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PubMed1978

Pesticide (hexachlorocyclohexane) inhibition of growth and nitrogen fixation in blue-green algae Anabaenopsis raciborskii and Anabaena aphanizomenoides.

The effects of the pesticide hexachlorocyclohexane (HCH) on the nitrogen fixing blue-green algae Anabaenopsis raciborskii and Anabaena aphanizomenoides commonly found as blooms in fish ponds were studied. These algae were very sensitive to HCH, and a distinct decrease in growth rate was observed on prolonged incubation. Lower concentrations (10 microgram/ml) were algistatic and higher concentrations (60 microgram/ml) were algicidal. The inhibition of nitrogen fixation indicated that the presence of HCH might affect overall nitrogen economy of inland waters.

Cyanobacteria↗
PubMed1996

Azotobacter vinelandii NIFL is a flavoprotein that modulates transcriptional activation of nitrogen-fixation genes via a redox-sensitive switch.

The NIFL regulatory protein controls transcriptional activation of nitrogen fixation (nif) genes in Azotobacter vinelandii by direct interaction with the enhancer binding protein NIFA. Modulation of NIFA activity by NIFL, in vivo occurs in response to external oxygen concentration or the level of fixed nitrogen. Spectral features of purified NIFL and chromatographic analysis indicate that it is a flavoprotein with FAD as the prosthetic group, which undergoes reduction in the presence of sodium dithionite. Under anaerobic conditions, the oxidized form of NIFL inhibits transcriptional activation by NIFA in vitro, and this inhibition is reversed when NIFL is in the reduced form. Hence NIFL is a redox-sensitive regulatory protein and may represent a type of flavoprotein in which electron transfer is not coupled to an obvious catalytic activity. In addition to its ability to act as a redox sensor, the activity of NIFL is also responsive to adenosine nucleotides, particularly ADP. This response overrides the influence of redox status on NIFL and is also observed with refolded NIFL apoprotein, which lacks the flavin moiety. These observations suggest that both energy and redox status are important determinants of nif gene regulation in vivo.

Adenosine Diphosphate↗
PubMed1990

Characterisation of the Klebsiella pneumoniae nitrogen-fixation regulatory proteins NIFA and NIFL in vitro.

Transcriptional activation by the Klebsiella pneumoniae nitrogen-fixation-specific positive control protein, NIFA, (nifA gene product) has been demonstrated in vitro in S30 extracts from cells which overproduce this protein. The activity of NIFA was dramatically reduced in vitro in the presence of the negative regulatory protein NIFL (nifL gene product) but was not inhibited by the presence of a mutant NIFL protein, NIFL2404. Transcriptional activation from the nifH promoter by NIFA was dependent on the alternative sigma factor, sigma 54, and also required the presence of an upstream activator sequence. NIFA activity was temperature-sensitive in vitro (as it is in vivo) which is due, at least in part, to the intrinsic lability of the protein itself. The majority of overproduced NIFA and NIFL was insoluble after low-speed centrifugation and was inactive in vitro. A low level of less aggregated NIFA protein present in cell extracts was responsible for in vitro activity and this fraction was partially purified.

Bacterial Proteins↗
PubMed1981

Complementation analysis of glnA-linked mutations which affect nitrogen fixation in Klebsiella pneumoniae.

A number of mutants have been isolated which affect regulation of the nitrogen fixation (nif) gene cluster in Klebsiella pneumoniae and all of which are linked to glnA, the structural gene for glutamine synthetase (G.S.). These mutants were classified on the basis of their G.S. and nitrogenase activities in conditions of nitrogen limitation and excess. The plasmid R68.45 was then used to generate a number of R-primes carrying the glnA region of the K. pneumoniae chromosome. One of these R-primes (pGE10) was subsequently used in complementation analysis and by isolation of transposon-induced insertion mutations in pGE10 we have demonstrated the existence of a gene, glnG, closely linked to glnA. Mutations in glnG have a similar phenotype to glnG mutants described in Escherichia coli (Pahel and Tyler 1979) and Salmonella typhimurium (Kustu et al. 1979) in that substantially reduce G.S. activity but are not glutamine auxotrophs. GlnG mutants have very low nitrogenase activity indicating that the glnG product may be involved regulation of the nif gene cluster in K. pneumoniae.

Genes↗
PubMed1965

PYRUVATE METABOLISM, CARBON DIOXIDE ASSIMILATION, AND NITROGEN FIXATION BY AN ACHROMOBACTER SPECIES.

