Nif Promoters Research Articles

Promoter-specific involvement of the FixJ receiver domain in transcriptional activation

The “two-component” FixLJ system activates nitrogen fixation genes via nifA and fixK in Sinorhizobium meliloti. Like other response regulators, the FixJ protein can be decomposed into an N-terminal phosphorylatable “receiver” domain FixJN and a C-terminal transcriptional activator domain FixJC. The FixJN receiver domain was known to regulate activity of FixJC negatively at the nifA promoter. Here we show a different situation at the fixK promoter where FixJN also contributes positively to transcriptional activation. This promoter-specific effect was mapped by alanine-scanning mutagenesis to the β2 strand of the receiver domain. This interaction with FixJN is required for the recruitment of RNA polymerase at the fixK promoter by phosphorylated FixJ. Altogether the FixJ receiver domain appears to carry at least four functions, some of which can be separated by mutation: (1) autophosphorylation; (2) inhibition of FixJC; (3) dimerization; (4) transcriptional activation at pfixK. This example illustrates the formidable functional plasticity of receiver domains.

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.

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.

Regulation of Azospirillum brasilense nifA gene expression by ammonium and oxygen

The structure and activity of the nifA promoter of Azospirillum brasilense was studied using deletion analysis. An essential region for nifA promoter activity was identified between nucleotides −67 and −47 from the identified transcription start site. A sequence resembling a σ 70 recognition site occurs in this region and may constitute the nifA gene promoter. The regulation of the nifA gene was studied in plasmid and chromosomal nifA::lacZ fusions. Full expression was obtained under low oxygen levels and in the absence of ammonium ions. Repression of nifA expression involves a synergistic effect between oxygen and ammonium.

Regulation of Azospirillum brasilense nifA gene expression by ammonium and oxygen.

The structure and activity of the nifA promoter of Azospirillum brasilense was studied using deletion analysis. An essential region for nifA promoter activity was identified between nucleotides -67 and -47 from the identified transcription start site. A sequence resembling a sigma(70) recognition site occurs in this region and may constitute the nifA gene promoter. The regulation of the nifA gene was studied in plasmid and chromosomal nifA::lacZ fusions. Full expression was obtained under low oxygen levels and in the absence of ammonium ions. Repression of nifA expression involves a synergistic effect between oxygen and ammonium.

Control by light of whole-cell nitrogenase activity and of nitrogenase and bacteriochlorophyll a formation in Rhodobacter capsulatus strain B10S

Control of nitrogenase and bacteriochlorophyll a (BChl) by light was studied under steady-state conditions with continuous cultures of Rhodobacter capsulatus B10S supplied with malate and growth-limiting amounts of ammonium. Consumption of malate and, correspondingly, the C/N ratio at which malate and ammonium were consumed increased when illumination was increased from 3 to approximately 20 klx and became constant at higher illuminations of up to 40 klx. Essentially the same kinetics were observed with respect to nitrogenase activity of cells, contents of nitrogenase polypeptides, and nifH promoter activity. Substrate consumption was half-maximal at 8 klx and was independent of the presence of nitrogenase. Therefore, it is concluded that light controls the C/N ratio (a quantitative measure of the nitrogen status of cells), which in turn is involved in the control of nitrogenase at the level of nif promoter activity. Post-translational regulation of nitrogenase activity by ADP-ribosylation was not observed under steady-state conditions, but it took place when illumination was suddenly decreased to the range where malate consumption and, consequently, the C/N ratio decreased. Irrespective of the presence or absence of nitrogenase, specific BChl contents of the cultures were constant above 20 klx, and they increased at lower illuminations. These results do not confirm a recently proposed link between nitrogen fixation and photosynthesis as represented by BChl.

Purification and binding analysis of the nitrogen fixation regulatory NifA protein from Azospirillum brasilense.

NifA protein activates transcription of nitrogen fixation operons by the alternative sigma 54 holoenzyme form of RNA polymerase. This protein binds to a well-defined upstream activator sequence (UAS) located at the -200/-100 position of nif promoters with the consensus motif TGT-N10-ACA. NifA of Azospirillum brasilense was purified in the form of a glutathione-S-transferase (GST)-NifA fusion protein and proteolytic release of GST yielded inactive and partially soluble NifA. However, the purified NifA was able to induce the production of specific anti-A. brasilense NifA-antiserum that recognized NifA from A. brasilense but not from K. pneumoniae. Both GST-NifA and NifA expressed from the E. coli tac promoter are able to activate transcription from the nifHDK promoter but only in an A. brasilense background. In order to investigate the mechanism that regulates NifA binding capacity we have used E. coli total protein extracts expressing A. brasilense nifA in mobility shift assays. DNA fragments carrying the two overlapping, wild-type or mutated UAS motifs present in the nifH promoter region revealed a retarded band of related size. These data show that the binding activity present in the C-terminal domain of A. brasilense NifA protein is still functional even in the presence of oxygen.

