UNIPROT:P51532 - FACTA Search

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Query: UNIPROT:P51532 (transcriptional activator)
6,546 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

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.
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PMID:Enhanced nitrogen fixation in a rhizobium etli ntrC mutant that overproduces the bradyrhizobium japonicum symbiotic terminal oxidase cbb3 1022 93

In diazotrophic organisms, nitrogenase synthesis and activity are tightly regulated. Two genes, nifL and nifA, are implicated as playing a major role in this regulation. NifA is a transcriptional activator, and its activity is inhibited by NifL in response to availability of excess fixed nitrogen and high O(2) tension. It was postulated that NifL binds to NifA to inhibit NifA-mediated transcriptional activation of nif genes. Mutational analysis combined with transcriptional activation studies clearly is in agreement with the proposal that NifL interacts with NifA. However, several attempts to identify NifA-NifL interactions by using methods such as coimmunoprecipitations and chemical cross-linking experiments failed to detect direct interactions between these proteins. Here we have taken a genetic approach, the use of a yeast two-hybrid protein-protein interaction assay system, to investigate NifL interaction with NifA. A DNA fragment corresponding to the kinase-like domain of nifL was PCR amplified and was used to generate translation fusions with the DNA binding domain and the DNA activation domain of the yeast transcriptional activator GAL4 in yeast two-hybrid vectors. Similarly, a DNA fragment corresponding to the catalytic domain of nifA was PCR amplified and used to generate translation fusions with the DNA-binding domain and the DNA-activation domain of GAL4 in yeast two-hybrid vectors. After introducing appropriate plasmid combinations in yeast cells, the existance of direct interaction between NifA and NifL was analyzed with the MATCHMAKER yeast two-hybrid system by testing for the expression of lacZ and his3 genes. These analyses showed that the kinase-like domain of NifL directly interacts with the catalytic domain of NifA.
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PMID:Genetic analysis of nif regulatory genes by utilizing the yeast two-hybrid system detected formation of a NifL-NifA complex that is implicated in regulated expression of nif genes. 1051 47

Purple photosynthetic bacteria are capable of generating cellular energy from several sources, including photosynthesis, respiration, and H(2) oxidation. Under nutrient-limiting conditions, cellular energy can be used to assimilate carbon and nitrogen. This study provides the first evidence of a molecular link for the coregulation of nitrogenase and hydrogenase biosynthesis in an anoxygenic photosynthetic bacterium. We demonstrated that molybdenum nitrogenase biosynthesis is under the control of the RegB-RegA two-component regulatory system in Rhodobacter capsulatus. Footprint analyses and in vivo transcription studies showed that RegA indirectly activates nitrogenase synthesis by binding to and activating the expression of nifA2, which encodes one of the two functional copies of the nif-specific transcriptional activator, NifA. Expression of nifA2 but not nifA1 is reduced in the reg mutants up to eightfold under derepressing conditions and is also reduced under repressing conditions. Thus, although NtrC is absolutely required for nifA2 expression, RegA acts as a coactivator of nifA2. We also demonstrated that in reg mutants, [NiFe]hydrogenase synthesis and activity are increased up to sixfold. RegA binds to the promoter of the hydrogenase gene operon and therefore directly represses its expression. Thus, the RegB-RegA system controls such diverse processes as energy-generating photosynthesis and H(2) oxidation, as well as the energy-demanding processes of N(2) fixation and CO(2) assimilation.
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PMID:Expression of uptake hydrogenase and molybdenum nitrogenase in Rhodobacter capsulatus is coregulated by the RegB-RegA two-component regulatory system. 1078 52

A hybrid promoter consisting of the in tandem fusion of the Tn5 nptII and the Klebsiella pneumoniae nifH promoters was constructed to study the functionality of the nif genes transcriptional activator NifA from Bradyrhizobium japonicum in two different host bacteria. beta-galactosidase experiments in E. coli revealed that the hybrid nptII-nifH promoter can behave as a constitutive or a NifA-inducible promoter depending on the aeration conditions. Expression of the B. japonicum NifA from the hybrid nptII-nifH promoter (plasmid pBPF204) induced "in trans" lacZ transcription from the Azotobacter chroococcum nifH promoter in E. coli and A. diazotrophicus cells grown at low pO2. Similarly, the plasmid pBPF204 increased nitrogenase activity in A. diazotrophicus cells grown under microaerobic conditions. Based on these results, we suggest that the B. japonicum NifA could function as an efficient O2-sensitive transcriptional activator of nif genes in genetically distant diazotrophic bacteria.
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PMID:Functional Bradyrhizobium japonicum NifA expression under a hybrid nptII-nifH promoter in E. coli and Acetobacter diazotrophicus SRT4. 1093 42

