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PubMed1992

Functional analysis of the cysteine motifs in the ferredoxin-like protein FdxN of Rhizobium meliloti involved in symbiotic nitrogen fixation.

The Rhizobium meliloti fdxN gene, which is part of the nifA-nifB-fdxN operon, is absolutely required for symbiotic nitrogen fixation. The deduced sequence of the FdxN protein is characterized by two cysteine motifs typical of bacterial-type ferredoxins. The Fix-phenotype of an R. meliloti fdxN::[Tc] mutant could be rescued by the R. leguminosarum fdxN gene, whereas no complementation was observed with nif-associated genes encoding ferredoxins from Bradyrhizobium japonicum, Azotobacter vinelandii, A. chroococcum and Rhodobacter capsulatus. In addition to these heterologous genes, several R. meliloti fdxN mutant genes constructed by site-directed mutagenesis were analyzed. Not only a cysteine residue within the second cysteine motif (position 42), which is known to coordinate the Fe-S cluster in homologous proteins, but also a cysteine located down-stream of this motif (position 61), was found to be essential for the activity of the R. meliloti FdxN protein. Changing the amino acid residue proline in position 56 into methionine resulted in a FdxN mutant protein with decreased activity, whereas changes in positions 35 (Asp35Glu) and 45 (Gly45Glu) had no significant effect on the function of the FdxN mutant proteins. In contrast to bacterial-type ferredoxins, which contain two identical cysteine motifs of the form C-X2-C-X2-C-X3-C, nif-associated ferredoxins, including R. meliloti FdxN, are characterized by two different cysteine motifs. Six "additional" amino acids separate the second (Cys42) and the third cysteine (Cys51) in the C-terminal motif (C-X2-C-X8-C-X3-C).(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗

PubMed2004

Possible roles of nitrogen fixation and mineral uptake induced by rhizobacterial inoculation on salt tolerance of maize.

Pot experiments were conducted to evaluate the possible roles of nitrogen fixation and/or enhanced mineral uptake by Azospirillum lipoferum and Bacillus polymexa inoculation in improving salt tolerance of maize plants. Plants were inoculated and grown under salt stress (osmotic potential: -0.3, -0.6, -0.9 and -1.2 Mpa). Both microorganisms were able to fix nitrogen up to -0.9 Mpa salinity level accompanied with increased total N-yield compared with the control plants. In order to investigate the role of bacterial inoculation on enhanced mineral uptake, the growth and some physiological parameters of inoculated plants were compared with plants fertilized by K and P foliar application. Plant inoculation with the N2-fixers or plant spraying with KH2PO4 resulted in an increase in fresh and dry matter as well as water content of plants. Treated plants exhibited changed plant mineral content which was associated with increased Mg/K and decreased P/K, Ca/K and Na/K ratios. This was accompanied by accumulation of soluble sugars, amino acids in shoots and roots of plants resulting in a concomitant increase in the osmotic potential of the cell sap as a possible mechanism of adaptation to salinity.

Azospirillum lipoferum↗

PubMed2002

Regulation of nitrogen fixation in Klebsiella pneumoniae and Azotobacter vinelandii: NifL, transducing two environmental signals to the nif transcriptional activator NifA.

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.

Azotobacter vinelandii↗

PubMed1988

Nucleotide sequence of a 24,206-base-pair DNA fragment carrying the entire nitrogen fixation gene cluster of Klebsiella pneumoniae.

