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Nitric oxide (NO), synthesized in eukaryotes by the NO synthases, has multiple roles in signalling pathways and in protection against pathogens. Pathogenic microorganisms have apparently evolved defence mechanisms that counteract the effects of NO and related reactive nitrogen species. Regulatory proteins that sense NO mediate the primary response to NO and nitrosative stress. The only regulatory protein in enteric bacteria known to serve exclusively as an NO-responsive transcription factor is the enhancer binding protein NorR (refs 9, 10–11). In Escherichia coli, NorR activates the transcription of the norVW genes encoding a flavorubredoxin (FlRd) and an associated flavoprotein, respectively, which together have NADH-dependent NO reductase activity. The NO-responsive activity of NorR raises important questions concerning the mechanism of NO sensing. Here we show that the regulatory domain of NorR contains a mononuclear non-haem iron centre, which reversibly binds NO. Binding of NO stimulates the ATPase activity of NorR, enabling the activation of transcription by RNA polymerase. The mechanism of NorR reveals an unprecedented biological role for a non-haem mononitrosyl–iron complex in NO sensing. [ABSTRACT FROM AUTHOR]

The NifA protein from Azotobacter vinelandii belongs to a family of enhancer binding proteins (EBPs) that activate transcription by RNA polymerase containing the sigma factor σ54. These proteins have conserved AAA+ domains that catalyse ATP hydrolysis to drive conformational changes necessary for open complex formation by σ54-RNA polymerase. The activity of the NifA protein is highly regulated in response to redox and fixed nitrogen through interaction with the antiactivator protein NifL. Binding of NifL to NifA inhibits the ATPase activity of NifA, and this interaction is controlled by the amino-terminal GAF domain of NifA that binds 2-oxoglutarate. Mutations conferring resistance to NifL are located in both the GAF and the AAA+ domains of NifA. To investigate the mechanism by which the GAF domain regulates the activity of the AAA+ domain, we screened for second-site mutations that suppress the NifL-resistant phenotype of mutations in the AAA+ domain. One suppressor mutation, F119S, in the GAF domain restores inhibition by NifL to an AAA+ domain mutation, E356K, in response to fixed nitrogen but not in response to oxygen. The biochemical properties of this mutant protein are consistent with the in vivo phenotype and demonstrate that interdomain suppression results in sensitivity to inhibition by NifL in the presence of the signal transduction protein GlnK, but not to the oxidized form of NifL. In the absence of an AAA+ domain mutation, the F119S mutation confers hypersensitivity to repression by NifL. Isothermal titration calorimetry demonstrates that this mutation prevents binding of 2-oxoglutarate to the GAF domain. Our data support a model in which the GAF domain plays an essential role in preventing inhibition by NifL under conditions appropriate for nitrogen fixation. These observations are of general significance in considering how the activities of EBPs are controlled in response to environmental signals. [ABSTRACT FROM AUTHOR]

The Azotobacter vinelandii σ[sup 54]-dependent transcriptional activator protein NifA is regulated by the NifL protein in response to redox, carbon, and nitrogen status. Under conditions inappropriate for nitrogen fixation, NifL inhibits transcription activation by NifA through the formation of the NifL-NifA protein complex. NifL inhibits the ATPase activity of the central AAA+ domain of NifA required to drive open complex formation by σ[sup 54]-RNA polymerase and may also inhibit the activator-polymerase interaction. To analyze the mechanism of inhibition in greater detail, we isolated NifA mutants which are resistant to the inhibitory action of NifL. Mutations in both the amino-terminal GAF domain and the catalytic AAA+ domain of NifA were isolated. Several mutants blocked inhibition by NifL in response to both nitrogen and redox status, whereas some of the mutant NifA proteins were apparently able to discriminate between the forms of NifL present under different environmental conditions. One mutant protein, NifA-Y254N, was resistant to NifL under conditions of anaerobic nitrogen excess but was relatively sensitive to NifL under aerobic growth conditions. The properties of the purified mutant protein in vitro were consistent with the in vivo phenotype and indicate that NifA-Y254N is not responsive to the nitrogen signal conveyed by the interaction of NifL with A. vinelandii GlnK but is responsive to the oxidized form of NifL when ADP is present. Our observations suggest that different conformers of NifL may be generated in response to discrete signal transduction events and that both the GAF and AAA+ domains of NifA are involved in the response to NifL. [ABSTRACT FROM AUTHOR]

Examines the role of Escherichia coli in the response of Azotobacter vinelandii to molecular and fixed nitrogen. Regulation of signal transduction proteins in nitrogen control; Impact of nitrogen excess on intracellular 2-oxyglutarate levels; Expression of PII protein homologues in proteobacteria.

Explores protein-protein interactions in the complex between the enhancer binding protein NIFA and the sensor NIFL from Azotobacter vinelandii. Transduction via complex formation between two proteins; Free-living diazotrophs Klebsiella pneumoniae and Azotobacter vinelandii; Activation of expression of genes involved in nitrogen fixation.

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. Author summary: The NifL-NifA regulatory system provides dedicated signal transduction machinery to regulate nitrogen fixation in diverse Proteobacteria. Understanding how the balance of nitrogen and carbon resources is signalled via NifL-NifA for precise control of nitrogen fixation may lead to broadly applicable translational outputs. Here, we characterize a NifA variant that bypasses nitrogen regulation but is still dependent on the carbon status to enable ammonia excretion in soil diazotrophs. Disruption of the regulatory hierarchy in response to nitrogen and carbon suggests how the integration of environmental stimuli could be harnessed to engineer conditional release of fixed nitrogen for the benefit of cereal crops. [ABSTRACT FROM AUTHOR]

A main goal of biological nitrogen fixation research has been to expand the nitrogen-fixing ability to major cereal crops. In this work, we demonstrate the use of the efficient nitrogen-fixing rhizobacterium Pseudomonas protegens Pf-5 X940 as a chassis to engineer the transfer of nitrogen fixed by BNF to maize and wheat under non-gnotobiotic conditions. Inoculation of maize and wheat with Pf-5 X940 largely improved nitrogen content and biomass accumulation in both vegetative and reproductive tissues, and this beneficial effect was positively associated with high nitrogen fixation rates in roots. 15N isotope dilution analysis showed that maize and wheat plants obtained substantial amounts of fixed nitrogen from the atmosphere. Pf-5 X940-GFP-tagged cells were always reisolated from the maize and wheat root surface but never from the inner root tissues. Confocal laser scanning microscopy confirmed root surface colonization of Pf-5 X940-GFP in wheat plants, and microcolonies were mostly visualized at the junctions between epidermal root cells. Genetic analysis using biofilm formation-related Pseudomonas mutants confirmed the relevance of bacterial root adhesion in the increase in nitrogen content, biomass accumulation and nitrogen fixation rates in wheat roots. To our knowledge, this is the first report of robust BNF in major cereal crops. [ABSTRACT FROM AUTHOR]

