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PubMed2004

Azide resistance in Rhizobium ciceri linked with superior symbiotic nitrogen fixation.

Isolated azide resistant (AzR) native R. ciceri strain 18-7 was resistant to sodium azide at 10 microg/ml. To find if nif-reiteration is responsible for azide resistance and linked to superior symbiotic nitrogen fixation, transposon (Tn5) induced azide sensitive mutants were generated. Using 4 kb nif-reiterated Sinorhizobium meliloti DNA, a clone C4 that complemented azide sensitivity was isolated by DNA hybridization from genomic library of chickpea Rhizobium strain Rcd301. EcoRI restriction mapping revealed the presence of 7 recognition sites with a total insert size of 19.17 kb. Restriction analysis of C4 clone and nif-reiterated DNA (pRK 290.7) with EcoRI and XhoI revealed similar banding pattern. Wild type strain 18-7, mutant M126 and complemented mutant M126(C4) were characterized for symbiotic properties (viz., acetylene reduction assay, total nitrogen content, nodule number and fresh and dry weight of the infected plants) and explanta nitrogenase activity. Our results suggested that azide resistance, nif-reiteration, and superior symbiotic effectiveness were interlinked with no correlation between ex-planta nitrogenase activity and azide resistance in R. ciceri.

Cicer↗
PubMed1994

Genetic regulation of nitrogen fixation in Rhizobium meliloti.

The soil bacterium Rhizobium meliloti fixes dinitrogen when associated with root nodules formed on its plant host, Medicago sativa (alfalfa). The expression of most of the known genes required for nitrogen fixation (nif and fix genes), including the structural genes for nitrogenase, is induced in response to a decrease in oxygen concentration. Induction of nif and fix gene expression by low oxygen is physiologically relevant because a low-oxygen environment is maintained in root nodules to prevent inactivation of the highly oxygen-sensitive nitrogenase enzyme. The genes responsible for sensing and transducing the low oxygen signal, fixL and fixJ, encode proteins (FixL and FixJ, respectively) that are homologous to a large family of bacterial proteins involved in signal transduction, the two component regulatory system proteins. The two components consist of a sensor protein, to which FixL is homologous, and a response regulator protein, to which FixJ is homologous. The sensor protein respond to an activating signal by autophosphorylating and then transferring the phosphate to its cognate response regulator protein. The phosphorylated response regulator, which is often a transcriptional activator, is then able to activate its target. A cascade model of nif and fix gene regulation in R. meliloti has been proposed, whereby FixL acts as an oxygen sensor as the initial event in the cascade and transmits this information to FixJ. FixJ, which possesses a putative helix-turn-helix DNA-binding motif, then activates transcription of the nifA and fixK genes. The nifA and fixK gene products, are transcriptional activators of at least 14 other nif and fix genes.

Bacterial Proteins↗
PubMed1980

Energy requirement for nitrogen fixation in actinorhizal and legume root nodules.

The ratio of respiration to nitrogenase activity was measured in five species of actinorhizal root nodules and eight species of legume nodules. The two types of nodules could not be distinguished on the basis of this ratio; this evidence thus indicates that the energy cost of nitrogen fixation is similar for both.

Actinomycetales↗
PubMed1976

Nitrogen fixation by bacteria from the hindgut of termites.

Anaerobically grown bacteria isolated from the hindgut contents of the termites Coptotermes lacteus (Froggatt), Mastotermes darwiniensis Froggatt and Nasutitermes exitiosus (Hill) were nitrogenase-positive as assayed by acetylene reduction. Nitrogen fixation, confirmed with 15N2, was highest in the isolate from M. darwiniensis. All isolates were identified as Citrobacter freundii (Braak) Werkman & Gillen.

Anaerobiosis↗
PubMed1988

Further analysis of nitrogen fixation (nif) genes in Azotobacter chroococcum: identification and expression in Klebsiella pneumoniae of nifS, nifV, nifM, and nifB genes and localization of nifE/N-, nifU-, nifA- and fixABC-like genes.

