Posttranslational regulation of nitrogenase in Rhodospirillum rubrum strains overexpressing the regulatory enzymes dinitrogenase reductase ADP-ribosyltransferase and dinitrogenase reductase activating glycohydrolase

Grunwald, Sandra K.; Lies, Douglas P.; Roberts, Gary P.; Ludden, Paul W.

doi:10.1128/jb.177.3.628-635.1995

Cited by 43 publications

(35 citation statements)

References 41 publications

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“…To clone these genes into a broad host-range plasmid, EcoRVHindIII fragments from these plasmids were cloned into pRK404, and then transferred into an R. rubrum glnBJK mutant (UR812) (13) by the method described (27).…”

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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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 excised fragment includes the last 70 bp of draG, the entire draB gene, and a portion of the pEXT21 multicloning site. This deletion in draG has been shown to be sufficient to abrogate DRAG activity (7). The plasmid resulting from the HindIII fragment excision was designated pCH3.…”

Section: Methodsmentioning

confidence: 99%

“…This places DRAT and DRAG, at most, at levels similar to those described in R. rubrum (0.1 to 0.3 g per ml of cell culture at an OD of 1.0) (14,29). Note that DRAT and DRAG are also undetectable in R. rubrum UR2 (wild type) crude extracts by immunoblotting; the proteins are only detected after partial purification or in crude extracts of overexpressing strains (7).…”

Section: Determination Of [ 32 P]adp-ribose Turnover On Fe Proteinmentioning

confidence: 99%

Effects of Perturbations of the Nitrogenase Electron Transfer Chain on Reversible ADP-Ribosylation of Nitrogenase Fe Protein in Klebsiella pneumoniae Strains Bearing the Rhodospirillum rubrum dra Operon

et al. 2000

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The redox state of nitrogenase Fe protein is shown to affect regulation of ADP-ribosylation in Klebsiella pneumoniae strains transformed by plasmids carrying dra genes from Rhodospirillum rubrum. The dra operon encodes dinitrogenase reductase ADP-ribosyltransferase and dinitrogenase reductase-activating glycohydrolase, enzymes responsible for the reversible inactivation, via ADP-ribosylation, of nitrogenase Fe protein in R. rubrum. In bacteria containing the dra operon in their chromosomes, inactivation occurs in response to energy limitation or nitrogen sufficiency. The dra gene products, expressed at a low level in K. pneumoniae, enable transformants to reversibly ADP-ribosylate nitrogenase Fe protein in response to the presence of fixed nitrogen. The activities of both regulatory enzymes are regulated in vivo as described in R. rubrum. Genetic perturbations of the nitrogenase electron transport chain were found to affect the rate of inactivation of Fe protein. Strains lacking the electron donors to Fe protein (NifF or NifJ) were found to inactivate Fe protein more quickly than a strain with wild-type background. Deletion of nifD, which encodes a subunit of nitrogenase MoFe protein, was found to result in a slower inactivation response. No variation was found in the reactivation responses of these strains. It is concluded that the redox state of the Fe protein contributes to the regulation of the ADP-ribosylation of Fe protein.Biological nitrogen fixation is a capability found in diverse genera of bacteria, including plant root-associated symbionts, free-living bacteria, and cyanobacteria. The conversion of N 2 to NH 3 is catalyzed by the nitrogenase enzyme complex, which comprises two separable components. Nitrogenase MoFe protein is an ␣ 2 ␤ 2 tetramer containing the cofactor FeMoco, believed to be the site of nitrogen reduction. Nitrogenase Fe protein is the obligate electron donor to MoFe protein. Fe protein is a homodimer, containing a [Fe 4 S 4 ] cluster bound by cysteinyl ligands at the dimer interface (reviewed in reference 9). Nitrogen fixation is extremely expensive in terms of the biological energy requirement. No fewer than 16 high-energy phosphoanhydride bond cleavages (ATP energy equivalents) are required for the reduction of a single dinitrogen molecule (21). In response to this requirement in the unstable microbial environment, some nitrogen-fixing bacteria have adopted a posttranslational regulatory system in which the activity of the electron carrier, Fe protein, is regulated. Inhibition of Fe protein activity is achieved by ADP-ribosylation of a specific arginine residue located on the surface of Fe protein that interacts with MoFe protein (6, 26).The ADP-ribosylation system has been best characterized in the purple nonsulfur bacterium Rhodospirillum rubrum. The NAD ϩ -dependent ADP-ribosylation of R101 of the R. rubrum Fe protein is catalyzed by dinitrogenase reductase ADP-ribosyltransferase (DRAT) in response to energy limitation or nitrogen sufficiency (16). Upon relief of these negative ...

