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  • 1.
    Berthold, Catrine L.
    et al.
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Wang, He
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Nordlund, Stefan
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Högbom, Martin
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Mechanism of ADP-ribosylation removal revealed by the structure and ligand complexes of the dimanganese mono-ADP-ribosylhydrolase DraG2009In: Proceedings of the National Academy of Sciences of the United States of America, ISSN 0027-8424, E-ISSN 1091-6490, Vol. 106, no 34, p. 14247-14252Article in journal (Refereed)
    Abstract [en]

    ADP-ribosylation is a ubiquitous regulatory posttranslational modification involved in numerous key processes such as DNA repair, transcription, cell differentiation, apoptosis, and the pathogenic mechanism of certain bacterial toxins. Despite the importance of this reversible process, very little is known about the structure and mechanism of the hydrolases that catalyze removal of the ADP-ribose moiety. In the phototrophic bacterium Rhodospirillum rubrum, dinitrogenase reductase-activating glycohydrolase (DraG), a dimanganese enzyme that reversibly associates with the cell membrane, is a key player in the regulation of nitrogenase activity. DraG has long served as a model protein for ADP-ribosylhydrolases. Here, we present the crystal structure of DraG in the holo and ADP-ribose bound forms. We also present the structure of a reaction intermediate analogue and propose a detailed catalytic mechanism for protein de-ADP-ribosylation involving ring opening of the substrate ribose. In addition, the particular manganese coordination in DraG suggests a rationale for the enzyme's preference for manganese over magnesium, although not requiring a redox active metal for the reaction.

  • 2.
    Jonsson, Anders
    et al.
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Nordlund, Stefan
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    In vitro studies of the uridylylation of the three PII protein paralogs from Rhodospirillum rubrum: the transferase activity of R. rubrum GlnD is regulated by alpha-ketoglutarate and divalent cations but not by glutamine.2007In: J Bacteriol, ISSN 0021-9193, Vol. 189, no 9, p. 3471-8Article in journal (Refereed)
  • 3.
    Jonsson, Anders
    et al.
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Nordlund, Stefan
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Teixeira, Pedro Filipe
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Reduced activity of glutamine synthetase in Rhodospirillum rubrum mutants lacking the adenylyltransferase GlnE2009In: Research in Microbiology, ISSN 0923-2508, E-ISSN 1769-7123, Vol. 160, no 8, p. 581-4Article in journal (Refereed)
    Abstract [en]

    In the nitrogen-fixing bacterium Rhodospirillum rubrum, the GlnE adenylyltransferase (encoded by glnE) catalyzes reversible adenylylation of glutamine synthetase, thereby regulating nitrogen assimilation. We have generated glnE mutant strains that are unable to adenylylate glutamine synthetase (GS). Surprisingly, the activity of GS was lower in the mutants than in the wild type, even when grown in nitrogen-fixing conditions. Our results support the proposal that R. rubrum can only cope with the absence of an adenylylation system in the presence of lowered GS expression or activity. In general terms, this report also provides further support for the central role of GS in bacterial metabolism.

  • 4.
    Jonsson, Anders
    et al.
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Teixeira, Pedro F.
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Nordlund, Stefan
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    The activity of adenylyltransferase in Rhodospirillum rubrum is only affected by alpha-ketoglutarate and unmodified PII proteins, but not by glutamine, in vitro2007In: The FBS Journal, ISSN 1742-464X, Vol. 274, no 10, p. 2449-2460Article in journal (Refereed)
    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.

  • 5. Jonsson, Anders
    et al.
    Teixeira, Pedro Filipe
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Nordlund, Stefan
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    A novel peroxiredoxin activity is located within the C-terminal end of Rhodospirillum rubrum adenylyltransferase.2008In: Journal of Bacteriology, ISSN 0021-9193, E-ISSN 1098-5530, Vol. 190, no 1, p. 434-7Article in journal (Refereed)
    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.