The results presented extend previous investigations on the genetics of nitrogen fixation in Azotobacter chroococcum and indicate that nif- and fix-like DNA is located in at least five different regions of the genome. Region I contains functional copies of nifS,V and M, as well as nifH, D and K, all of which complemented mutants of Klebsiella pneumoniae. In addition, nifE- and/or nifN-like and nifU-like DNA is located in this region. The organization of the nif cluster in region I closely resembles that of K. pneumoniae. though spread over 22 kb as compared with 14 kb. Region II contains a functional nifB gene, which complemented a K. pneumoniae nifB mutant, and seems to be adjacent to ap nifA-like gene. Region III harbours nifH*, encoding a second nitrogenase Fe-protein. Region IV contains a reiteration of nifE- on and/or nifN-like sequences, and DNA homologous to Rhizobium meliloti fixABC is present in region V. The apparent complexity of nifDNA in A. chroococcum is probably related to the two systems for N2-fixation pr present in this organism.

Azotobacter↗
PubMed2001

An interdisciplinary research strategy to improve symbiotic nitrogen fixation and yield of common bean (Phaseolus vulgaris) in salinised areas of the Mediterranean basin.

The main findings of a cooperative research group of agronomists, plant breeders, microbiologists, physiologists and molecularists to improve the symbiotic nitrogen fixation (SNF) and N2-dependent yield of common bean under moderate salinity in the Mediterranean basin are summarised. Agronomic surveys in reference production areas show large spatial and temporal variations in plant nodulation and growth, and in efficiency of utilisation of the rhizobial symbiosis. The latter was associated with a large rhizobial diversity, including new bean nodulating species. Macrosymbiont diversity in SNF and adaptation to NaCl was found. However, contrasts between plant genotypes could be altered by specific interactions with some native rhizobia. Therefore, variations in soil rhizobial population, in addition to agronomic practices and environmental constraints, may have contributed to erratic results observed in field inoculations. At the mechanistic level, nodule C and N metabolisms, and abcissic acid content, were related to SNF potential and tolerance to NaCl. Their relation with nodule conductance to O2 diffusion was addressed by in situ hybridisation of candidate carbonic anhydrase and aquaporin genes in nodule cortex. The limits and prospects of the cooperative strategy are discussed.

Gene Expression Regulation↗
PubMedDate not supplied

[Effect of plant illumination and humidity on nitrogen fixation in the phyllosphere].

The object of this work was to study the effect of illumination and humidity of air on the activity of nitorgenase in epiphytic microorganisms from the phyllosphere of timothy. An increase in humidity and illumination stimulated nitrogen fixation, apparently due to a higher rate of photosynthesis in the plants and of transport of the leaf excretion products.

Humidity↗
PubMed1982

ISRm1: A Rhizobium meliloti insertion sequence that transposes preferentially into nitrogen fixation genes.

After transposon Tn5 mutagenesis, a high proportion of Rhizobium meliloti symbiotic mutants do not contain Tn5 insertions in symbiotic genes. Instead, the mutations in these strains are correlated with the presence of an endogenous insertion sequence (ISRm1) in nitrogen fixation (nif) or symbiotic genes which are adjacent to the nif genes. ISRm1 is 1.4 kb and transposes to at least three restriction fragments in the nif region at a frequency between 10(-2) and 10(-3). A nif region restriction fragment containing ISRm1 was cloned from one of the mutant strains unable to fix nitrogen symbiotically (Fix-) and the resulting plasmid was used as a hybridization probe. ISRm1 is present at least ten times in the R. meliloti genome but is not present in any other R. meliloti strains, E. coli strains, or Rhizobium species tested. We demonstrated that the Fix- phenotype correlated with ISRm1 transposition is indeed caused by ISRm1 insertion by conjugating a cloned fragment containing ISRm1 into a wild type Fix+ R. meliloti host and replacing the normal genomic nif fragment with the nif::ISRm1 fragment. The resulting strain was Fix-.

DNA Transposable Elements↗
PubMed1985

Mapping and expression of a regulatory nitrogen fixation gene (fixD) of Rhizobium meliloti.

A 3.5-kb HindIII fragment from the main nif/fix (nitrogen fixation) gene cluster of Rhizobium meliloti was characterized by studying its expression in Escherichia coli minicells. A coding region for two polypeptides of 68 K and 66 K was mapped using Tn5 insertions and hybrid fusion polypeptides. DNA sequence analysis of this region revealed the presence of an open reading frame capable of coding for a polypeptide of 59.9 K mol. wt. This coding region was designated fixD. Plasmids, constitutively expressing this fixD gene from vector promoters, activated a nifHD-lacZ fusion in E. coli at a low level. Higher levels of activation were obtained following an enhanced expression of the fixD gene in plasmid pRmW541 which was achieved by inducing deletions between the vector promoter and the fixD gene. Sequencing of these deletion mutants showed that, in most cases, fusion polypeptides of the fixD gene product and the aphI (aminoglycoside-3'-phosphotransferase) gene product were sufficient for activation. In E. coli the activation is strictly dependent upon a functional glnF (ntrA) gene.

