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Quantitative shotgun proteomics of enriched heterocysts from Nostoc sp. PCC 7120 using 8-Plex isobaric peptide tags
Department of Chemical and Process Engineering, The University of Sheffield. (Biological & Environmental Systems Group)
Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Photochemistry and Molecular Science, Molecular Biomimetics.
Virginia Commonwealth University. (Center for the Study of Biological Complexity)
Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Photochemistry and Molecular Science, Molecular Biomimetics.
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2008 (English)In: Journal of Proteome Research, ISSN 1535-3893, E-ISSN 1535-3907, Vol. 7, no 4, 1615-1628 p.Article in journal (Refereed) Published
Abstract [en]

The filamentous cyanobacterium Nostoc sp. strain PCC 7120 is capable of fixing atmospheric nitrogen. The labile nature of the core process requires the terminal differentiation of vegetative cells to form heterocysts, specialized cells with altered cellular and metabolic infrastructure to mediate the N2-fixing process. We present an investigation targeting the cellular proteomic expression of the heterocysts compared to vegetative cells of a population cultured under N2-fixing conditions. New 8-plex iTRAQ reagents were used on enriched replicate heterocyst and vegetative cells, and replicate N2-fixing and non-N2-fixing filaments to achieve accurate measurements. With this approach, we successfully identified 506 proteins, where 402 had confident quantifications. Observations provided by purified heterocyst analysis enabled the elucidation of the dominant metabolic processes between the respective cell types, while emphasis on the filaments enabled an overall comparison. The level of analysis provided by this investigation presents various tools and knowledge that are important for future development of cyanobacterial biohydrogen production.

Place, publisher, year, edition, pages
2008. Vol. 7, no 4, 1615-1628 p.
Keyword [en]
8-plex, iTRAQ, proteomics, Nostoc sp PCC 7120, Tandem-MS, nitrogen fixation, cyanobacteria, nitrogen fixation, heterocyst, Proteome
National Category
Biochemistry and Molecular Biology
Research subject
Biochemistry
Identifiers
URN: urn:nbn:se:uu:diva-108461DOI: 10.1021/pr700604vISI: 000254711000026OAI: oai:DiVA.org:uu-108461DiVA: diva2:235947
Available from: 2012-05-08 Created: 2009-09-18 Last updated: 2017-12-13
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

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Cardona, TanaiMagnuson, AnnLindblad, PeterStensjö, Karin

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