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PII proteins as global regulators of bacterial nitrogen metabolism
Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
2009 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Nitrogen is an essential element to sustain life, being a component of most biological macromolecules. In spite of the abundance of gaseous N2, the availability of nitrogen compounds that can be readily used by most microorganisms is scarce and its production energetically demanding. Due to the central importance of nitrogen metabolism, most microorganisms evolved elaborate mechanisms to ensure efficient regulation, balancing substrate availability, product formation and energy expenditure.

In most bacteria, many archaea and some plants, the different aspects of nitrogen metabolism are coordinated by members of the PII family of signal transduction proteins, acting as fundamental molecular messengers controlling several cellular processes. In proteobacteria, including the nitrogen fixing organism Rhodospirillum rubrum, these proteins are involved in regulation at different levels: they regulate gene expression, modulating the activity of several transcription factors; they control the flux through the ammonium transport protein (AmtB); they influence the activity of key metabolic enzymes, e.g. glutamine synthetase (GS) and nitrogenase. The signal sensing and integration by these proteins is achieved in two different yet interdependent strategies: allosteric regulation (by the binding of metabolites like ATP, ADP, 2-oxoglutarate) and reversible post-translational modification. Signal integration likely results in different conformations of the proteins, influencing the direct protein-protein interaction with the cellular targets.

In the present work, using R. rubrum as a model organism, we have studied some aspects of the biochemistry of PII proteins in terms of regulatory interactions with the ammonium transport protein AmtB1 and the adenylyltransferase GlnE (involved in GS regulation). Additionally, we have investigated the post-translational modification of PII proteins, showing for the first time in vivo in addition in vitro selectivity in the modification of different PII proteins.

Our results contributed to elucidate several new aspects in the regulation by PII proteins and also strengthened the idea that these proteins act as global regulators in the context of bacterial nitrogen metabolism.

Place, publisher, year, edition, pages
Stockholm: Department of Biochemistry and Biophysics, Stockholms universitet , 2009. , 68 p.
Keyword [en]
Rhodospirillum rubrum, nitrogen metabolism, signal transduction, PII proteins
National Category
Biochemistry and Molecular Biology
Research subject
Biochemistry
Identifiers
URN: urn:nbn:se:su:diva-30815ISBN: 978-91-7155-963-0 (print)OAI: oai:DiVA.org:su-30815DiVA: diva2:274284
Public defence
2009-12-07, Magnélisalen, Svante Arrhenius väg 16 B, Stockholm, 10:00 (English)
Opponent
Supervisors
Note
At the time of the doctoral defence the following papers were unpublished and had a status as follows: Paper 1: Manuscript. Paper 4: Manuscript.Available from: 2009-11-15 Created: 2009-10-27 Last updated: 2010-01-12Bibliographically approved
List of papers
1. Molecular basis of the divalent cation selectivity of the uridylyltransferase GlnD towards the signal transduction proteins GlnJ and GlnB.
Open this publication in new window or tab >>Molecular basis of the divalent cation selectivity of the uridylyltransferase GlnD towards the signal transduction proteins GlnJ and GlnB.
(English)Manuscript (preprint) (Other academic)
Abstract [en]

PII proteins have a fundamental role in the control of nitrogen metabolism in bacteria, with the PII-target interaction being controlled by metabolite binding and post-translational modification. In the photosynthetic bacterium Rhodospirillum rubrum, the PII proteins GlnB and GlnJ were shown, in spite of their high degree of similarity, to have different requirements for post-translational uridylylation, with respect to the divalent cations, Mg2+ and Mn2+. Given the importance of uridylylation in the functional interactions of PII proteins, we have addressed the molecular basis for that difference and identified two amino acid residues that influence the divalent cation selectivity.

Research subject
Biochemistry
Identifiers
urn:nbn:se:su:diva-30810 (URN)
Available from: 2009-10-27 Created: 2009-10-27 Last updated: 2010-01-12Bibliographically approved
2. The activity of adenylyltransferase in Rhodospirillum rubrum is only affected by alpha-ketoglutarate and unmodified PII proteins, but not by glutamine, in vitro
Open this publication in new window or tab >>The activity of adenylyltransferase in Rhodospirillum rubrum is only affected by alpha-ketoglutarate and unmodified PII proteins, but not by glutamine, in vitro
2007 (English)In: The FBS Journal, ISSN 1742-464X, Vol. 274, no 10, 2449-2460 p.Article in journal (Refereed) Published
Abstract [en]

Ammonium assimilation is tightly regulated in nitrogen-fixing bacteria; the target of regulation is primarily the activity of the key enzyme glutamine synthetase that is regulated by reversible covalent modification by AMP groups in reactions catalysed by the bifunctional adenylyltransferase (ATase). The properties and regulation of ATase from Escherichia coli have been studied in great detail. We have investigated the regulation of ATase from Rhodospirillum rubrum, a photosynthetic nitrogen-fixing bacterium. In this diazotroph, nitrogenase is regulated at the metabolic level in addition to the transcriptional regulation operating in all diazotrophic bacteria, which makes understanding the regulatory features of nitrogen assimilation even more interesting. We show that in R. rubrum, in contrast to the E. coli system, ATase is primarily regulated by α-ketoglutarate and that glutamine has no effect on neither the adenylylation nor the deadenylylation of glutamine synthetase. Furthermore, the role of the regulatory PII proteins is only to stimulate the adenylylation reaction, as there is no effect on the reverse reaction. We propose that in R. rubrum and possibly other diazotrophs α-ketoglutarate plays the central role in the regulation of ATase and thus glutamine synthetase activity.

