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PubMed1978

6-cyanopurine, a color indicator useful for isolating mutations in the nif (nitrogen fixation) genes of Klebsiella pneumoniae.

6-Cyanopurine (6-CP) can be used as a color indicator for certain classes of nif (N2 fixation) mutations in Klebsiella pneumoniae. Under N2-fixing conditions, Nif+ colonies and most Nif- colonies are purple on media containing 6-CP. Twenty-two Nif- mutants with altered color on medium containing 6-CP were isolated. All white mutants contained mutations in the regulatory genes, nifAA-nifL. Mutants which were more darkly colored than the wild type had mutations distributed among six nif genes. Medium with 6-CP was used to isolate Nif- mutants with deletions internal to the nif genes, and 6-CP was used to identify strains depressed for nitrogenase synthesis in the presence of NH4+.

Chromosome Mapping↗

PubMed1978

Hydrogen metabolism and nitrogen fixation in wild type and Nif- mutants of Rhodopseudomonas acidophila.

N2 fixation, C2H2 reduction and H2 production in Rhodopseudomonas acidophila DSM 137 were shown to be stoichiometrically related in ratios of 1:2.8:2.8. The highest possible H2 oxidation rate has been calculated to be about 6 fold higher in Rhodopseudomonas acidophila DSM 137 than the maximum rate of H2 production. Nif- mutants were isolated and tested; all of them had lost their ability of reduce C2H2 and to produce H2. In two nif- mutants hydrogenase activity and the capacity for autotrophic growth with H2 were also strongly diminished. Nif+ revertants not only regained their ability for C2H2 reduction and H2 production but also their full capacity for autotrophic growth with H2.

Hydrogen↗

PubMed1982

Clustering of nitrogen fixation (nif) genes in Rhizobium meliloti.

A cloned 17.3-kilobase region of the Rhizobium meliloti genome with homology to the Klebsiella pneumoniae nitrogenase structural genes was studied. Limits on the extent of homology were determined. Transposon mutagenesis of this region of the genome verified that it contained functional nif genes, Some transposon insertions resulted in a defective symbiotic phenotype, whereas others had no noticeable effect on symbiotic competence. The relative position of insertions yielding these two phenotypic classes suggested that at least three distinct units of gene expression are present in this region. Hybridization of RNA from alfalfa root nodules and from vegetatively grown Rhizobium to this cloned DNA showed that at least 11.1 kilobases of the region was transcribed actively and that transcription was specific for the symbiotic state.

Cloning, Molecular↗

PubMed1987

Nitrogen fixation by Azotobacter vinelandii in tungsten-containing medium.

Nitrogenase was isolated and purified from wild-type and a tungsten-resistant mutant (LM2) of Azotobacter vinelandii strain OP derepressed on medium containing 1-10 mM W. While the enzyme from the wild-type strain contained the polypeptides of the conventional enzyme, metal analysis of component 1 demonstrated the existence of one atom each of molybdenum and tungsten. Furthermore, the ESR spectrum of this protein contained three signals, two of which originated from S = 3/2 spin states. One of these signals is nearly identical to that of the conventional MoFe-protein while the other is hypothesized to originate from a W-containing cofactor. In spite of the presence of W, the substrate reduction pattern of this enzyme is the same as that of the conventional enzyme.

Azotobacter↗

PubMed1990

The product of the Klebsiella pneumoniae nifX gene is a negative regulator of the nitrogen fixation (nif) regulon.

An insertional mutation was made in the nifX gene of Klebsiella pneumoniae. This mutation had little effect on the nitrogenase activity of the strain, as measured by acetylene reduction. However, on the addition of NH4+ or O2 (conditions which block nif protein synthesis by transcriptional and posttranscriptional mechanisms), the NifX- mutant synthesized nitrogenase proteins longer and had more accumulated nifHDKTY mRNA than did the wild-type K. pneumoniae at all time points tested. Conversely, overexpression of the wild-type nifX region blocked nif protein synthesis, protein accumulation, and nifHDKTY mRNA accumulation. These complementary results indicate that a product of the nifX region has a role in the negative regulation of nif regulation in response to NH4+ and O2.

Acetylene↗

PubMed1980

The use of translocatable genetic elements to construct a fine-structure map of the Klebsiella pneumoniae nitrogen fixation (nif) gene cluster.

