Comparison studies of dinitrogenase reductase ADP-ribosyl transferase/dinitrogenase reductase activating glycohydrolase regulatory systems in Rhodospirillum rubrum and Azospirillum brasilense

Zhang, Yaoping; Burris, R. H.; Ludden, Paul W.; Roberts, Gary P.

doi:10.1128/jb.177.9.2354-2359.1995

Cited by 30 publications

(36 citation statements)

References 34 publications

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“…R. rubrum was grown in rich SMN medium and then inoculated into MG or N-free MN Ϫ medium for derepression of nitrogenase activity under illumination. The whole-cell nitrogenase activity assay and darkness/NH 4 Cl treatments have been described previously (58).…”

Section: Methodsmentioning

confidence: 99%

GlnD Is Essential for NifA Activation, NtrB/NtrC-Regulated Gene Expression, and Posttranslational Regulation of Nitrogenase Activity in the Photosynthetic, Nitrogen-Fixing Bacterium Rhodospirillum rubrum

2005

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GlnD is a bifunctional uridylyltransferase/uridylyl-removing enzyme and is thought to be the primary sensor of nitrogen status in the cell. It plays an important role in nitrogen assimilation and metabolism by reversibly regulating the modification of P II proteins, which in turn regulate a variety of other proteins. We report here the characterization of glnD mutants from the photosynthetic, nitrogen-fixing bacterium Rhodospirillum rubrum and the analysis of the roles of GlnD in the regulation of nitrogen fixation. Unlike glnD mutations in Azotobacter vinelandii and some other bacteria, glnD deletion mutations are not lethal in R. rubrum. Such mutants grew well in minimal medium with glutamate as the sole nitrogen source, although they grew slowly with ammonium as the sole nitrogen source (MN medium) and were unable to fix N 2 . The slow growth in MN medium is apparently due to low glutamine synthetase activity, because a ⌬glnD strain with an altered glutamine synthetase that cannot be adenylylated can grow well in MN medium. Various mutation and complementation studies were used to show that the critical uridylyltransferase activity of GlnD is localized to the N-terminal region. Mutants with intermediate levels of uridylyltransferase activity are differentially defective in nif gene expression, the posttranslational regulation of nitrogenase, and NtrB/NtrC function, indicating the complexity of the physiological role of GlnD. These results have implications for the interpretation of results obtained with GlnD in many other organisms.

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Section: Methodsmentioning

confidence: 99%

GlnD Is Essential for NifA Activation, NtrB/NtrC-Regulated Gene Expression, and Posttranslational Regulation of Nitrogenase Activity in the Photosynthetic, Nitrogen-Fixing Bacterium Rhodospirillum rubrum

2005

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“…R. rubrum was grown in rich SMN (supplemented malate-NH 4 ϩ ) medium or malate-glutamate medium (MG) as described (13). Whole-cell nitrogenase activity assay has been described (24).…”

Section: Methodsmentioning

confidence: 99%

Identification of critical residues in GlnB for its activation of NifA activity in the photosynthetic bacterium Rhodospirillum rubrum

Zhang

Pohlmann

Roberts

2004

Proc. Natl. Acad. Sci. U.S.A.

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The PII regulatory protein family is unusually widely distributed, being found in all three domains of life. Three P II homologs called GlnB, GlnK, and GlnJ have been identified in the photosynthetic bacterium Rhodospirillum rubrum. These have roles in at least four distinct functions, one of which is activation of the nitrogen fixation-specific regulatory protein NifA. The activation of NifA requires only the covalently modified (uridylylated) form of GlnB. GlnK and GlnJ are not involved. However, the basis of specificity for different P II homologs in different processes is poorly understood. We examined this specificity by altering GlnJ to support NifA activation. A small number of amino acid substitutions in GlnJ were important for this ability. Two (affecting residues 45 and 54) are in a loop called the T-loop, which contains the site of uridylylation and is believed to be very important for contacts with other proteins, but other critical residues lie in the C terminus (residues 95-97 and 109 -112) and near the N terminus (residues 3-5 and 17). Because many of the residues important for P II-NifA interaction lie far from the T-loop in the known x-ray crystal structures of P II proteins, our results lead to the hypothesis that the T-loop of GlnB is flexible enough to come into proximity with both the C-and N-terminal regions of the protein to bind NifA. Finally, the results show that the level of P II accumulation is also an important factor for NifA activation.

