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PubMed1990

The purification, characterization and role of the d-type cytochrome oxidase of Klebsiella pneumoniae during nitrogen fixation.

Klebsiella pneumoniae synthesized only b-type and d-type cytochromes under the wide range of growth conditions tested, and reaction with CO revealed two potential oxidases. The o-type oxidase was produced only in the presence of O2 and appeared to be repressed by glucose. The d-type oxidase was, by contrast, produced only in the absence of measurable O2 (less than 1 microM), and was the only oxidase expressed in nitrogen-fixing conditions. It was extracted from the membrane, purified and shown to be similar to that from E. coli in being a heterodimer (subunits of Mr 52,000 and 35,000), in containing two distinguishable b haems and haem d (one or two molecules per molecule of oxidase), and in being able to react with O2 to give a stable oxygenated intermediate. The purified d-type cytochrome oxidase had a very high affinity for O2 (Km 20 nM; measured by the spectral properties of leghaemoglobin). It is proposed that this provides a role for this oxidase in lowering the O2 concentration to allow nitrogenase synthesis and function, and to provide a terminal oxidase to permit electron-transport-coupled ATP synthesis which supports the increase in efficiency of nitrogen fixation observed under microaerobic conditions.

Amino Acids↗
PubMed2006

Overexpression of BetS, a Sinorhizobium meliloti high-affinity betaine transporter, in bacteroids from Medicago sativa nodules sustains nitrogen fixation during early salt stress adaptation.

Sinorhizobium meliloti possesses several betaine transporters to cope with salt stress, and BetS represents a crucial high-affinity glycine and proline betaine uptake system involved in the rapid acquisition of betaines by cells subjected to osmotic upshock. Using a transcriptional lacZ (beta-galactosidase) fusion, we showed that betS is expressed during the establishment of the symbiosis and in mature nitrogen-fixing nodules. However, neither Nod nor Fix phenotypes were impaired in a betS mutant. BetS is functional in isolated bacteroids, and its activity is strongly activated by high osmolarity. In bacteroids from a betS mutant, glycine betaine and proline betaine uptake was reduced by 85 to 65%, indicating that BetS is a major component of the overall betaine uptake activity in bacteroids in response to osmotic stress. Upon betS overexpression (strain UNA349) in free-living cells, glycine betaine transport was 2.3-fold higher than in the wild-type strain. Interestingly, the accumulation of proline betaine, the endogenous betaine synthesized by alfalfa plants, was 41% higher in UNA349 bacteroids from alfalfa plants subjected to 1 week of salinization (0.3 M NaCl) than in wild-type bacteroids. In parallel, a much better maintenance of nitrogen fixation activity was observed in 7-day-salinized plants nodulated with the overexpressing strain than in wild-type nodulated plants. Taken altogether, these results are consistent with the major role of BetS as an emergency system involved in the rapid uptake of betaines in isolated and in planta osmotically stressed bacteroids of S. meliloti.

Adaptation, Physiological↗
PubMed2005

DNA microarray analysis of nitrogen fixation and Fe(III) reduction in Geobacter sulfurreducens.

A DNA microarray representing the genome of Geobacter sulfurreducens was constructed for use in global gene expression profiling of cells under steady-state conditions with acetate as the electron donor and Fe(III) or fumarate as the electron acceptor. Reproducible differences in transcript levels were also observed in comparisons between cells grown with ammonia and those fixing atmospheric nitrogen. There was a high correlation between changes in transcript levels determined with microarray analyses and an evaluation of a subset of the genome with quantitative PCR. As expected, cells required to fix nitrogen had higher levels of transcripts of genes associated with nitrogen fixation, further demonstrating that the microarray approach could reliably detect important physiological changes. Cells grown with Fe(III) as the electron acceptor had higher levels of transcripts for omcB, a gene coding for an outer membrane c-type cytochrome that is essential for Fe(III) reduction. Several other c-type cytochrome genes also appeared to be up-regulated. An unexpected result was significantly higher levels of transcripts for genes which have a role in metal efflux, potentially suggesting the importance of maintaining metal homeostasis during release of soluble metals when reducing Fe(III). A substantial proportion (30%) of significantly expressed genes during Fe(III) reduction were genes of unknown function or hypothetical proteins, suggesting differences in Fe(III) reduction physiology among microorganisms which perform this metabolic process.

Ferric Compounds↗
PubMed2000

[Improvement of nitrogen fixation efficiency and plasmid stability in Bradyrhizobium japonicum by the introduction of dctABD and parCBA/DE genes].

