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Influence of NifL and Av GlnK on limited proteolysis of NifA in the presence of 2-oxoglutarate. A, examples of the original data. All reactions contained 3.5 mM ADP. NifA (5 M dimer), NifL His6 (2.5 M tetramer), Av GlnK (10 M trimer), and 2-oxoglutarate (4 mM) were added as indicated below. Samples were taken at t 0 (lanes 2, 5, 8, 11, 14, and 17), t 6 (lanes 3, 6, 9, 12, 15, and 18), and t 30 (lanes 4, 7, 10, 13, 16, and 19). Samples contained NifL His6 (lanes 2– 4), NifA plus 2-oxoglutarate (lanes 5–7), NifA plus 2-oxoglutarate plus Av GlnK (lanes 8 –10), NifL His6 plus NifA (lanes 11–13), NifL His6 plus NifA plus 2-oxoglutarate (lanes 14 –16), and NifL His6 plus NifA plus 2-oxoglutarate plus Av GlnK (lanes 17–19). Lane 1 shows molecular mass markers in kilodaltons. B, quantitative analysis of NifA digestion. The percentage of full-length NifA remaining undigested at the indicated times was quantified using the MacBas Version 2.0 image analysis software (Fuji Photo Film Company Ltd). Reactions contained NifA plus 2-oxoglutarate (triangles), NifA plus 2-oxoglutarate plus Av GlnK (squares), NifL His6 plus NifA (inverted triangles), NifL His6 plus NifA plus 2-oxoglutarate (diamonds), and NifL His6 plus NifA plus 2-oxoglutarate plus Av GlnK (circles).
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The expression of genes required for the synthesis of molybdenum nitrogenase in Azotobacter vinelandii is controlled by the NifL-NifA transcriptional regulatory complex in response to nitrogen, carbon, and redox status. Activation of nif gene expression by the transcriptional activator NifA is inhibited by direct protein-protein interaction with Ni...
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A long-held goal of synthetic biology has been the transfer of a bacterial nitrogen-fixation pathway into plants to reduce the use of chemical fertiliser on crops such as rice, wheat and maize. There are three classes of bacterial nitrogenases, named after their unique metalloclusters containing either Mo-, V- or Fe, that convert N 2 gas to ammonia...
Nitrogenase catalyzes the multi-electron reduction of dinitrogen to ammonia. Electron transfer in the catalytic protein (MoFeP) proceeds through a unique [8Fe-7S] cluster (P-cluster) to the active site (FeMoco). In the reduced, all-ferrous (PN) state, the P-cluster is coordinated by six cysteine residues. Upon two-electron oxi-dation to the P2+ sta...
Azotobacter vinelandii has been studied for over 100 years since its discovery as an aerobic nitrogen-fixing organism. This species has proved useful for the study of many different biological systems, including enzyme kinetics and the genetic code. It has been especially useful in working out the structures and mechanisms of different nitrogenase...
The ubiquitous diazotrophic soil bacterium Azotobacter vinelandii has been extensively studied as a model organism for biological nitrogen fixation (BNF). In A. vinelandii , BNF is regulated by the NifL-NifA two-component system, where NifL acts as an anti-activator that tightly controls that activity of the nitrogen fixation specific transcription...
A long-held goal of synthetic biology has been the transfer of a bacterial nitrogen-fixation pathway into plants to reduce the use of chemical fertiliser on crops such as rice, wheat and maize. There are three classes of bacterial nitrogenase, named after their metal requirements, containing either a MoFe-, VFe- or FeFe-cofactor, that converts N2 g...
Citations
... In Proteobacteria, including the free-living associative diazotroph Azotobacter vinelandii, nitrogen fixation-related (nif) gene expression is controlled by the NifL-NifA two-component system (1)(2)(3). NifA is a σ 54 -dependent transcriptional activator that stimulates the expression of nif genes (4)(5)(6)(7)(8). While NifL is homologous to sensor histidine kinases (SHKs) in canonical two-component systems, NifL does not hydrolyze ATP or function as a kinase (6). ...