Hamilton, I. R. (University of Wisconsin, Madison), R. H. Burris, P. W. Wilson, and C. H. Wang. Pyruvate metabolism and carbon dioxide assimilation by an Achromobacter species. J. Bacteriol. 89:647-653. 1965.-Carbon dioxide fixation by washed whole cells of Achromobacter N4-B has been observed during anaerobic pyruvate metabolism with both nitrogen- and NH(4) (+)-grown cells. Labeled sodium bicarbonate-C(14) was assimilated into cells by a mechanism requiring pyruvate under conditions of nitrogen fixation, nitrogenase induction, and assimilation of NH(4) (+). Of the assimilated radioactivity, 89% appeared in six amino acids and two ninhydrin-positive unknown compounds, with the distribution of the label essentially independent of the nitrogen nutritional state of the organism. Aspartic and glutamic acids were the most highly labeled, with lesser amounts in glycine, alanine, ornithine, arginine, and the unknowns. All of the radioactivity extracted from these cells by ethanol-boiling water appeared in a protein fraction precipitated by 20% trichloroacetic acid. Radiorespirometric experiments with individually labeled pyruvate substrates demonstrated the preferential decarboxylation of the C-1 of pyruvate by this organism in a flowing helium gas phase. This decarboxylation was almost completely inhibited by using flowing nitrogen in place of helium; the addition of 0.5% CO(2) to the flowing nitrogen prevented inhibition and allowed 70% of the expected CO(2) evolution. These results, coupled with those from growth experiments, indicate a carbon dioxide requirement for anaerobic growth and pyruvate metabolism, which appears to be coupled to the formation of protein precursors.

Achromobacter↗
PubMed1982

Genetic analysis of nitrogen fixation in a tropical fast-growing Rhizobium.

The Rhizobium strain ORS571, which is associated with the tropical legume Sesbania rostrata, has the property of growing in the free-living state at the expense of ammonia or N(2) as sole nitrogen source. Five mutants, isolated as unable to form colonies on plates under conditions of nitrogen fixation, were studied. All of them, which appear as Fixin planta, are nif mutants. With mutant 5740, nitrogenase activity of the crude extract was restored by addition of pure Mo-Fe protein of Klebsiella pneumoniae. A 13-kb BamHI DNA fragment from the wild-type strain, which hybridized with a probe carrying the nifHDK genes of K. pneumoniae, was cloned in vector pRK290 to yield plasmid pRS1. The extent of homology between the probe and the BamHI fragment was estimated at 4 kb and hybridization with K. pneumoniae nifH, nifK, and possibly nifD was detected. The pRS1 plasmid was introduced into the sesbania rhizobium nif mutants. Genetic complementation was observed with strain 5740(pRS1) both in the free-living state and in planta. It thus appears that biochemistry and genetics of nitrogen fixation in this particular Rhizobium strain can be performed with bacteria grown under non-symbiotic conditions.

Journal Article↗
PubMed2000

The Rhizobium leguminosarum bv. viciae glnD gene, encoding a uridylyltransferase/uridylyl-removing enzyme, is expressed in the root nodule but is not essential for nitrogen fixation.

A Rhizobium leguminosarum bv. viciae VF39 gene (glnD) encoding the uridylyltransferase/uridylyl-removing enzyme, which constitutes the sensory component of the nitrogen regulation (ntr) system, was identified, cloned and characterized. The deduced amino acid sequence contains the conserved active site motif of the nucleotidyltransferase superfamily and is highly homologous to the glnD gene products of other bacterial species. Downstream of the VF39 glnD resides an open reading frame with similarity to the Salmonella typhimurium virulence factor gene mviN. Mutation of the glnD gene abolished the ability to use nitrate as a sole nitrogen source but not glutamine. In addition, neither uridylylation of P(II) nor induction of the ntr-regulated glnII gene (encoding glutamine synthetase II) under ammonium deficiency could be observed in mutant strains. This strongly suggests that glnD mutants harbour a permanently deuridylylated P(II) protein and as a consequence are unable to activate transcription from NtrC-dependent promoters. The glnD gene itself is expressed constitutively, irrespective of the nitrogen content of the medium. A functional GlnD protein is not essential for nitrogen fixation in R. leguminosarum bv. viciae, but in situ detection of glnD expression in the symbiotic and infection zone of the root nodule and quantitative measurements suggest that at least part of the ntr system functions in symbiosis. The results also indicate that the N-terminal part of GlnD is essential for the cell, as deletions in the 5'-region of the gene appear to be lethal and mutations possibly affecting the expression of the first half of the protein have a significant effect on the vitality of the mutant strain.

Bacterial Proteins↗
PubMed1987

Identification and characterization of the Rhizobium meliloti ntrC gene: R. meliloti has separate regulatory pathways for activation of nitrogen fixation genes in free-living and symbiotic cells.