The transposons Tn5, Tn7 and Tn10 and bacteriophage Mu have been used to derive insertion mutations in the Klebsiella pneumoniae nif gene cluster. A large number of deletion mutants have been derived by imprecise excision of insertion mutations and these deletions have been used to construct a fine-structure map of the nif cluster. Comparison of this genetic map with a physical map of the nif cluster derived by Reidel et al. (1979) showed a very good correlation between genetic and physical mapping methods. A new complementation group, designated nifU, has been identified and mapped between nifN and nifS. Polarity studies on the 14 nif cistrons now identified suggests that they are organized in at least seven transcriptional units and that all the multicistronic units are transcribed in the same direction.

Chromosome Deletion↗

PubMed2002

Nitrate reduction and nitrogen fixation in symbiotic association Rhizobium-legumes.

The inhibitory effect of nitrate on nitrogenase activity in root nodules of legume plants has been known for a long time. The major factor inducing changes in nitrogenase activity is the concentration of free oxygen inside nodules. Oxygen availability in the infected zone of nodule is limited, among others, by the gas diffusion resistance in nodule cortex. The presence of nitrate may cause changes in the resistance to O2 diffusion. The aim of this paper is to review literature data concerning the effect of nitrate on the symbiotic association between rhizobia and legume plants, with special emphasis on nitrogenase activity. Recent advances indicate that symbiotic associations of Rhizobium strains characterized by a high nitrate reductase activity are less susceptible to inhibition by nitrate. A thesis may be put forward that dissimilatory nitrate reduction, catalyzed by bacteroid nitrate reductase, significantly facilitates the symbiotic function of bacteroids.

Biological Transport↗

PubMed1977

Nitrogen fixation, denitrification, and pleomorphic growth in a highly pigmented Spirillum lipoferum.

A strain of Spirillum lipoferum with intense red pigmentation was isolated from the roots of Cynodon dactylon "Coastal." This isolate vigorously reduced acetylene when grown in N-free, Na-malate, semisolid agar, and it was identical to S. lipoferum strain SP7 by standard taxonomic tests. Both S. lipoferum SP7 and the C. dactylon root isolate displayed the unique features of being denitrifiers as well as N2 fixers. The N2-dependent growth curve was biphasic: cells in younger cultures showed the characteristic spiral shape and motility, but those in older cultures developed larger, nonmotile, cystlike forms. Nitrogenase activity seemed associated only with younger spiral forms. The red pigment may be a b- or c-type cytochrome. The strong red color, which this strain develops, could be used as a marker in evaluating soil inoculation experiments.

Acetylene↗

PubMed1976

Factors affecting growth and nitrogen fixation of Spirillum lipoferum.

Spirillum lipoferum grows vigorously on malate, succinate, lactate, or pyruvate, moderately on galactose or acetate, and poorly on glucose or citrate. It reduces 15N2. Acetylene reduction rates decrease rapidly when the pH of the culture rises above 7.8. The organism is highly aerobic and had doubling times as low as 2 h when grown on NH4+. However, S. lipoferum reduces N2 well only under microaerophilic conditions. The optimal pO2 for acetylene reduction by stagnant cultures was 0.006 to 0.02 atm depending upon the cell density; aerated cultures grew well at dissolved O2 concentration corresponding to a pO2 of about 0.008 atm. Shaking S. lipoferum with air temporarily inactivates its nitrogenase; reactivation is inhibited by chloramphenicol. The organism assimilated 20 to 24 mg of N/g of organic acid oxidized during growth. The strains studied can be placed in two groups based upon their morphology and physiological characteristics.

Acetylene↗

PubMed1981

The use of cloned nif (nitrogen fixation) DNA to investigate transcriptional regulation of nif expression in Klebsiella pneumoniae.

Some restriction endonuclease fragments of nif DNA, when carried on small multicopy plasmids, inhibited nif expression in Klebsiella pneumoniae. A study of this inhibitory effect revealed, (1) that overproduction of the nifL gene product inhibited transcription of two nif operons examined, nifJ and nifHDKY and, (2) that when transcription was initiated from the promoter of the nifHDKY operon on multicopy plasmids there was a corresponding decrease in the transcription rates of the chromosomally located nifJ and nifHDKY but not the nifLA operon. Studies of transcription in vivo also showed that the nifA gene product was essential for transcription initiation from the nifHDKY and nifBQ promoters. These results, taken with earlier observations (see Discussion) provide evidence that the nifL and nifA gene products are respectively a repressor and activator of nif transcription initiation from all nif promoters except that of the nifLA operon.