σ54-dependent transcription controls a wide range of stress-related genes in bacteria and is tightly regulated. In contrast to σ70, the σ54- RNA polymerase holoenzyme forms a stable closed complex at the promoter site that rarely isomerises into transcriptionally competent open complexes. The conversion into open complexes requires the ATPase activity of activator proteins that bind remotely upstream of the transcriptional start site. These activators belong to the large AAA protein family and the majority of them consist of an N-terminal regulatory domain, a central AAA domain and a C-terminal DNA binding domain. Here we use a functional variant of the NorR activator, a dedicated NO sensor, to provide the first structural and functional characterisation of a full length AAA activator in complex with its enhancer DNA. Our data suggest an inter-dependent and synergistic relationship of all three functional domains and provide an explanation for the dependence of NorR on enhancer DNA. Our results show that NorR readily assembles into higher order oligomers upon enhancer binding, independent of activating signals. Upon inducing signals, the N-terminal regulatory domain relocates to the periphery of the AAA ring. Together our data provide an assembly and activation mechanism for NorR. [ABSTRACT FROM AUTHOR]

To modulate the expression of genes involved in nitrogen assimilation, the cyanobacterial PII-interacting protein X (PipX) interacts with the global transcriptional regulator NtcA and the signal transduction protein PII, a protein found in all three domains of life as an integrator of signals of the nitrogen and carbon balance. PipX can form alternate complexes with NtcA and PII, and these interactions are stimulated and inhibited, respectively, by 2-oxoglutarate, providing a mechanistic link between PII signaling and NtcA-regulated gene expression. Here, we demonstrate that PipX is involved in a much wider interaction network. The effect of pipX alleles on transcript levels was studied by RNA sequencing of S. elongatus strains grown in the presence of either nitrate or ammonium, followed by multivariate analyses of relevant mutant/control comparisons. As a result of this process, 222 genes were classified into six coherent groups of differentially regulated genes, two of which, containing either NtcA-activated or NtcA-repressed genes, provided further insights into the function of NtcA–PipX complexes. The remaining four groups suggest the involvement of PipX in at least three NtcA-independent regulatory pathways. Our results pave the way to uncover new regulatory interactions and mechanisms in the control of gene expression in cyanobacteria. [ABSTRACT FROM AUTHOR]

Most biological nitrogen fixation is catalyzed by molybdenum-dependent nitrogenase, an enzyme complex comprising two component proteins that contains three different metalloclusters. Diazotrophs contain a common core of nitrogen fixation nif genes that encode the structural subunits of the enzyme and components required to synthesize the metalloclusters. However, the complement of nif genes required to enable diazotrophic growth varies significantly amongst nitrogen fixing bacteria and archaea. In this study, we identified a minimal nif gene cluster consisting of nine nif genes in the genome of Paenibacillus sp. WLY78, a gram-positive, facultative anaerobe isolated from the rhizosphere of bamboo. We demonstrate that the nif genes in this organism are organized as an operon comprising nifB, nifH, nifD, nifK, nifE, nifN, nifX, hesA and nifV and that the nif cluster is under the control of a σ70 (σA)-dependent promoter located upstream of nifB. To investigate genetic requirements for diazotrophy, we transferred the Paenibacillus nif cluster to Escherichia coli. The minimal nif gene cluster enables synthesis of catalytically active nitrogenase in this host, when expressed either from the native nifB promoter or from the T7 promoter. Deletion analysis indicates that in addition to the core nif genes, hesA plays an important role in nitrogen fixation and is responsive to the availability of molybdenum. Whereas nif transcription in Paenibacillus is regulated in response to nitrogen availability and by the external oxygen concentration, transcription from the nifB promoter is constitutive in E. coli, indicating that negative regulation of nif transcription is bypassed in the heterologous host. This study demonstrates the potential for engineering nitrogen fixation in a non-nitrogen fixing organism with a minimum set of nine nif genes. [ABSTRACT FROM AUTHOR]

Abstract: Nitrogen fixation in Herbaspirillum seropedicae is transcriptionally regulated by NifA, a σ54 transcriptional activator with three structural domains: an N-terminal GAF domain, a catalytic AAA+ domain and a C-terminal DNA-binding domain. NifA is only active in H. seropedicae when cultures are grown in the absence of fixed nitrogen and at low oxygen tensions. There is evidence that the inactivation of NifA in response to fixed nitrogen is mediated by the regulatory GAF domain. However, the mechanism of NifA repression by the GAF domain, as well as the transduction of nitrogen status to NifA, is not understood. In order to study the regulation of NifA activity by fixed nitrogen independently of oxygen regulation, we constructed a chimeric protein containing the GAF domain of H. seropedicae NifA fused to the AAA+ and C-terminal domains of Azotobacter vinelandii NifA. This chimeric protein (NifAQ1) lacks the cysteine motif found in oxygen sensitive NifA proteins and is not oxygen responsive in vivo. Our results demonstrate that NifAQ1 responds to fixed nitrogen and requires GlnK protein for activity, a behavior similar to H. seropedicae NifA. In addition, protein footprinting analysis indicates that this response probably involves a protein–protein contact between the GAF domain and the GlnK protein. [Copyright &y& Elsevier]

Background: The metabolic capacity for nitrogen fixation is known to be present in several prokaryotic species scattered across taxonomic groups. Experimental detection of nitrogen fixation in microbes requires species-specific conditions, making it difficult to obtain a comprehensive census of this trait. The recent and rapid increase in the availability of microbial genome sequences affords novel opportunities to re-examine the occurrence and distribution of nitrogen fixation genes. The current practice for computational prediction of nitrogen fixation is to use the presence of the nifH and/or nifD genes. Results: Based on a careful comparison of the repertoire of nitrogen fixation genes in known diazotroph species we propose a new criterion for computational prediction of nitrogen fixation: the presence of a minimum set of six genes coding for structural and biosynthetic components, namely NifHDK and NifENB. Using this criterion, we conducted a comprehensive search in fully sequenced genomes and identified 149 diazotrophic species, including 82 known diazotrophs and 67 species not known to fix nitrogen. The taxonomic distribution of nitrogen fixation in Archaea was limited to the Euryarchaeota phylum; within the Bacteria domain we predict that nitrogen fixation occurs in 13 different phyla. Of these, seven phyla had not hitherto been known to contain species capable of nitrogen fixation. Our analyses also identified protein sequences that are similar to nitrogenase in organisms that do not meet the minimum-gene-set criteria. The existence of nitrogenase-like proteins lacking conserved co-factor ligands in both diazotrophs and non-diazotrophs suggests their potential for performing other, as yet unidentified, metabolic functions. Conclusions: Our predictions expand the known phylogenetic diversity of nitrogen fixation, and suggest that this trait may be much more common in nature than it is currently thought. The diverse phylogenetic distribution of nitrogenase-like proteins indicates potential new roles for anciently duplicated and divergent members of this group of enzymes. [ABSTRACT FROM AUTHOR]

