| 1. |
- Devine, Ellenor, 1977-
(författare)
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Cyanobacterial Hydrogen Metabolism : Regulation and Maturation of Hydrogenases
- 2011
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- In times with elevated CO2 levels and global warming there is a need of finding alternatives to carbon based energy carriers. One such environmental friendly solution could be H2 produced by living organisms. Cyanobacteria are good candidates since they can produce H2 from sunlight and water through the combination of photosynthesis and H2 producing enzymes i.e. nitrogenases and/or [NiFe]-hydrogenases. This thesis investigates the maturation and transcriptional regulation of [NiFe]-hydrogenases in cyanobacteria, with a special focus on hydrogenase specific proteases. The core of all hydrogenases consists of the small and large subunit. The large subunit in which the catalytic site is located goes through an extenstive maturation process which ends with a proteolytic cleavage performed by a hydrogenase specific protease (HupW/HoxW). This thesis shows that within the maturation process of hydrogenases, the proteolytic cleavage is probably the only step that is specific with respect to different types of hydrogenases i.e. one type of protease cleaves only one type of hydrogenase. Further in-silico analysis revealed that these proteases and the hydrogenases might have co-evolved since ancient time and that the specificity observed could be the result of a conserved amino acid sequence which differs between the two types of proteases (HupW/HoxW). A number of different transcription factors were revealed and shown to interact with the promoter regions of several of the genes encoding maturation proteins. The results indicate that the hydrogenase specific proteases are regulated on a transcriptional level in a similar manner as the hydrogenases they cleave. This thesis contributes with knowledge concerning transcriptional regulation and protein regulation of hydrogenases which will be useful for designing genetically engineered cyanobacteria with an improved and adjustable H2 production.
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| 2. |
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| 3. |
- Lopes Pinto, Fernando, 1974-
(författare)
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Development of Molecular Biology and Bioinformatics Tools : From Hydrogen Evolution to Cell Division in Cyanobacteria
- 2009
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- The use of fossil fuels presents a particularly interesting challenge - our society strongly depends on coal and oil, but we are aware that their use is damaging the environment. Currently, this awareness is gaining momentum, and pressure to evolve towards an energetically cleaner planet is very strong. Molecular hydrogen (H2) is an environmentally suitable energy carrier that could initially supplement or even substitute fossil fuels. Ideally, the primary energy source to produce hydrogen gas should be renewable, and the process of conversion back to energy without polluting emissions, making this cycle environmentally clean. Photoconversion of water to hydrogen can be achieved using the following strategies: 1) the use of photochemical fuel cells, 2) by applying photovoltaics, or 3) by promoting production of hydrogen by photosynthetic microorganisms, either phototrophic anoxygenic bacteria and cyanobacteria or eukaryotic green algae. For photobiological H2 production cyanobacteria are among the ideal candidates since they: a) are capable of H2 evolution, and b) have simple nutritional requirements - they can grow in air (N2 and CO2), water and mineral salts, with light as the only energy source. As this project started, a vision and a set of overall goals were established. These postulated that improved H2 production over a long period demanded: 1) selection of strains taking in consideration their specific hydrogen metabolism, 2) genetic modification in order to improve the H2 evolution, and 3) cultivation conditions in bioreactors should be exmined and improved. Within these goals, three main research objectives were set: 1) update and document the use of cyanobacteria for hydrogen production, 2) create tools to improve molecular biology work at the transcription analysis level, and 3) study cell division in cyanobacteria. This work resulted in: 1) the publication of a review on hydrogen evolution by cyanobacteria, 2) the development of tools to assist understanding of transcription, and 3) the start of a new fundamental research approach to ultimately improve the yield of H2 evolution by cyanobacteria.
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| 4. |
- Lopes Pinto, Fernando, et al.
(författare)
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Generation of non-genomic oligonucleotide tag sequences for RNA template-specific PCR
- 2006
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Ingår i: BMC Biotechnology. - : Springer Science and Business Media LLC. - 1472-6750. ; 6, s. 31-
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Tidskriftsartikel (refereegranskat)abstract
- BackgroundIn order to overcome genomic DNA contamination in transcriptional studies, reverse template-specific polymerase chain reaction, a modification of reverse transcriptase polymerase chain reaction, is used. The possibility of using tags whose sequences are not found in the genome further improves reverse specific polymerase chain reaction experiments. Given the absence of software available to produce genome suitable tags, a simple tool to fulfill such need was developed.ResultsThe program was developed in Perl, with separate use of the basic local alignment search tool, making the tool platform independent (known to run on Windows XP and Linux). In order to test the performance of the generated tags, several molecular experiments were performed. The results show that Tagenerator is capable of generating tags with good priming properties, which will deliberately not result in PCR amplification of genomic DNA.ConclusionThe program Tagenerator is capable of generating tag sequences that combine genome absence with good priming properties for RT-PCR based experiments, circumventing the effects of genomic DNA contamination in an RNA sample.