Cloning, Molecular↗

PubMed1981

Nitrogen fixation and methane metabolism in a stream sediment-water system amended with leaf material.

The reduction of acetylene and the production and oxidation of methane in a stream sediment--water system amended with either fresh leaves or autumn-shed leaves in the presence and absence of air were studied. Net methane production by the sediment--water system occurred only when leaf material was added, with fresh leaves giving 2.2 times the methane accumulation as autumn-shed leaves. Static incubation in the presence of air had little effect on net methane production, with such production being about the same as (with fresh leaves) or 79% of (with autumn-shed leaves) the anaerobic rates. Acetylene reduction was more affected by the presence of air, with anaerobic rates being at very low levels. The presence of fresh leaves was again stimulatory, with the acetylene reduction rate being 5.5-fold greater than that occurring with autumn-shed leaves.

Air↗

PubMed1975

Involvement of oxyleghaemoglobin and cytochrome P-450 in an efficient oxidative phosphorylation pathway which supports nitrogen fixation in Rhizobium.

Cellular ATP level, ATP/ADP ratio and nitrogenase activity rise when oxyleghaemoglobin is added to respiring suspensions of Rhizobium japonicum bacteroids from soybean root nodules. Increased gaseous O2 tension is much less efficient than oxyleghaemoglobin in stimulation of bacteroid ATP production. Studies with the inhibitor carbonyl cyanide m-chlorophenylhydrazone show this ATP to be generated as a consequence of oxidative phosphorylation. N-Phenylimidazole, a specific cytochrome P-450 inhibitor, also lowers the efficiency of bacteroid oxidative phosphorylation. An approximately linear relationship is observed between ATP/ADP ratio and nitrogenase activity as N-phenylimidazole concentration is lowered. It is suggested that cytochrome P-450 is a component of the leghaemoglobin-facilitated respiration pathway and that it may act as intracellular O2 carrier rather than terminal oxidase. A less efficient oxidase appears to function when cytochrome P-450 is inhibited.

Adenosine Diphosphate↗

PubMed1981

Large plasmids of fast-growing rhizobia: homology studies and location of structural nitrogen fixation (nif) genes.

A single large plasmid was isolated from multiplasmid-harboring strains Rhizobium leguminosarum 1001 and R. trifolii 5. These single plasmids, as well as the largest plasmid detectable in R. phaseoli 3622, hybridized with part of the nif structural genes of Klebsiella pneumoniae. In contrast, the plasmids of R. meliloti strains V7 and L5-30 did not show hybridization with the nif genes of K. pneumoniae, indicating that these genes might be located either on the chromosome or on a much larger plasmid which as yet has not been isolated. Studies of the homology between plasmids of fast-growing Rhizobium species showed that a specific deoxyribonucleic acid sequence, which carries the structural genes for nitrogenase, is highly conserved on a plasmid in R. leguminosarum, R. trifolii, and R. phaseoli. Furthermore, it was found that this type of plasmid in the different species shares extensive deoxyribonucleic acid homology, suggesting that strains in the R. leguminosarum cluster have preserved a nif plasmid.

Base Sequence↗

PubMed2003

Cysteine proteases in nodulation and nitrogen fixation.

The cysteine proteinases or cysteine endopeptidases (EC 3.4.22) are known to occur widely in plant cells. They are involved in almost all aspects of plant growth and development including germination, circadian rhythms, senescence and programmed cell death. They are also involved in mediating plant cell responses to environmental stress (such as water stress, salinity, low temperature, wounding, ethylene, and oxidative conditions) and plant-microbe interactions (including nodulation). In the development and function of legume root nodules, cysteine proteases could be involved in several important processes:-(i) a defence response to root invasion by microorganisms; (ii) protein turnover required during the formation of new tissue; (iii) cellular homeostasis and metabolism; (iv) adaptation of host cells to physiological stresses; (v) control of nodule senescence. Because of their central importance to plant physiology, cysteine proteases could serve as important targets for the study of nodule development and functioning at the molecular level. Because of their widespread occurrence in nodulating plants they could also serve as candidate genes for targeted plant breeding programmes.