Summary The Per-ARNT-Sim (PAS) domain is a conserved α/β fold present within a plethora of signalling proteins from all kingdoms of life. PAS domains are often dimeric and act as versatile sensory and interaction modules to propagate environmental signals to effector domains. The NifL regulatory protein from Azotobacter vinelandii senses the oxygen status of the cell via an FAD cofactor accommodated within the first of two amino-terminal tandem PAS domains, termed PAS1 and PAS2. The redox signal perceived at PAS1 is relayed to PAS2 resulting in conformational reorganization of NifL and consequent inhibition of NifA activity. We have identified mutations in the cofactor-binding cavity of PAS1 that prevent 'release' of the inhibitory signal upon oxidation of FAD. Substitutions of conserved β-sheet residues on the distal surface of the FAD-binding cavity trap PAS1 in the inhibitory signalling state, irrespective of the redox state of the FAD group. In contrast, substitutions within the flanking A'α-helix that comprises part of the dimerization interface of PAS1 prevent transmission of the inhibitory signal. Taken together, these results suggest an inter-subunit pathway for redox signal transmission from PAS1 that propagates from core to the surface in a conformation-dependent manner requiring a flexible dimer interface. [ABSTRACT FROM AUTHOR]

NblS, the most conserved histidine kinase in cyanobacteria, regulates photosynthesis and acclimatization to a variety of environmental conditions. We used in silico, in vivo and in vitro approaches to identify RpaB and SrrA as the cognate response regulators of NblS and to characterize relevant interactions between components of this signalling system. While genetic analysis showed the importance of the NblS to RpaB phosphorylation branch for culture viability in Synechococcus elongatus PCC 7942, in vitro assays indicated a strong preference for NblS to phosphorylate SrrA. This apparent discrepancy can be explained by environmental insulation of the RpaB pathway, achieved by RpaB-dependent repression of srrA under standard, low light culture conditions. After a strong but transient increase in srrA expression upon high light exposure, negative regulation of srrA and other high light inducible genes takes place, suggesting cooperation between pathways under environmental conditions in which both RpaB and SrrA are present. Complex regulatory interactions between RpaB and SrrA, two response regulators with a common evolutionary origin that are controlled by a single histidine kinase, are thus emerging. Our results provide a paradigm for regulatory interactions between response regulators in a branched two-component system. [ABSTRACT FROM AUTHOR]

Bacterial enhancer binding proteins (bEBPs) are specialized transcriptional activators that assemble as hexameric rings in their active forms and utilize ATP hydrolysis to remodel the conformation of RNA polymerase containing the alternative sigma factor σ. Transcriptional activation by the NorR bEBP is controlled by a regulatory GAF domain that represses the ATPase activity of the central AAA+ domain in the absence of nitric oxide. Here, we investigate the mechanism of interdomain repression in NorR by characterizing substitutions in the AAA+ domain that bypass repression by the regulatory domain. Most of these substitutions are located in the vicinity of the surface-exposed loops that engage σ during the ATP hydrolysis cycle or in the highly conserved GAFTGA motif that directly contacts σ. Biochemical studies suggest that the bypass mutations in the GAFTGA loop do not influence the DNA binding properties of NorR or the assembly of higher order oligomers in the presence of enhancer DNA, and as expected these variants retain the ability to activate open complex formation in vitro. We identify a crucial arginine residue in the GAF domain that is essential for interdomain repression and demonstrate that hydrophobic substitutions at this position suppress the bypass phenotype of the GAFTGA substitutions. These observations suggest a novel mechanism for negative regulation in bEBPs in which the GAF domain targets the σ-interaction surface to prevent access of the AAA+ domain to the sigma factor. [ABSTRACT FROM AUTHOR]

Per-Arnt-Sim (PAS) domains play a critical role in signal transduction in multidomain proteins by sensing diverse environmental signals and regulating the activity of output domains. Multiple PAS domains are often found within a single protein. The NifL regulatory protein from Azotobacter vinelandii contains tandem PAS domains, the most N-terminal of which, PAS1, contains a FAD cofactor and is responsible for redox sensing, whereas the second PAS domain, PAS2, has no apparent cofactor and its function is unknown. Amino acid substitutions in PAS2 were identified that either lock NifL in a form that constitutively inhibits NifA or that fail to respond to the redox status, suggesting that PAS2 plays a pivotal role in transducing the redox signal from PAS1 to the C-terminal output domains. The isolated PAS2 domain is a homodimer in solution and the subunits are in rapid exchange. PAS2 dimerization is maintained in the redox signal transduction mutants, but is inhibited by substitutions in PAS2 that lock NifL in the inhibitory conformer. Our results support a model for signal transduction in NifL, whereby redox-dependent conformational changes in PAS1 are relayed to the C-terminal domains via changes in the quaternary structure of the PAS2 domain. [ABSTRACT FROM AUTHOR]

Pseudomonas putida KT2440 is a model strain for studying bacterial biodegradation processes. However, very little is known about nitrogen regulation in this strain. Here, we show that the nitrogen regulatory NtrC proteins from P. putida and Escherichia coli are functionally equivalent and that substitutions leading to partially active forms of enterobacterial NtrC provoke the same phenotypes in P. putida NtrC. P. putida has only a single PII-like protein, encoded by glnK, whose expression is nitrogen regulated. Two contiguous NtrC binding sites located upstream of the σ N-dependent glnK promoter have been identified by footprinting analysis. In vitro experiments with purified proteins demonstrated that glnK transcription was directly activated by NtrC and that open complex formation at this promoter required integration host factor. Transcription of genes orthologous to enterobacterial codB, dppA, and ureD genes, whose transcription is dependent on σ 70 and which are activated by Nac in E. coli, has also been analyzed for P. putida . Whereas dppA does not appear to be regulated by nitrogen via NtrC, the codB and ureD genes have σ N-dependent promoters and their nitrogen regulation was exerted directly by NtrC, thus avoiding the need for Nac, which is missing in this bacterial species. Based upon these results, we propose a simplified nitrogen regulatory network in P. putida (compared to that in enterobacteria), which involves an indirect-feedback autoregulation of glnK using NtrC as an intermediary. [ABSTRACT FROM AUTHOR]

