NifA Activity Research Articles

Functional characterization of three GlnB homologs in the photosynthetic bacterium Rhodospirillum rubrum: roles in sensing ammonium and energy status.

The GlnB (P(II)) protein, the product of glnB, has been characterized previously in the photosynthetic bacterium Rhodospirillum rubrum. Here we describe identification of two other P(II) 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.

Inter-domain cross-talk controls the NifA protein activity of Herbaspirillum seropedicae

Herbaspirillum seropedicae is an endophytic diazotroph, which colonizes sugar cane, wheat, rice and maize. The activity of NifA, a transcriptional activator of nif genes in H. seropedicae, is controlled by ammonium ions through a mechanism involving its N-terminal domain. Here we show that this domain interacts specifically in vitro with the N-truncated NifA protein, as revealed by protection against proteolysis, and this interaction caused an inhibitory effect on both the ATPase and DNA-binding activities of the N-truncated NifA protein. We suggest that the N-terminal domain inhibits NifA-dependent transcriptional activation by an inter-domain cross-talk between the catalytic domain of the NifA protein and its regulatory N-terminal domain in response to fixed nitrogen.

Recent developments in the structural organization and regulation of nitrogen fixation genes in Herbaspirillum seropedicae

Herbaspirillum seropedicae is a nitrogen-fixing bacterium found in association with economically important gramineae. Regulation of nitrogen fixation involves the transcriptional activator NifA protein. The regulation of NifA protein and its truncated mutant proteins is described and compared with that of other nitrogen fixation bacteria. Nitrogen fixation control in H. seropedicae, of the β-subgroup of Proteobacteria, has regulatory features in common with Klebsiella pneumoniae, of the γ-subgroup, at the level of nifA expression and with rhizobia and Azospirillum brasilense, of the α-subgroup, at the level of control of NifA by oxygen.

Rhodobacter capsulatus nifA mutants mediating nif gene expression in the presence of ammonium

Expression of nitrogen fixation genes in Rhodobacter capsulatus is repressed by ammonium at different regulatory levels including an NtrC-independent mechanism controlling NifA activity. In contrast to R. capsulatus NifA, heterologous NifA proteins of Klebsiella pneumoniae and Rhizobium meliloti, respectively, were not subjected to this posttranslational ammonium control in R. capsulatus. The characterization of ammonium-tolerant R. capsulatus NifA1 mutants indicated that the N-terminal domain of NifA was involved in posttranslational regulation. Analysis of a double mutant carrying amino acid substitutions in both the N-terminal domain and the C-terminal DNA-binding domain gave rise to the hypothesis that an interaction between these two domains might be involved in ammonium regulation of NifA activity. Western analysis demonstrated that both constitutively expressed wild-type and ammonium-tolerant NifA1 proteins exhibited high stability and accumulated to comparable levels in cells grown in the presence of ammonium excluding the possibility that proteolytic degradation was responsible for ammonium-dependent inactivation of NifA.

Rhodobacter capsulatus nifA mutants mediating nif gene expression in the presence of ammonium.

Expression of nitrogen fixation genes in Rhodobacter capsulatus is repressed by ammonium at different regulatory levels including an NtrC-independent mechanism controlling NifA activity. In contrast to R. capsulatus NifA, heterologous NifA proteins of Klebsiella pneumoniae and Rhizobium meliloti, respectively, were not subjected to this posttranslational ammonium control in R. capsulatus. The characterization of ammonium-tolerant R. capsulatus NifA1 mutants indicated that the N-terminal domain of NifA was involved in posttranslational regulation. Analysis of a double mutant carrying amino acid substitutions in both the N-terminal domain and the C-terminal DNA-binding domain gave rise to the hypothesis that an interaction between these two domains might be involved in ammonium regulation of NifA activity. Western analysis demonstrated that both constitutively expressed wild-type and ammonium-tolerant NifA1 proteins exhibited high stability and accumulated to comparable levels in cells grown in the presence of ammonium excluding the possibility that proteolytic degradation was responsible for ammonium-dependent inactivation of NifA.

