, s. 149-163
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Tidskriftsartikel (refereegranskat)abstract
- Plants and algae have developed various light-harvesting mechanisms for optimal delivery of excitation energy to the photosystems. Cryptophyte algae have evolved a novel soluble light-harvesting antenna utilizing phycobilin pigments to complement the membrane-intrinsic Chl a/c-binding LHC antenna. This new antenna consists of the plastid-encoded β-subunit, a relic of the ancestral phycobilisome, and a novel nuclear-encoded α-subunit unique to cryptophytes. Together, these proteins form the active α1β·α2β-tetramer. In all cryptophyte algae investigated so far, the α-subunits have duplicated and diversified into a large gene family. Although there is transcriptional evidence for expression of all these genes, the X-ray structures determined to date suggest that only two of the α-subunit genes might be significantly expressed at the protein level. Using proteomics, we show that in phycoerythrin 545 (PE545) of Guillardia theta, the only cryptophyte with a sequenced genome, all 20 α-subunits are expressed when the algae grow under white light. The expression level of each protein depends on the intensity of the growth light, but there is no evidence for a specific light-dependent regulation of individual members of the α-subunit family under the growth conditions applied. GtcpeA10 seems to be a special member of the α-subunit family, because it consists of two similar N- and C-terminal domains, which likely are the result of a partial tandem gene duplication. The proteomics data of this study have been deposited to the ProteomeXchange Consortium and have the dataset identifiers PXD006301 and 10.6019/PXD006301.
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| 15. |
- Mähler, Niklas, et al.
(författare)
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Synergy : a web resource for exploring gene regulation in Synechocystis sp. PCC6803
- 2014
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Ingår i: PLOS ONE. - : Public Library of Science (PLoS). - 1932-6203. ; 9:11
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Tidskriftsartikel (refereegranskat)abstract
- Despite being a highly studied model organism, most genes of the cyanobacterium Synechocystis sp. PCC 6803 encode proteins with completely unknown function. To facilitate studies of gene regulation in Synechocystis, we have developed Synergy (http://synergy.plantgenie.org), a web application integrating co-expression networks and regulatory motif analysis. Co-expression networks were inferred from publicly available microarray experiments, while regulatory motifs were identified using a phylogenetic footprinting approach. Automatically discovered motifs were shown to be enriched in the network neighborhoods of regulatory proteins much more often than in the neighborhoods of non-regulatory genes, showing that the data provide a sound starting point for studying gene regulation in Synechocystis. Concordantly, we provide several case studies demonstrating that Synergy can be used to find biologically relevant regulatory mechanisms in Synechocystis. Synergy can be used to interactively perform analyses such as gene/motif search, network visualization and motif/function enrichment. Considering the importance of Synechocystis for photosynthesis and biofuel research, we believe that Synergy will become a valuable resource to the research community.
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