Keyword
adenylyltransferase, ammonium assimilation, glutamine synthetase, Rhodospirillum rubrum
Identifiers
urn:nbn:se:su:diva-10353 (URN)10.1111/j.1742-4658.2007.05778.x (DOI)000246029800002 ()17419734 (PubMedID)
Available from: 2007-12-28 Created: 2007-12-28 Last updated: 2010-01-14Bibliographically approved
3. Interaction of the signal transduction protein GlnJ with the cellular targets AmtB1, GlnE and GlnD in Rhodospirillum rubrum: dependence on manganese, 2-oxoglutarate and the ADP/ATP ratio.
Open this publication in new window or tab >>Interaction of the signal transduction protein GlnJ with the cellular targets AmtB1, GlnE and GlnD in Rhodospirillum rubrum: dependence on manganese, 2-oxoglutarate and the ADP/ATP ratio.
Show others...
2008 (English)In: Microbiology, ISSN 1350-0872, E-ISSN 1465-2080, Vol. 154, no Pt 8, 2336-47 p.Article in journal (Refereed) Published
Abstract [en]

The PII family of signal transduction proteins is widespread amongst the three domains of life, and its members have fundamental roles in the general control of nitrogen metabolism. These proteins exert their regulatory role by direct protein-protein interaction with a multitude of cellular targets. The interactions are dependent on the binding of metabolites such as ATP, ADP and 2-oxoglutarate (2-OG), and on whether or not the PII protein is modified. In the photosynthetic nitrogen-fixing bacterium Rhodospirillum rubrum three PII paralogues have been identified and termed GlnB, GlnJ and GlnK. In this report we analysed the interaction of GlnJ with known cellular targets such as the ammonium transporter AmtB1, the adenylyltransferase GlnE and the uridylyltransferase GlnD. Our results show that the interaction of GlnJ with cellular targets is regulated in vitro by the concentrations of manganese and 2-OG and the ADP : ATP ratio. Furthermore, we show here for the first time, to our knowledge, that in the interactions of GlnJ with the three different partners, the energy signal (ADP : ATP ratio) in fact overrides the carbon/nitrogen signal (2-OG). In addition, by generating specific amino acid substitutions in GlnJ we show that the interactions with different cellular targets are differentially affected, and the possible implications of these results are discussed. Our results are important to further the understanding of the regulatory role of PII proteins in R. rubrum, a photosynthetic bacterium in which the nitrogen fixation process and its intricate control mechanisms make the regulation of nitrogen metabolism even more complex than in other studied bacteria.

Identifiers
urn:nbn:se:su:diva-30682 (URN)10.1099/mic.0.2008/017533-0 (DOI)000258860200016 ()18667566 (PubMedID)
Available from: 2009-10-27 Created: 2009-10-22 Last updated: 2017-12-12Bibliographically approved
4. A novel peroxiredoxin activity is located within the C-terminal end of Rhodospirillum rubrum adenylyltransferase.
Open this publication in new window or tab >>A novel peroxiredoxin activity is located within the C-terminal end of Rhodospirillum rubrum adenylyltransferase.
2008 (English)In: Journal of Bacteriology, ISSN 0021-9193, E-ISSN 1098-5530, Vol. 190, no 1, 434-7 p.Article in journal (Refereed) Published
Abstract [en]

Adenylyltransferase (GlnE) catalyzes the reversible adenylylation of glutamine synthetase. In this report we present, for the first time, evidence for a peroxiredoxin activity of Rhodospirillum rubrum GlnE, through the carboxyl-terminal AhpC/thiol-specific antioxidant (TSA) domain. The combination of GlnE and AhpC/TSA domains within the same polypeptide constitutes a unique domain architecture that has not previously been identified among proteobacteria.

Identifiers
urn:nbn:se:su:diva-30808 (URN)10.1128/JB.01058-07 (DOI)000252080400041 ()17951375 (PubMedID)
Available from: 2009-10-27 Created: 2009-10-27 Last updated: 2017-12-12Bibliographically approved
5. Expression of the PII-AmtB encoding operons in Rhodospirillum rubrum and studies of the functional role(s) of GlnB, GlnJ and AmtB1 in nitrogen metabolism
Open this publication in new window or tab >>Expression of the PII-AmtB encoding operons in Rhodospirillum rubrum and studies of the functional role(s) of GlnB, GlnJ and AmtB1 in nitrogen metabolism
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(English)Manuscript (preprint) (Other academic)
Abstract [en]

In Rhodospirillum rubrum and many other diazotrophs, PII proteins and the ammonium transport protein AmtB have been shown to play central roles in the regulation of nitrogen metabolism. In this report we have used Real-time RT-PCR to study the transcription of the genes encoding three PII proteins and the AmtB proteins in R. rubrum. We have generated amtB1 and amtB2 mutants and in the amtB1 mutant strains ammonium nitrogenase switch-off is lost although the rate of ammonium uptake is not affected compared to wild type.  In contrast darkness switch-off is unaffected. Most interestingly, we also show that the uridylylation status of GlnB is different from that of GlnJ under certain conditions in the amtB1 mutant strain, which is the first demonstration of physiological selectivity in PII modification in vivo, supporting the proposed different functions for these paralogs in the cell.  We suggest that the primary role of AmtB1 in this diazotroph, is as a central component together with GlnJ in signal transduction regulating nitrogen metabolism.

Research subject
Biochemistry
Identifiers
urn:nbn:se:su:diva-30812 (URN)
Available from: 2009-10-27 Created: 2009-10-27 Last updated: 2010-01-12Bibliographically approved

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