Abstract: Herbaspirillum seropedicae is an endophytic diazotrophic bacterium that associates with economically important crops. NifA protein, the transcriptional activator of nif genes in H. seropedicae, binds to nif promoters and, together with RNA polymerase-σ54 holoenzyme, catalyzes the formation of open complexes to allow transcription initiation. The activity of H. seropedicae NifA is controlled by ammonium and oxygen levels, but the mechanisms of such control are unknown. Oxygen sensitivity is attributed to a conserved motif of cysteine residues in NifA that spans the central AAA+ domain and the interdomain linker that connects the AAA+ domain to the C-terminal DNA binding domain. Here we mutagenized this conserved motif of cysteines and assayed the activity of mutant proteins in vivo. We also purified the mutant variants of NifA and tested their capacity to bind to the nifB promoter region. Chimeric proteins between H. seropedicae NifA, an oxygen-sensitive protein, and Azotobacter vinelandii NifA, an oxygen-tolerant protein, were constructed and showed that the oxygen response is conferred by the central AAA+ and C-terminal DNA binding domains of H. seropedicae NifA. We conclude that the conserved cysteine motif is essential for NifA activity, although single cysteine-to-serine mutants are still competent at binding DNA. [Copyright &y& Elsevier]

The Escherichia coil nitric oxide sensor NorR was shown to bind to the promoter region of the norVW transcription unit, forming at least two distinct complexes detectable by gel retardation. Three binding sites for NorR and two integration host factor binding sites were identified in the norR-norV intergenic region. The derived consensus sequence for NorR binding sites was used to search for novel members of the E. coli NorR regulon and to show that NorR binding sites are partially conserved in other members of the proteobacteria. [ABSTRACT FROM AUTHOR]

In the postgenomic era it is essential that protein sequences are annotated correctly in order to help in the assignment of their putative functions. Over 1300 proteins in current protein sequence databases are predicted to contain a PAS domain based upon amino acid sequence alignments. One of the problems with the current annotation of the PAS domain is that this domain exhibits limited similarity at the amino acid sequence level. It is therefore essential, when using proteins with low-sequence similarities, to apply profile hidden Markov model searches for the PAS domain-containing proteins, as for the PFAM database. From recent 3D X-ray and NMR structures, however, PAS domains appear to have a conserved 3D fold as shown here by structural alignment of the six representative 3D-structures from the PDB database. Large-scale modelling of the PAS sequences from the PFAM database against the 3D-structures of these six structural prototypes was performed. All 3D models generated (> 5700) were evaluated using prosaii. We conclude from our large-scale modelling studies that the PAS and PAC motifs (which are separately defined in the PFAM database) are directly linked and that these two motifs form the PAS fold. The existing subdivision in PAS and PAC motifs, as used by the PFAM and SMART databases, appears to be caused by major differences in sequences in the region connecting these two motifs. This region, as has been shown by Gardner and coworkers for human PAS kinase (Amezcua, C.A., Harper, S.M., Rutter, J. & Gardner, K.H. (2002) Structure 10, 1349–1361, [ ]), is very flexible and adopts different conformations depending on the bound ligand. Some PAS sequences present in the PFAM database did not produce a good structural model, even after realignment using a structure-based alignment method, suggesting that these representatives are unlikely to have a fold resembling any of the structural prototypes of the PAS domain superfamily. [ABSTRACT FROM AUTHOR]

The Azotobacter vinelandii NIFL regulatory flavoprotein responds to the redox, energy and nitrogen status of the cell to inhibit transcriptional activation by the σN-dependent enhancer binding protein, NIFA, via the formation of a NIFL–NIFA protein complex. The NIFA protein contains three domains: an N-terminal domain of unknown function; a central catalytic domain required to couple nucleotide hydrolysis to activation of the σN-RNA polymerase holoenzyme; and a C-terminal DNA-binding domain. We report that truncated NIFA proteins that either lack the amino-terminal domain or contain only the isolated central domain remain responsive to inhibition by NIFL but, in contrast to native NIFA, continue to hydrolyse nucleotides when NIFL is present. We also report that NIFL is competent to inhibit the DNA-binding function of NIFA. Taken together, these results suggest that NIFL inhibits NIFA via a concerted mechanism in which DNA binding, catalytic activity and, potentially, interaction with the polymerase are controlled by NIFL in order to prevent transcriptional activation under detrimental environmental conditions. [ABSTRACT FROM AUTHOR]

Azotobacter vinelandii NIFL is a nitrogen fixation-specific regulatory flavoprotein that modulates the activity of the transcriptional activator NIFA in response to oxygen and fixed nitrogen in vivo. NIFL is also responsive to ADP in vitro. Limited proteolysis of NIFL indicates that it comprises a relatively stable N-terminal domain and a C-terminal domain that is protected from trypsin digestion in the presence of adenosine nucleotides. ATP protects the protein from cleavage in the vicinity of potential nucleotide-binding sites in the C-terminus, whereas ADP protects the entire C-terminal domain. NIFL has an apparent K[sub d] of 130 μM for ATP and 16 μM for ADP. The purified N-terminal domain has an identical UV/visible absorption spectrum to the wild-type protein and is reduced by sodium dithionite, demonstrating that it is a flavin-binding domain. The isolated N-terminal domain does not inhibit NIFA activity. A subdomain fragment containing 160 residues of the C-terminal region, including the nucleotide-binding sites, is also not competent to inhibit NIFA. Removal of the first 146 residues of NIFL, which includes a conserved S-motif (PAS-like domain), found in a large family of sensory proteins from eubacteria, archea and eukarya eliminates the redox response. However, this truncated protein remains competent to inhibit NIFA activity in response to ADP in vitro and to the level of fixed nitrogen in vivo. The redox and nitrogen-sensing functions of A. vinelandii NIFL are therefore separable and are discrete functions of the protein. [ABSTRACT FROM AUTHOR]

