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PubMed2001

A glutamine-amidotransferase-like protein modulates FixT anti-kinase activity in Sinorhizobium meliloti.

BACKGROUND: Nitrogen fixation gene expression in Sinorhizobium meliloti, the alfalfa symbiont, depends on a cascade of regulation that involves both positive and negative control. On top of the cascade, the two-component regulatory system FixLJ is activated under the microoxic conditions of the nodule. In addition, activity of the FixLJ system is inhibited by a specific anti-kinase protein, FixT. The physiological significance of this negative regulation by FixT was so far unknown. RESULTS: We have isolated by random Tn5 mutagenesis a S. meliloti mutant strain that escapes repression by FixT. Complementation test and DNA analysis revealed that inactivation of an asparagine synthetase-like gene was responsible for the phenotype of the mutant. This gene, that was named asnO, encodes a protein homologous to glutamine-dependent asparagine synthetases. The asnO gene did not appear to affect asparagine biosynthesis and may instead serve a regulatory function in S. meliloti. We provide evidence that asnO is active during symbiosis. CONCLUSIONS: Isolation of the asnO mutant argues for the existence of a physiological regulation associated with fixT and makes it unlikely that fixT serves a mere homeostatic function in S. meliloti. Our data suggest that asnO might control activity of the FixT protein, in a way that remains to be elucidated. A proposed role for asnO might be to couple nitrogen fixation gene expression in S. meliloti to the nitrogen needs of the cells.

Anthranilate Synthase↗

PubMed1983

Nitrogenase activity (acetylene reduction) of root-associated, cold-climate azospirillum, enterobacter, Klebsiella, and pseudomonas species during growth on various carbon sources and at various partial pressures of oxygen.

A comprehensive view of the diazotrophic bacterial flora of plants requires that attention be paid to the appropriate carbon and oxygen requirements during isolation of the bacteria. Twenty compounds (monosaccharides, disaccharides, polyols, and organic acids) were therefore examined as carbon and energy sources for nitrogenase activity in semisolid stab cultures at pO(2) values of 0.21, 0.02, and </=0.002 with 12 strains of diazotrophic root-associated bacteria. With the facultatively anaerobic bacteria of the genera Klebsiella and Enterobacter, the best substrate was sucrose, followed by fructose and mannitol, whereas among the organic acids, only malic and fumaric acids supported any activity. With the obligately aerobic bacteria of the genera Azospirillum and Pseudomonas, disaccharides were not utilized for nitrogen fixation, but several organic acids were accepted in addition to monosaccharides and polyols; malate and glucose were the best substrates. The patterns of the carbon sources utilized for nitrogen fixation were coherent within the species, with the exception of one Klebsiella pneumoniae and one Enterobacter agglomerans strain, both isolated from the same individual grass plant, which were unable to utilize lactose. Anaerobic conditions (pO(2) value of </=0.002) were required for maximum nitrogenase activity with the facultatively anaerobic bacteria, with the exception of one strain of E. agglomerans, which required atmospheric oxygen (pO(2) value of 0.21). Also, the obligately aerobic diazotrophs required atmospheric oxygen for maximum nitrogenase activity. The maximum specific nitrogenase activities (expressed as micromoles of C(2)H(4) . milligram of bacterial protein . hour) noted during the exponential growth phase of the bacteria were the following: 2.68 with Azospirillum lipoferum on malate, 2.41 with K. pneumoniae and 1.58 with E. agglomerans on sucrose, and 0.95 with Pseudomonas sp. on malate.

Journal Article↗

PubMed1997

The Sesbania Root Symbionts Sinorhizobium saheli and S. teranga bv. sesbaniae Can Form Stem Nodules on Sesbania rostrata, although They Are Less Adapted to Stem Nodulation than Azorhizobium caulinodans.