NifL is a conformationally dynamic flavoprotein responsible for regulating the activity of the σ54-dependent activator NifA to control the transcription of nitrogen fixation (nif) genes in response to intracellular oxygen, cellular energy, or nitrogen availability. The NifL-NifA two-component system is the master regulatory system for nitrogen fixation. NifL serves as a sensory protein, undergoing signal-dependent conformational changes that modulate its interaction with NifA, forming the NifL-NifA complex, which inhibits NifA activity in conditions unsuitable for nitrogen fixation. While NifL-NifA regulation is well understood, these conformationally flexible proteins have eluded previous attempts at structure determination. In work described here, we advance a structural model of the NifL dimer supported by a combination of scattering techniques and mass spectrometry (MS)-coupled structural analyses that report on the average structure in solution. Using a combination of small angle X-ray scattering-derived electron density maps and MS-coupled surface labeling, we investigate the conformational dynamics responsible for NifL oxygen and energy responses. Our results reveal conformational differences in the structure of NifL under reduced and oxidized conditions that provide the basis for a model for modulating NifLA complex formation in the regulation of nitrogen fixation in response to oxygen in the model diazotroph, Azotobacter vinelandii.
... The bacterial enhancer-binding protein NifA is the protein partner of NifL and is the master transcription activator of gene clusters associated with Mo-dependent nitrogenase (Figure 23(B)). The regulatory GAF domain of A. vinelandii NifA binds the TCA cycle intermediate 2-oxoglutarate (Little and Dixon 2003), serving as an important signaling metabolite at the intersection of carbon and nitrogen metabolism (Huergo and Dixon 2015). A higher-order oligomer is formed when NifA is in its active state. ...
Understanding how Nature accomplishes the reduction of inert nitrogen gas to form metabolically tractable ammonia at ambient temperature and pressure has challenged scientists for more than a century. Such an understanding is a key aspect toward accomplishing the transfer of the genetic determinants of biological nitrogen fixation to crop plants as well as for the development of improved synthetic catalysts based on the biological mechanism. Over the past 30 years, the free-living nitrogen-fixing bacterium Azotobacter vinelandii emerged as a preferred model organism for mechanistic, structural, genetic, and physiological studies aimed at understanding biological nitrogen fixation. This review provides a contemporary overview of these studies and places them within the context of their historical development.
... The isothermal titration calorimetry (ITC) results showed that both the full-length NifA protein and GAF domain alone could bind 2-OG, and the affinity for either one of them is almost 60 µmol/L. The deletion of the GAF domain loses the ability to bind to 2-OG [43]. Limited protease hydrolysis experiments showed that 2-OG bound to the GAF domain increases the sensitivity of the GAF domain to trypsin digestion and inhibits the protection of these digestion sites by NifL [43]. ...
... The deletion of the GAF domain loses the ability to bind to 2-OG [43]. Limited protease hydrolysis experiments showed that 2-OG bound to the GAF domain increases the sensitivity of the GAF domain to trypsin digestion and inhibits the protection of these digestion sites by NifL [43]. This suggests that the binding of 2-OG probably leads to the allosteric reaction of the GAF domain, interrupting the inhibition of NifA by NifL. ...
Nitrogen–fixing bacteria execute biological nitrogen fixation through nitrogenase, converting inert dinitrogen (N2) in the atmosphere into bioavailable nitrogen. Elaborating the molecular mechanisms of orderly and efficient biological nitrogen fixation and applying them to agricultural production can alleviate the “nitrogen problem”. Azotobacter vinelandii is a well–established model bacterium for studying nitrogen fixation, utilizing nitrogenase encoded by the nif gene cluster to fix nitrogen. In Azotobacter vinelandii, the NifA–NifL system fine–tunes the nif gene cluster transcription by sensing the redox signals and energy status, then modulating nitrogen fixation. In this manuscript, we investigate the transcriptional regulation mechanism of the nif gene in autogenous nitrogen–fixing bacteria. We discuss how autogenous nitrogen fixation can better be integrated into agriculture, providing preliminary comprehensive data for the study of autogenous nitrogen–fixing regulation.
