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PubMed1975

Regulation of nitrogen fixation. Nitrogenase-derepressed mutants of Klebsiella pneumoniae.

1. A new procedure is described for selecting nitrogenase-derepressed mutants based on the method of Brenchley et al. (Brenchley, J.E., Prival, M.J. and Magasanik, B. (1973) J. Biol. Chem. 248, 6122-6128) for isolating histidase-constitutive mutants of a non-N2-fixing bacterium. 2. Nitrogenase levels of the new mutants in the presence of NH4+ were as high as 100% of the nitrogenase activity detected in the absence of NH4+. 3. Biochemical characterization of these nitrogen fixation (nif) derepressed mutants reveals that they fall into three classes. Three mutants (strains SK-24, 28 and 29), requiring glutamate for growth, synthesize nitrogenase and glutamine synthetase constitutively (in the presence of NH4+). A second class of mutants (strains SK-27 and 37) requiring glutamine for growth produces derepressed levels of nitrogenase activity and synthesized catalytically inactive glutamine synthetase protein, as determined immunologically. A third class of glutamine-requiring, nitrogenase-derepressed mutants (strain SK-25 and 26) synthesizes neither a catalytically active glutamine synthetase enzyme nor an immunologically cross-reactive glutamine synthetase protein. 4. F-prime complementation analysis reveals that the mutant strains SK-25, 26, 27, 37 map in a segment of the Klebsiella chromosome corresponding to the region coding for glutamine synthetase. Since the mutant strains SK-27 and SK-37 produce inactive glutamine synthetase protein, it is concluded that these mutations map within the glutamine synthetase structural gene.

Ammonia↗
PubMed2002

[A study of the effects on the symbiotic nitrogen fixation of Sinorhizobium fredii with the introduction of dctABD and nifA genes].

A recombinant plasmid pHN307 containing C4-dicarboxylic acid transport genes (dctABD) from Sinorhizobium meliloti, nifA genes from Klebsiella pneumoniae and reporter genes luxAB from pDB30 was constructed by using pTR102 as the vector. The pHN307 was then introduced into the S. fredii HN01, YC4 and GR3 by tri-parental mating, and the stability of pHN307 in the transconjugants under free-living and symbiotic condition was also investigated. The results of plant pot experiment indicated that the introduction of pHN307 in the transconjugants could significantly increase the nodule fresh weight, shoot dry weight (biomass) and total nitrogen content of the symbiotic plants with soybean variety of Heilong 33. When the transconjugants were in symbiotic with soybean variety of Chuanzao No. 1, HN01 (pHN307) could significantly increase its root nodule number and fresh weight; GR3 (pHN307) could significantly increase its root nodule number, nodule fresh weight, shoot dry weight and total nitrogen content, but YC4(pHN307) demonstrated negative effect under the same condition. The results of this study suggested that the introduction of dctABD and nifA could improve the symbiotic nitrogen fixation efficiency and nodulation ability of the rhizobia tested, respectively, and its effect was relevant to the characteristics of recipient rhizobia and soybean variety.

Bacterial Proteins↗
PubMed1987

Bradyrhizobium japonicum mutants defective in nitrogen fixation and molybdenum metabolism.

Bradyrhizobium japonicum JH mutants deficient in molybdenum metabolism into the enzymes nitrogenase and nitrate reductase were isolated by using the vector pSUP1011, which carries transposon Tn5 (streptomycin and kanamycin resistance). Mutants in Mo metabolism were obtained at a frequency of 3.6 X 10(-3) (per Kan Strr colony). The mutants were detected by their poor ability to grow in nitrate-containing medium without added Mo. One of the mutant types required 10(5) times more molybdate than the wild type to obtain maximal nitrogen fixation activity. Double-reciprocal plots of Mo uptake versus concentration indicated that the wild-type strain had a high- and a lower-affinity component for Mo binding. Mutant strains JH-90 and JH-119 lacked the high-affinity Mo uptake component and were also clearly deficient in Mo accumulation into a nonexchangeable form. Nitrogenase activity as well as Mo uptake ability could be restored in strains JH-90 and JH-119 by the addition of the sterile supernatant fraction of the wild type. Therefore, mutant strains JH-90 and JH-119 appeared to be deficient in an extracellular Mo-binding factor produced by the wild type. Mutant strains JH-14 and JH-143 had Mo uptake kinetics like those of the wild type (both high- and low-affinity binding for Mo) and appeared to be deficient in intracellular Mo metabolism processes. The addition of the wild-type supernatant did not restore Mo uptake or nitrogenase activity in these strains.