  • 6. Moure, Vivian R.
    et al.
    Siöberg, Catrine L. B.
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Valdameri, Glaucio
    Nji, Emmanuel
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Oliveira, Marco Aurelio S.
    Gerdhardt, Edileusa C. M.
    Pedrosa, Fabio O.
    Mitchell, David A.
    Seefeldt, Lance C.
    Huergo, Luciano F.
    Högbom, Martin
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Nordlund, Stefan
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Souza, Emanuel M.
    The ammonium transporter AmtB and the PII signal transduction protein GlnZ are required to inhibit DraG in Azospirillum brasilense2019In: The FEBS Journal, ISSN 1742-464X, E-ISSN 1742-4658, Vol. 286, no 6, p. 1214-1229Article in journal (Refereed)
    Abstract [en]

    The ammonium-dependent posttranslational regulation of nitrogenase activity in Azospirillum brasilense requires dinitrogenase reductase ADPribosyl transferase (DraT) and dinitrogenase reductase ADP-glycohydrolase (DraG). These enzymes are reciprocally regulated by interaction with the PII proteins, GlnB and GlnZ. In this study, purified ADP-ribosylated Fe-protein was used as substrate to study the mechanism involved in the regulation of A. brasilense DraG in vitro. The data show that DraG is partially inhibited by GlnZ and that DraG inhibition is further enhanced by the simultaneous presence of GlnZ and AmtB. These results are the first to demonstrate experimentally that DraG inactivation requires the formation of a ternary DraG-GlnZ-AmtB complex in vitro. Previous structural data have revealed that when the DraG-GlnZ complex associates with AmtB, the flexible T-loops of the trimeric GlnZ bind to AmtB and become rigid; these molecular events stabilize the DraG-GlnZ complex, resulting in DraG inactivation. To determine whether restraining the flexibility of the GlnZ T-loops is a limiting factor in DraG inhibition, we used a GlnZ variant that carries a partial deletion of the T-loop (GlnZD42-54). However, although the GlnZD42-54 variant was more effective in inhibiting DraG in vitro, it bound to DraG with a slightly lower affinity than does wild-type GlnZ and was not competent to completely inhibit DraG activity either in vitro or in vivo. We, therefore, conclude that the formation of a ternary complex between DraG-GlnZ-AmtB is necessary for the inactivation of DraG.

  • 7.
    Nordlund, Stefan
    et al.
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Högbom, Martin
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    ADP-ribosylation, a mechanism regulating nitrogenase activity2013In: The FEBS Journal, ISSN 1742-464X, E-ISSN 1742-4658, Vol. 280, no 15, p. 3484-3490Article, review/survey (Refereed)
    Abstract [en]

    Nitrogen fixation is the vital biochemical process in which atmospheric molecular nitrogen is made available to the biosphere. The process is highly energetically costly and thus tightly regulated. The activity of the key enzyme, nitrogenase, is controlled by reversible mono-ADP-ribosylation of one of its components, the Fe protein. This protein provides the other component, the MoFe protein, with the electrons required for the reduction of molecular nitrogen. The Fe-protein is ADP-ribosylated and de-ADP-ribosylated by dinitrogenase reductase ADP-ribosyl transferase and dinitrogenase reductase activating glycohydrolase, respectively. Here we review the current biochemical and structural knowledge of this central regulatory reaction.

  • 8.
    Selao, Tiago T.
    et al.
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Nordlund, Stefan
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Norén, Agneta
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Comparative proteomic studies in Rhodospirillum rubrum grown under different nitrogen conditions2008In: Journal of Proteome Research, ISSN 1535-3893, E-ISSN 1535-3907, Vol. 7, no 8, p. 3267-3275Article in journal (Refereed)
    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.

  • 9.
    Selao, Tiago Toscano
    et al.
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Branca, Rui
    Chae, Pil Seok
    Lehtio, Janne
    Gellman, Samuel H.
    Rasmussen, Sören G. F.
    Nordlund, Stefan
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Norén, Agneta
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Identification of Chromatophore Membrane Protein Complexes Formed under Different Nitrogen Availability Conditions in Rhodospirillum rubrum2011In: Journal of Proteome Research, ISSN 1535-3893, E-ISSN 1535-3907, Vol. 10, no 6, p. 2703-2714Article in journal (Refereed)
    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.