... Several attempts have been made to establish intracellular biosensors for α-KG by focusing on its important roles in balancing the nutritional status of nitrogen and carbon, such as nitrogen assimilation reactions. Candidate proteins that may act as α-KG sensors include the P II protein family which is widely distributed in bacteria and plants [19], the nitrogenase transcriptional regulator protein NifA [16,20], the NtcA transcriptional regulator belonging to the Crp-Fnr family in Cyanobacteria, the archaeal transcriptional repressor NprR, the carbohydrate phosphotransferase system in bacteria species, the Bacillus subtilis transcriptional regulator GltC, and the KguS/KguR in uropathogenic E. coli (reviewed in [1]). Among these, we employed NifA protein for establishing an intra-nuclear FRET biosensor by adding multiple NLSs to the previously reported sensor which contains the α-KG binding domain of the NifA protein from Azotobacter vinelandii and is highly selective for α-KG and did not exhibit an apparent response to the related metabolites such as citrate, isocitrate, glutamate, and glutamine [16,21]. ...
... NifA is a transcriptional activator of nitrogen fixation (nif) genes in response to the levels of fixed nitrogen by the regulation of P II proteins [1,22]. Its N-terminal GAF domain regulates the catalytic activity by binding directly to α-KG with a dissociation constant of ~60 μM [20]. Firstly, the DNA sequence coding GAF-AAA+ domains (400 a.a.) of NifA protein from Azotobacter vinelandii was tagged with EYFP on the 5' end and ECFP on the 3' end (YFP-GAF-AAA+-CFP: NifA-FRET-NLS(-)) to prepare an expression plasmid as previously reported. ...
... When expressed in 3T3-L1 preadipocytes, the FRET ratio of this nuclear α-KG probe decreased in a dose-dependent manner with dimethyl-2-OG or citrate treatment. Considering that the reported dissociation constant for α-KG of NifA protein is ~60 μM [20], the sensitivity of our probe to exogenous dimethyl-2-OG is lower (above mM range). There are several possible reasons for the lower sensitivity of this probe to dimethyl-2-OG. ...
α-Ketoglutarate (α-KG) also known as 2-oxoglutarate (2-OG) is an intermediate metabolite in the tricarboxylic acid (TCA) cycle and is also produced by the deamination of glutamate. It is an indispensable cofactor for a series of 2-oxoglutarate-dependent oxygenases including epigenetic modifiers such as ten-eleven translocation DNA demethylases (TETs) and JmjC domain-containing histone demethylases (JMJDs). Since these epigenetic enzymes target genomic DNA and histone in the nucleus, the nuclear concentration of α-KG would affect the levels of transcription by modulating the activity of the epigenetic enzymes. Thus, it is of great interest to measure the nuclear concentration of α-KG to elucidate the regulatory mechanism of these enzymes. Here, we report a novel fluorescence resonance energy transfer (FRET)-based biosensor with multiple nuclear localization signals (NLSs) to measure the nuclear concentration of α-KG. The probe contains the α-KG-binding GAF domain of NifA protein from Azotobacter vinelandii fused with EYFP and ECFP. Treatment of 3T3-L1 preadipocytes expressing this probe with either dimethyl-2-oxoglutarate (dimethyl-2-OG), a cell-permeable 2-OG derivative, or citrate elicited time- and dose-dependent changes in the FRET ratio, proving that this probe functions as an α-KG sensor. Measurement of the nuclear α-KG levels in the 3T3-L1 cells stably expressing the probe during adipocyte differentiation revealed that the nuclear concentration of α-KG increased in the early stage of differentiation and remained high thereafter. Thus, this nuclear-localized α-KG probe is a powerful tool for real-time monitoring of α-KG concentrations with subcellular resolution in living cells and is useful for elucidating the regulatory mechanisms of epigenetic enzymes.
