Control of Nitrogenase Reactivation by the GlnZ Protein in
            <i>Azospirillum brasilense</i>

Klassen, Giseli; Souza, Emanuel Maltempi de; Yates, M. G.; Rigo, L. U.; Inaba, Juliana; Pedrosa, Fábio de Oliveira

doi:10.1128/jb.183.22.6710-6713.2001

Cited by 27 publications

(27 citation statements)

References 29 publications

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“…This model is also consistent with the previous data on the system: (i) DRAG has been detected in association with the membrane fraction of R. rubrum cells in which DRAG activity is downregulated (Norén & Nordlund, 1997); (ii) in A. brasilense, the reactivation of DRAG activity was altered in a mutant lacking GlnZ, a GlnK-like protein (Klassen et al, 2001); (iii) in R. capsulatus, DRAT interacts with both GlnB and GlnK in the yeast two-hybrid system (Pawlowski et al, 2003) and we have detected similar interactions between DRAT and all three P II homologues of R. rubrum (Zhu et al, 2006). Thus, although we specifically propose this scheme for the R. rubrum system, it is consistent with results seen with Azoarcus, R. capsulatus and Azospirillum brasilense as well, in which the DRAT-DRAG regulation is altered in mutants lacking P II or AmtB Klassen et al, 2001;Martin & Reinhold-Hurek, 2002;Yakunin & Hallenbeck, 2002). We note that rather different results have been seen in the case of R. capsulatus, where mutants lacking two AmtB homologues (AmtB and AmtY) affected the DRAT-DRAG response to NH z 4 , but not to darkness; the basis for this difference is unclear (Yakunin & Hallenbeck, 2002).…”

Section: Discussionsupporting

confidence: 80%

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

et al. 2006

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The AmtB protein transports uncharged NH 3 into the cell, but it also interacts with the nitrogen regulatory protein P II , which in turn regulates a variety of proteins involved in nitrogen fixation and utilization. Three P II 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 NH + 4 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 AmtB 1 are defective in their response to both NH + 4 addition and darkness, while mutants lacking AmtB 2 show little effect on the regulation of nitrogenase activity. These responses to darkness and NH + 4 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 AmtB 1 is necessary to sequester detectable amounts GlnJ to the cell membrane. These results suggest that some element of the AmtB 1 -P II regulatory system senses energy deprivation and a consistent model for the integration of nitrogen, carbon and energy signals by P II is proposed. Other results demonstrate a degree of specificity in interaction of AmtB 1 with the different P II homologues in R. rubrum. Such interaction specificity might be important in explaining the way in which P II proteins regulate processes involved in nitrogen acquisition and utilization.

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Section: Discussionsupporting

confidence: 80%

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

et al. 2006

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“…After ammonium exhaustion, DraG becomes active, whereupon the modifying group is removed and nitrogenase activity is recovered. Recently, an involvement of PII-like proteins in the mechanism for the regulation of DraT and DraG has been demonstrated (32,40,64,65,66).…”

Section: Resultsmentioning

confidence: 99%

Yeast Two-Hybrid Studies on Interaction of Proteins Involved in Regulation of Nitrogen Fixation in the Phototrophic BacteriumRhodobacter capsulatus

et al. 2003

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Rhodobacter capsulatus contains two PII-like proteins, GlnB and GlnK, which play central roles in controlling the synthesis and activity of nitrogenase in response to ammonium availability. Here we used the yeast two-hybrid system to probe interactions between these PII-like proteins and proteins known to be involved in regulating nitrogen fixation. Analysis of defined protein pairs demonstrated the following interactions: GlnBNtrB, GlnB-NifA1, GlnB-NifA2, GlnB-DraT, GlnK-NifA1, GlnK-NifA2, and GlnK-DraT. These results corroborate earlier genetic data and in addition show that PII-dependent ammonium regulation of nitrogen fixation in R. capsulatus does not require additional proteins, like NifL in Klebsiella pneumoniae. In addition, we found interactions for the protein pairs GlnB-GlnB, GlnB-GlnK, NifA1-NifA1, NifA2-NifA2, and NifA1-NifA2, suggesting that fine tuning of the nitrogen fixation process in R. capsulatus may involve the formation of GlnBGlnK heterotrimers as well as NifA1-NifA2 heterodimers. In order to identify new proteins that interact with GlnB and GlnK, we constructed an R. capsulatus genomic library for use in yeast two-hybrid studies. Screening of this library identified the ATP-dependent helicase PcrA as a new putative protein that interacts with GlnB and the Ras-like protein Era as a new protein that interacts with GlnK.