Biological Transport↗
PubMed1997

The Rhizobium leguminosarum prsDE genes are required for secretion of several proteins, some of which influence nodulation, symbiotic nitrogen fixation and exopolysaccharide modification.

NodO is a secreted protein from Rhizobium leguminosarum bv. viciae with a role in signalling during legume nodulation. A Tn5-induced mutant was identified that was defective in NodO secretion. As predicted, the secretion defect decreased pea and vetch nodulation but only when the nodE gene was also mutated. This confirms earlier observations that NodO plays a particularly important role in nodulation when Nod factors carrying C18:1 (but not C18:4) acyl groups are the primary signalling molecules. In addition to NodO secretion and nodulation, the secretion mutant had a number of other characteristics. Several additional proteins including at least three Ca2+-binding proteins were not secreted by the mutant and this is thought to have caused the pleiotropic phenotype. The nodules formed by the secretion mutant were unable to fix nitrogen efficiently; this was not due to a defect in invasion because the nodule structures appeared normal and nodule cells contained many bacteroids. The mutant formed sticky colonies and viscous liquid cultures; analysis of the acidic exopolysaccharide revealed a decrease in the ratio of reducing sugars to total sugar content, indicating a longer chain length. The use of a plate assay showed that the mutant was defective in an extracellular glycanase activity. DNA sequencing identified the prsDE genes, which are homologous to genes encoding protease export systems in Erwinia chrysanthemi and Pseudomonas aeruginosa. An endoglycanase (Egl) from Azorhizobium caulinodans may be secreted from R. leguminosarum bv. viciae in a prsD-dependent manner. We conclude that the prsDE genes encode a Type I secretion complex that is required for the secretion of NodO, a glycanase and probably a number of other proteins, at least one of which is necessary for symbiotic nitrogen fixation.

ATP-Binding Cassette Transporters↗
PubMedDate not supplied

A critical role for aniA in energy-carbon flux and symbiotic nitrogen fixation in Sinorhizobium meliloti.

During free-living reproductive growth, Sinorhizobium meliloti accumulates poly-beta-hydroxybutyrate (PHB) and glycogen, and produces and excretes exopolysaccharides and beta-1,2-glucan. In previous investigations, PHB-minus mutants of S. meliloti 41 were obtained and studied; and the genes for PHB biosynthesis, phaAB and phaC, were described. In this work, the role of an open reading frame (orf) upstream of phaAB is studied. This orf is designated aniA because the gene was found to be expressed during anaerobic growth. Under low oxygen conditions, glycogen decreases and the production of extracellular polymeric substances (EPS) is partially repressed. When the aniA mutant is incubated under oxygen-limiting conditions, the only significant change observed is an overproduction of EPS. Subsequent in planta tests showed that although the mutant strain produced abundant nodules, only very low acetylene-reduction activity was detected, indicating that nitrogen fixation was not adequately supported by endogenous substrates.

Alcohol Oxidoreductases↗
PubMed1986

The use of nickel to probe the role of hydrogen metabolism in cyanobacterial nitrogen fixation.