A recombinant plasmid pHN207 containing C4-dicarboxylic acid transport genes (dctABD) from Sinorhizobium meliloti, parCBA/DE genes from pTR102 and reporter genes luxAB from pDB30 was constructed by using pLAFR3 as the vector. The pHN207 was then introduced into the Bradyrhizobium japonicum TA11 and CB1809 by bi-parental mating. It was confirmed that parCBA/DE genes could increase the stability of pLAFR3 in the transconjugants under both free-living and symbiotic condition. The results of plant pot experiment indicated that the introduction of dctABD genes could significantly improve the symbiotic nitrogen fixation efficiency of TA11 and CB1809 with soybean varieties of Heilong 33, Ningzhen No. 1 and Yudou No. 1. Compared with the control, the shoot dry weight (biomass) and total nitrogen content of the plants tested were significantly increased.

Bacterial Proteins↗
PubMed1991

Regulation of nitrogen fixation in Azorhizobium caulinodans: identification of a fixK-like gene, a positive regulator of nifA.

The nucleotide sequence of a 1 kb fragment upstream of Azorhizobium caulinodans fixL was established. An open reading frame of 744 bp was identified as a fixK homologue. A kanamycin cartridge was inserted into the cloned fixK-like gene and recombined into the host genome. The resulting mutant was Nif-Fix-, suggesting that FixK was required for nitrogen fixation both in symbiotic conditions and in the free-living state. Using a pfixK-lacZ fusion, the FixLJ products were shown to control the expression of fixK. Using a pnifA-lacZ fusion, the FixK product was shown to regulate positively the transcription of nifA in bacteria grown in the free-living state. In addition, a double ntrC-fixL mutant was constructed and was shown to be completely devoid of nitrogenase activity. A model of regulation, based on these data, is presented and might explain the unusual ability of A. caulinodans to fix nitrogen both under symbiotic conditions and in the free-living state.

Amino Acid Sequence↗
PubMed2004

Engineering the nifH promoter region and abolishing poly-beta-hydroxybutyrate accumulation in Rhizobium etli enhance nitrogen fixation in symbiosis with Phaseolus vulgaris.

Rhizobium etli, as well as some other rhizobia, presents nitrogenase reductase (nifH) gene reiterations. Several R. etli strains studied in this laboratory showed a unique organization and contained two complete nifHDK operons (copies a and b) and a truncated nifHD operon (copy c). Expression analysis of lacZ fusion demonstrated that copies a and b in strain CFN42 are transcribed at lower levels than copy c, although this copy has no discernible role during nitrogen fixation. To increase nitrogenase production, we constructed a chimeric nifHDK operon regulated by the strong nifHc promoter sequence and expressed it in symbiosis with the common bean plant (Phaseolus vulgaris), either cloned on a stably inherited plasmid or incorporated into the symbiotic plasmid (pSym). Compared with the wild-type strain, strains with the nitrogenase overexpression construction assayed in greenhouse experiments had, increased nitrogenase activity (58% on average), increased plant weight (32% on average), increased nitrogen content in plants (15% at 32 days postinoculation), and most importantly, higher seed yield (36% on average), higher nitrogen content (25%), and higher nitrogen yield (72% on average) in seeds. Additionally, expression of the chimeric nifHDK operon in a poly-beta-hydroxybutyrate-negative R. etli strain produced an additive effect in enhancing symbiosis. To our knowledge, this is the first report of increased seed yield and nutritional content in the common bean obtained by using only the genetic material already present in Rhizobium.

Gene Expression Regulation, Bacterial↗
PubMed1977

Nitrogen fixation in nitrate reductase-deficient mutants of cultured rhizobia.

Forty-eight mutants unable to reduce nitrate were isolated from "cowpea" Rhizobium sp. strain 32Hl and examined for nitrogenase activity in culture. All but two of the mutants had nitrogenase activity comparable with the parental sttain and two nitrogenase-defective strains showed alterations in their symbiotic properties. One strain was unable to nodulate either Macroptilium atropurpureum or Vigna uguiculata and, with the other, nodules appeared promptly, but effective nitrogen fixation was delayed. These results, and the relatively low proportion of nitrate reductase mutants with impaired nitrogenase activity, do not support the proposed commanality between nitrogenase and nitrate reductase in cowpea rhizobia. Inhibition studies of the effect of nitrate and its reduction products on the nitrogenase activity in cultured strains 32Hl and the nitrate reductase-deficient, Nif+ strains, indicated that nitrogenase activity was sensitive to nitrite rather than to nitrate.

Anaerobiosis↗
PubMed2004

Dark chilling inhibition of photosynthesis and symbiotic nitrogen fixation in soybean during pod filling.