Sesbania species can establish symbiotic interactions with rhizobia from two taxonomically distant genera, including the Sesbania rostrata stem-nodulating Azorhizobium sp. and Azorhizobium caulinodans and the newly described Sinorhizobium saheli and Sinorhizobium teranga bv. sesbaniae, isolated from the roots of various Sesbania species. A collection of strains from both groups were analyzed for their symbiotic properties with different Sesbania species. S. saheli and S. teranga bv. sesbaniae strains were found to effectively stem nodulate Sesbania rostrata, showing that stem nodulation is not restricted to Azorhizobium. Sinorhizobia and azorhizobia, however, exhibited clear differences in other aspects of symbiosis. Unlike Azorhizobium, S. teranga bv. sesbaniae and S. saheli did not induce effective stem nodules on plants previously inoculated on the roots, although stem nodulation was arrested at different stages. For Sesbania rostrata root nodulation, Sinorhizobium appeared more sensitive than Azorhizobium to the presence of combined nitrogen. S. saheli and S. teranga bv. sesbaniae were effective symbionts with all Sesbania species tested, while Azorhizobium strains fixed nitrogen only in symbiosis with Sesbania rostrata. In a simple screening test, S. saheli and S. teranga bv. sesbaniae were incapable of asymbiotic nitrogenase activity. Thus, Azorhizobium can easily be distinguished from Sinorhizobium among Sesbania symbionts on the basis of symbiotic and free-living nitrogen fixation. The ability of Azorhizobium to overcome the systemic plant control appears to be a stem adaptation function. This last property, together with its host-specific symbiotic nitrogen fixation, makes Azorhizobium highly specialized for stem nodulation of the aquatic legume Sesbania rostrata.

Journal Article↗

PubMed1982

De Novo Purine Synthesis in Nitrogen-Fixing Nodules of Cowpea (Vigna unguiculata [L.] Walp.) and Soybean (Glycine max [L.] Merr.).

Partially purified, cell-free extracts from nodules of cowpea (Vigna unguiculata L. Walp. cv. Caloona) and soybean (Glycine max L. Merr. cv. Bragg) showed high rates of de novo purine nucleotide and purine base synthesis. Activity increased with rates of nitrogen fixation and ureide export during development of cowpea plants; maximum rates (equivalent to 1.2 micromoles N(2) per hour per gram fresh nodule) being similar to those of maximum nitrogen fixation (1-2 micromoles N(2) per hour per gram fresh nodule). Extracts from actively fixing nodules of a symbiosis not producing ureides, Lupinus albus L. cv. Ultra, showed rates of de novo purine synthesis 0.1% to 0.5% those of cowpea and soybean. Most (70-90%) of the activity was associated with the particulate components of the nodule, but up to 50% was released from this fraction by osmotic shock. The accumulated end products with particulate fractions were inosine monophosphate and aminoimidazole carboxamide ribonucleotide. Further metabolism to purine bases and ureides was restricted to the soluble fraction of the nodule extract. High rates of inosine monophosphate synthesis were supported by glutamine as amide donor, lower rates (10-20%) by ammonia, and negligible rates with asparagine as substrate.

Journal Article↗

PubMed1987

Increased Effectiveness of Competitive Rhizobium Strains upon Inoculation of Cajanus cajan.

A field study was conducted in lysimeters containing N-enriched soil to determine the effects of four competitive rhizobium strains upon yield parameters of pigeon peas (Cajanus cajan). The greatest differences observed were in seed yields; strain P132 effected the highest seed yield (121 +/- 20 g per plant), and the control strain (indigenous rhizobia) effected the lowest yield (43.9 +/- 8 g per plant). With the exception of seeds and pods, the dry matter weights were not different. Although there appeared to be no effect by inoculum strains on the fractional content of N derived from biological nitrogen fixation when the total plant biomass was considered, strains P132 and 401 partitioned more of the N derived from fixation into seeds and leaves than did the other strains. Because the seeds comprised the major portion of plant N, more total N and more N derived from biological nitrogen fixation (about half of total N) were found in plants inoculated with P132, whereas the smallest amount was found in the uninoculated controls. P132 was also the best competitor with respect to indigenous rhizobia and acounted for all of the nodules found on the plants in which it was inoculated.

Journal Article↗

PubMed1981

Physiological Characteristics of Cowpea Rhizobia: Evaluation of Symbiotic Efficiency in Vigna unguiculata.

