SEARCH · PubMed Health

Results for “NtrC”

Explore indexed PubMed citations for clinical trials, systematic reviews and public health research. Read source abstracts and follow each citation to its original PubMed record.

Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.

At least 91 records · Page 5Linked to original sources

PubMed1995

The bacterial enhancer-binding protein NTRC is a molecular machine: ATP hydrolysis is coupled to transcriptional activation.

NTRC is a prokaryotic enhancer-binding protein that activates transcription by sigma 54-holoenzyme. NTRC has an ATPase activity that is required for transcriptional activation, specifically for isomerization of closed complexes between sigma 54-holoenzyme and a promoter to open complexes. In the absence of ATP hydrolysis, there is known to be a kinetic barrier to open complex formation (i.e., the reaction proceeds so slowly that the polymerase synthesizes essentially no transcripts even from a supercoiled template). We show here that open complex formation is also thermodynamically unfavorable. In the absence of ATP hydrolysis the position of equilibrium between closed and open complexes favors the closed ones. Use of linear templates with a region of heteroduplex around the transcriptional start site--"preopened" templates--does not bypass the requirement for either NTRC or ATP hydrolysis, providing evidence that the rate-limiting step in open complex formation does not lie in DNA strand denaturation per se. These results are in contrast to recent findings regarding the ATP requirement for initiation of transcription by eukaryotic RNA polymerase II; in the latter case, the ATP requirement is circumvented by use of a supercoiled plasmid template or a preopened linear template.

Adenosine Triphosphate↗

PubMed1993

nif gene expression studies in Rhodobacter capsulatus: ntrC-independent repression by high ammonium concentrations.

The expression of nif genes in Rhodobacter capsulatus depends on the two regulatory genes, rpoN and nifA, encoding a nif-specific alternative sigma factor of RNA polymerase and a nif-specific transcriptional activator, respectively. The expression of the rpoN gene itself is also RPON/NIFA dependent. In order to better characterize the regulation of nif gene induction, chromosomal nifH-, rpoN-, nifA1- and nifA2- lacZ fusions were constructed and the expression of these different nif-lacZ fusions was determined under photoheterotrophic conditions at different starting ammonium concentrations. The two nifA genes were found to be induced first, followed by nifH and finally by rpoN upon weak, medium and strong nitrogen starvation, respectively. This induction profile and the correlation between the expression of the different nif genes suggested that nifA1 expression is the limiting factor for nif gene induction. This hypothesis was tested by construction of different nifA1 overexpressing mutants. Contrary to the current model of nif gene expression in R. capsulatus, which predicted constitutive nif gene expression in such mutants, a strong repression of nifH and rpoN was found at high ammonium concentration. The low nifH expression under these conditions is unaffected by nifA2 and is not increased in a ntrC mutant, ruling out any role of NTRC as a mediator of this repression. This finding implies an additional, so far unidentified, regulation by fixed nitrogen in R. capsulatus. Changing the expression level of rpoN indicated that low levels of RPON are already sufficient for full nifH induction. The nifA1 and rpoN expression mutants were also tested for diazotrophic growth. Similar generation times were determined for the mutants and for the wild type, but diazotrophic growth of the nifA1 over-expressing ntrC mutant RCM14 did not start until after a prolonged lag phase of two to three days.

Bacterial Proteins↗

PubMed1997

Mutation in ntrC gene leading to the derepression of nitrogenase synthesis in Rhodobacter sphaeroides.

The Rhodobacter sphaeroides mutants Drn12 and Drn21 derepressed for nitrogenase synthesis in the presence of ammonia and impaired in utilization of certain nitrogen sources have been analyzed. Both mutants show a low level of expression of the glnBA operon. The DNA fragment restoring the wild-type phenotype to these mutants contains the 3'-portion of ntrB gene and the entire ntrC gene. Sequence analysis showed that Drn12 bears a missense mutation in the ntrC gene. The mutation results in the replacement of a glycine residue by aspartate within the N-terminal domain of the NtrC protein. Pleiotropic phenotypes of Drn12 and Drn21 appear to be associated with an alteration in the regulation of glnBA expression.