  • 10.
    Selao, Tiago Toscano
    et al.
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Edgren, Tomas
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Wang, He
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Norén, Agneta
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Nordlund, Stefan
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Effect of pyruvate on the metabolic regulation of nitrogenase activity in Rhodospirillum rubrum in darkness2011In: Microbiology, ISSN 1350-0872, E-ISSN 1465-2080, Vol. 157, p. 1834-1840Article in journal (Refereed)
    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 response to darkness. We have previously shown that the switch-off response and metabolic state differ between cells grown with dinitrogen or glutamate as the nitrogen source, although both represent poor nitrogen sources. In this study we show that pyruvate affects the response to darkness in cultures grown with glutamate as nitrogen source, leading to a response similar to that in cultures grown with dinitrogen. The effects are related to protein uridylylation and glutamine synthetase activity. We also show that pyruvate induces de novo protein synthesis and that inhibition of pyruvate formate-lyase leads to loss of nitrogenase activity in the dark.

  • 11.
    Selao, Tiago Toscano
    et al.
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Edgren, Tomas
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Wang, He
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Norén, Agneta
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Nordlund, Stefan
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    The effect of pyruvate on the metabolic regulation of nitrogenase activity in Rhodospirillum rubrum with darkness as switch-off effector2010In: Microbiology, ISSN 1350-0872, E-ISSN 1465-2080Article in journal (Refereed)
    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.

  • 12.
    Teixeira, Pedro Filipe
    et al.
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Dominguez-Martin, Maria A.
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Nordlund, Stefan
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Molecular basis for the distinct divalent cation requirement in the uridylylation of the signal transduction proteins GlnJ and GlnB from Rhodospirillum rubrum2012In: BMC Microbiology, ISSN 1471-2180, E-ISSN 1471-2180, Vol. 12, p. 136-Article in journal (Refereed)
    Abstract [en]

    Background: PII proteins have a fundamental role in the control of nitrogen metabolism in bacteria, through interactions with different PII targets, controlled by metabolite binding and post-translational modification, uridylylation in most organisms. 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+. Results: Given the importance of uridylylation in the functional interactions of PII proteins, we have hypothesized that the difference in the divalent cation requirement for the uridylylation is related to efficient binding of Mg/Mn-ATP to the PII proteins. We concluded that the amino acids at positions 42 and 85 in GlnJ and GlnB (in the vicinity of the ATP binding site) influence the divalent cation requirement for uridylylation catalyzed by GlnD. Conclusions: Efficient binding of Mg/Mn-ATP to the PII proteins is required for uridylylation by GlnD. Our results show that by simply exchanging two amino acid residues, we could modulate the divalent cation requirement in the uridylylation of GlnJ and GlnB. Considering that post-translational uridylylation of PII proteins modulates their signaling properties, a different requirement for divalent cations in the modification of GlnB and GlnJ adds an extra regulatory layer to the already intricate control of PII function.

  • 13.
    Teixeira, Pedro Filipe
    et al.
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Jonsson, Anders
    Frank, Martina
    Wang, He
    Nordlund, Stefan
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    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.2008In: Microbiology, ISSN 1350-0872, E-ISSN 1465-2080, Vol. 154, no Pt 8, p. 2336-47Article in journal (Refereed)
    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.

  • 14.
    Teixeira, Pedro Filipe
    et al.
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Selao, Tiago Toscano
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Henriksson, Veronika
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Wang, He
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Norén, Agneta
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Nordlund, Stefan
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Diazotrophic growth of Rhodospirillum rubrum with 2-oxoglutarate as sole carbon source affects the regulation of nitrogen metabolism as well as the soluble proteome2010In: Research in Microbiology, ISSN 0923-2508, E-ISSN 1769-7123, Vol. 161, no 8, p. 651-659Article in journal (Refereed)
    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.