The oxygen-responsive NIFL-NIFA complex: a novel two-component regulatory system controlling nitrogenase synthesis in gamma-proteobacteria.

The high energetic requirements for nitrogen fixation and the extreme oxygen sensitivity of the nitrogenase enzyme impose physiological constraints on diazotrophy that necessitate stringent control of nitrogen fixation (nif) gene expression at the transcriptional level. In the gamma-subdivision of the Proteobacteria, this control is maintained by a regulatory complex comprising an enhancer-binding protein (NIFA), which activates transcription at sigmaN-dependent nif (nitrogen fixation) promoters, and a sensor protein (NIFL), which inhibits NIFA activity in response to fixed nitrogen and external concentrations of molecular oxygen. Inhibition of NIFA activity by NIFL apparently requires stoichiometric amounts of the two proteins, implying direct protein-protein interaction rather than catalytic modulation of NIFA activity. NIFL contains FAD as a prosthetic group and is a novel type of flavoprotein in which the oxidation state of the bound flavin acts as a molecular switch to control transcriptional activation by NIFA. The FAD-binding domain of NIFL contains a motif common to a large family of redox sensory proteins. In addition to its ability to act as a redox sensor, the activity of NIFL is also responsive to adenosine nucleotides, particularly ADP, suggesting that formation of the inhibitory complex might be regulated by the ATP/ ADP ratio. Proposed mechanisms for the inhibition of NIFA activity by NIFL are beginning to emerge.

Structural organization and regulation of the nif genes of Herbaspirillum seropedicae

The promoter regions of nifA and nifH genes of Herbaspirillom seropedicae have been isolated and investigated, and the nifA promoter has been characterized in detail. Both regions contain a −24 −12 type promoter and NifA-binding sites; the nifA promoter also has an NtrC-binding site. The nifA expression is activated by NtrC and repressed by ammonium ions but not by oxygen. When the NtrC-binding site was deleted nifA expression was NifA-dependent. The native NifA protein of H. seropedicae was unable to activate nifH expression in Escherichia coli whereas a truncated protein, lacking the N-terminal domain was able to activate nifH expression in the absence of oxygen and presence of iron, indicating that the N-terminal domain regulates NifA activity. The truncated protein also activates nifH in Azospirillum brasilense in the presence or absence of ammonium ions. This suggests that the N-terminal domain senses the nitrogen status of the cell.

Interactive regulation of Azorhizobium nifA transcription via overlapping promoters

The Azorhizobium nifA promoter (PnifA) is positively regulated by two physiological signal transduction pathways, NtrBC, which signals anabolic N status, and FixLJK, which signals prevailing O2 status. Yet, PnifA response (gene product per unit time) to these two activating signals together is more than twice that of the summed, individual signals. In the absence of NIFA, a negative PnifA autoregulator, the fully induced PnifA response is more than 10-fold greater than that of summed, individual signals. Given this synergism, these two signal transduction pathways must interactively regulate PnifA activity. PnifA carries three cis-acting elements, an anaerobox, which presumably binds FIXK, a NIFAbox, which presumably binds NIFA itself, and a sigma 54 box, which presumably binds sigma 54 initiator, a subunit of RNA polymerase. For combinatorial analysis, single, double, and triple promoter mutations were constructed in these cis-acting elements, and PnifA activities were measured in six different trans-acting background, i.e., fixK, fixJ, nifA, ntrC, rpoF, and wild type. Under all physiological conditions studied, high-level PnifA activity required both FIXK in trans and the anaerobox element in cis. Surprisingly, because PnifA was hyperactive with a mutated sigma 54box, this cis-acting element mediates both negative and positive control. Because PnifA hyperactivity also required a wild-type upstream NIFAbox element, even in the absence of NIFA, a second upstream nifA transcription start superimposed on the NIFAbox element was hypothesized. When nifA mRNA 5' start points were mapped by primer extension, both a minor upstream transcript(s) starting 45 bp distal to the anaerobox and a major downstream transcript starting 10 bp distal to the sigma 54 box were observed. In Azorhizobium, RNA polymerase sigma 54 initiator subunits are encoded by a multigene family, which includes rpoF and rpoN genes. Because rpoF mutants show an Ntr+ phenotype, whereas rpoN mutants are Ntr-, multiple sigma 54 initiators are functionally distinct. Two independent rpoF mutants both show a tight Nif- phenotype. Moreover, rpoF product sigma 54F is absolutely required for high-level PnifA activity. In summary, the Azorhizobium nifA gene carries overlapping housekeeping-type and sigma 54-type promoters which interactively respond to different signals. Effectively, the upstream, housekeeping-type promoter responds to FIXK and positively regulates the downstream, sigma 54-type promoter. The downstream, sigma 54-type promoter responds to NTRC and negatively regulates the upstream, housekeeping-type promoter. In terms of transcript yield, the upstream, housekeeping-type promoter is therefore weak, and the downstream, sigma 54-type promoter is strong.