Bacterial Proteins↗

PubMed1999

Enhanced nitrogen fixation in a rhizobium etli ntrC mutant that overproduces the bradyrhizobium japonicum symbiotic terminal oxidase cbb3

The ntrC gene codes for a transcriptional activator protein that modulates gene expression in response to nitrogen. The cytochrome production pattern of a Rhizobium etli ntrC mutant (CFN2012) was studied. CO difference spectral analysis of membranes showed that CFN2012 produced a terminal oxidase similar to the symbiotic terminal oxidase of bacteroids in free-living cells under aerobic conditions, with a characteristic trough at 553 nm. CFN2012 produced two c-type cytochromes with molecular masses of 27 and 32 kDa, in contrast with the wild-type strain, which produced only a 32-kDa c-type cytochrome. The expression levels of the R. etli fixNOQP operon, which codes for terminal oxidase cbb3, were not affected by the ntrC mutation. However, the production levels of the two c-type cytochromes (27 and 32 kDa) were enhanced at least eightfold when the Bradyrhizobium japonicum fixNOQP operon was expressed in CFN2012 from the nptII promoter (pMSfixc), suggesting that these proteins are subunits FixO (27 kDa) and FixP (32 kDa) of cbb3 and that CFN2012/pMSfixc overproduced this terminal oxidase. CFN2012/pMSfixc showed a significant increase in its symbiotic performance as judged by the determination of nitrogenase activities of plants inoculated with this strain, suggesting that the overproduction of cbb3 terminal oxidase correlates with an enhancement in symbiotic nitrogen fixation.

Journal Article↗

PubMed1989

Bradyrhizobium japonicum glnB, a putative nitrogen-regulatory gene, is regulated by NtrC at tandem promoters.

The glnB gene from Bradyrhizobium japonicum, the endosymbiont of soybeans (Glycine max), was isolated and sequenced, and its expression was examined under various culture conditions and in soybean nodules. The B. japonicum glnB gene encodes a 12,237-dalton polypeptide that is highly homologous to the glnB gene products from Klebsiella pneumoniae and Escherichia coli. The gene is located directly upstream from glnA (encoding glutamine synthetase), a linkage not observed in enteric bacteria. The glnB gene from B. japonicum is expressed from tandem promoters, which are differentially regulated in response to the nitrogen status of the medium. Expression from the downstream promoter involves the B. japonicum ntrC gene product (NtrC) in both free-living and symbiotic cells. Thus, glnB, a putative nitrogen-regulatory gene in B. japonicum, is itself Ntr regulated, and NtrC is active in B. japonicum cells in their symbiotic state.

Amino Acid Sequence↗

PubMed1994

The Rhodobacter capsulatus glnB gene is regulated by NtrC at tandem rpoN-independent promoters.

The protein encoded by glnB of Rhodobacter capsulatus is part of a nitrogen-sensing cascade which regulates the expression of nitrogen fixation genes (nif). The expression of glnB was studied by using lacZ fusions, primer extension analysis, and in vitro DNase I footprinting. Our results suggest that glnB is transcribed from two promoters, one of which requires the R. capsulatus ntrC gene but is rpoN independent. Another promoter upstream of glnB is repressed by NtrC; purified R. capsulatus NtrC binds to sites that overlap this distal promoter region.

Bacterial Proteins↗

PubMed1996

Promoters controlling expression of the alternative nitrogenase and the molybdenum uptake system in Rhodobacter capsulatus are activated by NtrC, independent of sigma54, and repressed by molybdenum.

The alternative nitrogenase of Rhodobacter capsulatus is expressed only under conditions of nitrogen and molybdenum depletion. The analysis of anfA-lacZ fusions demonstrated that this dual control occurred at the level of transcription of anfA, which encodes a transcriptional activator specific for the alternative nitrogenase. The anfA promoter was found to be activated under nitrogen-limiting conditions by NtrC in a sigma54-independent manner. In addition, anfA transcription was repressed by traces of molybdenum. This molybdenum-dependent repression of anfA was released in R. capsulatus mutants carrying either lesions in the high-affinity molybdenum uptake system (modABCD) or a double deletion of mopA and mopB, two genes encoding molybdenum-pterin-binding proteins. The expression of the molybdenum transport system itself was shown to be negatively regulated by molybdenum and, unexpectedly, to be also regulated by NtrC. This finding is in line with the presence of two tandemly arranged DNA motifs located in front of the R. capsulatus mopA-modABCD operon, which are homologous to R. capsulatus NtrC binding sites. Mapping of the transcriptional initiation sites of mopA and anfA revealed promoter sequences exhibiting significant homology to each other but no homology to known prokaryotic promoters. In addition, a conserved DNA sequence of dyad symmetry overlapping the transcriptional initiation sites of mopA and anfA was found. Deletions within this element resulted in molybdenum-independent expression of anfA, indicating that this DNA sequence may be the target of MopA/MopB-mediated repression.