... The protein partner of NifL, the prokaryotic enhancer binding protein NifA, which activates nif transcription, is comprised of an N-terminal regulatory domain (GAF), a central AAA+ sigma-54 activation domain and a C-terminal DNA binding domain. The regulatory GAF domain of A. vinelandii NifA binds 2-oxoglutarate [14,15], a TCA cycle intermediate at the interface of carbon and nitrogen metabolism [16]. NifA can only escape inhibition by NifL, when the GAF domain is saturated with 2-oxoglutarate, thus potentially providing a mechanism for the NifL-NifA system to respond to the carbon status. ...
... Random mutagenesis of nifA followed by screening the activity of the A. vinelandii NifL-NifA system in E. coli identified various NifA variants able to escape regulation by NifL under nitrogen excess conditions [21]. One of these mutations, resulting in a charge-change substitution, E356K, located in the central catalytic domain of NifA, (hereafter named NifA-E356K), was found to require binding of 2-oxoglutarate to the GAF domain to escape NifL repression in response to excess fixed nitrogen [15,22]. ...
... Although the potential for carbon regulation, signalled via binding of 2-oxoglutarate to the NifA GAF domain, has been well established in vitro, the physiological relevance of 2-oxoglutarate in NifL-NifA regulation has not been clearly demonstrated in vivo. NifA-E356K requires 2-oxoglutarate in order to escape inhibition by NifL in the presence of GlnK in vitro [15,22], suggesting that its ability to bypass nitrogen regulation in vivo might be regulated by carbon source availability. To facilitate correlation of nitrogenase activity with expression of the nitrogenase structural genes when strains were grown on different carbon sources we constructed strains containing a translational nifH::lacZ fusion located at a neutral site in the chromosome (see Materials and Methods and S1 Table). ...
The energetic requirements for biological nitrogen fixation necessitate stringent regulation of this process in response to diverse environmental constraints. To ensure that the nitrogen fixation machinery is expressed only under appropriate physiological conditions, the dedicated NifL-NifA regulatory system, prevalent in Proteobacteria, plays a crucial role in integrating signals of the oxygen, carbon and nitrogen status to control transcription of nitrogen fixation ( nif ) genes. Greater understanding of the intricate molecular mechanisms driving transcriptional control of nif genes may provide a blueprint for engineering diazotrophs that associate with cereals. In this study, we investigated the properties of a single amino acid substitution in NifA, (NifA-E356K) which disrupts the hierarchy of nif regulation in response to carbon and nitrogen status in Azotobacter vinelandii . The NifA-E356K substitution enabled overexpression of nitrogenase in the presence of excess fixed nitrogen and release of ammonia outside the cell. However, both of these properties were conditional upon the nature of the carbon source. Our studies reveal that the uncoupling of nitrogen fixation from its assimilation is likely to result from feedback regulation of glutamine synthetase, allowing surplus fixed nitrogen to be excreted. Reciprocal substitutions in NifA from other Proteobacteria yielded similar properties to the A . vinelandii counterpart, suggesting that this variant protein may facilitate engineering of carbon source-dependent ammonia excretion amongst diverse members of this family.
... Measurements of nitrogenase activity and N-fixation are therefore routinely performed in the absence of a host plant. Critically, these measurements may not reflect activity in the rhizosphere where the availability and forms of nutrients such as carbon (C) and N, which are key regulators of N fixation (Little et al., 2000;Little and Dixon, 2003;Ninfa and Jiang, 2005; Bueno Batista and Dixon, 2019), may vary widely. As we are continually isolating novel putative plant-associative diazotrophic bacteria from the environment, a high-throughput assay to confirm and compare nitrogenase activity in an environment more reflective of plant root and rhizosphere would be of significant value to assess their use as cereal inoculants. ...