The complete nucleotide sequence (24,206 base-pairs) of the Klebsiella pneumoniae gene region for nitrogen fixation (nif) is presented. Coding regions corresponding to the 19 known nif genes (including nifW and nifZ) could be identified. An additional open reading frame of 216 base-pairs, called nifT, was detected between nifK and nifY. Search for transcriptional signal structures revealed some unusual features: (1) several possible NifA-binding motifs are present in the intergenic regions between nifJ and nifH as well as between nifX and nifU; (2) a perfect NifA-binding motif, preceding the nifENX promoter, is located within an inverted repeat structure; (3) structures resembling the consensus nif promoter are found within the coding regions of nifW and nifZ and, together with a NifA-binding motif, in nifN. Typical rho-independent termination structures were detected only downstream from the nifHDKTY and the nifBQ operons. Analysis of the deduced amino acid sequences revealed the presence of two Cys-X2-Cys-X2-Cys-X3-Cys-Pro clusters in the pyruvate-flavodoxin oxidoreductase NifJ. This arrangement of cysteine residues is normally present only in ferredoxins. A high degree of homology between the two gene products (NifE and NifN) involved in iron-molybdenum cofactor biosynthesis and the two nitrogenase component I structural proteins (NifD and NifK) was found. All four proteins are characterized by the conserved motif His-Gly-X2-Gly-Cys, which may play a role in binding the iron-molybdenum cofactor.

Amino Acid Sequence↗

PubMed1982

Nitrogen Fixation Associated with Rinsed Roots and Rhizomes of the Eelgrass Zostera marina.

Nitrogen fixation was associated with the rinsed roots and rhizomes of the seagrass, Zostera marina L. Nitrogenase activity (acetylene reduction) was greater on rhizomes compared to roots, and on older roots and rhizomes relative to younger tissue. Compared to aerobic assays, anaerobic or microaerobic conditions enhanced the rate of acetylene reduction by rhizomes with attached roots, with the highest activity (100 nanomoles per gram dry weight per hour) occurring at pO(2) = 0.01 atmosphere. Addition of glucose, sucrose, or succinate also increased the rate of acetylene reduction under anaerobic conditions, with glucose providing the most stimulation. In one experiment, comparison of acetylene reduction assays with (15)N(2) incorporation yielded a ratio of about 2.6:1. Seagrass communities are thought to be limited by the availability of nitrogen and, therefore, nitrogenase activity directly associated with their roots and rhizomes suggests the possibility of a N(2)-fixing flora which may subsidize their nutritional demand for nitrogen.

Journal Article↗

PubMed2006

Isocitrate dehydrogenase of Bradyrhizobium japonicum is not required for symbiotic nitrogen fixation with soybean.

A mutant strain of Bradyrhizobium japonicum USDA110 lacking isocitrate dehydrogenase activity was created to determine whether this enzyme was required for symbiotic nitrogen fixation with soybean (Glycine max cv. Williams 82). The isocitrate dehydrogenase mutant, strain 5051, was constructed by insertion of a streptomycin resistance gene cassette. The mutant was devoid of isocitrate dehydrogenase activity and of immunologically detectable protein, indicating there is only one copy in the genome. Strain 5051 grew well on a variety of carbon sources, including arabinose, pyruvate, succinate, and malate, but, unlike many microorganisms, was a glutamate auxotroph. Although the formation of nodules was slightly delayed, the mutant was able to form nodules on soybean and reduce atmospheric dinitrogen as well as the wild type, indicating that the plant was able to supply sufficient glutamate to permit infection. Combined with the results of other citric acid cycle mutants, these results suggest a role for the citric acid cycle in the infection and colonization stage of nodule development but not in the actual fixation of atmospheric dinitrogen.

Arabinose↗

PubMed1994

Purification and in vitro activities of the native nitrogen fixation control proteins NifA and NifL.

The prokaryotic enhancer-binding protein NifA stimulates transcription at a distance by binding to sequences upstream of nitrogen fixation (nif) promoters and catalyzing the formation of open promoter complexes by RNA polymerase containing the alternative sigma factor, sigma 54. The activity of NifA in vivo is modulated by the negative regulatory protein NifL in response to environmental oxygen and fixed nitrogen. To date, a detailed biochemical analysis of these proteins from the model diazotroph Klebsiella pneumoniae has been hindered by their insolubility. We have now purified NifA and NifL from Azotobacter vinelandii in their native form. NifA is competent in specific DNA binding, transcriptional activation, and response to negative regulation by NifL in vitro. In contrast to the conserved mechanism of phosphotransfer demonstrated by other two-component regulatory systems, our results support a model in which NifL regulates the activity of NifA via a protein-protein steric block interaction rather than a catalytic modification of NifA.