Cysteine Endopeptidases↗

PubMed2006

Phage typing of indigenous soybean-rhizobia and relationship of a phage group strains for their asymbiotic and symbiotic nitrogen fixation.

A total of 354 indigenous bradyrhizobia were isolated from soybean nodules collected from five major crop grown regions. Host-specific 12 phages, each active on particular strains were selected. Factors, which influence the interaction between the host and phage, were examined. Four different types of plaques were detected. Nearly 17% of isolates were found resistant to all phages. Phage sensitivity patterns revealed a total of 32 distinct phage genotype groups. Different set of phage combinations expressed variation in specificity for parasitizing against particular group of rhizobia. Distributions of isolates in each phage types differed markedly between regions. Interestingly, nine strains belonging to phage group 16 exhibited high ex planta nitrogenase activity in culture. However, no correlation could be established between high ex planta nitrogenase activity and their symbiotic effectiveness with soybean cultivars. Soybean cv. JS335 showed relatively superior performance than Bragg and Lee with indigenous bradyrhizobial strains. Phage typing revealed the existence of large genetic diversity among native rhizobia and selection of the superior bradyrhizobial strains can also be possible for a given soil-climate-cultivar complex.

Bacteriophages↗

PubMed1982

Nitrogen fixation system of tungsten-resistant mutants of Azotobacter vinelandii.

Mutants of Azotobacter vinelandii ATCC 12837 were isolated which could fix N2 in the presence of high tungsten concentrations. The most studied of these mutants (WD2) grew well in N-free modified Burk broth containing 10 mM W, whereas the wild type would not grow in this medium. WD2 would also grow in Burk N-free broth at about the same rate as the wild type. WD2 in broth containing W exhibited 22% of the whole cell acetylene reduction activity of the wild type in broth containing Mo and showed a lowered affinity for acetylene. Two-dimensional gel electrophoresis experiments showed that N2-fixing cells of WD2 from broth containing W or Mo did not produce significant amounts of component I of native nitrogenase protein. Electron spin resonance spectra of whole cells and cell-free extracts of WD2 from broth containing W lacked any trace of the g = 3.6 resonance associated with FeMoCo.

Acetylene↗

PubMed1978

Genetic and biochemical analysis of mutants induced by bacteriophage Mu DNA integration into Klebsiella pneumoniae nitrogen fixation genes.

Polar mutations were obtained by integration of bacteriophage Mu c+ or Mu cts DNA into the Klebsiella pneumoniae nif genes located on plasmid pCE1, a derivative of pRD1. In addition, nif deletions were isolated from nif::Mu cts plasmids. Complementation data allowed the characterization of twelve nif cistrons, nine corresponding to previously identified genes. Polar effect of Mu DNA insertions suggested the existence of at least six transcription units: 1) nif K, nif D and nif H--2)nif A and nif L--3) nif E and a new gene--4) nif B--5) nif F--6) nif J. Nif K, nif D and nif H, which are most probably the structural genes for nitrogenase, seem to belong to the same operon transcribed from nif H to nif K. This was confirmed by SDS gel autoradiography of pulse labelled proteins. Moreover it was possible to identify, on the autoradiograms, a polypeptide which likely is the product of nif J and whose biosynthesis is under the control of nif A.

Coliphages↗

PubMed1978

Transfer of nitrogen fixation genes from a bacterium with the characteristics of both Rhizobium and Agrobacterium.

Strain T1K, reported to be Rhizobium trifolii strain T1 carrying the drug resistance plasmid RU-1drd, was able to transfer a cluster of nif+ genes to Escherichia coli K-12. Additional genetic material, resembling the gal-chlA region of E. coli, was also transferred from strain T1K. The segregation pattern of these transferred genes suggested that they were on a plasmid. Although strain TIK was able to nodulate red and white clover, it also formed very slow-growing galls on tomato stems and shared many physiological properties with Agrobacterium tumefaciens, to which it seemed more closely related than to R. trifolii. The R. trifolii hybrid T1 (R1-19drd), constructed by conjugation, did not share any of these properties of both A. tumefaciens. Thus, strain T1K appears to be a bacterium with properties of both A. tumefaciens and R. trifolii and with the capacity to transfer nif+ genes and other functions which it may have "cloned" from another bacterium such as Klebsiella.

Chromosomes, Bacterial↗