scholarly journals The role of NAD+ as a signal during nitrogenase switch-off in Rhodospirillum rubrum

1997 ◽  
Vol 322 (3) ◽  
pp. 829-832 ◽  
Author(s):  
Agneta NORÉN ◽  
Abdelhamid SOLIMAN ◽  
Stefan NORDLUND
Keyword(s):  
Metabolic Regulation ◽  
Opposite Effect ◽  
Rhodospirillum Rubrum ◽  
Nitrogenase Activity ◽  
Regulatory System ◽  
Glutamate Synthase ◽  
The Other ◽  
Dinitrogenase Reductase ◽  
Adp Ribosyltransferase

The role of NAD+ in the metabolic regulation of nitrogenase, the ‘switch-off’ effect, in Rhodospirillum rubrum has been studied. We now show that the decrease in nitrogenase activity upon addition of NAD+ to R. rubrum is due to modification of dinitrogenase reductase. There was no effect when NAD+ was added to a mutant of R. rubrumdevoid of dinitrogenase reductase ADP-ribosyltransferase, indicating that NAD+ ‘switch-off’ is an effect of the same regulatory system as ammonium ‘switch-off’. We also show that oxaloacetate and α-ketoglutarate function as ‘switch-off’ effectors. On the other hand β-hydroxybutyrate has the opposite effect by shortening the ‘switch-off’ period. Furthermore, by using an inhibitor of glutamate synthase the role of this enzyme in ‘switch-off’ was investigated. The results are discussed in relation to our proposal that changes in the concentration of NAD+ are involved in initiating ‘switch-off’.

scholarly journals Role of the Dinitrogenase Reductase Arginine 101 Residue in Dinitrogenase Reductase ADP-Ribosyltransferase Binding, NAD Binding, and Cleavage

2001 ◽  
Vol 183 (1) ◽  
pp. 250-256 ◽  
Author(s):  
Yan Ma ◽  
Paul W. Ludden
Keyword(s):  
Rhodospirillum Rubrum ◽  
Nitrogenase Activity ◽  
Site Directed Mutagenesis ◽  
Directed Mutagenesis ◽  
Adp Ribosylation ◽  
Dinitrogenase Reductase ◽  
Adp Ribosyltransferase

ABSTRACT Dinitrogenase reductase is posttranslationally regulated by dinitrogenase reductase ADP-ribosyltransferase (DRAT) via ADP-ribosylation of the arginine 101 residue in some bacteria.Rhodospirillum rubrum strains in which the arginine 101 of dinitrogenase reductase was replaced by tyrosine, phenylalanine, or leucine were constructed by site-directed mutagenesis of thenifH gene. The strain containing the R101F form of dinitrogenase reductase retains 91%, the strain containing the R101Y form retains 72%, and the strain containing the R101L form retains only 28% of in vivo nitrogenase activity of the strain containing the dinitrogenase reductase with arginine at position 101. In vivo acetylene reduction assays, immunoblotting with anti-dinitrogenase reductase antibody, and [adenylate-32P]NAD labeling experiments showed that no switch-off of nitrogenase activity occurred in any of the three mutants and no ADP-ribosylation of altered dinitrogenase reductases occurred either in vivo or in vitro. Altered dinitrogenase reductases from strains UR629 (R101Y) and UR630 (R101F) were purified to homogeneity. The R101F and R101Y forms of dinitrogenase reductase were able to form a complex with DRAT that could be chemically cross-linked by 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide. The R101F form of dinitrogenase reductase and DRAT together were not able to cleave NAD. This suggests that arginine 101 is not critical for the binding of DRAT to dinitrogenase reductase but that the availability of arginine 101 is important for NAD cleavage. Both DRAT and dinitrogenase reductase can be labeled by [carbonyl-14C]NAD individually upon UV irradiation, but most 14C label is incorporated into DRAT when both proteins are present. The ability of R101F dinitrogenase reductase to be labeled by [carbonyl-14C]NAD suggested that Arg 101 is not absolutely required for NAD binding.

scholarly journals Correlation of Activity Regulation and Substrate Recognition of the ADP-Ribosyltransferase That Regulates Nitrogenase Activity in Rhodospirillum rubrum

1999 ◽  
Vol 181 (5) ◽  
pp. 1698-1702 ◽  
Author(s):  
Kitai Kim ◽  
Yaoping Zhang ◽  
Gary P. Roberts
Keyword(s):  
Rhodospirillum Rubrum ◽  
Nitrogenase Activity ◽  
Substrate Recognition ◽  
Posttranslational Regulation ◽  
Activity Regulation ◽  
Adp Ribosylation ◽  
Dinitrogenase Reductase ◽  
Adp Ribosyltransferase

ABSTRACT In Rhodospirillum rubrum, nitrogenase activity is regulated posttranslationally through the ADP-ribosylation of dinitrogenase reductase by dinitrogenase reductase ADP-ribosyltransferase (DRAT). Several DRAT variants that are altered both in the posttranslational regulation of DRAT activity and in the ability to recognize variants of dinitrogenase reductase have been found. This correlation suggests that these two properties are biochemically connected.