Azotobacter vinelandii↗

PubMed1989

Molecular genetics of Rhizobium Meliloti symbiotic nitrogen fixation.

The application of recombinant DNA techniques to the study of symbiotic nitrogen fixation has yielded a growing list of Rhizobium meliloti genes involved in the processes of nodulation, infection thread formation and nitrogenase activity in nodules on the roots of the host plant, Medicago sativa (alfalfa). Interaction with the plant is initiated by genes encoding sensing and motility systems by which the bacteria recognizes and approaches the root. Signal molecules, such as flavonoids, mediate a complex interplay of bacterial and plant nodulation genes leading to entry of the bacteria through a root hair. As the nodule develops, the bacteria proceed inward towards the cortex within infection threads, the formation of which depends on bacterial genes involved in polysaccharide synthesis. Within the cortex, the bacteria enter host cells and differentiate into forms known as bacteroids. Genes which encode and regulate nitrogenase enzyme are expressed in the mature nodule, together with other genes required for import and metabolism of carbon and energy sources offered by the plant.

Journal Article↗

PubMed1983

Chromosomal gene controlling symbiotic nitrogen fixation in Rhizobium meliloti L5-30.

Auxotrophic Rhizobium meliloti strain RM 246 carries two independent mutations: in the biosynthesis of cysteine (cys) and symbiotic nitrogen fixation process (fix). These two mutations were mapped by transduction between his-240 and ade-4 markers. Cotransduction frequencies show the following order of genes: his-240 fix-1 cys-246 ade-4.

Chromosome Mapping↗

PubMed1984

Nitrogen fixation and nitrogenase activities in members of the family Rhodospirillaceae.

Strains of all 18 species of the family Rhodospirillaceae (nonsulfur photosynthetic bacteria) were studied for their comparative nitrogen-fixing abilities. All species, with the exception of Rhodocyclus purpureus, were capable of growth with N2 as the sole nitrogen source under photosynthetic (anaerobic) conditions. Most rapid growth on N2 was observed in strains of Rhodopseudomonas capsulata. Within the genus Rhodopseudomonas, the species R. capsulata, R. sphaeroides, R. viridis, R. gelatinosa, and R. blastica consistently showed the highest in vivo nitrogenase rates (with the acetylene reduction technique); nitrogenase rates in other species of Rhodopseudomonas and in most species of Rhodospirillum were notably lower. Chemotrophic (dark microaerobic) nitrogen fixation occurred in all species with the exception of one strain of Rhodospirillum fulvum; oxygen requirements for dark N2 fixation varied considerably among species and even within strains of the same species. We conclude that the capacity to fix molecular nitrogen is virtually universal among members of the Rhodospirillaceae but that the efficacy of the process varies considerably among species.

Culture Media↗

PubMedDate not supplied

[The intensification of biological nitrogen fixation by using ammonium carbonate compounds and the routes of their action on microorganisms and plants].

The intensification of activity of associative and symbiotic nitrogen fixation while using low amounts of ammonium-carbonate compounds, carbonic-ammonium salts, in particular, has been established. The main ways and mechanisms of the action of ammonium-carbonate salts as a new and perspective agrochemical on physiological and biochemical processes of microorganisms and agricultural plants were presented on the basis of the analysis of literary information and the author's investigations.

Agrochemicals↗

PubMed1989

The DNA sequence of the Rhodobacter capsulatus ntrA, ntrB and ntrC gene analogues required for nitrogen fixation.