Role of Escherichia coli nitrogen regulatory genes in the nitrogen response of the Azotobacter vinelandii NifL-NifA complex.

The redox-sensing flavoprotein NifL inhibits the activity of the nitrogen fixation (nif)-specific transcriptional activator NifA in Azotobacter vinelandii in response to molecular oxygen and fixed nitrogen. Although the mechanism whereby the A. vinelandii NifL-NifA system responds to fixed nitrogen in vivo is unknown, the glnK gene, which encodes a PII-like signal transduction protein, has been implicated in nitrogen control. However, the precise function of A. vinelandii glnK in this response is difficult to establish because of the essential nature of this gene. We have shown previously that A. vinelandii NifL is able to respond to fixed nitrogen to control NifA activity when expressed in Escherichia coli. In this study, we investigated the role of the E. coli PII-like signal transduction proteins in nitrogen control of the A. vinelandii NifL-NifA regulatory system in vivo. In contrast to recent findings with Klebsiella pneumoniae NifL, our results indicate that neither the E. coli PII nor GlnK protein is required to relieve inhibition by A. vinelandii NifL under nitrogen-limiting conditions. Moreover, disruption of both the E. coli glnB and ntrC genes resulted in a complete loss of nitrogen regulation of NifA activity by NifL. We observe that glnB ntrC and glnB glnK ntrC mutant strains accumulate high levels of intracellular 2-oxoglutarate under conditions of nitrogen excess. These findings are in accord with our recent in vitro observations (R. Little, F. Reyes-Ramirez, Y. Zhang, W. Van Heeswijk, and R. Dixon, EMBO J. 19:6041-6050, 2000) and suggest a model in which nitrogen control of the A. vinelandii NifL-NifA system is achieved through the response to the level of 2-oxoglutarate and an interaction with PII-like proteins under conditions of nitrogen excess.

Uridylylation of the PII protein from Herbaspirillum seropedicae.

The PII protein is apparently involved in the control of NifA activity in Herbaspirillum seropedicae. To evaluate the probable role of PII in signal transduction, uridylylation assays were conducted with purified H. seropedicae PII and Escherichia coli GlnD, or a cell-free extract of H. seropedicae as sources of uridylylating activity. The results showed that alpha-ketoglutarate and ATP stimulate uridylylation whereas glutamine inhibits uridylylation. Deuridylylation of PII-UMP was dependent on glutamine and inhibited by ATP and alpha-ketoglutarate. PII uridylylation and (or) deuridylylation in response to these effectors suggests that PII is a nitrogen level signal transducer in H. seropedicae.

Two Residues in the T-loop of GlnK Determine NifL-dependent Nitrogen Control of nif Gene Expression

X-ray crystallographic analysis of the Escherichia coli P(II) protein paralogues GlnB and GlnK has shown that they share a superimposable structural core but can differ in conformation of the T-loop, a region of the protein (residues 37-54) that has been shown to be important for interaction with other proteins. In Klebsiella pneumoniae GlnK has been shown to have a clearly defined function in regulating NifL-mediated inhibition of NifA activity in response to the nitrogen status, and GlnB, when expressed from the chromosome, does not substitute for GlnK. Because the T-loops of K. pneumoniae and E. coli GlnB and GlnK differ at just three residues, 43, 52, and 54, we have used a previously constructed heterologous system, in which K. pneumoniae nifLA is expressed in E. coli, to investigate the importance of GlnK residues 43, 52, and 54 for regulation of the NifLA interaction. By site-directed mutagenesis of glnB we have shown that residue 54 is the single most important amino acid in the T-loop in the context of the regulation of NifA activity. Furthermore, a combination of just two changes, in residues 54 and 43, allows GlnB to function as GlnK and completely relieve NifL inhibition of NifA activity.

Azospirillum, a free-living nitrogen-fixing bacterium closely associated with grasses: genetic, biochemical and ecological aspects.