One fast-growing and three slow-growing strains of Rhizobium (isolated from cowpeas) were evaluated for symbiotic performance on Vigna unguiculata (L.) Walp. cultivar California no. 5 blackeyes. Plants inoculated with slow-growing strains 176A22, 176A30, and 176A32 developed a maximum acetylene reduction activity of 24.6, 27.0, and 32 mumol of ethylene formed per plant per h, respectively, versus 6.4 mumol per plant per h in plants inoculated with the fast-growing strain 176A28. When inoculated with approximately equal proportions of rhizobia, the fast-growing strain 176A28 produced 95% of the nodules when challenged with the slow-growing strain 176A22, but formed only 6% of the nodules when challenged with the slow-growing strain 176A30. Consequently, there was no relation between the growth rate in vitro and the capability of rhizobia to compete for nodule-forming sites. Plants inoculated with strain 176A28 and subjected to drought during the vegetative growth period recovered to the same level of nitrogen fixation and nodulation as those that received adequate irrigation. On the other hand, plants inoculated with strains 176A22, 176A30, and 176A32 failed to achieve the same levels of nodulation and nitrogen fixation under drought as compared with irrigated conditions.

Journal Article↗

PubMed2005

Mutational analysis of the nucleotide-binding domain of the anti-activator NifL.

The NifL regulatory protein controls transcription of nitrogen fixation genes in Azotobacter vinelandii by modulating the activity of the transcriptional activator NifA through direct protein-protein interactions. The ability of NifL to integrate the antagonistic signals of redox and nitrogen status is achieved via the involvement of discrete domains in signalling specific environmental cues. NifL senses the redox status via an FAD co-factor located within the amino-terminal PAS domain and responds to the fixed nitrogen status by interaction with the signal transduction protein GlnK, which binds to the C-terminal GHKL domain of NifL. The GHKL domain binds adenosine nucleotides and is similar to the core catalytic domain of the histidine protein kinases. Binding of ADP to this domain increases the inhibitory activity of NifL and the formation of protein complexes with NifA. This inhibition is antagonised by the binding of 2-oxoglutarate, a key metabolic signal of the carbon status, to the amino-terminal GAF domain of NifA. In this study we have examined the properties of three mutations within conserved residues in the GHKL domain of NifL that impair signal transduction. All three mutations decrease the affinity of NifL for ADP significantly, but the mutant proteins exhibit discrete properties. The N419D mutation prevents inhibition of NifA activity by NifL both in vivo and in vitro. In contrast, the G455A and G480A mutations eliminate the redox response, but the mutant proteins retain some sensitivity to the fixed nitrogen status and the ability to interact with the GlnK signal transduction protein. Our data suggest that the absence of the redox switch in the G455A and G480A mutants is a consequence of their inability to override the allosteric effect of 2-oxoglutarate on NifA activity. Overall, these results demonstrate that the binding of adenosine nucleotides to the GHKL domain of NifL plays an important role in counteracting the response of NifA to 2-oxoglutarate, under conditions that are inappropriate for nitrogen fixation.

Adenosine Diphosphate↗

PubMed2004

Symbiotic autoregulation of nifA expression in Rhizobium leguminosarum bv. viciae.

NifA is the general transcriptional activator of nitrogen fixation genes in diazotrophic bacteria. In Rhizobium leguminosarum bv. viciae UPM791, the nifA gene is part of a gene cluster (orf71 orf79 fixW orf5 fixABCX nifAB) separated by 896 bp from an upstream and divergent truncated duplication of nifH (DeltanifH). Symbiotic expression analysis of genomic nifA::lacZ fusions revealed that in strain UPM791 nifA is expressed mainly from a sigma54-dependent promoter (P(nifA1)) located upstream of orf71. This promoter contains canonical NifA upstream activating sequences located 91 bp from the transcription initiation site. The transcript initiated in P(nifA1) spans 5.1 kb and includes nifA and nifB genes. NifA from Klebsiella pneumoniae was able to activate transcription from P(nifA1) in a heterologous Escherichia coli system. In R. leguminosarum, the P(nifA1) promoter is essential for effective nitrogen fixation in symbiosis with peas. In its absence, partially efficient nitrogen-fixing nodules were produced, and the corresponding bacteroids exhibited only low levels of nifA gene expression. The basal level of nifA expression resulted from a promoter activity originating upstream of the fixX-nifA intergenic region and probably from an incomplete duplication of P(nifA1) located immediately upstream of fixA.