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“…The signal pathway leading to ammonium-induced inhibition or activation of these proteins is dependent on members of the P II family of signal transduction proteins (1,2). In A. brasilense, signaling of ammonium levels to the DRAT/DRAG system involves a P II paralog, GlnZ, which is necessary for reactivation of the Fe protein following depletion of ammonium levels in ammonium-shocked cultures (15). In R. capsulatus, a glnB mutant fixed nitrogen but inhibition of nitrogenase by ammonium ions was impaired (16) and ADP-ribosylation of the Fe protein in this organism was found to be dependent on the ammonium transporter AmtB (31).…”

mentioning

confidence: 99%

“…The Escherichia coli and A. brasilense strains used in this work (Table 1) were grown in liquid LB (24) and NFbHPN (23), respectively. Nitrogenase activity was measured for A. brasilense cultures grown in 60-ml flasks containing NFbHP (10 ml) supplemented with ammonium chloride (5 mM) at 120 rpm and 30°C, as described previously (15). After 18 to 20 h of incubation (optical density at 600 nm, ϳ1.8) the cultures had zero ammonium and were derepressed for nitrogenase activity, which was assayed by the acetylene reduction method (26).…”

mentioning

confidence: 99%

Nitrogenase Switch-Off by Ammonium Ions in Azospirillum brasilense Requires the GlnB Nitrogen Signal-Transducing Protein

Klassen

Souza²,

Yates³

et al. 2005

Appl Environ Microbiol

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Nitrogenase activity in several diazotrophs is switched off by ammonium and reactivated after consumption. The signaling pathway to this system in Azospirillum brasilense is not understood. We show that ammoniumdependent switch-off through ADP-ribosylation of Fe protein was partial in a glnB mutant of A. brasilense but absent in a glnB glnZ double mutant. Triggering of inactivation by anaerobic conditions was not affected in either mutant. The results suggest that glnB is necessary for full ammonium-dependent nitrogenase switch-off in A. brasilense.Nitrogenase activity was first shown to be reversibly inhibited by low levels of ammonium ions or energy depletion in Rhodopseudomonas palustris by Zumft and Castillo (40); this effect, called nitrogenase switch-off/switch-on, was later shown for Rhodospirillum rubrum by Ludden and coworkers to be due to the ADP-ribosylation of an arginine residue of one of the Fe protein (dinitrogenase reductase) subunits by the enzyme dinitrogenase reductase ADP-ribosyl transferase (DRAT). Removal of the ADP-ribosyl moiety and reactivation of the Fe protein is catalyzed by the dinitrogenase reductase-activating glycohydrolase (DRAG) (12,13,19). Control of nitrogenase activity by ADP-ribosylation in response to the increase of ammonium concentration or energy depletion also occurs in other diazotrophs, such as Rhodobacter capsulatus, Azospirillum brasilense, and Azospirillum lipoferum, most of these belonging to the subclass of alpha-Proteobacteria (9,10,12,25,39). The exceptions are Azotobacter chroococcum (of gammaProteobacteria) and Azoarcus sp. strain BH72 (of beta-Proteobacteria) (20,21), although the presence of DRAT and DRAG has not been clearly established for these last two bacteria.The activities of the enzymes DRAG and DRAT in A. brasilense are opposingly regulated depending on the ammonium concentration (35,36). On addition of ammonium ions to a culture of A. brasilense fixing nitrogen, the DRAT protein is temporarily activated and DRAG is inactivated, resulting in a rapid increase in the

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Control of Nitrogenase Reactivation by the GlnZ Protein in Azospirillum brasilense

Cited by 27 publications

References 29 publications

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

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

Yeast Two-Hybrid Studies on Interaction of Proteins Involved in Regulation of Nitrogen Fixation in the Phototrophic BacteriumRhodobacter capsulatus

Nitrogenase Switch-Off by Ammonium Ions in Azospirillum brasilense Requires the GlnB Nitrogen Signal-Transducing Protein

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