Azospirillum represents the best characterized genus of plant growth-promoting rhizobacteria. Other free-living diazotrophs repeatedly detected in association with plant roots, include Acetobacter diazotrophicus, Herbaspirillum seropedicae, Azoarcus spp. and Azotobacter. Four aspects of the Azospirillum-plant root interaction are highlighted: natural habitat, plant root interaction, nitrogen fixation and biosynthesis of plant growth hormones. Each of these aspects is dealt with in a comparative way. Azospirilla are predominantly surface-colonizing bacteria, whereas A. diazotrophicus, H. seropedicae and Azoarcus sp. are endophytic diazotrophs. The attachment of Azospirillum cells to plant roots occurs in two steps. The polar flagellum, of which the flagellin was shown to be a glycoprotein, mediates the adsorption step. An as yet unidentified surface polysaccharide is believed to be essential in the subsequent anchoring phase. In Azoarcus sp. the attachment process is mediated by type IV pili. Nitrogen fixation structural genes (nif) are highly conserved among all nitrogen-fixing bacteria, and in all diazotrophic species of the class of proteobacteria examined, the transcriptional activator NifA is required for expression of other nif genes in response to two major environmental signals (oxygen and fixed N). However, the mechanisms involved in this control can vary in different organisms. In Azospirillum brasilense and H. seropedicae (alpha- and beta-subgroup, respectively), NifA is inactive in conditions of excess nitrogen. Activation of NifA upon removal of fixed N seems to involve, either directly or indirectly, the signal transduction protein P(II). The presence of four conserved cysteine residues in the NifA protein might be an indication that NifA is directly sensitive to oxygen. In Azotobacter vinelandii (gamma-subgroup) nifA is cotranscribed with a second gene nifL. The nifL gene product inactivates NifA in response to high oxygen tension and cellular nitrogen-status. NifL was found to be a redox-sensitive flavoprotein. The relief of NifL inhibition on NifA activity, in response to N-limitation, is suggested to involve a P(II)-like protein. Moreover, nitrogenase activity is regulated according to the intracellular nitrogen and O(2) level. In A. brasilense and Azospirillum lipoferum posttranslational control of nitrogenase, in response to ammonium and anaerobiosis, involves ADP-ribosylation of the nitrogenase iron protein, mediated by the enzymes DraT and DraG. At least three pathways for indole-3-acetic acid (IAA) biosynthesis in A. brasilense exist: two Trp-dependent (the indole-3-pyruvic acid and presumably the indole-3-acetamide pathway) and one Trp-independent pathway. The occurrence of an IAA biosynthetic pathway not using Trp (tryptophan) as precursor is highly unusual in bacteria. Nevertheless, the indole-3-pyruvate decarboxylase encoding ipdC gene is crucial in the overall IAA biosynthesis in Azospirillum. A number of genes essential for Trp production have been isolated in A. brasilense, including trpE(G) which codes for anthranilate synthase, the key enzyme in Trp biosynthesis. The relevance of each of these four aspects for plant growth promotion by Azospirillum is discussed.

Secondary Structure and DNA binding of the C-terminal Domain of the Transcriptional Activator NifA from Klebsiella pneumoniae.

Secondary Structure and DNA binding of the C-terminal Domain of the Transcriptional Activator NifA from <i>Klebsiella pneumoniae.</i>

Expression of the nifA gene of Herbaspirillum seropedicae: role of the NtrC and NifA binding sites and of the -24/-12 promoter element.

The nifA promoter of Herbaspirillum seropedicae contains potential NtrC, NifA and IHF binding sites together with a -12/-24 sigma(N)-dependent promoter. This region has now been investigated by deletion mutagenesis for the effect of NtrC and NifA on the expression of a nifA::lacZ fusion. A 5' end to the RNA was identified at position 641, 12 bp downstream from the -12/-24 promoter. Footprinting experiments showed that the G residues at positions -26 and -9 are hypermethylated, and that the region from -10 to +10 is partially melted under nitrogen-fixing conditions, confirming that this is the active nifA promoter. In H. seropedicae nifA expression from the sigma(N)-dependent promoter is repressed by fixed nitrogen but not by oxygen and is probably activated by the NtrC protein. NifA protein is apparently not essential for nifA expression but it can still bind the NifA upstream activating sequence.