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“…Derepression of R. rubrum for nitrogenase, whole-cell nitrogenase activity assay, and NH 4 ϩ and darkness treatments were done as described previously (41). Rapid extraction of protein from R. rubrum was accomplished by the trichloroacetic acid precipitation method (39,41). Low-crosslinker sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblotting of dinitrogenase reductase were done as described previously (12,14).…”

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Effect of an ntrBC mutation on the posttranslational regulation of nitrogenase activity in Rhodospirillum rubrum

et al. 1995

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Homologs of ntrB and ntrC genes from Rhodospirillum rubrum were cloned and sequenced. A mutant lacking ntrBC was constructed, and this mutant has normal nitrogenase activity under nif-derepressing conditions, indicating that ntrBC are not necessary for the expression of the nif genes in R. rubrum. However, the posttranslational regulation of nitrogenase activity by ADP-ribosylation in response to NH 4 ؉ was partially abolished in this mutant. More surprisingly, the regulation of nitrogenase activity in response to darkness was also affected, suggesting a physiological link between the ntr system and energy signal transduction in R. rubrum. The expression of glutamine synthetase, as well as its posttranslational regulation, was also altered in this ntrBC mutant.In enteric bacteria the general nitrogen regulation (ntr) system controls a variety of nitrogen assimilation pathways (29). The ntr system involves the products of at least five genes: ntrA, ntrB, ntrC, glnB, and glnD. The products of ntrB and ntrC belong to the family of two-component regulators: NTRB is a histidine kinase that phosphorylates NTRC under nitrogenlimiting conditions, and the phosphorylated form of NTRC then acts as transcriptional activator of glnA (encoding glutamine synthetase [GS]) and other operons involved in nitrogen assimilation.Homologs of the ntrBC genes have been found in many nitrogen-fixing bacteria, and their roles in nitrogen fixation have been best characterized in Klebsiella pneumoniae. In this organism, NTRB and NTRC are required for transcription of nifLA regulatory genes, with NIFA activating the transcription of other nif operons (25). In some diazotrophs, such as Azotobacter vinelandii, Bradyrhizobium japonicum, and Azospirillum brasilense, NTRB and NTRC are not essential for nif gene expression, but the mutations in ntrBC have detectable effects on some other aspects of nitrogen assimilation, such as nitrate utilization and GS activity (20,22,35).Rhodospirillum rubrum is a purple nonsulfur, photosynthetic, nitrogen-fixing bacterium, and the posttranslational regulatory system that regulates nitrogenase activity is best characterized in this organism. This regulatory system involves reversible mono-ADP-ribosylation of dinitrogenase reductase, and regulation is performed by two enzymes: dinitrogenase reductase ADP-ribosyltransferase (DRAT) and dinitrogenase reductaseactivating glycohydrolase (DRAG). DRAT modifies dinitrogenase reductase and thereby inactivates the enzyme. DRAG removes the ADP-ribose from dinitrogenase reductase and restores its activity (21).The DRAT-DRAG system has been characterized in other nitrogen-fixing bacteria, such as A. brasilense (9, 42) and Rhodobacter capsulatus (24), and it negatively regulates nitrogenase activity in response to exogenous NH 4 ϩ or to energy limitation in the form of darkness (in the cases of R. rubrum and R. capsulatus) or anaerobiosis (in A. brasilense). Both DRAT and DRAG activities are themselves subject to posttranslational regulation under these conditions (10,14,19,...

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Comparison studies of dinitrogenase reductase ADP-ribosyl transferase/dinitrogenase reductase activating glycohydrolase regulatory systems in Rhodospirillum rubrum and Azospirillum brasilense

Cited by 30 publications

References 34 publications

GlnD Is Essential for NifA Activation, NtrB/NtrC-Regulated Gene Expression, and Posttranslational Regulation of Nitrogenase Activity in the Photosynthetic, Nitrogen-Fixing Bacterium Rhodospirillum rubrum

GlnD Is Essential for NifA Activation, NtrB/NtrC-Regulated Gene Expression, and Posttranslational Regulation of Nitrogenase Activity in the Photosynthetic, Nitrogen-Fixing Bacterium Rhodospirillum rubrum

Identification of critical residues in GlnB for its activation of NifA activity in the photosynthetic bacterium Rhodospirillum rubrum

Effect of an ntrBC mutation on the posttranslational regulation of nitrogenase activity in Rhodospirillum rubrum

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