The growth stage of a soybean [Glycine max (L.) Merrill] plant may influence its physiological response to dark chilling. Opposed to vegetative development, the intense nutrient and energy requirements of the developing seeds during pod filling could cause additional chilling damage and decreased recovery capacity. Previously, we investigated dark chilling tolerance during vegetative development in two soybean genotypes, 'Maple Arrow' and 'Fiskeby V' and consistently found that photosynthesis and symbiotic nitrogen fixation (SNF) was less affected by dark chilling in 'Maple Arrow'. In this study we describe the dark chilling response of the same genotypes during pod filling. Our aim was to establish whether the potential selection criteria for dark chilling tolerance, identified during vegetative development, was equally sensitive during pod filling. The results indicate that photosynthesis is less affected by dark chilling in 'Maple Arrow' than in 'Fiskeby V', not only during vegetative development, but also during the critical reproductive stage of pod filling. 'Fiskeby V' also lacks the ability to restore normal photosynthetic capacity during an extended recovery treatment. The decrease of nodule ureide content indicates that SNF was inhibited to a similar extent in both genotypes. Nodule ureide content was reduced more than stem ureide content, suggesting that the former is a more sensitive indicator of chilling stress effects on SNF. The results indicate that certain photosynthetic and fluorescence parameters are sensitive indicators of dark chilling tolerance throughout plant development and should prove valuable in future breeding programmes aimed at increasing the chilling tolerance of soybean.

Carbon Dioxide↗
PubMed2002

Relative efficacy of different distillery effluents on growth, nitrogen fixation and yield of groundnut.

A field experiment with groundnut as test crop was conducted to evaluate the manurial potential of three distillery effluents: raw spent wash (RSW), biomethanated spent wash (BSW) and lagoon sludge (LS) vis-à-vis recommended fertilizers (NPK + farm yard manure (FYM)) and a control (no fertilizer or distillery effluent). It was found that all the three distillery effluents increased total chlorophyll content, crop growth rate (CGR), total dry matter, nutrient uptake (N, P and K) and finally seed yield compared to the control but inhibited nodulation and decreased nitrogen fixation. Among the three distillery effluents, BSW produced the highest seed yield (619 kg ha(-1)) twice that of control (3.10 kg ha(-1)), followed by RSW (557 kg ha(-1)) and LS (472 kg ha(-1)). However, the distillery effluents did not influence protein and oil contents. It was concluded that these distillery effluents because of their high manurial potential could supply nutrients, particularly potassium, nitrogen and sulphur, to the crops and thus reduce the fertilizer requirement of crops. Nevertheless, the crop performance and yield with three distillery effluents were overall less than that produced by recommended NPK + FYM probably on account of failure of the effluents to supply balanced nutrition to the plants for achieving their potential growth capacity.

Fertilizers↗
PubMed1994

The FeSII protein of Azotobacter vinelandii is not essential for aerobic nitrogen fixation, but confers significant protection to oxygen-mediated inactivation of nitrogenase in vitro and in vivo.

The FeSII protein of Azotobacter vinelandii has been proposed to mediate the 'conformational protection' of the molybdenum-dependent nitrogenase components against oxygen inactivation. We have cloned and characterized the structural gene for the FeSII protein (the fesII locus). Hybridization studies did not reveal the presence of fesII-like genes in a number of diverse species of well-studied nitrogen-fixing bacteria, with the exception of Azotobacter chroococcum. The fesII locus is transcriptionally expressed during both nitrogen fixing and non-nitrogen fixing conditions, although the level of its message is upregulated by approximately 2.5-fold during nitrogen fixation. The promoter region was identified by primer extension analysis, and is similar to other sigma 70-type promoters. Mutants devoid of the FeSII protein were constructed. These mutants possessed growth characteristics on a variety of carbon substrates during non-diazotrophic as well as diazotrophic growth that were essentially indistinguishable from the wild-type strain. Nevertheless, the nitrogenase activity in cell-free extracts is significantly more sensitive to irreversible oxygen inactivation in the mutants as compared with the wild type. When treated with 250 mM NaCl (a condition known to dissociate FeSII from nitrogenase components), the wild-type and mutant extracts were equally hypersensitive to oxygen inactivation. Upon energy starvation, conditions in which 'respiratory protection' is inoperable, the MoFe and Fe proteins of nitrogenase are degraded much more rapidly in vivo in the deletion mutants, compared to the wild type. Strains relying on either the vanadium or the 'iron-only' alternative nitrogenases exhibited similar growth rates irrespective of the presence or absence of the FeSII protein, and the in vitro inactivation of the vanadium nitrogenase components was not affected by the lack of the FeSII protein. All in all, these results are consistent with a model whereby 'respiratory protection' is the major physiological mechanism responsible for the protection of all three nitrogenases during energy-supplemented growth. Upon energy starvation, however, 'conformational protection', mediated by the FeSII protein is capable of temporarily protecting the conventional molybdenum nitrogenase components from inactivation and subsequent degradation.