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| 5. |
- Miranda, Hélder, et al.
(författare)
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Sll1783, a monooxygenase associated with polysaccharide processing in the unicellular cyanobacterium Synechocystis PCC 6803
- 2017
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Ingår i: Physiologia Plantarum. - : Wiley. - 0031-9317 .- 1399-3054. ; 161:2, s. 182-195
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Tidskriftsartikel (refereegranskat)abstract
- Cyanobacteria play a pivotal role as the primary producer in many aquatic ecosystems. The knowledge on the interacting processes of cyanobacteria with its environment - abiotic and biotic factors - is still very limited. Many potential exocytoplasmic proteins in the model unicellular cyanobacterium Synechocystis PCC 6803 have unknown functions and their study is essential to improve our understanding of this photosynthetic organism and its potential for biotechnology use. Here we characterize a deletion mutant of Synechocystis PCC 6803, Δsll1783, a strain that showed a remarkably high light resistance which is related with its lower thylakoid membrane formation. Our results suggests Sll1783 to be involved in a mechanism of polysaccharide degradation and uptake and we hypothesize it might function as a sensor for cell density in cyanobacterial cultures.
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| 6. |
- Axelsson, Rikard, 1970-
(författare)
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Cyanobacterial Hydrogen Metabolism : Transcriptional Regulation of the Hydrogenases in Filamentous Strains
- 2003
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Cyanobacteria are a heterogeneous group of phototrophic microorganisms. Many cyanobacteria have the capacity to fix atmospheric nitrogen. During the process of nitrogen fixation, molecular hydrogen is produced. Three enzymes are directly involved in hydrogen metabolism in cyanobacteria. A nitrogenase, evolving hydrogen during nitrogen-fixation, an uptake hydrogenase, recycling the hydrogen produced by nitrogenase, and a bidirectional hydrogenase that has the capacity to both take up and produce hydrogen. The main objective in this thesis was to examine the transcriptional regulation of both the uptake and the bidirectional hydrogenase in filamentous cyanobacteria.The transcriptional regulation of the uptake hydrogenase was demonstrated to be influenced by external conditions in Nostoc muscorum and Nostoc punctiforme. Nickel, molecular hydrogen, and anaerobic conditions all induced the relative amount of uptake hydrogenase transcript. In addition, a transcript could be detected in nitrogen-fixing, but not in non-nitrogen fixing conditions.The transcriptional regulation of the bidirectional hydrogenase in N. muscorum and Anabaena PCC 7120 was also examined. The relative amount of transcript from the bidirectional hydrogenase in both strains was demonstrated to increase during anaerobic conditions. Moreover, experiments using N. muscorum demonstrated that addition of nickel also increase the amount of transcript. However, no change in the relative amount of transcript from the bidirectional hydrogenase could be observed by additional hydrogen or during a shift from non-nitrogen fixing to nitrogen fixing conditions.The genes responsible for maturation of the hydrogenase were identified, cloned and sequenced in N. punctiforme. The transcription of the genes was examined and all genes were located on a single transcript. Like the uptake hydrogenase, a transcript could be detected under nitrogen-fixing but not under non-nitrogen fixing conditions. Initial studies, using microarrays, were used to analyse and compare the transcription of a large set of Anabaena PCC 7120 genes under non-nitrogen and nitrogen-fixing conditions. Both up- and down-regulated genes could be identified.This thesis advances the knowledge about the transcriptional regulation of the hydrogenases in filamentous cyanobacteria and can be used as a platform for further experiments aiming at a modified hydrogen metabolism.
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| 7. |
- Cardona, Tanai, 1983-, et al.