scholarly journals Functional Characterization of Three GlnB Homologs in the Photosynthetic Bacterium Rhodospirillum rubrum: Roles in Sensing Ammonium and Energy Status

2001 ◽  
Vol 183 (21) ◽  
pp. 6159-6168 ◽  
Author(s):  
Yaoping Zhang ◽  
Edward L. Pohlmann ◽  
Paul W. Ludden ◽  
Gary P. Roberts
Keyword(s):  
Metabolic Regulation ◽  
Rhodospirillum Rubrum ◽  
Functional Characterization ◽  
Regulatory System ◽  
Normal Growth ◽  
Photosynthetic Bacterium ◽  
Critical Element ◽  
Energy Status ◽  
Rich Media ◽  
Dinitrogenase Reductase

ABSTRACT The GlnB (PII) protein, the product ofglnB, has been characterized previously in the photosynthetic bacterium Rhodospirillum rubrum. Here we describe identification of two other PII homologs in this organism, GlnK and GlnJ. Although the sequences of these three homologs are very similar, the molecules have both distinct and overlapping functions in the cell. While GlnB is required for activation of NifA activity in R. rubrum, GlnK and GlnJ do not appear to be involved in this process. In contrast, either GlnB or GlnJ can serve as a critical element in regulation of the reversible ADP ribosylation of dinitrogenase reductase catalyzed by the dinitrogenase reductase ADP-ribosyl transferase (DRAT)/dinitrogenase reductase-activating glycohydrolase (DRAG) regulatory system. Similarly, either GlnB or GlnJ is necessary for normal growth on a variety of minimal and rich media, and any of the proteins is sufficient for normal posttranslational regulation of glutamine synthetase. Surprisingly, in their regulation of the DRAT/DRAG system, GlnB and GlnJ appeared to be responsive not only to changes in nitrogen status but also to changes in energy status, revealing a new role for this family of regulators in central metabolic regulation.

scholarly journals Mutagenesis and Functional Characterization of theglnB, glnA, and nifA Genes from the Photosynthetic Bacterium Rhodospirillum rubrum

2000 ◽  
Vol 182 (4) ◽  
pp. 983-992 ◽  
Author(s):  
Yaoping Zhang ◽  
Edward L. Pohlmann ◽  
Paul W. Ludden ◽  
Gary P. Roberts
Keyword(s):  
Nitrogen Fixation ◽  
Rhodospirillum Rubrum ◽  
Nitrogenase Activity ◽  
Functional Characterization ◽  
Photosynthetic Bacterium ◽  
Wild Type ◽  
Adp Ribosylation ◽  
Dinitrogenase Reductase ◽  
Adp Ribosyltransferase

ABSTRACT Nitrogen fixation is tightly regulated in Rhodospirillum rubrum at two different levels: transcriptional regulation ofnif expression and posttranslational regulation of dinitrogenase reductase by reversible ADP-ribosylation catalyzed by the DRAT-DRAG (dinitrogenase reductase ADP-ribosyltransferase–dinitrogenase reductase-activating glycohydrolase) system. We report here the characterization ofglnB, glnA, and nifA mutants and studies of their relationship to the regulation of nitrogen fixation. Two mutants which affect glnB (structural gene for PII) were constructed. While PII-Y51F showed a lower nitrogenase activity than that of wild type, a PIIdeletion mutant showed very little nif expression. This effect of PII on nif expression is apparently the result of a requirement of PII for NifA activation, whose activity is regulated by NH4 + in R. rubrum. The modification of glutamine synthetase (GS) in theseglnB mutants appears to be similar to that seen in wild type, suggesting that a paralog of PII might exist inR. rubrum and regulate the modification of GS. PII also appears to be involved in the regulation of DRAT activity, since an altered response to NH4 + was found in a mutant expressing PII-Y51F. The adenylylation of GS plays no significant role in nif expression or the ADP-ribosylation of dinitrogenase reductase, since a mutant expressing GS-Y398F showed normal nitrogenase activity and normal modification of dinitrogenase reductase in response to NH4 + and darkness treatments.

scholarly journals Effect of PII and Its Homolog GlnK on Reversible ADP-Ribosylation of Dinitrogenase Reductase by Heterologous Expression of the Rhodospirillum rubrum Dinitrogenase Reductase ADP-Ribosyl Transferase–Dinitrogenase Reductase-Activating Glycohydrolase Regulatory System inKlebsiella pneumoniae

2001 ◽  
Vol 183 (5) ◽  
pp. 1610-1620 ◽  
Author(s):  
Yaoping Zhang ◽  
Edward L. Pohlmann ◽  
Cale M. Halbleib ◽  
Paul W. Ludden ◽  
Gary P. Roberts
Keyword(s):  
Klebsiella Pneumoniae ◽  
Heterologous Expression ◽  
Rhodospirillum Rubrum ◽  
Nitrogenase Activity ◽  
Expression System ◽  
Regulatory System ◽  
Wild Type ◽  
Heterologous Expression System ◽  
Adp Ribosylation ◽  
Dinitrogenase Reductase