Aerobiosis↗
PubMed2000

Sinorhizobium meliloti nfe (nodulation formation efficiency) genes exhibit temporal and spatial expression patterns similar to those of genes involved in symbiotic nitrogen fixation.

The nfe genes (nfeA, nfeB, and nfeD) are involved in the nodulation efficiency and competitiveness of the Sinorhizobium meliloti strain GR4 on alfalfa roots. The nfeA and nfeB genes are preceded by functional nif consensus sequences and NifA binding motifs. Here, we determined the temporal and spatial expression patterns of the nfe genes in symbiosis with alfalfa. Translational fusions of the nfe promoters with the gusA gene and reverse transcription-polymerase chain reaction analyses indicate that they are expressed and translated within mature nitrogen-fixing nodules and not during early steps of nodule development. Within the nodules the three nfe genes exhibit a spatial expression pattern similar to that of genes involved in symbiotic nitrogen fixation. We show that nfeB and nfeD genes are expressed not only from their own promoters but also from the upstream nfe promoter sequences. Furthermore, with the use of specific antibodies the NfeB and NfeD proteins were detected within the root nodule bacteroid fraction. Finally, NfeB was inmunolocalized in the bacteroid cell membrane whereas NfeD was detected in the bacteroid cytoplasm.

Bacterial Proteins↗
PubMed2004

Associative nitrogen fixation, C4 photosynthesis, and the evolution of spittlebugs (Hemiptera: Cercopidae) as major pests of neotropical sugarcane and forage grasses.

Neotropical grass-feeding spittlebugs of several genera are important pests of pasture grasses from the southeastern USA to northern Argentina, and of sugarcane from southern Mexico to southern Brazil, causing estimated reductions of up to 70% in yield and estimated monetary losses of 840-2100 million US dollars annually. With few exceptions, the species badly damaged by these spittlebugs are introduced C4 grasses that exhibit associative nitrogen fixation. This study synthesizes evidence that the pest status of many tropical and subtropical grass-feeding spittlebugs is linked to associative N-fixation in their C4 hosts. Recognition that associative N-fixation is a major factor in spittlebug host preferences should deepen understanding of spittlebug agricultural ecology and facilitate efforts to combat spittlebug pests. In particular, spittlebugs should be susceptible to manipulation of xylem transport solutes. However, reduction of nitrate fertilizer rates, increase in ammonium fertilizer rates, or enhancement of associative N-fixation as a consequence of genetic engineering could make hosts more susceptible to spittlebug attack. Because of their predilection for C4 grasses, spittlebugs present a clear counterexample to the hypothesis that herbivores prefer C3 plants to C4 plants. Finally, it appears that declines in atmospheric carbon dioxide levels during recent geological history promoted the proliferation of C4 grasses. This, compounded by human agricultural activities, has driven an ecological and evolutionary radiation of grass-feeding spittlebugs that presents continuing opportunities for the evolution of spittlebug pests.

Animals↗
PubMed2002

Host plant nodule parameters associated with nitrogen fixation efficiency in French bean (Phaseolus vulgaris L) cultivars.

Two cultivars of French bean (Phaseolus vulgaris L.) viz. contender and arka komal were planted in polythene bags containing sand and grown under glasshouse conditions. The nodulation status, shoot/root biomass, activities of several nodule enzymes, total soluble protein and leghaemoglobin contents were monitored over the entire growth period. Allantoinase activity in leaves was measured to monitor the ureide degrading capacity. Significant genotype difference was observed in both the cultivars. All the parameters showed a decline after flowering except uricase, which declined before flowering. Malate dehydrogenase and isocitrate dehydrogenase showed a constant decline throughout the growth period. Degree of decline varied with the genotype for all the parameters. Leghaemoglobin content, PEP carboxylase activity and ureide degrading capacity of leaves did not show an appreciable decline in contender and were significantly higher than in arka komal. These factors can be used to increase nitrogen fixation in French bean.

Ammonia↗
PubMed1993

High-resolution mapping of genetic loci of Anabaena PCC 7120 required for photosynthesis and nitrogen fixation.