Journal Article↗
PubMed1979

Physical map of chromosomal nitrogen fixation (nif) genes of Klebsiella pneumoniae.

We describe a method for the rapid determination of the physical location of mutations caused by insertion of transposable elements. We used this method to construct a detailed physical map of the nitrogen fixation (nif) gene cluster of Klebsiella pneumoniae and to correlate it with the genetic map. Total cellular DNA was isolated from individual strains, each carrying an insertion in 1 of 15 different nif genes. The DNA was digested with a restriction endonuclease, fractionated by agarose gel electrophoresis, denatured, and blotted onto nitrocellulose filter paper. The DNA on the filters was hybridized with (32)P-labeled DNA fragments derived from amplifiable plasmids carrying cloned nif DNA fragments from K. pneumoniae. Altered hybridization patterns caused by insertions into nif genes allowed us to map nif mutations with respect to the previously mapped cleavage sites for various restriction endonucleases. We have used the same method to map the end points of nif deletions. Using this procedure, we assigned physical locations on the K. pneumoniae chromosome to 86 nif insertion mutations and 13 nif deletion end points. This mapping procedure provides a convenient alternative to deletion mapping as a definitive method for mapping insertion mutations within a gene or for ordering genes within a gene cluster. This procedure will be especially useful for mapping mutations conferring phenotypes that are difficult to monitor and for mapping mutations in bacterial species in which techniques for conducting deletion mapping have not been devised.

Bacteriophages↗
PubMed2005

Nitrogen fixation by corona discharge on the early precambrian Earth.

We report the first experimental study of nitrogen fixation by corona discharge on the anoxic primitive Earth. The energy yields of nitric oxide (NO) and nitrous oxide (N(2)O) were experimentally determined over a wide range of CO(2)-N(2) mixtures simulating the evolution of the Earth's atmosphere during the Hadean and Archean eras (from 4.5 ba to 2.5 ba). NO, the principal form of fixed nitrogen in lightning and coronal discharge in early Earth, is produced ten times less efficiently in the latter type of electrical discharge with an estimated maximum annual production rate of the order of 10(10) g yr(-1). For N(2)O the maximum production rate was estimated to be approximately 10(9) g yr(-1). These low rates of syntheses indicate that corona discharges as point discharges on the clouds and ground did not play a significant role in the overall pool of reactive nitrogen needed for the emergence and sustainability of life.

Atmosphere↗
PubMed1999

Biological nitrogen fixation and future challenges of agriculture. The endophytic connection.

Feeding the growing global population, anticipated to be 8 billion by the year 2020, is one of the most important recent challenges of agriculture. The increase in cereal grain yield, to cope with this demand, directly implies a dramatic increase in the use of nitrogen-based fertilizers and agrochemicals. Some of these intensive agricultural practices have progressive detrimental effects on the environment. This review is focused on some novel insights gained into the understanding of associative and symbiotic interactions of plants with nitrogen-fixing organisms that makes Biological Nitrogen Fixation (BNF) a viable answer to this compelling dilemma.

Agriculture↗
PubMed1988

Dicarboxylic acid transport in Bradyrhizobium japonicum: use of Rhizobium meliloti dct gene(s) to enhance nitrogen fixation.

A recombinant plasmid encoding Rhizobium meliloti sequences involved in dicarboxylic acid transport (plasmid pRK290:4:46) (E. Bolton, B. Higgisson, A. Harrington, and F. O'Gara, Arch. Microbiol. 144:142-146, 1986) was used to study the relationship between dicarboxylic acid transport and nitrogen fixation in Bradyrhizobium japonicum. The expression of the dct sequences on plasmid pRK290:4:46 in B. japonicum CJ1 resulted in increased growth rates in media containing dicarboxylic acids as the sole source of carbon. In addition, strain CJ1(pRK290:4:46) exhibited enhanced succinate uptake activity when grown on dicarboxylic acids under aerobic conditions. Under free-living nitrogen-fixing conditions, strain CJ1(pRK290:4:46) exhibited higher nitrogenase (acetylene reduction) activity compared with that of the wild-type strain. This increase in nitrogenase activity also correlated with an enhanced dicarboxylic acid uptake rate under these microaerobic conditions. The regulation of dicarboxylic acid transport by factors such as metabolic inhibitors and the presence of additional carbon sources was similar in both the wild-type and the engineered strains. The implications of increasing nitrogenase activity through alterations in the dicarboxylic acid transport system are discussed.

