Posttranslational regulatory system for nitrogenase activity in Azospirillum spp

Fu, Haian; Hartmann, Anton; Lowery, Robert G.; Fitzmaurice, Wayne P.; Roberts, Gary P.; Burris, R. H.

doi:10.1128/jb.171.9.4679-4685.1989

Cited by 63 publications

(49 citation statements)

References 37 publications

(35 reference statements)

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“…For the photosynthetic purple nonsulfur bacterium Rhodospirillum rubrum and other members of the Alphaproteobacteria, like Azospirillum brasilense, Azospirillum lipoferum, and Rhodobacter capsulatus, it has been shown that nitrogenase activity is rapidly switched off as a consequence of either the addition of ammonium or energy depletion (10,26,32,35,54). One subunit of the nitrogenase Fe protein is modified under switch-off conditions, with the modification leading to the inactivation of the enzyme.…”

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Characterization of the DraT/DraG System for Posttranslational Regulation of Nitrogenase in the Endophytic Betaproteobacterium Azoarcus sp. Strain BH72

Oetjen

Reinhold‐Hurek

2009

J Bacteriol

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DraT/DraG-mediated posttranslational regulation of the nitrogenase Fe protein by ADP-ribosylation has been described for a few diazotrophic bacteria belonging to the class Alphaproteobacteria. Here we present for the first time the DraT/DraG system of a betaproteobacterium, Azoarcus sp. strain BH72, a diazotrophic grass endophyte. Its genome harbors one draT ortholog and two physically unlinked genes coding for ADP-ribosylhydrolases. Northern blot analysis revealed cotranscription of draT with two genes encoding hypothetical proteins. Furthermore, draT and draG2 were expressed under all studied conditions, whereas draG1 expression was nitrogen regulated. By using Western blot analysis of deletion mutants and nitrogenase assays in vivo, we demonstrated that DraT is required for the nitrogenase Fe protein modification but not for the physiological inactivation of nitrogenase activity. A second mechanism responsible for nitrogenase inactivation must operate in this bacterium, which is independent of DraT. Fe protein demodification was dependent mainly on DraG1, corroborating the assumption from phylogenetic analysis that DraG2 might be mostly involved in processes other than the posttranslational regulation of nitrogenase. Nitrogenase in vivo reactivation was impaired in a draG1 mutant and a mutant lacking both draG alleles after anaerobiosis shifts and subsequent adjustment to microaerobic conditions, suggesting that modified dinitrogenase reductase was inactive. Our results demonstrate that the DraT/DraG system, despite some differences, is functionally conserved in diazotrophic proteobacteria.

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“…In A. brasilense, A. lipoferum, and R. capsulatus, draT and draG orthologs were discovered as well (10,35). Moreover, in various genome projects (http://www.ncbi.nlm.nih.gov/genomes /lproks.cgi/), draG-like sequences and few draT orthologs can be identified.…”

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Characterization of the DraT/DraG System for Posttranslational Regulation of Nitrogenase in the Endophytic Betaproteobacterium Azoarcus sp. Strain BH72

Oetjen

Reinhold‐Hurek

2009

J Bacteriol

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“…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.…”

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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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“…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 (10,14,19,39,42), but neither the mechanisms of their regulation nor the signal transduction system that controls it is understood.…”

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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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Posttranslational regulatory system for nitrogenase activity in Azospirillum spp

Cited by 63 publications

References 37 publications

Characterization of the DraT/DraG System for Posttranslational Regulation of Nitrogenase in the Endophytic Betaproteobacterium Azoarcus sp. Strain BH72

Characterization of the DraT/DraG System for Posttranslational Regulation of Nitrogenase in the Endophytic Betaproteobacterium Azoarcus sp. Strain BH72

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

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

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