Characterization of an fdxN mutant of Rhodobacter capsulatus indicates that ferredoxin I serves as electron donor to nitrogenase

A mutant of Rhodobacter capsulatus, carrying an insertion into the fdxN gene encoding ferredoxin I (FdI), has been studied by biochemical analysis and genetic complementation experiments. When compared to the wild-type strain, the fdxN mutant exhibited altered nitrogen fixing ability and 20-fold lower levels of nitrogenase activity as assayed in vivo. When assayed in vitro with an artificial reductant, nitrogenase activity was only 3- to 4-fold lower than in the wild type. These results suggested that the FdI-deleted mutant had impaired electron transport to nitrogenase. Immunochemical assay of both nitrogenase components showed that the fdxN mutant contained about 4-fold less enzyme than wild-type cells. Results of pulse-chase labeling experiments using [ 35S]methionine indicated that nitrogenase was significantly less stable in the FdI-deleted mutant. When a copy of fdxN was introduced in the mutant in trans, the resulting strain appeared to be fully complemented with respect to both diazotrophic growth and nitrogenase activity. Depending on whether fdxN expression was driven by a nif promoter or a fructose-inducible promoter. FdI was synthesized either at wild-type level or in 10-fold lower amounts. The strain producing 10-fold less FdI did, however, display normal N 2-fixing ability. Analysis of cytosolic proteins by bidimensional electrophoresis revealed that the fdxN mutant produced a 14 kDa polypeptide in amounts about 3-fold greater than wild-type cells. This protein was identified by N-terminal microsequencing as a recently purified [2Fe-2S] ferredoxin, called FdV, which cannot reduce nitrogenase. It is concluded that FdI serves as the main electron donor to nitrogenase in R. capsulatus and that an ancillary electron carrier, distinct of FdV, is responsible for the residual nitrogenase activity observed in the FdI-deleted mutant.

Transcriptional activation of the nitrogenase promoter in vitro: adenosine nucleotides are required for inhibition of NIFA activity by NIFL.

The enhancer-binding protein NIFA is required for transcriptional activation of nif promoters by the alternative holoenzyme form of RNA polymerase, which contains the sigma factor sigma 54 (sigma N). NIFA hydrolyzes nucleoside triphosphates to catalyze the isomerization of closed promoter complexes to transcriptionally competent open complexes. The activity of NIFA is antagonized by the regulatory protein NIFL in response to oxygen and fixed nitrogen in vivo. We have investigated the requirement for nucleotides in the formation and stability of open promoter complexes by NIFA and inhibition of its activity by NIFL at the Klebsiella pneumoniae nifH promoter. Open complexes formed by sigma 54-containing RNA polymerase are considerably more stable to heparin challenge in the presence of GTP than in the presence of ATP. This differential stability is most probably a consequence of GTP being the initiating nucleotide at this promoter. Adenosine nucleosides are specifically required for Azotobacter vinelandii NIFL to inhibit open complex formation by native NIFA, and the nucleoside triphosphatase activity of NIFA is strongly inhibited by NIFL under these conditions. We propose a model in which NIFL modulates the activity of NIFA via an adenosine nucleotide switch.

Chaperonins as potential gene regulatory factors. In vitro interaction and solubilization of NifA, the nif transcriptional activator, with GroEL.