Among other attributes, the Betaproteobacterial genus Azoarcus has biotechnological importance for plant growth-promotion and remediation of petroleum waste-polluted water and soils. It comprises at least two phylogenetically distinct groups. The "plant-associated" group includes strains that are isolated from the rhizosphere or root interior of the C4 plant Kallar Grass, but also strains from soil and/or water; all are considered to be obligate aerobes and all are diazotrophic. The other group (now partly incorporated into the new genus Aromatoleum) comprises a diverse range of species and strains that live in water or soil that is contaminated with petroleum and/or aromatic compounds; all are facultative or obligate anaerobes. Some are diazotrophs. A comparative genome analysis of 32 genomes from 30 Azoarcus-Aromatoleum strains was performed in order to delineate generic boundaries more precisely than the single gene, 16S rRNA, that has been commonly used in bacterial taxonomy. The origin of diazotrophy in Azoarcus-Aromatoleum was also investigated by comparing full-length sequences of nif genes, and by physiological measurements of nitrogenase activity using the acetylene reduction assay. Based on average nucleotide identity (ANI) and whole genome analyses, three major groups could be discerned: (i) Azoarcus comprising Az. communis, Az. indigens and Az. olearius, and two unnamed species complexes, (ii) Aromatoleum Group 1 comprising Ar. anaerobium, Ar. aromaticum, Ar. bremense, and Ar. buckelii, and (iii) Aromatoleum Group 2 comprising Ar. diolicum, Ar. evansii, Ar. petrolei, Ar. toluclasticum, Ar. tolulyticum, Ar. toluolicum, and Ar. toluvorans. Single strain lineages such as Azoarcus sp. KH32C, Az. pumilus, and Az. taiwanensis were also revealed. Full length sequences of nif-cluster genes revealed two groups of diazotrophs in Azoarcus-Aromatoleum with nif being derived from Dechloromonas in Azoarcus sensu stricto (and two Thauera strains) and from Azospira in Aromatoleum Group 2. Diazotrophy was confirmed in several strains, and for the first time in Az. communis LMG5514, Azoarcus sp. TTM-91 and Ar. toluolicum TT. In terms of ecology, with the exception of a few plant-associated strains in Azoarcus (s.s.), across the group, most strains/species are found in soil and water (often contaminated with petroleum or related aromatic compounds), sewage sludge, and seawater. The possession of nar, nap, nir, nor, and nos genes by most Azoarcus-Aromatoleum strains suggests that they have the potential to derive energy through anaerobic nitrate respiration, so this ability cannot be usefully used as a phenotypic marker to distinguish genera. However, the possession of bzd genes indicating the ability to degrade benzoate anaerobically plus the type of diazotrophy (aerobic vs. anaerobic) could, after confirmation of their functionality, be considered as distinguishing phenotypes in any new generic delineations. The taxonomy of the Azoarcus-Aromatoleum group should be revisited; retaining the generic name Azoarcus for its entirety, or creating additional genera are both possible outcomes. [ABSTRACT FROM AUTHOR]

Herbaspirillum seropedicae is a plant growth promoting bacterium that is able to fix nitrogen and to colonize the surface and internal tissues of important crops. Nitrogen fixation in H. seropedicae is regulated at the transcriptional level by the prokaryotic enhancer binding protein NifA. The activity of NifA is negatively affected by oxygen and positively stimulated by interaction with GlnK, a PII signaling protein that monitors intracellular levels of the key metabolite 2-oxoglutarate (2-OG) and functions as an indirect sensor of the intracellular nitrogen status. GlnK is also subjected to a cycle of reversible uridylylation in response to intracellular levels of glutamine. Previous studies have established the role of the N-terminal GAF domain of NifA in intramolecular repression of NifA activity and the role of GlnK in relieving this inhibition under nitrogen-limiting conditions. However, the mechanism of this control of NifA activity is not fully understood. Here, we constructed a series of GlnK variants to elucidate the role of uridylylation and effector binding during the process of NifA activation. Our data support a model whereby GlnK uridylylation is not necessary to activate NifA. On the other hand, binding of 2-OG and MgATP to GlnK are very important for NifA activation and constitute the most important signal of cellular nitrogen status to NifA. • GlnK activates H. seropedicae NifA. • GlnK uridylylation is not necessary for NifA activation. • GlnK allosteric effectors ATP and 2-OG are necessary for NifA activation. [ABSTRACT FROM AUTHOR]

Uncoupling of biological nitrogen fixation from ammonia assimilation is a prerequisite step for engineering ammonia excretion and improvement of plant-associative nitrogen fixation. In this study, we have identified an amino acid substitution in glutamine synthetase, which provides temperature sensitive biosynthesis of glutamine, the intracellular metabolic signal of the nitrogen status. As a consequence, negative feedback regulation of genes and enzymes subject to nitrogen regulation, including nitrogenase is thermally controlled, enabling ammonia excretion in engineered Escherichia coli and the plant-associated diazotroph Klebsiella oxytoca at 23 °C, but not at 30 °C. We demonstrate that this temperature profile can be exploited to provide diurnal oscillation of ammonia excretion when variant bacteria are used to inoculate cereal crops. We provide evidence that diurnal temperature variation improves nitrogen donation to the plant because the inoculant bacteria have the ability to recover and proliferate at higher temperatures during the daytime., (© 2023. The Author(s).)

Introducing nitrogen fixation ( nif    ) genes into eukaryotic genomes and targeting Nif components to mitochondria or chloroplasts is a promising strategy for engineering nitrogen-fixing plants. A prerequisite for achieving nitrogen fixation in crops is stable and stoichiometric expression of each component in organelles. Previously, we designed a polyprotein-based nitrogenase system depending on Tobacco Etch Virus protease (TEVp) to release functional Nif components from five polyproteins. Although this system satisfies the demand for specific expression ratios of Nif components in Escherichia coli , we encountered issues with TEVp cleavage of polyproteins targeted to yeast mitochondria. To overcome this obstacle, a version of the Nif polyprotein system was constructed by replacing TEVp cleavage sites with minimal peptide sequences, identified by knowledge-based engineering, that are susceptible to cleavage by the endogenous mitochondrial-processing peptidase. This replacement not only further reduces the number of genes required, but also prevents potential precleavage of polyproteins outside the target organelle. This version of the polyprotein-based nitrogenase system achieved levels of nitrogenase activity in E. coli , comparable to those observed with the TEVp-based polyprotein nitrogenase system. When applied to yeast mitochondria, stable and balanced expression of Nif components was realized. This strategy has potential advantages, not only for transferring nitrogen fixation to eukaryotic cells, but also for the engineering of other metabolic pathways that require mitochondrial compartmentalization.

NifL is a conformationally dynamic flavoprotein responsible for regulating the activity of the σ 54 -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 .

