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Isolation and characterization of thylakoid membranes from the filamentous cyanobacterium Nostoc punctiforme
Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Photochemistry and Molecular Science, Molecular Biomimetics.
Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Photochemistry and Molecular Science, Molecular Biomimetics.
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2007 (English)In: Physiologia Plantarum: An International Journal for Plant Biology, ISSN 0031-9317, E-ISSN 1399-3054, Vol. 131, no 4, 622-634 p.Article in journal (Refereed) Published
Abstract [en]

Nostoc punctiforme strain Pasteur Culture Collection (PCC) 73102, a sequenced filamentous cyanobacterium capable of nitrogen fixation, is used as a model organism for characterization of bioenergetic processes during nitrogen fixation in Nostoc. A protocol for isolating thylakoid membranes was developed to examine the biochem. and biophys. aspects of photosynthetic electron transfer. Thylakoids were isolated from filaments of N. punctiforme by pneumatic pressure-drop lysis. The activity of photosynthetic enzymes in the isolated thylakoids was analyzed by measuring oxygen evolution activity, fluorescence spectroscopy and ESR spectroscopy. Electron transfer was found functional in both PSII and PSI. Electron transfer measurements in PSII, using diphenylcarbazide as electron donor and 2,6-dichlorophenolindophenol as electron acceptor, showed that 80% of the PSII centers were active in water oxidn. in the final membrane prepn. Anal. of the membrane protein complexes was made by 2D gel electrophoresis, and identification of representative proteins was made by mass spectrometry. The ATP synthase, several oligomers of PSI, PSII and the NAD(P)H dehydrogenase (NDH)-1L and NDH-1M complexes, were all found in the gels. Some differences were noted compared with previous results from Synechocystis sp. PCC 6803. Two oligomers of PSII were found, monomeric and dimeric forms, but no CP43-less complexes. Both dimeric and monomeric forms of Cyt b6/f could be obsd. In all, 28 different proteins were identified, of which 25 are transmembrane proteins or membrane associated ones.

Place, publisher, year, edition, pages
2007. Vol. 131, no 4, 622-634 p.
Keyword [en]
Nostoc punctiforme, isolation, membranes, thylakoids, proteomics, photosystem II, photosystem I
National Category
Chemical Sciences Biological Sciences
Research subject
Biochemistry
Identifiers
URN: urn:nbn:se:uu:diva-12478DOI: 10.1111/j.1399-3054.2007.00982.xISI: 000250763500010PubMedID: 18251853OAI: oai:DiVA.org:uu-12478DiVA: diva2:40247
Available from: 2012-05-08 Created: 2007-12-27 Last updated: 2017-12-11Bibliographically approved
In thesis
1. The Heterocysts of Nostoc punctiforme: From Proteomics to Energy Transfer
Open this publication in new window or tab >>The Heterocysts of Nostoc punctiforme: From Proteomics to Energy Transfer
2009 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

The aim of this thesis is to provide a thorough characterization of the photosynthetic machinery from the heterocysts of Nostoc punctiforme strain ATCC 29133. In this thesis I describe the protocols I have optimized for the isolation of thylakoids from vegetative cells, the purification of heterocysts and the isolation of thylakoids from the purified heterocysts. The composition of the thylakoid membranes was studied by two dimensional electrophoresis and mass-spectrometry. Further insight into the functionality of the photosynthetic complexes was obtained by EPR, electron transport measurements through Photosystem II (PSII), and fluorescence spectroscopy. The proteome of the heterocysts thylakoids compared to that of the vegetative cell was found to be dominated by Photosystem I (PSI) and ATP-synthase complexes, both essential for keeping high nitrogenase activities. Surprisingly, we found a significant amount of assembled monomeric PSII complexes in the heterocysts thylakoid membranes. We measured in vitro light-driven electron transfer from PSII in heterocysts using an artificial electron donor, suggesting that under certain circumstances heterocysts might activate PSII. Parallel to my main research I also worked in a collaboration to elucidate the total proteome of Nostoc sp. strain 7120 and Nostoc punctiforme using quantitative shotgun proteomics. Several hundred proteins were quantified for both species. It was possible to trace the detailed changes that occurred in the energy and nitrogen metabolism of a heterocyst after differentiation. Moreover, the presence of PSII proteins identified in our membrane proteome was also confirmed and extended. Lastly, I studied how the heterocysts are capable of responding to variations in light quality as compared to vegetative cells. Using 77 K fluorescence spectroscopy on heterocysts and vegetative cells previously illuminated with light at specific wavelengths, I was able to demonstrate that heterocysts still possess a possibly modified but functional antenna system, capable of harvesting light and transferring energy preferentially to PSI. The characterization of the membrane and total proteome permitted to draw a more comprehensive and integrated picture of the interplay between the distinct metabolic processes that are carried out in each cell type at the same time; from oxygenic photosynthesis and carbon fixation in the vegetative cells to the anoxygenic cyclic photophosphorylation essential to power nitrogen assimilation in the heterocysts.