We have determined the DNA sequence for the genes nifR1, nifR2 and nifR4 in the photosynthetic bacterium Rhodobacter capsulatus. These genes regulate transcription of the nifHDK operon and so limit the expression of nitrogen fixation activity to periods of low environmental concentrations of both oxygen and fixed nitrogen. The sequences of these three genes are similar to components of the ntr regulation system in Escherichia coli and Klebsiella pneumoniae. The two-component regulatory system of ntrB and ntrC in E. coli is represented by nifR2 and nifR1 in R. capsulatus and nifR4 in R. capsulatus is the equivalent of the E. coli ntr-related sigma factor ntrA.

Amino Acid Sequence↗

PubMed1991

The product of the nitrogen fixation regulatory gene nfrX of Azotobacter vinelandii is functionally and structurally homologous to the uridylyltransferase encoded by glnD in enteric bacteria.

We sequenced the nitrogen fixation regulatory gene nfrX from Azotobacter vinelandii, mutations in which cause a Nif- phenotype, and found that it encodes a 105-kDa protein (NfrX), the N terminus of which is highly homologous to that of the uridylyltransferase-uridylyl-removing enzyme encoded by glnD in Escherichia coli. In vivo complementation experiments demonstrate that the glnD and nfrX products are functionally interchangeable. A vinelandii nfrX thus appears to encode a uridylyltransferase-uridylyl-removing enzyme, and in this paper we report the first sequence of such a protein. The Nif- phenotype of nfrX mutants can be suppressed by a second mutation in a recently identified nifL-like gene immediately upstream of nifA in A. vinelandii. NifL mediates nif regulation in response to the N status in A. vinelandii, presumably by inhibiting NifA activator function as occurs in Klebsiella pneumoniae; thus, one role of NfrX is to modify, either directly or indirectly, the activity of the nifL product.

Adenine↗

PubMed1996

The cytochrome bc1 complex but not CycM is necessary for symbiotic nitrogen fixation by Rhizobium leguminosarum.

Following Tn5 mutagenesis of Rhizobium leguminosarum biovar viciae, two mutants in one complementation group were identified as being unable to fix nitrogen in pea nodules. Spectroscopic analysis revealed that the mutants had lowered levels of c-type cytochromes and cytochromes aa3, but increased levels of cytochrome d. Cells of the mutants were greatly reduced in their ability to oxidize the artificial electron donor N,N,N',N'-tetramethyl-p-phenylenediamine but membranes prepared from them had increased levels of succinate- and NADH-dependent respiration. NADH oxidation by the mutants was insensitive to the respiratory inhibitor antimycin A, that targets the cytochrome bc1 complex. Molecular analysis of the mutants revealed that they were affected in the cytochrome bc1 complex. One of the mutants contained Tn5 in a gene homologous to that encoding cytochrome c1, and in the other the Tn5 was in DNA homologous to that encoding the cytochrome b component of the cytochrome bc1 complex. Haem staining revealed that haem proteins of M(r)31,000 and M(r)23,000 were absent from membranes from the mutants whereas an additional soluble c-type cytochrome protein of M(r)23,000 was present. We conclude that the larger of these two haem proteins corresponds to cytochrome c1 and, in its absence, the protein of M(r)23,000 does not remain associated with the membrane. Formation of this M(r)23,000 component was specifically blocked in a third respiratory-defective mutant which contained Tn5 in a region of DNA showing homology to a Bradyrhizobium Japonicum gene previously shown to encode the membrane-bound c-type cytochrome CycM. Although the cytochrome bc1 complex is essential for symbiotic nitrogen fixation, the other membrane-bound c-type cytochrome (CycM) is not.

Amino Acid Sequence↗

PubMed1995

In vitro studies of the domains of the nitrogen fixation regulatory protein NIFA.