Most diazotrophs fix nitrogen only under nitrogen-limiting conditions, for example, in the presence of relatively low concentrations of NH 4 + (0 to 2 mM). However, Paenibacillus sabinae T27 exhibits an unusual pattern of nitrogen regulation of nitrogen fixation, since although nitrogenase activities are high under nitrogen-limiting conditions (0 to 3 mM NH 4 + ) and are repressed under conditions of nitrogen sufficiency (4 to 30 mM NH 4 + ), nitrogenase activity is reestablished when very high levels of NH 4 + (30 to 300 mM) are present in the medium. To further understand this pattern of nitrogen fixation regulation, we carried out transcriptome analyses of P. sabinae T27 in response to increasing ammonium concentrations. As anticipated, the nif genes were highly expressed, either in the absence of fixed nitrogen or in the presence of a high concentration of NH 4 + (100 mM), but were subject to negative feedback regulation at an intermediate concentration of NH 4 + (10 mM). Among the differentially expressed genes, ald1 , encoding alanine dehydrogenase (ADH1), was highly expressed in the presence of a high level of NH 4 + (100 mM). Mutation and complementation experiments revealed that ald1 is required for nitrogen fixation at high ammonium concentrations. We demonstrate that alanine, synthesized by ADH1 from pyruvate and NH 4 + , inhibits GS activity, leading to a low intracellular glutamine concentration that prevents feedback inhibition of GS and mimics nitrogen limitation, enabling activation of nif transcription by the nitrogen-responsive regulator GlnR in the presence of high levels of extracellular ammonium.

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.

All diazotrophic bacteria and archaea isolated so far utilise a nitrogenase enzyme-containing molybdenum in the active site co-factor to fix atmospheric dinitrogen to ammonia. However, in addition to the Mo-dependent nitrogenase, some nitrogen-fixing prokaryotes also express genetically distinct alternative nitrogenase isoenzymes, namely the V-dependent and Fe-only nitrogenases, respectively. Nitrogenase isoenzymes are expressed hierarchically according to metal availability and catalytic efficiency. In proteobacteria, this hierarchy is maintained via stringent transcriptional regulation of gene clusters by dedicated bacterial enhancer-binding proteins (bEBPs). The model diazotroph Azotobacter vinelandii contains two paralogs of the vanadium nitrogenase activator VnfA (henceforth, VnfA1), designated VnfA2 and VnfA3, with unknown functions. Here we demonstrate that the VnfA1 and VnfA3 bEBPs bind to the same target promoters in the Azotobacter vinelandii genome and co-activate a subset of genes in the absence of V, including the structural genes for the Fe-only nitrogenase. Co-activation is inhibited by the presence of V and is dependent on an accessory protein VnfZ that is co-expressed with VnfA3. Our studies uncover a plethora of interactions between bEBPs required for nitrogen fixation, revealing the unprecedented potential for fine-tuning the expression of alternative nitrogenases in response to metal availability., (© 2022 The Authors. Molecular Microbiology published by John Wiley & Sons Ltd.)

Due to the costly energy demands of nitrogen (N) fixation, diazotrophic bacteria have evolved complex regulatory networks that permit expression of the catalyst nitrogenase only under conditions of N starvation, whereas the same condition stimulates upregulation of high-affinity ammonia (NH3) assimilation by glutamine synthetase (GS), preventing excess release of excess NH3 for plants. Diazotrophic bacteria can be engineered to excrete NH3 by interference with GS, however control is required to minimise growth penalties and prevent unintended provision of NH3 to non-target plants. Here, we tested two strategies to control GS regulation and NH3 excretion in our model cereal symbiont Azorhizobium caulinodans AcLP, a derivative of ORS571. We first attempted to recapitulate previous work where mutation of both PII homologues glnB and glnK stimulated GS shutdown but found that one of these genes was essential for growth. Secondly, we expressed unidirectional adenylyl transferases (uATs) in a ΔglnE mutant of AcLP which permitted strong GS shutdown and excretion of NH3 derived from N2 fixation and completely alleviated negative feedback regulation on nitrogenase expression. We placed a uAT allele under control of the NifA-dependent promoter PnifH, permitting GS shutdown and NH3 excretion specifically under microaerobic conditions, the same cue that initiates N2 fixation, then deleted nifA and transferred a rhizopine nifAL94Q/D95Q-rpoN controller plasmid into this strain, permitting coupled rhizopine-dependent activation of N2 fixation and NH3 excretion. This highly sophisticated and multi-layered control circuitry brings us a step closer to the development of a "synthetic symbioses" where N2 fixation and NH3 excretion could be specifically activated in diazotrophic bacteria colonising transgenic rhizopine producing cereals, targeting delivery of fixed N to the crop while preventing interaction with non-target plants., Competing Interests: The authors have declared that no competing interests exist.

Engineering biological nitrogen fixation in eukaryotic cells by direct introduction of nif genes requires elegant synthetic biology approaches to ensure that components required for the biosynthesis of active nitrogenase are stable and expressed in the appropriate stoichiometry. Previously, the NifD subunits of nitrogenase MoFe protein from Azotobacter vinelandii and Klebsiella oxytoca were found to be unstable in yeast and plant mitochondria, respectively, presenting a bottleneck to the assembly of active MoFe protein in eukaryotic cells. In this study, we have delineated the region and subsequently a key residue, NifD-R98, from K. oxytoca that confers susceptibility to protease-mediated degradation in mitochondria. The effect observed is pervasive, as R98 is conserved among all NifD proteins analyzed. NifD proteins from four representative diazotrophs, but not their R98 variants, were observed to be unstable in yeast mitochondria. Furthermore, by reconstituting mitochondrial-processing peptidases (MPPs) from yeast, Oryza sativa , Nicotiana tabacum , and Arabidopsis thaliana in Escherichia coli , we demonstrated that MPPs are responsible for cleavage of NifD. These results indicate a pervasive effect on the stability of NifD proteins in mitochondria resulting from cleavage by MPPs. NifD-R98 variants that retained high levels of nitrogenase activity were obtained, with the potential to stably target active MoFe protein to mitochondria. This reconstitution approach could help preevaluate the stability of Nif proteins for plant expression and paves the way for engineering active nitrogenase in plant organelles., Competing Interests: The authors declare no competing interest., (Copyright © 2020 the Author(s). Published by PNAS.)

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., (© 2019 The Author(s).)

Re-engineering of complex biological systems (CBS) is an important goal for applications in synthetic biology. Efforts have been made to simplify CBS by refactoring a large number of genes with rearranged polycistrons and synthetic regulatory circuits. Here, a posttranslational protein-splicing strategy derived from RNA viruses was exploited to minimize gene numbers of the classic nitrogenase system, where the expression stoichiometry is particularly important. Operon-based nif genes from Klebsiella oxytoca were regrouped into giant genes either by fusing genes together or by expressing polyproteins that are subsequently cleaved with Tobacco Etch Virus protease. After several rounds of selection based on protein expression levels and tolerance toward a remnant C-terminal ENLYFQ-tail, a system with only five giant genes showed optimal nitrogenase activity and supported diazotrophic growth of Escherichia coli This study provides an approach for efficient translation from an operon-based system into a polyprotein-based assembly that has the potential for portable and stoichiometric expression of the complex nitrogenase system in eukaryotic organisms., Competing Interests: The authors declare no conflict of interest., (Copyright © 2018 the Author(s). Published by PNAS.)