Bacterial Proteins↗

PubMed1997

NtrC is required for control of Klebsiella pneumoniae NifL activity.

In response to molecular oxygen and/or fixed nitrogen, the product of the Klebsiella pneumoniae nitrogen fixation L (nifL) gene inhibits NifA-mediated transcriptional activation. Nitrogen regulation of NifL function occurs at two levels: transcription of the nifLA operon is regulated by the general Ntr system, and the activity of NifL is controlled by an unknown mechanism. We have studied the regulation of NifL activity in Escherichia coli and Salmonella typhimurium by monitoring its inhibition of NifA-mediated expression of a K. pneumoniae phi(nifH'-'lacZ) fusion. The activity of the NifL protein transcribed from the tac promoter is regulated well in response to changes of oxygen and/or nitrogen status, indicating that no nif- or K. pneumoniae-specific product is required. Unexpectedly, strains carrying ntrC (glnG) null alleles failed to release NifL inhibition, despite the fact that synthesis of NifL was no longer under Ntr control. Additional evidence indicated that it is indeed the transcriptional activation capacity of NtrC, rather than its repression capacity, that is needed, and hence it is a plausible hypothesis that NtrC activates transcription of a gene(s) whose product(s) in turn functions to relieve NifL inhibition under nitrogen-limiting conditions.

Bacterial Proteins↗

PubMed2003

Global mutational analysis of NtrC-like activators in Myxococcus xanthus: identifying activator mutants defective for motility and fruiting body development.

The multicellular developmental cycle of Myxococcus xanthus requires large-scale changes in gene transcription, and recent findings indicate that NtrC-like activators play a prominent role in regulating these changes. In this study, we made insertions in 28 uncharacterized ntrC-like activator (nla) genes and found that eight of these insertions cause developmental defects. Hence, these results are consistent with the idea that M. xanthus uses a series of different NtrC-like activators during fruiting body development. Four of the eight developmental mutants we identified have motility defects. The nla1, nla19, and nla23 mutants show S-motility defects, while the nla24 mutant shows defects in both S-motility and A-motility. During development, aggregation of the nla1, nla19, and nla23 mutants is delayed slightly and the nla24 mutant shows no signs of aggregation or sporulation. The nla4, nla6, nla18, and nla28 mutants have no appreciable loss in motility, but they fail to aggregate and to sporulate normally. The nla18 mutant belongs to a special class of developmental mutants whose defects can be rescued when they are codeveloped with wild-type cells, suggesting that nla18 fails to produce a cell-cell signal required for development. The three remaining activator mutants, nla4, nla6, and nla28, appear to have complex developmental phenotypes that include deficiencies in cell-cell developmental signals.

Bacterial Proteins↗

PubMed1985

Expression of Rhizobium japonicum nifH and nifDK operons can be activated by the Klebsiella pneumonia nifA protein but not by the product of ntrC.

Rhizobium japonicum nifH'- and nifD'-'lacZ fusions were constructed using the translational fusion vector pMC1403. beta-Galactosidase activities from these fusion plasmids were measured in wild-type, ntrA- and delta(ntrBC) Escherichia coli strains carrying plasmids which overproduced the Klebsiella pneumoniae nifA or ntrC gene products. In contrast to results reported in R. meliloti (ref. in the text) neither nifH nor nifD promoters were activated by the ntrC product. In the presence of nifA gene product, however, beta-galactosidase activity from both nifH and nifD fusion plasmids increased substantially. NifA-mediated activation of these Rhizobium promoters was temperature sensitive and was dependent on the host ntrA product. In order to determine the point at which the fusion transcripts were initiated, RNA was extracted from the wild-type E. coli strain carrying each of the R. japonicum fusion plasmids plus the nifA overproducing plasmid. This RNA was used to perform S1 mapping experiments. NifA-mediated transcription from both R. japonicum promoters, began at the same point as previously determined in soybean root-nodule bacteroids (ref. in the text). The results obtained suggest that there may be differences in the mode of regulation between members of the fast- and slow-growing rhizobia. Also, the results of the S1 mapping experiments indicate that activation of the R. japonicum nitrogenase structural genes may be similar to the activation of nif genes in K. pneumoniae thus raising the possibility that R. japonicum may contain nifA and ntrA-like genes.

Amino Acid Sequence↗

PubMed1992

Phenotype of a Rhizobium leguminosarum ntrC mutant.