Azospirillum represents the best characterized genus of plant growth-promoting rhizobacteria. Other free-living diazotrophs repeatedly detected in association with plant roots, include Acetobacter diazotrophicus, Herbaspirillum seropedicae, Azoarcus spp. and Azotobacter. Four aspects of the Azospirillum-plant root interaction are highlighted: natural habitat, plant root interaction, nitrogen fixation and biosynthesis of plant growth hormones. Each of these aspects is dealt with in a comparative way. Azospirilla are predominantly surface-colonizing bacteria, whereas A. diazotrophicus, H. seropedicae and Azoarcus sp. are endophytic diazotrophs. The attachment of Azospirillum cells to plant roots occurs in two steps. The polar flagellum, of which the flagellin was shown to be a glycoprotein, mediates the adsorption step. An as yet unidentified surface polysaccharide is believed to be essential in the subsequent anchoring phase. In Azoarcus sp. the attachment process is mediated by type IV pili. Nitrogen fixation structural genes (nif) are highly conserved among all nitrogen-fixing bacteria, and in all diazotrophic species of the class of proteobacteria examined, the transcriptional activator NifA is required for expression of other nif genes in response to two major environmental signals (oxygen and fixed N). However, the mechanisms involved in this control can vary in different organisms. In Azospirillum brasilense and H. seropedicae (alpha- and beta-subgroup, respectively), NifA is inactive in conditions of excess nitrogen. Activation of NifA upon removal of fixed N seems to involve, either directly or indirectly, the signal transduction protein P(II). The presence of four conserved cysteine residues in the NifA protein might be an indication that NifA is directly sensitive to oxygen. In Azotobacter vinelandii (gamma-subgroup) nifA is cotranscribed with a second gene nifL. The nifL gene product inactivates NifA in response to high oxygen tension and cellular nitrogen-status. NifL was found to be a redox-sensitive flavoprotein. The relief of NifL inhibition on NifA activity, in response to N-limitation, is suggested to involve a P(II)-like protein. Moreover, nitrogenase activity is regulated according to the intracellular nitrogen and O(2) level. In A. brasilense and Azospirillum lipoferum posttranslational control of nitrogenase, in response to ammonium and anaerobiosis, involves ADP-ribosylation of the nitrogenase iron protein, mediated by the enzymes DraT and DraG. At least three pathways for indole-3-acetic acid (IAA) biosynthesis in A. brasilense exist: two Trp-dependent (the indole-3-pyruvic acid and presumably the indole-3-acetamide pathway) and one Trp-independent pathway. The occurrence of an IAA biosynthetic pathway not using Trp (tryptophan) as precursor is highly unusual in bacteria. Nevertheless, the indole-3-pyruvate decarboxylase encoding ipdC gene is crucial in the overall IAA biosynthesis in Azospirillum. A number of genes essential for Trp production have been isolated in A. brasilense, including trpE(G) which codes for anthranilate synthase, the key enzyme in Trp biosynthesis. The relevance of each of these four aspects for plant growth promotion by Azospirillum is discussed.
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PMID:Azospirillum, a free-living nitrogen-fixing bacterium closely associated with grasses: genetic, biochemical and ecological aspects. 1097 48

Eight genes have been identified that function in the regulation, biosynthesis, and transport of rhizobactin 1021, a hydroxamate siderophore produced under iron stress by Sinorhizobium meliloti. The genes were sequenced, and transposon insertion mutants were constructed for phenotypic analysis. Six of the genes, named rhbABCDEF, function in the biosynthesis of the siderophore and were shown to constitute an operon that is repressed under iron-replete conditions. Another gene in the cluster, named rhtA, encodes the outer membrane receptor protein for rhizobactin 1021. It was shown to be regulated by iron and to encode a product having 61% similarity to IutA, the outer membrane receptor for aerobactin. Transcription of both the rhbABCDEF operon and the rhtA gene was found to be positively regulated by the product of the eighth gene in the cluster, named rhrA, which has characteristics of an AraC-type transcriptional activator. The six genes in the rhbABCDEF operon have interesting gene junctions with short base overlaps existing between the genes. Similarities between the protein products of the biosynthesis genes and other proteins suggest that rhizobactin 1021 is synthesized by the formation of a novel siderophore precursor, 1,3-diaminopropane, which is then modified and attached to citrate in steps resembling those of the aerobactin biosynthetic pathway. The cluster of genes is located on the pSyma megaplasmid of S. meliloti 2011. Reverse transcription-PCR with RNA isolated from mature alfalfa nodules yielded no products for rhbF or rhtA at a time when the nifH gene was strongly expressed, indicating that siderophore biosynthesis and transport genes are not strongly expressed when nitrogenase is being formed in root nodules. Mutants having transposon insertions in the biosynthesis or transport genes induced effective nitrogen-fixing nodules on alfalfa plants.
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PMID:Genetic organization of the region encoding regulation, biosynthesis, and transport of rhizobactin 1021, a siderophore produced by Sinorhizobium meliloti. 1127 18