Metabolic regulation of Rhodospirillum rubrum nitrogenase is mediated at the post-translational level by the enzymes DraT and DraG when subjected to changes in nitrogen or energy status. DraT is activated during switch-off, while DraG is inactivated by reversible membrane association. We confirm here that the ammonium transporter, AmtB1, rather than its paralog AmtB2, is required for ammonium induced switch-off. Amongst several substitutions at the N100 position in DraG, only N100K failed to locate to the membrane following ammonium shock, suggesting loss of interaction through charge repulsion. When switch-off was induced by lowering energy levels, either by darkness during photosynthetic growth or oxygen depletion under respiratory conditions, reversible membrane sequestration of DraG was independent of AmtB proteins and occurred even under non-diazotrophic conditions. We propose that under these conditions, changes in redox status or possibly membrane potential induce interactions between DraG and another membrane protein in response to the energy status., (© FEMS 2018.)

Bacteria adjust the composition of their electron transport chain (ETC) to efficiently adapt to oxygen gradients. This involves differential expression of various ETC components to optimize energy generation. In Herbaspirillum seropedicae, reprogramming of gene expression in response to oxygen availability is controlled at the transcriptional level by three Fnr orthologs. Here, we characterised Fnr regulons using a combination of RNA-Seq and ChIP-Seq analysis. We found that Fnr1 and Fnr3 directly regulate discrete groups of promoters (Groups I and II, respectively), and that a third group (Group III) is co-regulated by both transcription factors. Comparison of DNA binding motifs between the three promoter groups suggests Group III promoters are potentially co-activated by Fnr3-Fnr1 heterodimers. Specific interaction between Fnr1 and Fnr3, detected in two-hybrid assays, was dependent on conserved residues in their dimerization interfaces, indicative of heterodimer formation in vivo. The requirements for co-activation of the fnr1 promoter, belonging to Group III, suggest either sequential activation by Fnr3 and Fnr1 homodimers or the involvement of Fnr3-Fnr1 heterodimers. Analysis of Fnr proteins with swapped activation domains provides evidence that co-activation by Fnr1 and Fnr3 at Group III promoters optimises interactions with RNA polymerase to fine-tune transcription in response to prevailing oxygen concentrations.

The ability of bacteria to produce polyhydroxyalkanoates such as poly(3-hydroxybutyrate) (PHB) enables provision of a carbon storage molecule that can be mobilized under demanding physiological conditions. However, the precise function of PHB in cellular metabolism has not been clearly defined. In order to determine the impact of PHB production on global physiology, we have characterized the properties of a Δ phaC1 mutant strain of the diazotrophic bacterium Herbaspirillum seropedicae . The absence of PHB in the mutant strain not only perturbs redox balance and increases oxidative stress, but also influences the activity of the redox-sensing Fnr transcription regulators, resulting in significant changes in expression of the cytochrome c -branch of the electron transport chain. The synthesis of PHB is itself dependent on the Fnr1 and Fnr3 proteins resulting in a cyclic dependency that couples synthesis of PHB with redox regulation. Transcriptional profiling of the Δ phaC1 mutant reveals that the loss of PHB synthesis affects the expression of many genes, including approximately 30% of the Fnr regulon.

Overcoming the inhibitory effects of excess environmental ammonium on nitrogenase synthesis or activity and preventing ammonium assimilation have been considered strategies to increase the amount of fixed nitrogen transferred from bacterial to plant partners in associative or symbiotic plant-diazotroph relationships. The GlnE adenylyltransferase/adenylyl-removing enzyme catalyzes reversible adenylylation of glutamine synthetase (GS), thereby affecting the posttranslational regulation of ammonium assimilation that is critical for the appropriate coordination of carbon and nitrogen assimilation. Since GS is key to the sole ammonium assimilation pathway of Azotobacter vinelandii , attempts to obtain deletion mutants in the gene encoding GS ( glnA ) have been unsuccessful. We have generated a glnE deletion strain, thus preventing posttranslational regulation of GS. The resultant strain containing constitutively active GS is unable to grow well on ammonium-containing medium, as previously observed in other organisms, and can be cultured only at low ammonium concentrations. This phenotype is caused by the lack of downregulation of GS activity, resulting in high intracellular glutamine levels and severe perturbation of the ratio of glutamine to 2-oxoglutarate under excess-nitrogen conditions. Interestingly, the mutant can grow diazotrophically at rates comparable to those of the wild type. This observation suggests that the control of nitrogen fixation-specific gene expression at the transcriptional level in response to 2-oxoglutarate via NifA is sufficiently tight to alone regulate ammonium production at levels appropriate for optimal carbon and nitrogen balance. IMPORTANCE In this study, the characterization of the glnE knockout mutant of the model diazotroph Azotobacter vinelandii provides significant insights into the integration of the regulatory mechanisms of ammonium production and ammonium assimilation during nitrogen fixation. The work reveals the profound fidelity of nitrogen fixation regulation in providing ammonium sufficient for maximal growth but constraining energetically costly excess production. A detailed fundamental understanding of the interplay between the regulation of ammonium production and assimilation is of paramount importance in exploiting existing and potentially engineering new plant-diazotroph relationships for improved agriculture., (Copyright © 2017 American Society for Microbiology.)

A large number of genes are necessary for the biosynthesis and activity of the enzyme nitrogenase to carry out the process of biological nitrogen fixation (BNF), which requires large amounts of ATP and reducing power. The multiplicity of the genes involved, the oxygen sensitivity of nitrogenase, plus the demand for energy and reducing power, are thought to be major obstacles to engineering BNF into cereal crops. Genes required for nitrogen fixation can be considered as three functional modules encoding electron-transport components (ETCs), proteins required for metal cluster biosynthesis, and the "core" nitrogenase apoenzyme, respectively. Among these modules, the ETC is important for the supply of reducing power. In this work, we have used Escherichia coli as a chassis to study the compatibility between molybdenum and the iron-only nitrogenases with ETC modules from target plant organelles, including chloroplasts, root plastids, and mitochondria. We have replaced an ETC module present in diazotrophic bacteria with genes encoding ferredoxin-NADPH oxidoreductases (FNRs) and their cognate ferredoxin counterparts from plant organelles. We observe that the FNR-ferredoxin module from chloroplasts and root plastids can support the activities of both types of nitrogenase. In contrast, an analogous ETC module from mitochondria could not function in electron transfer to nitrogenase. However, this incompatibility could be overcome with hybrid modules comprising mitochondrial NADPH-dependent adrenodoxin oxidoreductase and the Anabaena ferredoxins FdxH or FdxB. We pinpoint endogenous ETCs from plant organelles as power supplies to support nitrogenase for future engineering of diazotrophy in cereal crops.