A Tn5 insertion mutant, strain CFN2012, of Rhizobium leguminosarum biovar phaseoli devoid of glutamine synthetase II (GSII) activity was analysed. It was shown to contain Tn5 within an 11-kb BamHI DNA fragment, which was isolated (pSM261) from the wild-type strain and, when introduced into strain CFN2012, was shown to complement the absence of GSII activity. The DNA sequence of the corresponding region from the wild-type allele revealed the presence of an ntrC regulatory gene, and restriction analysis indicated that the mutant allele carried the Tn5 insertion within it. Further analysis of strain CFN2012 indicated that this mutant has reduced levels of the PII regulatory protein and that, in contrast to ntrC mutants of other Rhizobiaceae, it grows on nitrate as the sole nitrogen source.

Bacterial Proteins↗

PubMed1998

Evolution of new protein function: recombinational enhancer Fis originated by horizontal gene transfer from the transcriptional regulator NtrC.

New protein function is thought to evolve mostly by gene duplication and divergence. Here we present phylogenetic evidence that the multifunctional protein Fis of the gamma proteobacterial species derived from the COOH-terminal domain of an ancestral alpha proteobacterial NtrC transcriptional regulatory protein. All of the known enterobacterial fis genes are preceded by an open reading frame, named yhdG, that is highly similar to nifR3, a gene that forms an operon with ntrC in several alpha proteobacterial species. Thus, we propose that yhdG and fis were acquired by a lineage ancestral to the gamma proteobacteria in a single horizontal gene transfer event, and later diverged to their present functions.

Amino Acid Sequence↗

PubMed1998

Properties of a mutant form of the prokaryotic enhancer binding protein, NTRC, which hydrolyses ATP in the absence of effectors.

The mutation S170A in the proposed nucleotide binding site of the transcriptional activator protein NTRC abolishes its ability to catalyse open promoter complex formation by the sigma(N)-RNA polymerase holoenzyme. NTRC(S170A) has significant ATPase activity, which, in contrast to the wild-type protein, is unaffected by phosphorylation or binding to enhancer sites on DNA. The mutant protein appears to oligomerise normally on DNA in response to phosphorylation but the ATPase activity is apparently not responsive to changes in oligomerisation state. The defect in transcriptional activation is discussed in relation to mutations in other sigma(N)-dependent activators.

Adenosine Triphosphatases↗

PubMed1983

Promoters regulated by the glnG (ntrC) and nifA gene products share a heptameric consensus sequence in the -15 region.

We have determined the nucleotide sequences of the Klebsiella pneumoniae nifL (regulation of N2 fixation genes) and the Escherichia coli glnA (glutamine synthetase) promoters. We compared these sequences with the published sequences of three other promoters that, like the nifL and glnA promoters, are activated by the general nitrogen regulators glnF (ntrA) and glnG (ntrC). The three promoters are the argTr (arginine transport) and dhuA (histidine transport) promoters of Salmonella typhimurium and the nifH (nitrogenase) promoter of Rhizobium meliloti. All five sequences (with at most one mismatch) contain the heptameric consensus sequence T-T-T-T-G-C-A. In the R. meliloti nifH and K. pneumoniae nifL promoters, in which the transcription initiation sites have been determined, the consensus sequence is situated in the -15 region. We recently reported that the K. pneumoniae nifA product, which activates nif genes, can substitute for the glnG (ntrC) product in activating promoters of several genes involved in nitrogen assimilation, including the nifL, the glnA, and the R. meliloti nifH promoters. It is likely that nifA also activates the S. typhimurium argTr and dhuA promoters. In contrast, the glnG product cannot substitute for the nifA product in the activation of the K. pneumoniae nifH (nitrogenase) promoter. Consistent with this latter observation, and supporting the conclusion that the T-T-T-T-G-C-A sequence is a regulatory site for glnG product activation, the K. pneumoniae nifH promoter (C-C-C-T-G-C-A) has only partial similarity with the T-T-T-T-G-C-A consensus sequence in the -15 region.

Amino Acid Sequence↗

PubMed1985

Products of nitrogen regulatory genes ntrA and ntrC of enteric bacteria activate glnA transcription in vitro: evidence that the ntrA product is a sigma factor.