ABSTRACT Reversible ADP-ribosylation of dinitrogenase reductase, catalyzed by the dinitrogenase reductase ADP-ribosyl transferase–dinitrogenase reductase-activating glycohydrolase (DRAT-DRAG) regulatory system, has been characterized in Rhodospirillum rubrum and other nitrogen-fixing bacteria. To investigate the mechanisms for the regulation of DRAT and DRAG activities, we studied the heterologous expression of R. rubrum draTG in Klebsiella pneumoniae glnB and glnK mutants. In K. pneumoniae wild type, the regulation of both DRAT and DRAG activity appears to be comparable to that seen in R. rubrum. However, the regulation of both DRAT and DRAG activities is altered in a glnB background. Some DRAT escapes regulation and becomes active under N-limiting conditions. The regulation of DRAG activity is also altered in a glnBmutant, with DRAG being inactivated more slowly in response to NH4 + treatment than is seen in wild type, resulting in a high residual nitrogenase activity. In aglnK background, the regulation of DRAT activity is similar to that seen in wild type. However, the regulation of DRAG activity is completely abolished in the glnK mutant; DRAG remains active even after NH4 + addition, so there is no loss of nitrogenase activity. The results with this heterologous expression system have implications for DRAT-DRAG regulation inR. rubrum.

Metabolic regulation of nitrogen fixation in Rhodospirillum rubrum

2006 ◽  
Vol 34 (1) ◽  
pp. 160-161 ◽  
Author(s):  
H. Wang ◽  
A. Norén
Keyword(s):  
Nitrogen Fixation ◽  
Metabolic Regulation ◽  
Rhodospirillum Rubrum ◽  
Nitrogenase Activity ◽  
Nitrogen Concentration ◽  
Sudden Increase ◽  
Fixed Nitrogen ◽  
Energy Depletion ◽  
Adp Ribosylation ◽  
Dinitrogenase Reductase

Nitrogenase activity in Rhodospirillum rubrum is post-translationally regulated by DRAG (dinitrogenase reductase glycohydrolase) and DRAT (dinitrogenase reductase ADP-ribosylation transferase). When a sudden increase in fixed nitrogen concentration or energy depletion is sensed by the cells, DRAG is inactivated and DRAT activated. We propose that the regulation of DRAG is dependent on its location in the cell and the presence of an ammonium-sensing protein.

scholarly journals Effect of AmtB homologues on the post-translational regulation of nitrogenase activity in response to ammonium and energy signals in Rhodospirillum rubrum

Microbiology ◽  
2006 ◽  
Vol 152 (7) ◽  
pp. 2075-2089 ◽  
Author(s):  
Yaoping Zhang ◽  
David M. Wolfe ◽  
Edward L. Pohlmann ◽  
Mary C. Conrad ◽  
Gary P. Roberts
Keyword(s):  
Translational Regulation ◽  
Rhodospirillum Rubrum ◽  
Nitrogenase Activity ◽  
Regulatory Protein ◽  
Regulatory System ◽  
Poor Response ◽  
Photosynthetic Bacterium ◽  
Nitrogen Acquisition ◽  
Consistent Model ◽  
Energy Deprivation

The AmtB protein transports uncharged NH3 into the cell, but it also interacts with the nitrogen regulatory protein PII, which in turn regulates a variety of proteins involved in nitrogen fixation and utilization. Three PII homologues, GlnB, GlnK and GlnJ, have been identified in the photosynthetic bacterium Rhodospirillum rubrum, and they have roles in at least four overlapping and distinct functions, one of which is the post-translational regulation of nitrogenase activity. In R. rubrum, nitrogenase activity is tightly regulated in response to addition or energy depletion (shift to darkness), and this regulation is catalysed by the post-translational regulatory system encoded by draTG. Two amtB homologues, amtB 1 and amtB 2, have been identified in R. rubrum, and they are linked with glnJ and glnK, respectively. Mutants lacking AmtB1 are defective in their response to both addition and darkness, while mutants lacking AmtB2 show little effect on the regulation of nitrogenase activity. These responses to darkness and appear to involve different signal transduction pathways, and the poor response to darkness does not seem to be an indirect result of perturbation of internal pools of nitrogen. It is also shown that AmtB1 is necessary to sequester detectable amounts GlnJ to the cell membrane. These results suggest that some element of the AmtB1-PII regulatory system senses energy deprivation and a consistent model for the integration of nitrogen, carbon and energy signals by PII is proposed. Other results demonstrate a degree of specificity in interaction of AmtB1 with the different PII homologues in R. rubrum. Such interaction specificity might be important in explaining the way in which PII proteins regulate processes involved in nitrogen acquisition and utilization.

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