Bacterial Proteins↗

PubMed2005

Role of oxyR from Sinorhizobium meliloti in regulating the expression of catalases.

The process of symbiotic nitrogen fixation results in the generation of reactive oxygen species such as the superoxide anion (O2-) and hydrogen peroxide (H2O2). The response of rhizobia to these toxic oxygen species is an important factor in nodulation and nitrogen fixation. In Sinorhizobium meliloti, one oxyR homologue and three catalase genes, katA, katB, and katC were detected by sequence analysis. This oxyR gene is located next to and divergently from katA on the chromosome. To investigate the possible roles of oxyR in regulating the expression of catalases at the transcriptional level in S. meliloti, an insertion mutant of this gene was constructed. The mutant was more sensitive and less adaptive to H2O2 than the wild type strain, and total catalase/peroxidase activity was reduced approximately fourfold with the OxyR mutation relative to controls. The activities of KatA and KatB and the expression of katA::lacZ and katB::lacZ promoter fusions were increased in the mutant strain compared with the parental strain grown in the absence of H2O2, indicating that katA and katB are repressed by OxyR. However, when exposed to H2O2, katA expression was also increased in both S. meliloti and Escherichia coli. When exposed to H2O2, OxyR is converted from a reduced to an oxidized form in E. coli. We concluded that the reduced form of OxyR functions as a repressor of katA and katB expression. Thus, in the presence of H2O2, reduced OxyR is converted to the oxidized form of OxyR that then results in increased katA expression. We further showed that oxyR expression is autoregulated via negative feedback.

Catalase↗

PubMed2002

A mass spectrometry method for measuring 15N incorporation into pheophytin.

The 15N content of pheophytin, the magnesium-free derivative of chlorophyll, can be measured with great accuracy and precision using positive-ion atmospheric pressure ionization electrospray mass spectroscopy following a simple solvent extraction of small amounts of plant tissue. The molecular weight of pheophytin prepared from Chlamydomonas reinhardtii grown in different ratios of 14N/15N showed linear regression with the isotopic input, with a precision of 0.5-1%. Using an isotope dilution strategy, we have shown that nitrogen fixation can contribute substantial 14N to pheophytin isolated from Medicago truncatula plants grown in symbiosis with Sinorhizobium meliloti. The assay is sensitive, precise, rapid, simple, and robust. These features suggest that it could become an important tool for measuring the contribution of symbiotic and associative nitrogen fixation to plant metabolism.

Animals↗

PubMed1983

Nitrogenase structural genes are unlinked in the nonlegume symbiont Parasponia rhizobium.

Several Rhizobium strains are capable of biological nitrogen fixation in symbiotic association with nonleguminous plants. The gene encoding the iron-protein component of nitrogenase (nifH) from one such strain, Parasponia Rhizobium sp. ANU289, has been isolated and completely sequenced. Unlike previously studied nitrogen-fixing prokaryotes, the Fe-protein subunit is encoded on a separate operon from other components of the nitrogenase enzyme complex. Comparative analysis of Fe-protein amino acid sequences indicates that the symbiotic nitrogen fixation phenotype in Rhizobium may have arisen on at least two separate occasions during its evolution.

Amino Acid Sequence↗

PubMed1989

Identification and sequence analysis of the Rhizobium meliloti dctA gene encoding the C4-dicarboxylate carrier.