A previous study (Govezensky, D., Greener, T., and Zamir, A. (1991) J. Bacteriol. 20, 6339-6346) indicated that the chaperonin GroEL was required for maximal expression from nif promoters in Klebsiella pneumoniae and nif-transformed Escherichia coli. That this requirement stemmed from the ability of GroEL to properly fold NifA, the nif transcriptional activator, was first supported by co-immunoprecipitation of NifA in K. pneumoniae extracts with anti-GroEL antibodies. In the present in vitro study, NifA, partially purified from E. coli overexpressing the protein, was diluted from a 6 M urea solution into a refolding buffer in the presence or absence of GroEL. Dilution in the absence of GroEL caused the complete precipitation of NifA. When present in the dilution buffer, GroEL bound NifA and maintained it in a soluble state. GroEL was also found to bind NifA newly synthesized in an in vitro translation system. For both NifA preparations, cochaperonin GroES and ATP promoted release of NifA from GroEL. These results provide evidence for the association of NifA with GroEL and for the role of both GroEL and GroES in the solubilization and thereby folding of the nif transcriptional activator.

Intramolecular signal transduction within the FixJ transcriptional activator: in vitro evidence for the inhibitory effect of the phosphorylatable regulatory domain.

FixJ is a phosphorylatable 'response regulator' controlling the transcription of the key nitrogen fixation genes nifA and fixK in Rhizobium meliloti. Sequence and genetic analyses indicated that FixJ comprises an N-terminal phosphorylatable regulatory domain, FixJN, and a C-terminal transcriptional activator domain, FixJC. We have now overexpressed and purified the FixJC protein and show that it is fully active in an in vitro transcription system with purified RNA polymerase. FixJC appeared to act synergistically with RNA polymerase at the nifA promoter. Furthermore FixJC was more active in vitro than the full-length dephosphorylated FixJ protein. Therefore activity of FixJC is inhibited by FixJN within the FixJ protein. This inhibition is relieved by phosphorylation of FixJN. Such a negative mode of intramolecular signal transduction may be generalizable to other response regulators.

Mutants of the two-component regulatory protein FixJ of Rhizobium meliloti that have increased activity at the nifA promoter

FixL and FixJ belong to a two-component regulatory system in Rhizobium meliloti that induces the expression of numerous nitrogen-fixation genes during symbiosis with alfalfa. FixJ is a positive activator required for transcription of the regulatory genes nifA and fixK, while FixL is an oxygen-binding hemoprotein capable of regulating the phosphorylation status of both itself and FixJ, in response to oxygen availability. In this study, we isolated four FixJ mutants that display increased activity at the nifA promoter ( PnifA) in Escherichia coli. All four mutants possess amino acid changes in a domain of FixJ that is conserved in other response regulator proteins, and all exhibit increased activity at PnifA in R. meliloti that is dependent on the presence of FixL. One of the mutant proteins, while less efficient at accepting phosphate from a truncated derivative of FixL (FixL∗), nevertheless has a phosphorylated form that is more stable than the phosphorylated form of wild-type (wt) FixJ and is more resistant to the phosphatase activity of FixL∗. The wt FixJ-phosphate was found to have a half-life of approximately 4 h, which makes it an unusually long-lived response regulator protein. The exceptional stability of wt FixJ-phosphate and the altered phosphorylation properties observed for the mutant are discussed in relation to signal transduction in the FixLJ system.

Oxygen regulation of nifA transcription in vitro.

In Rhizobium meliloti, transcription of the key nitrogen-fixation regulatory genes nifA and fixK is induced in response to microaerobiosis through the action of the FixL and FixJ proteins. These two proteins are sensor and regulator homologues, respectively, of a large family of bacterial two-component systems involved in sensing and responding to environmental changes. A soluble, truncated form of the membrane protein FixL, FixL*, has been shown to be a hemoprotein that phosphorylates and dephosphorylates FixJ in response to oxygen tension. Here we use an in vitro transcription system to prove that FixJ is a transcriptional activator of both nifA and fixK and that phosphorylation of FixJ markedly increases its activity. Phosphorylation was achieved either by preincubating FixJ with FixL* and ATP or by exposing FixJ to the inorganic phospho donor ammonium hydrogen phosphoramidate. Both FixJ and FixJ-phosphate formed heparin-resistant complexes under the assay conditions used. Lastly, we were able to show that anaerobiosis, in the presence of FixL* and ATP, greatly stimulates FixJ activity at the nifA promoter with either Escherichia coli or R. meliloti RNA polymerase. This use of atmospheric oxygen to control nifA transcription in vitro represents a reconstitution of a bacterial two-component signal transduction system in its entirety, from effector to ultimate target, by the use of purified components.

The Klebsiella pneumoniae nifJ promoter: analysis of promoter elements regulating activation by the NifA promoter.