The enzymatic reduction of molecular nitrogen to ammonia requires high amounts of energy, and the presence of oxygen causes the catalyzing nitrogenase complex to be irreversible inactivated. Thus nitrogen-fixing microorganisms tightly control both the synthesis and activity of nitrogenase to avoid the unnecessary consumption of energy. In the free-living diazotrophs Klebsiella pneumoniae and Azotobacter vinelandii, products of the nitrogen fixation nifLA operon regulate transcription of the other nifoperons. NifA activates transcription of nif genes by the alternative form of RNA-polymerase, sigma54-holoenzyme; NifL modulates the activity of the transcriptional activator NifA in response to the presence of combined nitrogen and molecular oxygen. The translationally-coupled synthesis of the two regulatory proteins, in addition to evidence from studies of NifL/NifA complex formation, imply that the inhibition of NifA activity by NifL occurs via direct protein-protein interaction in vivo. The inhibitory function of the negative regulator NifL appears to lie in the C-terminal domain, whereas the N-terminal domain binds FAD as a redox-sensitive cofactor, which is required for signal transduction of the internal oxygen status. Recently it was shown, that NifL acts as a redox-sensitive regulatory protein, which modulates NifA activity in response to the redox-state of its FAD cofactor, and allows NifA activity only in the absence of oxygen. In K. pneumoniae, the primary oxygen sensor appears to be Fnr (fumarate nitrate reduction regulator), which is presumed to transduce the signal of anaerobiosis towards NifL by activating the transcription of gene(s) whose product(s) function to relieve NifL inhibition through reduction of the FAD cofactor. In contrast, the reduction of A. vinelandii-NifL appears to occur unspecifically in response to the availability of reducing equivalents in the cell. Nitrogen status of the cells is transduced towards the NifL/NifA regulatory system by the GlnK protein, a paralogue PII-protein, which appears to interact with the NifL/NifA regulatory system via direct protein-protein interaction. It is not currently known whether GlnK interacts with NifL alone or affects the NifL/NifA-complex; moreover the effects appear to be the opposite in K. pneumoniae and A. vinelandii. In addition to these environmental signals, adenine nucleotides also affect the inhibitory function of NifL; in the presence of ATP or ADP the inhibitory effect on NifA activity in vitro is increased. The NifL proteins from the two organisms differ, however, in that stimulation of K. pneumoniae-NifL occurs only when synthesized under nitrogen excess, and is correlated with the ability to hydrolyze ATP. In general, transduction of environmental signals to the nif regulatory system appears to involve a conformational change of NifL or the NifL/NifA complex. However, experimental data suggest that K. pneumoniae and A. vinelandii employ significantly different species-specific mechanisms of signal transduction.
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PMID:Regulation of nitrogen fixation in Klebsiella pneumoniae and Azotobacter vinelandii: NifL, transducing two environmental signals to the nif transcriptional activator NifA. 1193 53

