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Regulation of nitrogen fixation in Rhodospirillum rubrum: Through proteomics and beyond
Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
2010 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Adaptability is one of the reasons for the success of bacteria, allowing them to survive in conditions where no other organisms would be able to thrive. Nitrogen deficiency, for example, can be a limiting factor for the growth of micro-organisms, as this element is an essential part of almost all types of biomolecules. As such, some bacteria have evolved specific mechanisms to overcome nitrogen limitation. Nitrogen fixing bacteria, or diazotrophs, use a specific enzyme complex, nitrogenase, in order to harness this element from the enormous reservoir that is the Earth’s atmosphere. However, nitrogen fixation is a demanding process for the cells, requiring vast amounts of energy and tight regulation.

In this thesis we explore the mechanisms regulating nitrogen fixation in Rhodospirillum rubrum, a purple non-sulphur photosynthetic bacterium. Using proteomics tools, we show how the regulation of both the nitrogen and carbon fixation processes is interconnected, possibly in order to maintain the intracellular redox balance. Using a new detergent molecule, we also demonstrate how nitrogen availability affects the chromatophore membrane proteome.

Our studies have revealed the crucial role of the cellular pool of 2-oxoglutarate (2OG) for adequate signaling through the PII proteins and the effects resulting from artificially manipulating this metabolite’s concentration. In R. rubrum nitrogenase is also subjected to post-translational control (the “switch-off” effect) and this work shows for the first time that the enzyme modifying nitrogenase (Dinitrogenase Reductase ADP-ribsosyl Transferase or DRAT) forms a complex with the PII protein GlnB. This complex allows DRAT activation and its formation – and, therefore, DRAT activity – is regulated by binding of ADP:ATP and 2OG to GlnB.

Upon light withdrawal, nitrogenase activity anaerobically in the dark is also here demonstrated to be dependent on the activity of the pathway starting in pyruvate formate-lyase and we show how different nitrogen sources influence the switch-off response. This response can, in some conditions, be modified by addition of pyruvate and we have studied how this metabolite influences nitrogenase activity and switch-off regulation.

This study allows a better understanding of the underlying processes controlling the metabolic routes in R. rubrum and also provides new insights into regulation of enzyme activity, paving the road for the complete establishment of the mechanisms regulating nitrogenase switch-off.

Place, publisher, year, edition, pages
Stockholm: Department of Biochemistry and Biophysics, Stockholm University , 2010. , 71 p.
Keyword [en]
Rhodospirillum rubrum, nitrogen fixation, redox balance, switch-off, DRAT
National Category
Biochemistry and Molecular Biology
Research subject
Biochemistry
Identifiers
URN: urn:nbn:se:su:diva-42101ISBN: 978-91-7447-125-0 (print)OAI: oai:DiVA.org:su-42101DiVA: diva2:344106
Public defence
2010-10-08, Magnélisalen, Kemiska övningslaboratoriet, Svante Arrhenius väg 16 B, Stockholm, 10:00 (English)
Opponent
Supervisors
Note

At the time of the doctoral defense, the following papers were unpublished and had a status as follows: Paper 2: In press. Paper 3: Submitted. Paper 4: Manuscript. Paper 5: Submitted.

Available from: 2010-09-16 Created: 2010-08-16 Last updated: 2012-10-03Bibliographically approved
List of papers
1. Comparative proteomic studies in Rhodospirillum rubrum grown under different nitrogen conditions
Open this publication in new window or tab >>Comparative proteomic studies in Rhodospirillum rubrum grown under different nitrogen conditions
2008 (English)In: Journal of Proteome Research, ISSN 1535-3893, E-ISSN 1535-3907, Vol. 7, no 8, 3267-3275 p.Article in journal (Refereed) Published
Abstract [en]

Forty-four differentially expressed proteins have been identified in the photosynthetic diazotroph Rhodospirillum rubrum grown anaerobic and photoheterotrophically, with different nitrogen sources, using 2D-PAGE and MALDI-TOF, from gels containing an average of 679 ± 52 (in N+) and 619 ± 37 (in N−) protein spots for each gel. A higher level of expression was found under nitrogen-rich growth, for proteins involved in carbon metabolism (reductive tricarboxylic acid cycle, CO2 fixation, and poly-β-hydroxybutyrate metabolism) and amino acid metabolism. The key enzymes RuBisCO and α-ketoglutarate synthase were found to be present in higher amounts in nitrogen-rich conditions. Ntr and Nif regulated proteins, such as glutamine synthetase and nitrogenase, were, as expected, induced under nitrogen-fixing conditions and glutamate dehydrogenase was down regulated. A novel 2Fe-2S ferredoxin with unknown function was identified from nitrogen-fixing cultures. In addition to differential expression, two of the identified proteins revealed variable pI values in response to the nitrogen source used.