Mutagenesis and functional characterization of the glnB, glnA, and nifA genes from the photosynthetic bacterium Rhodospirillum rubrum.

Nitrogen fixation is tightly regulated in Rhodospirillum rubrum at two different levels: transcriptional regulation of nif 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 of glnB, glnA, and nifA mutants and studies of their relationship to the regulation of nitrogen fixation. Two mutants which affect glnB (structural gene for P(II)) were constructed. While P(II)-Y51F showed a lower nitrogenase activity than that of wild type, a P(II) deletion mutant showed very little nif expression. This effect of P(II) on nif expression is apparently the result of a requirement of P(II) for NifA activation, whose activity is regulated by NH(4)(+) in R. rubrum. The modification of glutamine synthetase (GS) in these glnB mutants appears to be similar to that seen in wild type, suggesting that a paralog of P(II) might exist in R. rubrum and regulate the modification of GS. P(II) also appears to be involved in the regulation of DRAT activity, since an altered response to NH(4)(+) was found in a mutant expressing P(II)-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 NH(4)(+) and darkness treatments.

Two roles for integration host factor at an enhancer-dependent nifA promoter.

Control of transcription in prokaryotes often involves direct contact of regulatory proteins with RNA polymerase. For the sigma54 RNA polymerase, regulatory proteins bound to distally located enhancers engage the polymerase via DNA looping. The sigma54-dependent nifA promoter of Herbaspirillum seropedicae (Hs) is activated under nitrogen-limiting growth conditions. Potential enhancers for the nitrogen control activators NTRC and NIFA and binding sites for integration host factor (IHF) and sigma54-holoenzyme were identified. DNA footprinting experiments showed that these sites functioned for protein binding. Their involvement in the promoter regulation was explored. In vitro, activation of the Hs nifA promoter by NTRC is stimulated by the DNA bending protein IHF. In marked contrast, activation by NIFA is greatly reduced by IHF, thus diminishing potentially destabilizing autoactivation of the nifA promoter by NIFA. Additionally, high levels of NIFA appear to limit NTRC-dependent activation. This inhibition is IHF dependent. Therefore, IHF acts positively and negatively at the nifA promoter to restrict transcription activation to NTRC and one signal transduction pathway.

Studies on the roles of GlnK and GlnB in regulating Klebsiella pneumoniae NifL-dependent nitrogen control

In Klebsiella pneumoniae, nitrogen fixation ( nif) genes are regulated in response to fixed nitrogen and oxygen. The activity of the nif-specific transcriptional activator NifA is modulated by NifL, which mediates both oxygen and nitrogen control. The signal transduction protein GlnK is required to relieve the inhibitory effect of NifL on NifA that occurs when the intracellular N status is high and in a wild-type cell, the action of GlnK cannot be substituted by the structurally related protein PII. We have studied the modulation of NifA activity by NifL in an heterologous system in which the host organism is Escherichia coli. Using a Δ glnB,Δ glnK mutant, we have shown that the modulation of NifA activity by NifL is dependent on the concentration of GlnK in the cell and that when overproduced, PII can substitute for GlnK. Furthermore, our data suggest that PII can counteract the positive action of GlnK in relieving NifL-dependent inhibition of NifA activity. This negative effect of PII may be physiologically important in establishing repression of nif gene expression when the intracellular nitrogen status rises.

In- trans regulation of the N-truncated-NIFA protein of Herbaspirillum seropedicae by the N-terminal domain

The NifA protein is responsible for transcription activation of nif genes in the endophytic diazotroph Herbaspirillum seropedicae. When expressed in Escherichia coli this NifA protein is unable to activate the transcription of a Klebsiella pneumoniae nifH::lacZ fusion. However, a form of NifA lacking the N-terminal domain did activate transcription and its activity was not inhibited by ammonium. In this work we show that when expressed separately, the N-terminal domain of H. seropedicae NifA protein can restore ammonium control of the N-truncated NifA activity in E. coli. This effect is dependent on the relative concentrations of the N-terminal domain and the N-truncated protein and suggests that the N-terminal domain behaves in this respect in a manner similar to that of NifL of the γ proteobacteria.