The hydrogenase activities of the heterocystous cyanobacteria Anabaena cylindrica and Mastigocladus laminosus are nickel dependent, based on their inability to consume hydrogen with various electron acceptors or produce hydrogen with dithionite-reduced methyl viologen, after growth in nickel-depleted medium. Upon addition of nickel ions to nickel-deficient cultures of A. cylindrica, the hydrogenase activity recovered in a manner which was protein synthesis-dependent, the recovery being inhibited by chloramphenicol. We have used the nickel dependence of the hydrogenase as a probe of the possible roles of H2 consumption in enhancing nitrogen fixation, and particularly for protecting nitrogenase against oxygen inhibition. Although at the usual growth temperatures (25 degrees for A. cylindrica and 40 degrees for M. laminosus), the cells consume H2 vigorously in an oxyhydrogen reaction after growth in the presence of nickel ions, we have not found that the reaction confers any significant additional protection of nitrogenase, either at aerobic pO2 (for both organisms) or at elevated pO2 (for A. cylindrica). However, at elevated temperatures (e.g., 40 degrees for A. cylindrica and 48 degrees for M. laminosus) a definite protective effect was observed. At these temperatures both organisms rapidly lost acetylene reduction activity under aerobic conditions. When hydrogen gas (10%) was present, the cells retained approximately 50% of the nitrogenase activity observed under anaerobic conditions (argon gas phase). No such protection by hydrogen gas was observed with nickel-deficient cells. Studies with cell-free extracts of A. cylindrica showed that the predominant effect of temperature was not due to thermal inactivation of nitrogenase.

Acetylene↗
PubMed1996

The Azospirillum brasilense rpoN gene is involved in nitrogen fixation, nitrate assimilation, ammonium uptake, and flagellar biosynthesis.

The rpoN (ntrA) gene (encoding sigma 54) of Azospirillum brasilense Sp7 was isolated by using conserved rpoN primers and the polymerase chain reaction, and its nucleotide sequence was determined. The deduced amino acid sequence of the RpoN protein was found to share a high degree of homology with other members of the sigma 54 family. Two additional open reading frames were found in the Azospirillum brasilense rpoN region, with significant similarity to equivalent regions surrounding the rpoN locus in other bacteria. An rpoN mutant of Azospirillum brasilense Sp7 was constructed by gene replacement and found to be defective in nitrogen fixation, nitrate assimilation, and ammonium uptake. Lack of ammonium uptake was also found in previously isolated Azospirillum brasilense ntrB and ntrC mutants, further supporting the role of the ntr system in this process. In addition, the rpoN mutant was found to be nonmotile, suggesting a role of RpoN in Azospirillum brasilense flagellar biosynthesis.

Amino Acid Sequence↗
PubMed1974

Regulation of nitrogen fixation in Klebsiella pneumoniae: evidence for a role of glutamine synthetase as a regulator of nitrogenase synthesis.

Mutations causing constitutive synthesis of glutamine synthetase (GlnC(-) phenotype) were transferred from Klebsiella aerogenes into Klebsiella pneumoniae by P1-mediated transduction. Such GlnC(-) strains of K. pneumoniae have constitutive levels of glutamine synthetase. Two of three GlnC(-) strains of K. pneumoniae studied, each containing independently isolated mutations that confer the GlnC(-) phenotype, continue to synthesize nitrogenase in the presence of NH(4) (+). One strain, KP5069, produces 30% as much nitrogenase when grown in the presence of 15 mM NH(4) (+) as in its absence. The GlnC(-) phenotype allows the synthesis of nitrogenase to continue under conditions that completely repress nitrogenase synthesis in the wild-type strain. Glutamine auxotrophs of K. pneumoniae, that do not produce catalytically active glutamine synthetase, are unable to synthesize nitrogenase during nitrogen limited growth. Complementation of K. pneumoniae Gln(-) strains by an Escherichia coli episome (F'133) simultaneously restores glutamine synthetase activity and the ability to synthesize nitrogenase. These results indicate a role for glutamine synthetase as a positive control element for nitrogen fixation in K. pneumoniae.

Catalysis↗
PubMed2001

A functional myo-inositol dehydrogenase gene is required for efficient nitrogen fixation and competitiveness of Sinorhizobium fredii USDA191 to nodulate soybean (Glycine max [L.] Merr.).