The prokaryotic enhancer-binding protein NIFA is a multidomain transcriptional activator that catalyzes the formation of open complexes at nitrogen fixation (nif) promoters by a specialized form of RNA polymerase containing sigma 54. The NIFA protein from Klebsiella pneumoniae consists of three domains: the N-terminal domain of unknown function; the central catalytic domain, which is sufficient for transcriptional activation; and the C-terminal DNA-binding domain. Purified fusion proteins between maltose-binding protein (MBP) and NIFA deleted of its N-terminal domain (MBP-delta N-NIFA) or its C-terminal domain (MBP-NIFA-delta C) activated transcription from the K. pneumoniae nifH promoter both in vitro and in vivo. We previously showed that the same was true for a fusion between MBP and the central domain of NIFA. These results indicate that NIFA is sufficiently modular for all fusions carrying its catalytic domain to be active. Unexpectedly, however, simple predictions regarding the location of determinants of the heat lability and insolubility of NIFA, which were based on previous studies of its isolated central and C-terminal domains, were not borne out. Contrary to a previous report from this laboratory, we found that the in vitro start site of transcription for the K. pneumoniae nifH operon could be either of two adjacent G residues, as others had reported in vivo. This was true independent of the activator, i.e., with MBP-NIFA and MBP-delta N-NIFA and with the homologous activator NTRC. When open complexes were formed with GTP as the activating nucleotide, the upstream G residue was probably as a consequence of initiation of transcription.

Bacterial Proteins↗

PubMed2004

Nitrogen fixation in acidophile iron-oxidizing bacteria: the nif regulon of Leptospirillum ferrooxidans.

The Gram-negative iron-oxidizing bacterium Leptospirillum ferrooxidans contains all genes necessary for nitrogen fixation, from genes encoding the Mo-Fe nitrogenase, the specific regulator (nifA), global regulators like glnB and ntrC like genes, to other sensors and transport systems somehow related to nitrogen assimilation. We review current knowledge about the nif regulon and its connection with other metabolic functions in L. ferrooxidans.

Amino Acid Sequence↗

PubMed1997

Characterization of Azorhizobium caulinodans glnB and glnA genes: involvement of the P(II) protein in symbiotic nitrogen fixation.

The nucleotide sequence and transcriptional organization of Azorhizobium caulinodans ORS571 glnA, the structural gene for glutamine synthetase (GS), and glnB, the structural gene for the P(II) protein, have been determined. glnB and glnA are organized as a single operon transcribed from the same start site, under conditions of both nitrogen limitation and nitrogen excess. This start site may be used by two different promoters since the expression of a glnB-lacZ fusion was high in the presence of ammonia and enhanced under conditions of nitrogen limitation in the wild-type strain. The increase was not observed in rpoN or ntrC mutants. In addition, this fusion was overexpressed under both growth conditions, in the glnB mutant strain, suggesting that P(II) negatively regulates its own expression. A DNA motif, similar to a sigma54-dependent promoter consensus, was found in the 5' nontranscribed region. Thus, the glnBA operon seems to be transcribed from a sigma54-dependent promoter that operates under conditions of nitrogen limitation and from another uncharacterized promoter in the presence of ammonia. Both glnB and glnBA mutant strains derepress their nitrogenase in the free-living state, but only the glnBA mutant, auxotrophic for glutamine, does not utilize molecular nitrogen for growth. The level of GS adenylylation is not affected in the glnB mutant as compared to that in the wild type. Under symbiotic conditions, the glnB and glnBA mutant strains induced Fix- nodules on Sesbania rostrata roots. P(II) is the first example in A. caulinodans of a protein required for symbiotic nitrogen fixation but dispensable in bacteria growing in the free-living state.

Amino Acid Sequence↗

PubMed1980

Analysis of regulation of Klebsiella pneumoniae nitrogen fixation (nif) gene cluster with gene fusions.

Gene fusions in which the lac genes are under the control of each promoter in the Klebsiella pneumoniae, nitrogen fixation (nif) gene cluster have been constructed. These fusions have been used to examine positive control of the cluster and the response of individual genes to repression by ammonia and oxygen. De-repression of nif transcriptional units is coordinate and molybdate is required for maximal expression of the structural gene operon, which is autogenously regulated.

Ammonia↗