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“…Under derepressing (nitrogen-fixing) conditions, DRAG is active and DRAT is inactive (7,15,28,30). In A. brasilense, for example, shifting a culture from microaerobic to anaerobic conditions eliminates the preferred energy source.…”

mentioning

confidence: 99%

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

et al. 1995

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Reversible ADP ribosylation of dinitrogenase reductase, catalyzed by the dinitrogenase reductase ADPribosyl transferase (DRAT)/dinitrogenase reductase activating glycohydrolase (DRAG) regulatory system, has been characterized in both Rhodospirillum rubrum and Azospirillum brasilense. Although the general functions of DRAT and DRAG are very similar in these two organisms, there are a number of interesting differences, e.g., in the timing and extent of the regulatory response to different stimuli. In this work, the basis of these differences has been studied by the heterologous expression of either draTG or nifH from A. brasilense in R. rubrum mutants that lack these genes, as well as the expression of draTG from R. rubrum in an A. brasilense draTG mutant. In general, these hybrid strains respond to stimuli in a manner similar to that of the wild-type parent of the recipient strain rather than the wild-type source of the introduced genes. These results suggest that the differences seen in the regulatory response in these organisms are not primarily a result of different properties of DRAT, DRAG, or dinitrogenase reductase. Instead, the differences are likely the result of different signal pathways that regulate DRAG and DRAT activities in these two organisms. Our results also suggest that draT and draG are cotranscribed in A. brasilense.The posttranslational regulation of nitrogenase activity, which is also termed switch-off (31), has been found in many diverse nitrogen-fixing bacteria. However, this regulation has been well characterized only in Rhodospirillum rubrum, Azospirillum brasilense, Azospirillum lipoferum, and Rhodobacter capsulatus, in which it involves reversible mono-ADP ribosylation of dinitrogenase reductase, catalyzed by the dinitrogenase reductase ADP-ribosyl transferase (DRAT)/dinitrogenase reductase activating glycohydrolase (DRAG) regulatory system (5,6,17,30). DRAT (the gene product of draT) catalyzes the transfer of the ADP-ribose from NAD to the Arg-101 residue of one subunit of the dinitrogenase reductase dimer and thereby inactivates the enzyme. The ADP-ribose group attached to dinitrogenase reductase can be removed by another enzyme, DRAG (the gene product of draG), thus restoring nitrogenase activity (16).draT and draG have been cloned and sequenced from R. rubrum, R. capsulatus, and A. brasilense (5, 17, 30). The sequence comparison of draTG from these strains showed an extensive similarity, with some conserved regions (17). draTG mutants also were constructed and physiologically characterized in these organisms (15,17,30).In the cases of A. brasilense and R. rubrum, the activities of DRAG and DRAT themselves are subject to some form of posttranslational regulation by an unknown mechanism. Under derepressing (nitrogen-fixing) conditions, DRAG is active and DRAT is inactive (7,15,28,30). In A. brasilense, for example, shifting a culture from microaerobic to anaerobic conditions eliminates the preferred energy source. In response to this shift, DRAG activity ceases rapidly and DRAT activ...

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Posttranslational regulation of nitrogenase in Rhodospirillum rubrum strains overexpressing the regulatory enzymes dinitrogenase reductase ADP-ribosyltransferase and dinitrogenase reductase activating glycohydrolase

Cited by 43 publications

References 41 publications

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

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

Effects of Perturbations of the Nitrogenase Electron Transfer Chain on Reversible ADP-Ribosylation of Nitrogenase Fe Protein in Klebsiella pneumoniae Strains Bearing the Rhodospirillum rubrum dra Operon

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

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