Keyword
Rhodospirillum rubrum, nitrogen fixation, comparative proteome, 2D-PAGE
National Category
Medical Biotechnology (with a focus on Cell Biology (including Stem Cell Biology), Molecular Biology, Microbiology, Biochemistry or Biopharmacy)
Research subject
Biochemistry
Identifiers
urn:nbn:se:su:diva-14839 (URN)10.1021/pr700771u (DOI)000258200400017 ()18570453 (PubMedID)
Available from: 2008-11-05 Created: 2008-11-05 Last updated: 2010-09-06Bibliographically approved
2. Diazotrophic growth of Rhodospirillum rubrum with 2-oxoglutarate as sole carbon source affects the regulation of nitrogen metabolism as well as the soluble proteome
Open this publication in new window or tab >>Diazotrophic growth of Rhodospirillum rubrum with 2-oxoglutarate as sole carbon source affects the regulation of nitrogen metabolism as well as the soluble proteome
Show others...
2010 (English)In: Research in Microbiology, ISSN 0923-2508, E-ISSN 1769-7123, Vol. 161, no 8, 651-659 p.Article in journal (Refereed) Published
Abstract [en]

2-Oxoglutarate plays a central role as a signal in the regulation of nitrogen metabolism in the phototrophic diazotroph Rhodospirillum rubrum. In order to further study the role of this metabolite, we have constructed an R. rubrum strain that has the capacity to grow on 2-oxoglutarate as sole carbon source, in contrast to wild-type R. rubrum. This strain has the same growth characteristics as wild-type with malate as carbon source, but showed clear metabolic differences when 2-oxoglutarate was used. Among other things, the regulation of nitrogen metabolism is altered, which can be related to different modification profiles of the regulatory PII proteins.

Keyword
Ammonium assimilation, Nitrogen fixation, Regulation, Rhodospirillum rubrum
National Category
Biochemistry and Molecular Biology
Research subject
Biochemistry
Identifiers
urn:nbn:se:su:diva-42155 (URN)10.1016/j.resmic.2010.06.003 (DOI)000283686200003 ()
Available from: 2010-08-31 Created: 2010-08-18 Last updated: 2017-12-12Bibliographically approved
3. The activity of dinitrogenase reductase ADP-ribosyltransferase of Rhodospirillum rubrum is controlled through reversible complex formation with the PII protein GlnB
Open this publication in new window or tab >>The activity of dinitrogenase reductase ADP-ribosyltransferase of Rhodospirillum rubrum is controlled through reversible complex formation with the PII protein GlnB
2010 (English)Manuscript (preprint) (Other academic)
Keyword
ADP-ribosylation, nitrogenase, PII proteins
Identifiers
urn:nbn:se:su:diva-42158 (URN)
Available from: 2010-08-31 Created: 2010-08-18 Last updated: 2010-09-06Bibliographically approved
4. The effect of pyruvate on the metabolic regulation of nitrogenase activity in Rhodospirillum rubrum with darkness as switch-off effector
Open this publication in new window or tab >>The effect of pyruvate on the metabolic regulation of nitrogenase activity in Rhodospirillum rubrum with darkness as switch-off effector
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2010 (English)In: Microbiology, ISSN 1350-0872, E-ISSN 1465-2080Article in journal (Refereed) Submitted
Abstract [en]

Rhodospirillum rubrum, a photosynthetic diazotroph, is able to regulate nitrogenase activity in response to environmental factors such as ammonium ions or darkness – the so-called switch-off effect. This is due to reversible modification of the Fe-protein one of the two components of nitrogenase. The signal transduction pathway(s) in this regulatory mechanism is not fully understood, especially not in the response to darkness. We have previously shown that the switch-off response and metabolic state differ between cells grown with dinitrogen or glutamate as nitrogen source, although both represent poor nitrogen sources. In this study we show that addition of pyruvate to cultures grown with glutamate as nitrogen source will lead to a switch-off response that is similar to that in cultures grown with dinitrogen. The effects are related to PII protein uridylylation and GS activity. We also show that pyruvate induces de novo protein synthesis and that pyruvate formate-lyase activity is required for activity in the dark.

National Category
Biochemistry and Molecular Biology
Research subject
Biochemistry
Identifiers
urn:nbn:se:su:diva-42159 (URN)
Available from: 2010-09-16 Created: 2010-08-18 Last updated: 2017-12-12Bibliographically approved
5. Identification of Chromatophore Membrane Protein Complexes Formed under Different Nitrogen Availability Conditions in Rhodospirillum rubrum
Open this publication in new window or tab >>Identification of Chromatophore Membrane Protein Complexes Formed under Different Nitrogen Availability Conditions in Rhodospirillum rubrum
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2011 (English)In: Journal of Proteome Research, ISSN 1535-3893, E-ISSN 1535-3907, Vol. 10, no 6, 2703-2714 p.Article in journal (Refereed) Published
Abstract [en]

The chromatophore membrane of the photosynthetic diazotroph Rhodospirillum rubrum is of vital importance for a number of central processes, including nitrogen fixation. Using a novel amphiphile, we have identified protein complexes present under different nitrogen availability conditions by the use of two-dimensional Blue Native/SDS-PAGE and NSI-LC-LTQ-Orbitrap mass spectrometry. We have identified several membrane protein complexes, including components of the ATP synthase, reaction center, light harvesting, and NADH dehydrogenase complexes. Additionally, we have identified differentially expressed proteins, such as subunits of the succinate dehydrogenase complex and other TCA cycle enzymes that are usually found in the cytosol, thus hinting at a possible association to the membrane in response to nitrogen deficiency. We propose a redox sensing mechanism that can influence the membrane subproteome in response to nitrogen availability.

Keyword
Rhodospirillum rubrum, nitrogen metabolism, Blue Native, chromatophore subproteome, amphiphile, Orbitrap
National Category
Biological Sciences
Identifiers
urn:nbn:se:su:diva-67875 (URN)10.1021/pr100838x (DOI)000291186100002 ()
Note
authorCount :8Available from: 2012-01-02 Created: 2012-01-02 Last updated: 2017-12-08Bibliographically approved

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