Unlabelled: A fundamental question in microbial physiology concerns why organisms prefer certain nutrients to others. For example, among different nitrogen sources, ammonium is the preferred nitrogen source, supporting fast growth, whereas alternative nitrogen sources, such as certain amino acids, are considered to be poor nitrogen sources, supporting much slower exponential growth. However, the physiological/regulatory logic behind such nitrogen dietary choices remains elusive. In this study, by engineering Escherichia coli, we switched the dietary preferences toward amino acids, with growth rates equivalent to that of the wild-type strain grown on ammonia. However, when the engineered strain was cultured together with wild-type E. coli, this growth advantage was diminished as a consequence of ammonium leakage from the transport-and-catabolism (TC)-enhanced (TCE) cells, which are preferentially utilized by wild-type bacteria. Our results reveal that the nitrogen regulatory (Ntr) system fine tunes the expression of amino acid transport and catabolism components to match the flux through the ammonia assimilation pathway such that essential nutrients are retained, but, as a consequence, the fast growth rate on amino acids is sacrificed., Importance: Bacteria exhibit different growth rates under various nutrient conditions. These environmentally related behaviors reflect the coordination between metabolism and the underlying regulatory networks. In the present study, we investigated the intertwined nitrogen metabolic and nitrogen regulatory systems to understand the growth differences between rich and poor nitrogen sources. Although maximal growth rate is considered to be evolutionarily advantageous for bacteria (as remarked by François Jacob, who said that the "dream" of every cell is to become two cells), we showed that negative-feedback loops in the regulatory system inhibit growth rates on amino acids. We demonstrated that in the absence of regulatory feedback, amino acids are capable of supporting fast growth rates, but this results in ammonia leaking out from cells as "waste," benefiting the growth of competitors. These findings provide important insights into the regulatory logic that controls metabolic flux and ensures nutrient containment but consequently sacrifices growth rate., (Copyright © 2016 Wang et al.)

The metabolite 2-oxoglutarate (also known as α-ketoglutarate, 2-ketoglutaric acid, or oxoglutaric acid) lies at the intersection between the carbon and nitrogen metabolic pathways. This compound is a key intermediate of one of the most fundamental biochemical pathways in carbon metabolism, the tricarboxylic acid (TCA) cycle. In addition, 2-oxoglutarate also acts as the major carbon skeleton for nitrogen-assimilatory reactions. Experimental data support the conclusion that intracellular levels of 2-oxoglutarate fluctuate according to nitrogen and carbon availability. This review summarizes how nature has capitalized on the ability of 2-oxoglutarate to reflect cellular nutritional status through evolution of a variety of 2-oxoglutarate-sensing regulatory proteins. The number of metabolic pathways known to be regulated by 2-oxoglutarate levels has increased significantly in recent years. The signaling properties of 2-oxoglutarate are highlighted by the fact that this metabolite regulates the synthesis of the well-established master signaling molecule, cyclic AMP (cAMP), in Escherichia coli., (Copyright © 2015, American Society for Microbiology. All Rights Reserved.)

All diazotrophic organisms sequenced to date encode a molybdenum-dependent nitrogenase, but some also have alternative nitrogenases that are dependent on either vanadium (VFe) or iron only (FeFe) for activity. In Azotobacter vinelandii, expression of the three different types of nitrogenase is regulated in response to metal availability. The majority of genes required for nitrogen fixation in this organism are encoded in the nitrogen fixation (nif) gene clusters, whereas genes specific for vanadium- or iron-dependent diazotophy are encoded by the vanadium nitrogen fixation (vnf) and alternative nitrogen fixation (anf) genes, respectively. Due to the complexities of metal-dependent regulation and gene redundancy in A. vinelandii, it has been difficult to determine the precise genetic requirements for alternative nitrogen fixation. In this study, we have used Escherichia coli as a chassis to build an artificial iron-only (Anf) nitrogenase system composed of defined anf and nif genes. Using this system, we demonstrate that the pathway for biosynthesis of the iron-only cofactor (FeFe-co) is likely to be simpler than the pathway for biosynthesis of the molybdenum-dependent cofactor (FeMo-co) equivalent. A number of genes considered to be essential for nitrogen fixation by FeFe nitrogenase, including nifM, vnfEN, and anfOR, are not required for the artificial Anf system in E. coli. This finding has enabled us to engineer a minimal FeFe nitrogenase system comprising the structural anfHDGK genes and the nifBUSV genes required for metallocluster biosynthesis, with nifF and nifJ providing electron transport to the alternative nitrogenase. This minimal Anf system has potential implications for engineering diazotrophy in eukaryotes, particularly in compartments (e.g., organelles) where molybdenum may be limiting.

Bacterial enhancer binding proteins (bEBPs) are transcriptional activators that assemble as hexameric rings in their active forms and utilize ATP hydrolysis to remodel the conformation of RNA polymerase containing the alternative sigma factor σ(54). We present a comprehensive and detailed summary of recent advances in our understanding of how these specialized molecular machines function. The review is structured by introducing each of the three domains in turn: the central catalytic domain, the N-terminal regulatory domain, and the C-terminal DNA binding domain. The role of the central catalytic domain is presented with particular reference to (i) oligomerization, (ii) ATP hydrolysis, and (iii) the key GAFTGA motif that contacts σ(54) for remodeling. Each of these functions forms a potential target of the signal-sensing N-terminal regulatory domain, which can act either positively or negatively to control the activation of σ(54)-dependent transcription. Finally, we focus on the DNA binding function of the C-terminal domain and the enhancer sites to which it binds. Particular attention is paid to the importance of σ(54) to the bacterial cell and its unique role in regulating transcription.

The bacterial activator protein NorR binds to enhancer-like elements, upstream of the promoter site, and activates sigma(54)-dependent transcription of genes that encode nitric oxide detoxifying enzymes (NorVW), in response to NO stress. Unique to the norVW promoter in Escherichia coli is the presence of three enhancer sites associated with a binding site for sigma(54)-RNA polymerase. Here we show that all three sites are required for NorR-dependent catalysis of open complex formation by sigma(54)-RNAP holoenzyme (Esigma(54)). We demonstrate that this is essentially due to the need for all three enhancers for maximal ATPase activity of NorR, energy from which is used to remodel the closed Esigma(54) complex and allow melting of the promoter DNA. We also find that site-specific DNA binding per se promotes oligomerisation but the DNA flanking the three sites is needed to further stabilise the functional higher order oligomer of NorR at the enhancers.