The nifJ and nifH promoters of Klebsiella pneumoniae are divergently transcribed sigma 54-dependent promoters that are positively activated by the NifA protein. NifA binds to upstream activator sequences (UASs), usually located 60-200 bp upstream of the start of transcription. Bound NifA is presented to the RNA polymerase-sigma 54 complex (E sigma 54) via DNA loop formation, mediated by the binding of integration host factor protein (IHF) between E sigma 54 and NifA. The nifJ promoter sequence contains three potential NifA binding sites (UAS1, 2 and 3) and two potential RNA polymerase-sigma 54-binding sites (downstream promoter elements, DPEs 1 and 2). DPE2 is located 420 bp into the coding region and DPE1 overlaps UAS1 by 5 bp. Mutational and footprinting analyses have shown efficient activation of the nifJ promoter requires that NifA is bound at UAS 2 and 3. Transcription is initiated at DPE1. Only a weak interaction of NifA with the UAS overlapping DPE1 was detected. Footprints demonstrated that E sigma 54 forms a closed complex at DPE1 but not DPE2 and that bound E sigma 54 closely approaches the -15 region of DPE1. Stimulation of nifJ promoter activity by IHF was not as great as that observed for other nif promoters. In the absence of IHF nifH promoter sequences stimulated activation of the nifJ promoter. This appeared to require NifA bound at the nifH UAS. Thus, one additional role of IHF may be to partition NifA between the two promoters by constraining the topology of the DNA.

Autophosphorylation and phosphatase activities of the oxygen-sensing protein FixL of Rhizobium meliloti are coordinately regulated by oxygen.

The FixL and FixJ proteins of Rhizobium meliloti control the expression of other nif and fix genes in response to oxygen levels. FixL is a hemoprotein kinase that senses oxygen availability and responds to the absence of oxygen by activation of its autophosphorylating activity followed by transfer of the phosphate to FixJ. FixJ in turn activates the nifA and fixK promoters. In vitro studies reported here with a soluble truncated version of FixL (FixL*) indicate that, while low oxygen tension specifically increases the autophosphorylating activity of FixL*, the ability of phospho-FixL* to act as a phosphate donor to FixJ is not affected by the presence or absence of oxygen. FixL* is also shown to possess a phosphatase activity that is repressed under anaerobic conditions only when the protein is in the phosphorylated form. A fixL mutant that induces a higher level of nifA promoter activity in the presence of fixJ in vivo displayed both an increased autophosphorylating activity and a decreased phosphatase activity in vitro. These data provide evidence for a role for both autophosphorylation and phosphatase activities of FixL in the mechanism by which oxygen tension within the alfalfa nodule induces expression of bacterial nitrogen fixation genes during symbiosis.

The regulation of nitrogen utilization in enteric bacteria

The regulation of nitrogen utilization in enteric bacteria

Mutational analysis of the Rhizobium meliloti nifA promoter.

The nifA gene of Rhizobium meliloti, the bacterial endosymbiont of alfalfa, is a regulatory nitrogen fixation gene required for the induction of several key nif and fix genes. Transcription of nifA is strongly induced in planta and under microaerobic conditions ex planta. Induction of nifA, in turn, is positively controlled by the fixL and fixJ genes of R. meliloti, the sensor and regulator, respectively, of a two-component system responsible for oxygen sensing by this bacterium. This system is also responsible for the positive induction of fixK. Here, we report that chemical and oligonucleotide site-directed mutageneses of the nifA promoter (nifAp) were conducted to identify nucleotides essential for induction. Nineteen mutants, including 14 single-point mutants, were analyzed for microaerobic induction of nifAp in R. meliloti. Critical residues were identified in an upstream region between base pairs -54 and -39 relative to the transcription start site. Attempts at separating the upstream and downstream regions of the nifA promoter so as to maintain fixJ-dependent activity were unsuccessful. A 5' deletion of the fixK promoter (fixKp) to -67 indicates that sequences upstream of this position are not required for microaerobic induction. A sequence comparison of the -54 to -39 region of nifAp with the upstream sequences of fixKp does not reveal a block of identical nucleotides that could account for the fixJ-dependent microaerobic induction of both promoters. Many of the defective nifAp mutants in this region, however, are in residues with identity to fixKp in an alignment of the promoters according to their transcription start sites. Therefore, it is possible that there is a common sequence motif in the -54 to -39 region of the two promoters that is required for fixLJ-dependent microaerobic induction.