Inositol derivative compounds provide a nutrient source for soil bacteria that possess the ability to degrade such compounds. Rhizobium strains that are capable of utilizing certain inositol derivatives are better colonizers of their host plants. We have cloned and determined the nucleotide sequence of the myo-inositol dehydrogenase gene (idhA) of Sinorhizobium fredii USDA191, the first enzyme responsible for inositol catabolism. The deduced IdhA protein has a molecular mass of 34,648 Da and shows significant sequence similarity with protein sequences of Sinorhizobium meliloti IdhA and MocA; Bacillus subtilis IolG, YrbE, and YucG; and Streptomyces griseus StrI. S. fredii USDA191 idhA mutants revealed no detectable myo-inositol dehydrogenase activity and failed to grow on myo-inositol as a sole carbon source. Northern blot analysis and idhA-lacZ fusion expression studies indicate that idhA is inducible by myo-inositol. S. fredii USDA191 idhA mutant was drastically affected in its ability to reduce nitrogen and revealed deteriorating bacteroids inside the nodules. The number of bacteria recovered from such nodules was about threefold lower than the number of bacteria isolated from nodules initiated by S. fredii USDA191. In addition, the idhA mutant was also severely affected in its ability to compete with the wild-type strain in nodulating soybean. Under competitive conditions, nodules induced on soybean roots were predominantly occupied by the parent strain, even when the idhA mutant was applied at a 10-fold numerical advantage. Thus, we conclude that a functional idhA gene is required for efficient nitrogen fixation and for competitive nodulation of soybeans by S. fredii USDA191.

Amino Acid Sequence↗
PubMed1989

Rhizobium meliloti fixGHI sequence predicts involvement of a specific cation pump in symbiotic nitrogen fixation.

We present genetic and structural analyses of a fix operon conserved among rhizobia, fixGHI from Rhizobium meliloti. The nucleotide sequence of the operon suggests it may contain a fourth gene, fixS. Adjacent open reading frames of this operon showed an overlap between TGA stop codons and ATG start codons in the form of an ATGA motif suggestive of translational coupling. All four predicted gene products contained probable transmembrane sequences. FixG contained two cysteine clusters typical of iron-sulfur centers and is predicted to be involved in a redox process. FixI was found to be homologous with P-type ATPases, particularly with K+ pumps from Escherichia coli and Streptococcus faecalis but also with eucaryotic Ca2+, Na+/K+, H+/K+, and H+ pumps, which implies that FixI is a pump of a specific cation involved in symbiotic nitrogen fixation. Since prototrophic growth of fixI mutants appeared to be unimpaired, the predicted FixI cation pump probably has a specifically symbiotic function. We suggest that the four proteins FixG, FixH, FixI, and FixS may participate in a membrane-bound complex coupling the FixI cation pump with a redox process catalyzed by FixG.

Adenosine Triphosphatases↗
PubMed1981

Nitrogen fixation gene (nifL) involved in oxygen regulation of nitrogenase synthesis in K. pneumoniae.

The enzyme complex nitrogenase, which reduces N2 to NH+4, involves two redox proteins, both irreversibly damaged by O2 (ref. 1). Enzyme activity therefore requires anaerobic conditions, a source of reductant and a large amount of ATP (approximately 16 ATPs per N2). In both aerobic and facultative anaerobic N2-fixing bacteria, nitrogenase synthesis is regulated by O2 and NH+4, but in the aerobes there are also processes to protect the enzyme from O2 damage. The mechanisms of repression by O2 and NH+4 seem to be independent in the organisms so far examined. In the facultative anaerobe, Klebsiella pneumoniae, O2 was shown to repress nitrogenase synthesis in an NH+4-constitutive strain. The fusion of the Escherichia coli lacZ gene into each transcriptional unit of the nitrogen fixation (nif) gene cluster in K. pneumoniae has facilitated studies with O2, because expression from the various nif promoters results in an O2-stable product (beta-galactosidase). Notably, the nifHDK operon (the nitrogenase structural genes) was more sensitive to O2 repression than the nifLA operon (regulatory genes). The characterization of mutants, reported here, indicates the involvement of a nif-regulatory gene product in the mechanism of O2 control of nitrogenase synthesis.