A physical map of the Anabaena genome permitted the localization of its genes to chromosomal fragments generated by rarely cutting restriction endonucleases and separated by pulsed-field gel electrophoresis. We introduce a novel means of mapping more precisely to c. 20 kb by use of rare restriction sites within vectors bearing cloned sequences that undergo homologous recombination with the genome. We thereby localize and orient genes encoding principal photosynthetic pigments. The relative spacing of loci within a single restriction fragment was determined with even higher resolution, as illustrated for genes required for heterocyst development and nitrogen fixation that were marked with transposons. Small, newly visualized restriction fragments of the chromosome were also mapped.

Anabaena↗
PubMed1980

Regulation of Rhizobium nitrogen fixation by the unadenylylated glutamine synthetase I system.

Rhizobium sp. 32H1 glutamine auxotrophs have a complex phenotype: a highly adenylylated glutamine synthetase [GS; L-glutamate:ammonia ligase (ADP-forming), EC 6.3.1.2] I and an undetectable GS II (GS II(-)). They are defective in the adenylylation cascade system for GS I. Prototrophic revertants are of two classes: those (3204 type) which retain the adenylylated GS I phenotype but become GS II(+), and those (3205 type) which acquire a constitutive unadenylylated GS I but remain GS II(-). Like the parent auxotroph, 3204 remains incapable of nitrogen fixation both in culture and in root nodules of Macroptileum atropurpureum. In contrast, 3205 is nitrogenase constitutive. This implies that GS I or associated adenylylation proteins are involved in the control of Rhizobium 32H1 nif gene expression and that GS II is not so involved. Normally, rhizobia fix atmospheric N(2) only during symbiosis and, in so doing, only transiently synthesize nitrogenase. Moreover, whereas wild-type strains export ammonium, constitutive strains can assimilate ammonium produced by nitrogenase. This phenotype allows direct selection of nitrogen fixation-defective mutants in Rhizobium.

Journal Article↗
PubMed1987

Isolation and characterization of symbiotic mutants of bradyrhizobium sp. (Arachis) strain NC92: mutants with host-specific defects in nodulation and nitrogen fixation.

Random transposon Tn5 mutagenesis of Bradyrhizobium sp. (Arachis) strain NC92, a member of the cowpea cross-inoculation group, was carried out, and kanamycin-resistant transconjugants were tested for their symbiotic phenotype on three host plants: groundnut, siratro, and pigeonpea. Two nodulation (Nod- phenotype) mutants were isolated. One is unable to nodulate all three hosts and appears to contain an insertion in one of the common nodulation genes (nodABCD); the other is a host-specific nodulation mutant that fails to nodulate pigeonpea, elicits uninvaded nodules on siratro, and elicits normal, nitrogen-fixing nodules on groundnut. In addition, nine mutants defective in nitrogen fixation (Fix- phenotype) were isolated. Three fail to supply symbiotically fixed nitrogen to all three host plants. Surprisingly, nodules elicited by one of these mutants exhibit high levels of acetylene reduction activity, demonstrating the presence of the enzyme nitrogenase. Three more mutants have partially effective phenotypes (Fix +/-) in symbiosis with all three host plants. The remaining three mutants fail to supply fixed nitrogen to one of the host plants tested while remaining partially or fully effective on the other two hosts; two of these mutants are Fix- in pigeonpea and Fix +/- on groundnut and on siratro, whereas the other one is Fix- on groundnut but Fix+ on siratro and on pigeonpea. These latter mutants also retain significant nodule acetylene reduction activity, even in the ineffective symbioses. Such bacterial host-specific fixation (Hsf) mutants have not previously been reported.

DNA Transposable Elements↗
PubMed2007

Symbiotic nitrogen fixation in a tropical rainforest: 15N natural abundance measurements supported by experimental isotopic enrichment.

* Leguminous trees are very common in the tropical rainforests of Guyana. Here, species-specific differences in N(2) fixation capability among nodulating legumes growing on different soils and a possible limitation of N(2) fixation by a relatively high nitrogen (N) and low phosphorus (P) availability in the forest were investigated. * Leaves of 17 nodulating species and 17 non-nodulating reference trees were sampled and their delta(15)N values measured. Estimates of N(2) fixation rates were calculated using the (15)N natural abundance method. Pot experiments were conducted on the effect of N and P availability on N(2) fixation using the (15)N-enriched isotope dilution method. * Nine species showed estimates of > 33% leaf N derived from N(2) fixation, while the others had low or undetectable N(2) fixation rates. High N and low P availability reduced N(2) fixation substantially. * The results suggest that a high N and low P availability in the forest limit N(2) fixation. At the forest ecosystem level, N(2) fixation was estimated at c. 6% of total N uptake by the tree community. We conclude that symbiotic N(2) fixation plays an important role in maintaining high amounts of soil available N in undisturbed forest.

Ecosystem↗