Place, publisher, year, edition, pages
Uppsala: Acta Universitatis Upsaliensis, 2009. 78 p.
Series
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Science and Technology, ISSN 1651-6214 ; 671
Keyword
Nostoc punctiforme, heterocyst, photosynthesis, thylakoid, isolation, proteomics, photosystem, energy transfer, hydrogen
National Category
Biochemistry and Molecular Biology
Research subject
Biochemistry
Identifiers
urn:nbn:se:uu:diva-108413 (URN)978-91-554-7607-6 (ISBN)
Public defence
2009-10-30, Häggsalen, Lägerhyddsvägen 1, Uppsala, 10:15 (English)
Opponent
Supervisors
Available from: 2009-10-09 Created: 2009-09-17 Last updated: 2009-10-09
2. Maturation and Regulation of Cyanobacterial Hydrogenases
Open this publication in new window or tab >>Maturation and Regulation of Cyanobacterial Hydrogenases
2009 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Accelerated global warming plus an increasing need for energy is an equation not easily solved, thus new forms of sustainable energy production are urgently requested. In this context hydrogen production based on a cyanobacterial system offers an environmentally friendly alternative for energy capture and conversion. Cyanobacteria can produce hydrogen gas from sun light and water through the combination of photosystems and hydrogenases, and are suitable to cultivate in large scale.

In the present thesis the maturation process of [NiFe]-hydrogenases is investigated with special focus on transcription of the accessory genes encoding proteins needed for assembly of the large and possibly also for the small hydrogenase subunit. The cyanobacteria used are two N2-fixing, filamentous, heterocystous strains; Nostoc sp. strain PCC 7120 and Nostoc punctiforme PCC 73102.

For a biotechnological exploration of hydrogen production tools for regulatory purposes are important. The transcription factor CalA (cyanobacterial AbrB like) (Alr0946 in the genome) in Nostoc sp. strain PCC 7120 was found to be involved in hydrogen metabolism by regulating the transcription of the maturation protein HypC. Further the bidirectional hydrogenase activity was down-regulated in the presence of elevated levels of CalA, a result important to take into account when optimizing cyanobacteria for hydrogen production.

CalA regulates at least 25 proteins in Nostoc sp. strain PCC 7120 and one of the down-regulated proteins was superoxide dismutase, FeSOD. The characterization of FeSOD shows that it has a specific and important function in the oxidative stress tolerance of Nostoc sp. stain PCC 7120. Since CalA is involved in regulation of both the hydrogen metabolism as well as stress responses these findings indicate that Alr0946 is an important transcription factor in Nostoc sp. strain PCC 7120 active on a global level in the cell.

This thesis adds more knowledge concerning maturation and regulation of cyanobacterial hydrogenases which might be useful for future large scale hydrogen.

Place, publisher, year, edition, pages
Uppsala: Acta Universitatis Upsaliensis, 2009. 62 p.
Series
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Science and Technology, ISSN 1651-6214 ; 695
Keyword
Biohydrogen, Cyanobacteria, Nostoc sp. PCC 7120, Nostoc punctiforme PCC 73102, Hydrogenase, Maturation, Transcriptional regulation, CyAbrB, CalA, FeSOD
Identifiers
urn:nbn:se:uu:diva-110871 (URN)978-91-554-7673-1 (ISBN)
Public defence
2010-01-15, Häggsalen, The Ångström Laboratory, Lägerhyddsvägen 1, Polacksbacken, Uppsala, 13:00 (English)
Opponent
Supervisors
Available from: 2009-12-21 Created: 2009-11-27 Last updated: 2009-12-21Bibliographically approved

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Cardona, TanaiAgervald, ÅsaStyring, StenbjörnLindblad, PeterMagnuson, Ann

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