Aerobiosis↗
PubMed1991

Identification of a nifA-like regulatory gene of Azospirillum brasilense Sp7 expressed under conditions of nitrogen fixation and in the presence of air and ammonia.

A gene bank of Azospirillum lipoferum Br17 constructed in the vector lambda GEM11 was screened with a Bradyrhizobium japonicum nifA gene probe. A 7.3 kb EcoRI fragment carrying a nifA-like gene was thereby isolated and subsequently used to screen a gene bank of Azospirillum brasilense Sp7 constructed in pUC18. Two EcoRI fragments of 5.6 kb and 3.6 kb covering the nifA-homology region were found. Mutants with Nif- phenotype were obtained by site-directed Tn5 mutagenesis of the 5.6 kb fragment and subsequent recombination into the A. brasilense Sp7 genome. The mutations were clustered into two loci located at each extremity of the fragment. One of these loci corresponded to nifA and the other to nifB. The nucleotide sequence of nifA of A. brasilense Sp7 was determined. Comparison of the deduced amino acid sequences of NifA of A. brasilense Sp7 and NifA of B. japonicum, Rhizobium leguminosarum biovar trifolii and Klebsiella pneumoniae confirmed that it was a nifA-like gene. Construction of a nifA-lacZ fusion and mapping of the RNA transcriptional start site showed that the nifA-like gene was expressed from an unidentified promoter, under conditions of nitrogen fixation and in the presence of oxygen and ammonia.

Amino Acid Sequence↗
PubMed2004

Regulators of nonsulfur purple phototrophic bacteria and the interactive control of CO2 assimilation, nitrogen fixation, hydrogen metabolism and energy generation.

For the metabolically diverse nonsulfur purple phototrophic bacteria, maintaining redox homeostasis requires balancing the activities of energy supplying and energy-utilizing pathways, often in the face of drastic changes in environmental conditions. These organisms, members of the class Alphaproteobacteria, primarily use CO2 as an electron sink to achieve redox homeostasis. After noting the consequences of inactivating the capacity for CO2 reduction through the Calvin-Benson-Bassham (CBB) pathway, it was shown that the molecular control of many additional important biological processes catalyzed by nonsulfur purple bacteria is linked to expression of the CBB genes. Several regulator proteins are involved, with the two component Reg/Prr regulatory system playing a major role in maintaining redox poise in these organisms. Reg/Prr was shown to be a global regulator involved in the coordinate control of a number of metabolic processes including CO2 assimilation, nitrogen fixation, hydrogen metabolism and energy-generation pathways. Accumulating evidence suggests that the Reg/Prr system senses the oxidation/reduction state of the cell by monitoring a signal associated with electron transport. The response regulator RegA/PrrA activates or represses gene expression through direct interaction with target gene promoters where it often works in concert with other regulators that can be either global or specific. For the key CO2 reduction pathway, which clearly triggers whether other redox balancing mechanisms are employed, the ability to activate or inactivate the specific regulator CbbR is of paramount importance. From these studies, it is apparent that a detailed understanding of how diverse regulatory elements integrate and control metabolism will eventually be achieved.

Alphaproteobacteria↗
PubMed1989

Dual control of the Bradyrhizobium japonicum symbiotic nitrogen fixation regulatory operon fixR nifA: analysis of cis- and trans-acting elements.