Assessment of plant-associative bacterial nitrogen (N) fixation is crucial for selection and development of elite diazotrophic inoculants that could be used to supply cereal crops with nitrogen in a sustainable manner. Although diazotrophic bacteria possess diverse oxygen tolerance mechanisms, most require a sub 21% oxygen environment to achieve optimal stability and function of the N-fixing catalyst nitrogenase. Consequently, assessment of N fixation is routinely carried out on “free-living” bacteria grown in the absence of a host plant and such experiments may not accurately divulge activity in the rhizosphere where the availability and forms of nutrients such as carbon and N, which are key regulators of N fixation, may vary widely. Here, we present a modified in situ acetylene reduction assay (ARA), utilizing the model cereal barley as a host to comparatively assess nitrogenase activity in diazotrophic bacteria. The assay is rapid, highly reproducible, applicable to a broad range of diazotrophs, and can be performed with simple equipment commonly found in most laboratories that investigate plant-microbe interactions. Thus, the assay could serve as a first point of order for high-throughput identification of elite plant-associative diazotrophs.
... MxGacB harbors the GAF domain, which was first identified by Aravind and colleagues and named for its presence in cGMP-specific phosphodiesterases, Anabaena adenylate cyclases and Escherichia coli FhlA (Aravind and Ponting, 1997). Since then, GAF domains have been shown to respond to a diverse set of small molecule signals, including cyclic nucleotides, amino acids and metabolites, as well as redox and light (Little and Dixon, 2003;Sardiwal et al., 2005;Heikaus et al., 2009;Villapakkam et al., 2009;Enomoto et al., 2014;Tang et al., 2015). Our initial in vitro experiments with MxGacB included measuring enzyme activity in the presence of ATP, GTP and related analogs. ...
3’,3’‐cyclic GMP‐AMP (cGAMP) is the third cyclic dinucleotide (CDN) to be discovered in bacteria. No activators of cGAMP signaling have yet been identified, and the signaling pathways for cGAMP have been inferred to display a narrow distribution based upon the characterized synthases, DncV and Hypr GGDEFs. Here we report that the ubiquitous second messenger cyclic AMP (cAMP) is an activator of the Hypr GGDEF enzyme GacB from Myxococcus xanthus. Furthermore, we show that GacB is inhibited directly by cyclic di‐GMP, which provides evidence for cross‐regulation between different CDN pathways. Finally, we reveal that the HD‐GYP enzyme PmxA is a cGAMP‐specific phosphodiesterase (GAP) that promotes resistance to osmotic stress in M. xanthus. A signature amino acid change in PmxA was found to reprogram substrate specificity and was applied to predict the presence of non‐canonical HD‐GYP phosphodiesterases in many bacterial species, including phyla previously not known to utilize cGAMP signaling. There are two distinct functions and pathways for cGAMP signalling in bacteria. We have discovered the first regulatory mechanisms for the Hypr GGDEF pathway. The synthase GacB from Myxococcus xanthus is activated by cAMP through its GAF domain and inhibited by cyclic di‐GMP through the I‐site. We also discovered that some HD‐GYP enzymes with signature amino acid changes are cGAMP‐specific phosphodiesterases (GAPs). Using bioinformatics, we predict GAP enzymes in bacteria not known to utilize cGAMP.
... Aravind and colleagues first identified the GAF domain by bioinformatics and coined the name for its presence in cGMP-specific phosphodiesterases, Anabaena adenylate cyclases, and Escherichia coli FhlA (11). Since then, GAF domains have been shown to respond to a diverse set of small molecule signals, including cyclic nucleotides, amino acids, and metabolites, as well as redox and light (12)(13)(14)(15)(16)(17). Our initial in vitro experiments with MxGacB included measuring enzyme activity in the presence of ATP, GTP, and related analogs. ...