Biological Transport, Active↗
PubMed1993

Homology of pyridoxal-5'-phosphate-dependent aminotransferases with the cobC (cobalamin synthesis), nifS (nitrogen fixation), pabC (p-aminobenzoate synthesis) and malY (abolishing endogenous induction of the maltose system) gene products.

Bacterial deletion mutants have indicated that the gene products of cobC, nifS, pabC and malY participate in important metabolic pathways, i.e. cobalamin synthesis, nitrogen fixation, synthesis of p-aminobenzoate and the regulation of the maltose system, respectively. However, the proteins themselves and their specific functions have not yet been identified. In the course of our studies on the evolutionary relationships among aminotransferases, we have found that the above gene products are homologous to aminotransferases. Profile analysis [Gribskov, M., Lüthy, R. & Eisenberg, D. (1990) Methods Enzymol. 183, 146-159] based on the amino acid sequences of certain subgroups of aminotransferases as probes attributed significant Z scores in the range 5-20 SD to the deduced amino acid sequences of the above gene products as included in the protein data base. Reciprocal profile analyses confirmed the homologies. All known aminotransferases are pyridoxal-5'-phosphate-dependent enzymes and catalyze the reversible transfer of amino groups from amino acids to oxo acids. The sequence homologies suggest that the above gene products are aminotransferases or other closely related pyridoxal-5'-phosphate-dependent enzymes probably catalyzing transformations of amino acids involving cleavage of a bond at C alpha.

4-Aminobenzoic Acid↗
PubMed1991

Aspartate aminotransferase activity is required for aspartate catabolism and symbiotic nitrogen fixation in Rhizobium meliloti.

A mutant of Rhizobium meliloti, 4R3, which is unable to grow on aspartate has been isolated. The defect is specific to aspartate utilization, since 4R3 is not an auxotroph and grows as well as its parent strain on other carbon and nitrogen sources. The defect was correlated with an inability to fix nitrogen within nodules formed on alfalfa. Transport of aspartate into the mutant cells was found to be normal. Analysis of enzymes involved in aspartate catabolism showed a significantly lower level of aspartate aminotransferase activity in cell extracts of 4R3 than in the wild type. Two unrelated regions identified from a genomic cosmid bank each complemented the aspartate catabolism and symbiotic defects in 4R3. One of the cosmids was found to encode an aspartate aminotransferase enzyme and resulted in restoration of aspartate aminotransferase activity in the mutant. Analysis of the region cloned in this cosmid by transposon mutagenesis showed that mutations within this region generate the original mutant phenotypes. The second type of cosmid was found to encode an aromatic aminotransferase enzyme and resulted in highly elevated levels of aromatic aminotransferase activity. This enzyme apparently compensated for the mutation by its ability to partially utilize aspartate as a substrate. These findings demonstrate that R. meliloti contains an aspartate aminotransferase activity required for symbiotic nitrogen fixation and implicate aspartate as an essential substrate for bacteria in the nodule.

Aspartate Aminotransferases↗
PubMed1983

Nitrogen fixation by Klebsiella pneumoniae is inhibited by certain multicopy hybrid nif plasmids.

In our studies of nif gene regulation, we have observed that certain hybrid nif plasmids drastically inhibit the expression of the chromosomal nif genes of Klebsiella pneumonia. Wild-type (Nif+) K. pneumoniae strains that acquire certain hybrid nif plasmids also acquire the Nif- phenotype; these strains lose 90 to 99% of all detectable nitrogen fixation activity and grow poorly (or not at all) on solid media with N2 as the sole nitrogen source. We describe experiments which defined this inhibition of the Nif+ phenotype by hybrid nif plasmids and identify and characterize four nif DNA regions associated with this inhibition. We show that plasmids carrying these nif regions could recombine with, but not complement, nif chromosomal mutations. Our results suggest that inhibition of the Nif+ phenotype will provide a useful bioassay for some of the factors that mediate nif gene expression.

Chromosomes, Bacterial↗