Transposon Tn5-induced C4-dicarboxylate transport mutants of Rhizobium meliloti 2011 which could be complemented by cosmid pRmSC121 were subdivided into two classes. Class I mutants (RMS37 and RMS938) were defective in symbiotic C4-dicarboxylate transport and in nitrogen fixation. They were mutated in the structural gene dctA, which codes for the C4-dicarboxylate carrier. Class II mutants (RMS11, RMS16, RMS17, RMS24, and RMS31) expressed reduced activity in symbiotic C4-dicarboxylate transport and in nitrogen fixation. These mutants were mutated in regulatory dct genes which do not play an essential role in the symbiotic state. Thin sections of alfalfa nodules induced by the wild type and class I and class II mutants were analyzed by light microscopy. Class mutants induced typical Fix- nodules, showing a large senescent zone, whereas nodules induced by class II mutants only differed in an enhanced content of starch granules compared with wild-type nodules. Class I mutants could be complemented by a 2.1-kilobase SalI-HindIII subfragment of cosmid pRmSC121. DNA sequencing of this fragment resulted in the identification of an open reading frame, which was designated dctA because Tn5 insertion sites of the class I mutants mapped within this coding region. The dctA gene was preceded by a nif consensus promoter and an upstream NifA-binding element. Upstream of the dctA promoter, the 5' end of the R. meliloti dctB gene could be localized. The amino acid sequence of the N-terminal part of the R. meliloti DctB protein shared 49% homology with the corresponding part of the R. leguminosarum DctB protein. The DctA protein consisted of 441 or 453 amino acids due to two possible ATG start codons, with calculated molecular masses of 46.1 and 47.6 kilodaltons, respectively. The hydrophobicity plot suggests that DctA is a membrane protein with several membrane passages. The amino acid sequences of the R. meliloti and the R. leguminosarum DctA proteins were highly conserved (82%).

Amino Acid Sequence↗

PubMed2006

Transcriptional specificity of RpoN1 and RpoN2 involves differential recognition of the promoter sequences and specific interaction with the cognate activator proteins.

The four RpoN factors of Rhodobacter sphaeroides are functionally specialized. In this bacterium, RpoN1 and RpoN2 are specifically required for the transcription of the nitrogen fixation and flagellar genes, respectively. Analysis of the promoter sequences recognized by each of these RpoN proteins revealed some significant differences. To investigate the functional relevance of these differences, the flagellar promoter fliOp was sequentially mutagenized to resemble the nitrogen fixation promoter nifUp. Our results indicate that the promoter sequences recognized by these sigma factors have diverged enough so that particular positions of the promoter sequence are differentially recognized. In this regard, we demonstrate that the identity of the -11-position is critical for promoter discrimination by RpoN1 and RpoN2. Accordingly, purified RpoN proteins with a deletion of Region I, which has been involved in the recognition of the -11-position, did not show differential binding of fliOp and nifUp promoters. Substitution of the flagellar enhancer region located upstream fliOp by the enhancer region of nifUp allowed us to demonstrate that RpoN1 and RpoN2 interact specifically with their respective activator protein. In conclusion, two different molecular mechanisms underlie the transcriptional specialization of these sigma factors.

Base Sequence↗

PubMed1983

Bacterial activity associated with the decomposition of woody substrates in a stream sediment.

Ground bark and heartwood from Alnus rubra and Pseudotsuga menziesii were added to a muddy sediment from a small Oregon stream and incubated in situ. Carbon dioxide and methane production rates were increased by all amendments, the biggest increase being shown with A. rubra wood. Except for sediment amended with A. rubra wood, nitrogen fixation rates from all treatments (including the control) were approximately 0.1 nmol/g per h throughout the 6-month study period. Contrary to expectations, neither bark had a noticeable adverse effect on microbial activity, but the A. rubra wood promoted nitrogen fixation. These results help to explain the faster rate of decomposition of A. rubra wood in water compared with that of P. menziesii described in the literature. The uptake kinetics of glucose (V(max)) did not follow the same pattern as gas evolution.

Journal Article↗

PubMed2004

In vitro uridylylation of the Azospirillum brasilense N-signal transducing GlnZ protein.

Azospirillum brasilense is a diazotroph which associates with important agricultural crops. The nitrogen fixation process in this organism is highly regulated by ammonium and oxygen, and involves several proteins including the two PII-like proteins, GlnB and GlnZ. Although these proteins are structurally very similar, they play different roles in the control of nitrogen fixation. In this work, we describe the expression, purification, and uridylylation of the GlnZ protein of A. brasilense strain FP2. The amplified glnZ gene was sub-cloned and expressed as a His-tagged fusion protein. After purification, we obtained 30-40 mg of purified GlnZ per liter of culture. This protein was purified to 99% purity and assayed for in vitro uridylylation using a partially purified Escherichia coli GlnD as a source of uridylylyl-transferase activity. Analyses of the uridylylation reactions in non-denaturing and denaturing polyacrylamide gel electrophoresis showed that up to 74% of GlnZ monomers were modified after 30 min reaction. This covalent modification is strictly dependent on ATP and 2-ketoglutarate, while glutamine acts as an inhibitor and promotes deuridylylation.