NifA, the transcriptional activator of nitrogenase (nif) genes, has up to now been described to be regulated in its activity via the sensor NifL only for members of the gamma-subgroup of the PROTEOBACTERIA: This paper reports a functionally similar NifL-like protein outside this group in Azoarcus sp. strain BH72, a diazotrophic grass endophyte belonging to the beta-subgroup of the PROTEOBACTERIA: Its structural genes for nitrogenase (nifHDK) are regulated in response to combined nitrogen and O(2) and expressed endophytically inside rice roots. In order to characterize nitrogen-regulatory genes, an Azoarcus sp. BH72 genomic library was used to select cosmids that complemented a nifA mutation in Azotobacter vinelandii. Sequence analysis of the 3.4 kb genomic region complementing nifA showed two ORFs with sequence identities of 44% to NifL and 61% to NifA of Azotobacter vinelandii. According to Northern blot and reverse transcriptase PCR analysis, the nifLA transcript was more abundant at low combined nitrogen and O(2) levels, results which were corroborated by GUS (beta-glucuronidase) assays using a transcriptional nifL::gusA fusion. N(2) fixation was abolished in a NifLA(-) and a NifA(-) mutant, wild-type fixation being restored by nifLA in trans. The NifLA(-) mutant also failed to activate nifH::gus expression, indicating that NifA is the obligate transcriptional activator for nifHDK. A nifL mutant was diazotrophic and did not show repression of nifH::gusA by ammonium or O(2), suggesting that NifL of Azoarcus sp. strain BH72 has a similar role in inactivating NifA in response to O(2) and combined nitrogen as NifL in bacteria of the gamma-PROTEOBACTERIA:
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PMID:Identification of a NifL-like protein in a diazotroph of the beta-subgroup of the Proteobacteria, Azoarcus sp. strain BH72. 1236 54

The expression of genes required for the synthesis of molybdenum nitrogenase in Azotobacter vinelandii is controlled by the NifL-NifA transcriptional regulatory complex in response to nitrogen, carbon, and redox status. Activation of nif gene expression by the transcriptional activator NifA is inhibited by direct protein-protein interaction with NifL under conditions unfavorable for nitrogen fixation. We have recently shown that the NifL-NifA system responds directly to physiological concentrations of 2-oxoglutarate, resulting in relief of NifA activity from inhibition by NifL under conditions when fixed nitrogen is limiting. The inhibitory activity of NifL is restored under conditions of excess combined nitrogen through the binding of the signal transduction protein Av GlnK to the carboxyl-terminal domain of NifL. The amino-terminal domain of NifA comprises a GAF domain implicated in the regulatory response to NifL. A truncated form of NifA lacking this domain is not responsive to 2-oxoglutarate in the presence of NifL, suggesting that the GAF domain is required for the response to this ligand. Using isothermal titration calorimetry, we demonstrate stoichiometric binding of 2-oxoglutarate to both the isolated GAF domain and the full-length A. vinelandii NifA protein with a dissociation constant of approximately 60 microm. Limited proteolysis experiments indicate that the binding of 2-oxoglutarate increases the susceptibility of the GAF domain to trypsin digestion and also prevents NifL from protecting these cleavage sites. However, protection by NifL is restored when the non-modified (non-uridylylated) form of Av GlnK is also present. Our results suggest that the binding of 2-oxoglutarate to the GAF domain of NifA may induce a conformational change that prevents inhibition by NifL under conditions when fixed nitrogen is limiting.
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PMID:The amino-terminal GAF domain of Azotobacter vinelandii NifA binds 2-oxoglutarate to resist inhibition by NifL under nitrogen-limiting conditions. 1275 52

The phototrophic purple bacterium Rhodobacter capsulatus encodes two similar but functionally not identical molybdenum-dependent regulator proteins (MopA and MopB), which are known to replace each other in repression of the modABC genes (coding for an ABC-type high-affinity Mo transport system) and anfA (coding for the transcriptional activator of Fe-nitrogenase genes). We identified further Mo-regulated (mor) genes coding for a putative ABC-type transport system of unknown function (MorABC) and a putative Mo-binding protein (Mop). The genes coding for MopA and the ModABC transporter form part of a single transcriptional unit, mopA-modABCD, as shown by reverse transcriptase PCR. Immediately upstream of mopA and transcribed in the opposite direction is mopB. The genes coding for the putative MorABC transporter belong to two divergently transcribed operons, morAB and morC. Expression studies based on lacZ reporter gene fusions in mutant strains defective for either MopA, MopB, or both revealed that the regulators substitute for each other in Mo-dependent repression of morAB and morC. Specific Mo-dependent activation of the mop gene by MopA, but not MopB, was found to control the putative Mo-binding protein. Both MopA and MopB are thought to bind to conserved DNA sequences with dyad symmetry in the promoter regions of all target genes. The positions of these so-called Mo boxes relative to the transcription start sites (as determined by primer extension analyses) differed between Mo-repressed genes and the Mo-activated mop gene. DNA mobility shift assays showed that MopA and MopB require molybdenum to bind to their target sites with high affinity.
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PMID:Overlapping and specialized functions of the molybdenum-dependent regulators MopA and MopB in Rhodobacter capsulatus. 1702 78

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