3',3'-cyclic GMP-AMP (cGAMP) is the third cyclic dinucleotide (CDN) to be discovered in bacteria. No activators of cGAMP signaling have yet been identified, and the signaling pathways for cGAMP have appeared narrowly distributed based upon the characterized synthases, DncV and Hypr GGDEFs. Here we report that the ubiquitous second messenger cyclic AMP (cAMP) is an activator of the Hypr GGDEF enzyme GacB from Myxococcus xanthus . Furthermore, we show that GacB is inhibited directly by cyclic di-GMP, which provides evidence for cross-regulation between different CDN pathways. Finally, we reveal that the HD-GYP enzyme PmxA is a cGAMP-specific phosphodiesterase (GAP) that promotes resistance to osmotic stress in M. xanthus . A signature amino acid change in PmxA was found to reprogram substrate specificity and was applied to predict the presence of non-canonical HD-GYP phosphodiesterases in many bacterial species, including phyla previously not known to utilize cGAMP signaling.
... Under nitrogen-limiting conditions, uridylylation of GlnK prevents the interaction with NifL leading to dissociation of the ternary complex (Figure 2, right) and consequent activation of NifA. However, activation of NifA under low-nitrogen conditions is also dependent on the carbon status, since binding of 2-OG to the regulatory GAF domain of NifA is required to prevent the formation of the inhibitory NifL-NifA binary complex [51][52][53]. Therefore, in addition to the response of NifL to the oxygen and nitrogen status, the interaction of NifL with NifA is modulated by carbon availability, since the ability of NifA to escape from inhibition by NifL is dependent on the binding of 2-oxoglurate to the GAF domain. ...
Biological nitrogen fixation (BNF) is controlled by intricate regulatory mechanisms to ensure that fixed nitrogen is readily assimilated into biomass and not released to the environment. Understanding the complex regulatory circuits that couple nitrogen fixation to ammonium assimilation is a prerequisite for engineering diazotrophic strains that can potentially supply fixed nitrogen to non-legume crops. In this review, we explore how the current knowledge of nitrogen metabolism and BNF regulation may allow strategies for genetic manipulation of diazotrophs for ammonia excretion and provide a contribution towards solving the nitrogen crisis.
... This repression is further regulated by the NifA GAF domain which is required for NifL binding (Arnott, Sidoti, Hill, & Merrick, 1989). In A. vinelandii GAF binds 2oxoglutarate as a proxy for sensing nitrogen limiting conditions (Little & Dixon, 2003). Binding of 2-oxoglutarate prevents GAF from interacting with NifL, abolishing NifL-mediated oxygen repression. ...
Rhizobia are α- and β-proteobacteria that form a symbiotic partnership with legumes, fixing atmospheric dinitrogen to ammonia and providing it to the plant. Oxygen regulation is key in this symbiosis. Fixation is performed by an oxygen-intolerant nitrogenase enzyme but requires respiration to meet its high energy demands. To satisfy these opposing constraints the symbiotic partners cooperate intimately, employing a variety of mechanisms to regulate and respond to oxygen concentration. During symbiosis rhizobia undergo significant changes in gene expression to differentiate into nitrogen-fixing bacteroids. Legumes host these bacteroids in specialized root organs called nodules. These generate a near-anoxic environment using an oxygen diffusion barrier, oxygen-binding leghemoglobin and control of mitochondria localization. Rhizobia sense oxygen using multiple interconnected systems which enable a finely-tuned response to the wide range of oxygen concentrations they experience when transitioning from soil to nodules. The oxygen-sensing FixL-FixJ and hybrid FixL-FxkR two-component systems activate at relatively high oxygen concentration and regulate fixK transcription. FixK activates the fixNOQP and fixGHIS operons producing a high-affinity terminal oxidase required for bacterial respiration in the microaerobic nodule. Additionally or alternatively, some rhizobia regulate expression of these operons by FnrN, an FNR-like oxygen-sensing protein. The final stage of symbiotic establishment is activated by the NifA protein, regulated by oxygen at both the transcriptional and protein level. A cross-species comparison of these systems highlights differences in their roles and interconnections but reveals common regulatory patterns and themes. Future work is needed to establish the complete regulon of these systems and identify other regulatory signals.