Adenosine Triphosphate↗

PubMed1992

Crystallographic structure of the nitrogenase iron protein from Azotobacter vinelandii.

The nitrogenase enzyme system catalyzes the ATP (adenosine triphosphate)-dependent reduction of dinitrogen to ammonia during the process of nitrogen fixation. Nitrogenase consists of two proteins: the iron (Fe)-protein, which couples hydrolysis of ATP to electron transfer, and the molybdenum-iron (MoFe)-protein, which contains the dinitrogen binding site. In order to address the role of ATP in nitrogen fixation, the crystal structure of the nitrogenase Fe-protein from Azotobacter vinelandii has been determined at 2.9 angstrom (A) resolution. Fe-protein is a dimer of two identical subunits that coordinate a single 4Fe:4S cluster. Each subunit folds as a single alpha/beta type domain, which together symmetrically ligate the surface exposed 4Fe:4S cluster through two cysteines from each subunit. A single bound ADP (adenosine diphosphate) molecule is located in the interface region between the two subunits. Because the phosphate groups of this nucleotide are approximately 20 A from the 4Fe:4S cluster, it is unlikely that ATP hydrolysis and electron transfer are directly coupled. Instead, it appears that interactions between the nucleotide and cluster sites must be indirectly coupled by allosteric changes occurring at the subunit interface. The coupling between protein conformation and nucleotide hydrolysis in Fe-protein exhibits general similarities to the H-Ras p21 and recA proteins that have been recently characterized structurally. The Fe-protein structure may be relevant to the functioning of other biochemical energy-transducing systems containing two nucleotide-binding sites, including membrane transport proteins.

Adenosine Diphosphate↗

PubMed2000

Role of hsfA gene on host-specificity by Bradyrhizobium japonicum in a broad range of tropical legumes.

Bradyrhizobium japonicum mutant strain NAD163, containing a 30-kb deletion mutant encompassing the hsfA gene, was inoculated onto a broad range of legume species to test host-specificity. Most legume species formed ineffective nodules except Vigna angularis var. Chibopat and Glycine max var. Pureunkong. A hsfA insertion mutant, BjjC211, gave similar results to strain NAD163, implying that many legume species require HsfA for host-specific nitrogen fixation. To determine whether other genes in the deleted region of NAD163 are also necessary, the hsfA gene was conjugally transferred into the NAD163 mutant. The transconjugant formed effective nodules on the host legume plants, which earlier had formed ineffective nodules with mutant NAD163. Thus, we conclude that the hsfA gene in the 30-kb region is the only factor responsible for host-specific nitrogen fixation in legume plants.

Bacterial Proteins↗

PubMed1995

Programmed DNA rearrangement of a cyanobacterial hupL gene in heterocysts.

Programmed DNA rearrangements that occur during cellular differentiation are uncommon and have been described in only two prokaryotic organisms. Here, we identify the developmentally regulated rearrangement of a hydrogenase gene in heterocysts of the cyanobacterium Anabaena sp. strain PCC 7120. Heterocysts are terminally differentiated cells specialized for nitrogen fixation. Late during heterocyst differentiation, a 10.5-kb DNA element is excised from within the hupL gene by site-specific recombination between 16-bp direct repeats that flank the element. The predicted HupL polypeptide is homologous to the large subunit of [NiFe] uptake hydrogenases. hupL is expressed similarly to the nitrogen-fixation genes; hupL message was detected only during the late stages of heterocyst development. An open reading frame, named xisC, identified near one end of the hupL DNA element is presumed to encode the element's site-specific recombinase. The predicted XisC polypeptide is homologous with the Anabaena sp. strain PCC 7120 site-specific recombinase XisA. Neither XisC nor XisA shows sequence similarity to other proteins, suggesting that they represent a different class of site-specific recombinase.

Amino Acid Sequence↗