(författare)
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Electron transfer protein complexes in the thylakoid membranes of heterocysts from the cyanobacterium Nostoc punctiforme
- 2009
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Ingår i: Biochimica et Biophysica Acta - Bioenergetics. - : Elsevier. - 0005-2728 .- 1879-2650. ; 1787:4, s. 252-263
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Tidskriftsartikel (refereegranskat)abstract
- Filamentous, heterocystous cyanobacteria are capable of nitrogen fixation and photoautotrophic growth. Nitrogen fixation takes place in heterocysts that differentiate as a result of nitrogen starvation. Heterocysts uphold a microoxic environment to avoid inactivation of nitrogenase, e.g. by downregulation of oxygenic photosynthesis. The ATP and reductant requirement for the nitrogenase reaction is considered to depend on Photosystem I, but little is known about the organization of energy converting membrane proteins in heterocysts. We have investigated the membrane proteome of heterocysts from nitrogen fixing filaments of Nostoc punctiforme sp. PCC 73102, by 2D gel electrophoresis and mass spectrometry. The membrane proteome was found to be dominated by the Photosystem I and ATP-synthase complexes.We could identify asignificant amount of assembled Photosystem II complexes containing the D1, D2, CP43, CP47 and PsbO proteins from these complexes. We could also measure light-driven in vitro electron transfer from Photosystem II in heterocyst thylakoid membranes. We did not find any partially disassembled PhotosystemII complexes lacking the CP43 protein. Several subunits of the NDH-1 complex were also identified. The relative amount of NDH-1M complexes was found to be higher than NDH-1L complexes, which might suggest a role for this complex in cyclic electron transfer in the heterocysts of Nostoc punctiforme.
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| 8. |
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| 9. |
- Holmqvist, Marie, 1978-
(författare)
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The Cyanobacterial Uptake Hydrogenase : Regulation, Maturation and Function
- 2010
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- With accellerating global warming and pollution problems a change of energy regime is necessary. Solar energy offers a clean and unlimited energy source of enormous potential. Due to it’s intermittenet nature solar energy must be stored - ideally in the chemical bond of a carrier molecule. Hydrogen gas, H2, an energy carrier with water as only emission when used in a fuel cell, is considered to be the choise for the future. In this context cyanobacteria show promising potential as future H2 factories since they can produce H2 from solar energy and water. The main enzymes directly involved in cyanobacterial hydrogen metabolism are nitrogenases and hydrogenases. Cyanobacterial hydrogenases are either uptake hydrogenases or bidirectional hydrogenases and their maturation requires assistance of six maturation proteins and two hydrogenase specific proteases. In this thesis the transcriptional regulation, maturation and function of the cyanobacterial uptake hydrogenases were investigated in the filamentous, heterocyst forming strains Nostoc punctiforme ATCC 29133 and Nostoc sp. strain PCC 7120. Five genes, encoding proteins putatively involved in the maturation of the uptake hydrogenase were identified upstream the known maturation genes. Two transcription factors, CalA and CalB, were found interacting with the stretch of DNA forming the upstream regions of the uptake hydrogenase structural genes and the novel maturation genes. The expression of the uptake hydrogenase were heterocysts specific and the specificity mapped to a short promoter region starting -57 bp upstream the transcription start point. In addition, the function of the uptake hydrogenase was inserted in a metabolic context. Among the proteases, a conserved region was discovered possibly involved in determining the hydrogenase specificity. This thesis has given valuable information about the transcriptional regulation, maturation and function of the uptake hydrogenase in filamentous, heterocystous cyanobacteria and identified new targets for bioengineering of mutant strains with higher H2 production rates.
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| 10. |
- Maneeruttanarungroj, Cherdsak, et al.
(författare)
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A newly isolated green alga, Tetraspora sp. CU2551, from Thailand with efficient hydrogen production
- 2010
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Ingår i: International journal of hydrogen energy. - : Elsevier BV. - 0360-3199 .- 1879-3487. ; 35:24, s. 13193-13199
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Tidskriftsartikel (refereegranskat)abstract
- A novel unicellular hydrogen-producing green alga was isolated from fresh water pond in Pathumthani province, Thailand. Under light microscope, this alga was identified as belonging to the genus Tetraspora. Phylogenetic analysis of 18S rRNA sequence revealed that the green alga, identified as Tetraspora sp. CU2551, is closely related to other unicellular green algal species. Tetraspora sp. CU2551 had the shortest doubling time when grown in Tris-acetate-phosphate (TAP) medium under a light intensity of 48-92 mu E/m(2)/s and a temperature of 36 C. Hydrogen production increased with increasing pH from 5.75 to 9.30; however, almost no production was observed at a pH of 5.25. Addition of 0.5 mM P-mercaptoethanol to the TAP medium stimulated hydrogen production about two-fold. During the hydrogen production phase, the use of TAP medium lacking both nitrogen and sulfur resulted in about 50% increase in the hydrogen production. This was in contrast to only a small increase in the production when either nitrogen or sulfur was omitted in TAP medium. The stimulation of hydrogen production by 0.5 mM beta-mercaptoethanol under nitrogen- and sulfur-deprived conditions occurred only when the cells were grown at a light intensity lower than 5 mu E/m(2)/s with no effects at higher intensities. Maximal calculated hydrogen production, 17.3-61.7 mu mol/mg Chl a/h, is a very high production rate compared to other green algae and makes Tetraspora sp. CU2551 an interesting model strain for photobiological hydrogen production.
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