In enteric bacteria the products of two nitrogen regulatory genes, ntrA and ntrC, activate transcription of glnA, the structural gene encoding glutamine synthetase, both in vivo and in vitro. The ntrC product (gpntrC) is a DNA-binding protein, which binds to five sites in the glnA promoter-regulatory region and appears to activate transcription initiation. Using as an assay the stimulation of glnA transcription in a coupled in vitro transcription-translation system, we have partially purified the ntrA gene product (gpntrA). The following evidence is consistent with the view that gpntrA is a sigma subunit for RNA polymerase: (i) The gpntrA activity copurifies with the sigma 70 holoenzyme (E sigma 70) and core (E) forms of RNA polymerase through several steps but can be separated from them by chromatography on heparin agarose. (ii) After further purification by molecular sieve chromatography, the partially purified gpntrA fraction allows transcription of glnA from the same startpoint used in vivo; transcription is dependent on gpntrC and on added E. The gpntrA fraction does not allow transcription from promoters that we have used as controls, including lacUV5. E sigma 70 has the reverse specificity.

Bacterial Proteins↗

PubMed2003

Evidence for a second interaction between the regulatory amino-terminal and central output domains of the response regulator NtrC (nitrogen regulator I) in Escherichia coli.

Nitrogen limitation in Escherichia coli activates about 100 genes. Their expression requires the response regulator NtrC (also called nitrogen regulator I or NR(I)). Phosphorylation of the amino-terminal domain (NTD) of NtrC activates the neighboring central domain and leads to transcriptional activation from promoters that require sigma(54)-containing RNA polymerase. The NTD has five beta strands alternating with five alpha helices. Phosphorylation of aspartate 54 has been shown to reposition alpha helix 3 to beta strand 5 (the "3445 face") within the NTD. To further study the interactions between the amino-terminal and central domains, we isolated strains with alterations in the NTD that were able to grow on a poor nitrogen source in the absence of phosphorylation by the cognate sensor kinase. We identified strains with alterations located in the 3445 face and alpha helix 5. Both types of alterations stimulated central domain activities. The alpha helix 5 alterations differed from those in the 3445 face. They did not cause a large scale conformational change in the NTD, which is not necessary for transcriptional activation in these mutants. Yeast two-hybrid analysis indicated that substitutions in both alpha helix 5 and the 3445 face diminish the interaction between the NTD and the central domain. Our results suggest that alpha helix 5 of the NTD, in addition to the 3445 face, interacts with the central domain. We present a model of interdomain signal transduction that proposes different functions for alpha helix 5 and the 3445 face.

Bacterial Proteins↗

PubMed1985

Nitrogen fixation specific regulatory genes of Klebsiella pneumoniae and Rhizobium meliloti share homology with the general nitrogen regulatory gene ntrC of K. pneumoniae.

We have determined the complete nucleotide sequences of three functionally related nitrogen assimilation regulatory genes from Klebsiella pneumoniae and Rhizobium meliloti. These genes are: 1) The K. pneumoniae general nitrogen assimilation regulatory gene ntrC (formerly called glnG), 2) the K. pneumoniae nif-specific regulatory gene nifA, and 3) an R. meliloti nif-specific regulatory gene that appears to be functionally analogous to the K. pneumoniae nifA gene. In addition to the DNA sequence data, gel-purified K. pneumoniae nifA protein was used to determine the amino acid composition of the nifA protein. The K. pneumoniae ntrC and nifA genes code for proteins of 52,259 and 53,319 d respectively. The R. meliloti nifA gene codes for a 59,968 d protein. A central region within each polypeptide, consisting of approximately 200 amino acids, is between 52% and 58% conserved among the three proteins. Neither the amino termini nor the carboxy termini show any conserved sequences. Together with data that shows that the three regulatory proteins activate promoters that share a common consensus sequence in the -10 (5'-TTGCA-3') and -23 (5'-CTGG-3') regions, the sequence data presented here suggest a common evolutionary origin for the three regulatory genes.

Amino Acid Sequence↗

PubMed1992

Isolation and characterization of a ntrC mutant of Bradyrhizobium (Parasponia) sp. ANU289.

A mutant of Bradyrhizobium (Parasponia) sp. ANU289 affected in the regulation of nitrogen metabolism was isolated. The mutant, designated ANU293, was unable to induce ammonium transport (Amt), nitrate reductase (NR) or glutamine synthetase II (GSII) activities under conditions that induce these activities in the wild-type. However, glutamine synthetase I (GSI), which is expressed constitutively in the wild-type, was present at normal levels in the mutant. The mutant also retained the ability to fix nitrogen in vitro and in planta, although nodule development on siratro (Macroptilium atropurpureum) was retarded. Southern blot analysis showed that ntrC, the product of which is involved in regulation of nitrogen metabolism, is the site of pSUP1021 insertion in ANU293. These results indicate that the transcriptional activator NtrC is required for the expression of Amt, NR and GSII, but not GSI or nitrogenase in Bradyrhizobium (Parasponia) sp. ANU289.

Ammonia↗