  • 15.
    Teixeira, Pedro
    et al.
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Wang, He
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Stefan, Nordlund
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Nitrogenase switch-off and regulation of ammonium assimilation in response to light deprivation in Rhodospirillum rubrum are influnced by the nitrogen source used during growth2010In: Journal of Bacteriology, ISSN 0021-9193, E-ISSN 1098-5530, Vol. 192, no 5, p. 1463-1466Article in journal (Refereed)
    Abstract [en]

    Nitrogen fixation and ammonium assimilation in Rhodospirillum rubrum are regulated in response to changes in light availability, and we show that the response in terms of glutamine synthetase activity and PII modification is dependent on the nitrogen source used for growth, N2 or glutamate, although both lead to nitrogenase derepression.

  • 16.
    Wang, He
    et al.
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Franke, Claudia C.
    Nordlund, Stefan
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Norén, Agneta
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Reversible membrane association of dinitrogenase reductase activating glycohydrolase in the regulation of nitrogenase activity in Rhodospirillum rubrum; dependence on GlnJ and AmtB12005In: FEMS Microbiology Letters, ISSN 0378-1097, E-ISSN 1574-6968, Vol. 253, no 2, p. 273-279Article in journal (Refereed)
    Abstract [en]

    In the photosynthetic bacterium Rhodospirillum rubrum nitrogenase activity is regulated by reversible ADP-ribosylation of dinitrogenase reductase in response to external so called “switch-off” effectors. Activation of the modified, inactive form is catalyzed by dinitrogenase reductase activating glycohydrolase (DRAG) which removes the ADP-ribose moiety. This study addresses the signal transduction between external effectors and DRAG. R. rubrum, wild-type and PII mutant strains, were studied with respect to DRAG localization. We conclude that GlnJ clearly has an effect on the association of DRAG to the membrane in agreement with the effect on regulation of nitrogenase activity. Furthermore, we have generated a R. rubrum mutant lacking the putative ammonium transporter AmtB1 which was shown not to respond to “switch-off” effectors; no loss of nitrogenase activity and no ADP-ribosylation. Interestingly, DRAG was mainly localized to the cytosol in this mutant. Overall the results support our model in which association to the membrane is part of the mechanism regulating DRAG activity.

  • 17.
    Wang, He
    et al.
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Teixeira, Pedro Filipe
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Jonsson, Anders
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Vintila, Simina
    Stockholm University, Faculty of Science, Department of Botany.
    Norén, Agneta
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Nordlund, Stefan
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Expression of the PII-AmtB encoding operons in Rhodospirillum rubrum and studies of the functional role(s) of GlnB, GlnJ and AmtB1 in nitrogen metabolismManuscript (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.

  • 18.
    Wang, He
    et al.
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Teixeira, Pedro
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Jonsson, Anders
    Vintila, Simina
    Norén, Agneta
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Nordlund, Stefan
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Transcription of the PII-AmtB encoding operons in Rhodospirillum rubrum and studies of the functional role(s) of GlnB, GlnJ and AmtB1 in nitrogen metabolismArticle in journal (Refereed)
  • 19. Wang, Helen
    et al.
    Waluk, Dominik
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Dixon, Ray
    Nordlund, Stefan
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Norén, Agneta
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Energy shifts induce membrane sequestration of DraG in Rhodospirillum rubrum independent of the ammonium transporters and diazotrophic conditions2018In: FEMS Microbiology Letters, ISSN 0378-1097, E-ISSN 1574-6968, Vol. 365, no 16, article id fny176Article in journal (Refereed)
    Abstract [en]

    Metabolic regulation of Rhodospirillum rubrum nitrogenase is mediated at the post-translational level by the enzymes DraT and DraG when subjected to changes in nitrogen or energy status. DraT is activated during switch-off, while DraG is inactivated by reversible membrane association. We confirm here that the ammonium transporter, AmtB1, rather than its paralog AmtB2, is required for ammonium induced switch-off. Amongst several substitutions at the N100 position in DraG, only N100K failed to locate to the membrane following ammonium shock, suggesting loss of interaction through charge repulsion. When switch-off was induced by lowering energy levels, either by darkness during photosynthetic growth or oxygen depletion under respiratory conditions, reversible membrane sequestration of DraG was independent of AmtB proteins and occurred even under non-diazotrophic conditions. We propose that under these conditions, changes in redox status or possibly membrane potential induce interactions between DraG and another membrane protein in response to the energy status.

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