Aerobic expression of the fixR nifA operon in Bradyrhizobium japonicum was shown to depend on a cis-acting, promoter-upstream DNA sequence located between the -24/-12 promoter and position -86 relative to the transcription start site. An adenine at position -66 was essential for maximal expression. A chromosomal deletion of the upstream activator sequence (UAS) led to a symbiotically defective phenotype which was typical of nifA mutants. B. japonicum crude extracts contained a protein that bound to the UAS. By using chromosomally integrated fixR-lacZ fusions, the level of expression of the fixR nifA operon was found to be fivefold higher under reduced oxygen tension than under aerobiosis. This increase was due to autoactivation by the NifA protein and was partly independent of the UAS. Based on these data, we propose a model for the regulation of nitrogen fixation genes in B. japonicum that involves dual positive control of the fixR nifA operon. At high oxygen concentrations, the operon is expressed at a moderate level, subject to activation by the binding of a trans-acting factor to the UAS. Under such conditions, the nifA gene product is known to be inactive. At very low oxygen concentrations--a condition favorable to NifA activity--the NifA protein is the trans-acting factor which (i) enhances the level of fixR nifA expression (and hence its own synthesis) and (ii) activates other nif and fix genes.

Chromosome Deletion↗
PubMed1976

Acetylene reduction (nitrogen fixation) by enterobacteriaceae isolated from paper mill process waters.

Using selective media containing galactitol, over 130 Enterobacteriaceae have been isolated from paper mill process waters collected from different localities. These bacteria were extensively characterized and tested for acetylene-reducing (nitrogen-fixing) activity under anaerobic conditions. High activity was found in representatives of Klebsiella pneumoniae, Enterobacter aerogenes, Enterobacter cloacae, Erwinia herbicola, Citrobacter freundii, Citrobacter intermedius, and Escherichia coli. Under argon, nitrogenase synthesis was generally not repressed by 5 mM l-glutamate, l-aspartate, l-leucine or Casamino Acids (0.5 g/liter). In many strains, both the specific activities (nanomoles of C(2)H(4) per minute per milligram of protein) and the activities (nanomoles of C(2)H(4) per minute) had considerably declined after 24 h. In three selected strains, activity in intact cells grown under nitrogen was unaffected by the presence during assay of 10 mM l-amino acids or ammonium acetate. All of the strains examined were tolerant towards inactivation of nitrogen-fixing activity by 1.8% (vol/vol) oxygen during assay, and inactivation by up to 10% oxygen was partly reversible. Representatives of the six taxa synthesized nitrogenase in stirred aerobic cultures, though the protein concentrations attained were lower than under anaerobic conditions. It seems reasonable to suggest that under natural conditions, nitrogen fixation is able to contribute significantly to the nitrogen economy of the cells.

Journal Article↗
PubMed1994

Effects of elevated selenium concentration on selenium accumulation and nitrogen fixation symbiotic activity of Melilotus indica L.

Biological and soil factors which contribute to the successful colonization of an annual legume species. Melilotus indica L., in soils with elevated selenium (Se) were studied. This species was introduced into the Kesterson Reservoir in the fresh top soil that was brought in under the Kesterson Cleanup Action to fill lowering pond sites and prevent the formation of ephemeral pools containing hazardous levels of Se. In 4 years since its introduction, it has expanded its range of colonization from the fresh soil fill sites to the native soil sites and contributed 10 to 50% of biomass to the grassland communities. The plant and nodule tissue Se concentrations of the field grown plants were found to be negatively correlated with the soil sulfate concentration. Nutrient solution culture studies discovered that M. indica was able to accumulate 500 micrograms Se g-1 dry weight without a reduction of growth rate. Plants without nodulation were found to accumulate a greater amount of Se and more vulnerable to Se toxicity. Acetylene reduction rate measurements indicate that the nitrogen fixation symbiotic activity appears to be more susceptible to an elevated Se concentration than its host plant. M. indica is a winter weed, and it occurs naturally in the Se-rich soils. It grows actively over the winter and spring and complete its life cycle in May. If the root nodules and root tissues are incorporated into the soil, the rate of soil Se volatilization may be accelerated over the warm summer months. For disposal of the Se-rich plant materials the plant shoot tissues may be harvested for Se-deficient forage supplementation. Therefore, this species may be useful for field management and reclamation of Se-contaminated soils.

Biomass↗