Studies on the roles of GlnK and GlnB in regulating Klebsiella pneumoniae NifL-dependent nitrogen control.

In Klebsiella pneumoniae, nitrogen fixation (nif) genes are regulated in response to fixed nitrogen and oxygen. The activity of the nif-specific transcriptional activator NifA is modulated by NifL, which mediates both oxygen and nitrogen control. The signal transduction protein GlnK is required to relieve the inhibitory effect of NifL on NifA that occurs when the intracellular N status is high and in a wild-type cell, the action of GlnK cannot be substituted by the structurally related protein PII. We have studied the modulation of NifA activity by NifL in an heterologous system in which the host organism is Escherichia coli. Using a DeltaglnB, DeltaglnK mutant, we have shown that the modulation of NifA activity by NifL is dependent on the concentration of GlnK in the cell and that when overproduced, PII can substitute for GlnK. Furthermore, our data suggest that PII can counteract the positive action of GlnK in relieving NifL-dependent inhibition of NifA activity. This negative effect of PII may be physiologically important in establishing repression of nif gene expression when the intracellular nitrogen status rises.

In-trans regulation of the N-truncated-NIFA protein of Herbaspirillum seropedicae by the N-terminal domain.

The NifA protein is responsible for transcription activation of nif genes in the endophytic diazotroph Herbaspirillum seropedicae. When expressed in Escherichia coli this NifA protein is unable to activate the transcription of a Klebsiella pneumoniae nifH::lacZ fusion. However, a form of NifA lacking the N-terminal domain did activate transcription and its activity was not inhibited by ammonium. In this work we show that when expressed separately, the N-terminal domain of H. seropedicae NifA protein can restore ammonium control of the N-truncated NifA activity in E. coli. This effect is dependent on the relative concentrations of the N-terminal domain and the N-truncated protein and suggests that the N-terminal domain behaves in this respect in a manner similar to that of NifL of the gamma proteobacteria.

Control of Azospirillum brasilense NifA activity by P II: effect of replacing Tyr residues of the NifA N-terminal domain on NifA activity

It was previously reported that the N-terminal domain of Azospirillum brasilense NifA was a negative regulator of the NifA activity and that the P II protein prevented this inhibition under nitrogen fixing conditions. Here, we show that a mutation of a single Tyr residue at position 18 of the N-terminal domain of NifA led to an active NifA protein that did not require P II for activation under nitrogen fixation conditions.

Control of Azospirillum brasilense NifA activity by P(II): effect of replacing Tyr residues of the NifA N-terminal domain on NifA activity.

It was previously reported that the N-terminal domain of Azospirillum brasilense NifA was a negative regulator of the NifA activity and that the P(II) protein prevented this inhibition under nitrogen fixing conditions. Here, we show that a mutation of a single Tyr residue at position 18 of the N-terminal domain of NifA led to an active NifA protein that did not require P(II) for activation under nitrogen fixation conditions.

Isolation and properties of the complex between the enhancer binding protein NIFA and the sensor NIFL.

In Azotobacter vinelandii, activation of nif gene expression by the transcriptional regulatory enhancer binding protein NIFA is controlled by the sensor protein NIFL in response to changes in levels of oxygen and fixed nitrogen in vivo. NIFL is a novel redox-sensing flavoprotein which is also responsive to adenosine nucleotides in vitro. Inhibition of NIFA activity by NIFL requires stoichiometric amounts of the two proteins, implying that the mechanism of inhibition is by direct protein-protein interaction rather than by catalytic modification of the NIFA protein. The formation of the inhibitory complex between NIFL and NIFA may be regulated by the intracellular ATP/ADP ratio. We show that adenosine nucleotides promote complex formation between purified NIFA and NIFL in vitro, allowing isolation of the NIFL-NIFA complex. The complex can also be isolated from cell extracts containing coexpressed NIFL and NIFA in the presence of MgADP. Removal of the nucleotide causes dissociation of the complex. Experiments with truncated proteins demonstrate that the amino-terminal domain of NIFA and the C-terminal region of NIFL potentiate the ADP-dependent stimulation of NIFL-NIFA complex formation.