We show here that Rhizobium meliloti, the nitrogen-fixing endosymbiont of alfalfa (Medicago sativa), has a regulatory gene that is structurally homologous to previously characterized ntrC genes in enteric bacteria. DNA sequence analysis showed that R. meliloti ntrC is homologous to previously sequenced ntrC genes from Klebsiella pneumoniae and Bradyrhizobium sp. (Parasponia) and that an ntrB-like gene is situated directly upstream from R. meliloti ntrC. Similar to its counterparts in K. pneumoniae and Escherichia coli, R. meliloti ntrC is expressed when the cells are grown in nitrogen-limiting media. In addition, R. meliloti ntrC is required for growth on media containing nitrate as the sole nitrogen source and for the ex planta transcription of several R. meliloti nif genes. On the other hand, root nodules elicited by R. meliloti ntrC mutants fix nitrogen as well as nodules elicited by wild-type R. meliloti. These latter results indicate that R. meliloti has separate regulatory pathways for activating nif gene expression ex planta and during symbiotic nitrogen fixation.

Base Sequence↗
PubMed2003

Heterocyst-specific expression of patB, a gene required for nitrogen fixation in Anabaena sp. strain PCC 7120.

The patB gene product is required for growth and survival of the filamentous cyanobacterium Anabaena sp. strain PCC 7120 in the absence of combined nitrogen. A patB::gfp fusion demonstrated that this gene is expressed exclusively in heterocysts. patB mutants have a normal initial pattern of heterocyst spacing along the filament but differentiate excess heterocysts after several days in the absence of combined nitrogen. Expression of hetR and patS, two critical regulators of the heterocyst development cascade, are normal for patB mutants, indicating that patB acts downstream of them in the differentiation pathway. A patB deletion mutant suffers an almost complete cessation of growth and nitrogen fixation within 24 h of combined nitrogen removal. In contrast, a new PatB mutant that is defective in its N-terminal ferredoxin domain, or a previously described mutant that has a frameshift removing its C-terminal helix-turn-helix domain, grows very slowly and differentiates multiple contiguous heterocysts under nitrogen-deficient conditions.

Anabaena↗
PubMed1979

Temperature control of nitrogen fixation in Klebsiella pneumoniae.

At growth temperatures above 37 degrees C, Klebsiella pneumoniae does not grow in a medium containing N2 or NO3- as nitrogen sources. However, both the growth in the presence of other nitrogen sources as well as the in vitro nitrogenase activity are not affected at this temperature. The inability to fix N2 at high temperature is due to the failure of the cells to synthesize nitrogenase and other nitrogen fixation (nif) gene encoded proteins. When cells grown under nitrogen fixing conditions at 30 degrees C were shifted to 39 degrees C, there was a rapid decrease of the rate of de novo biosynthesis of nitrogenase (component 1), nitrogenase reductase (component 2), and the nifJ gene product. There was no degradation of nitrogenase at the elevated temperature since preformed enzyme remained stable over a period of at least 3 h at 39 degrees C. Thus, temperature seems to represent a third control system, besides NH4+ and O2, governing the expression of nif genes of K. pneumoniae.

Klebsiella pneumoniae↗
PubMed2002

Iron and marine nitrogen fixation: progress and future directions.

A synthesis of the current understanding of potential iron limitation of pelagic nitrogen fixation is given, considering biochemical bases of Fe requirements and empirical observations of growth and Fe quotas of cultures and field populations of Trichodesmium. The potential for iron limitation of heterotrophic diazotrophy in the marine environment is also evaluated.

Cyanobacteria↗
PubMed1996

A modular domain of NifU, a nitrogen fixation cluster protein, is highly conserved in evolution.

hnifU, a gene exhibiting similarity to nifU genes of nitrogen fixation gene clusters, was identified in the course of expressed sequence tag (EST) generation from a human fetal heart cDNA library. Northern blot of human tissues and polymerase chain reaction (PCR) using human genomic DNA verified that the hnifU gene represented a human gene rather than a microbial contaminant of the cDNA library. Conceptual translation of the hnifU cDNA yielded a protein product bearing 77% and 70% amino acid identity to NifU-like hypothetical proteins from Haemophilus influenzae and Saccharomyces cerevisiae, respectively, and 40-44% identity to the N-terminal regions of NifU proteins from several diazatrophs (i.e., nitrogen-fixing organisms). Pairwise determination of amino acid identities between the NifU-like proteins of nondiazatrophs showed that these NifU-like proteins exhibited higher sequence identity to each other (63-77%) than to the diazatrophic NifU proteins (40-48%). Further, the NifU-like proteins of non-nitrogen-fixing organisms were similar only to the N-terminal region of diazatrophic NifU proteins and therefore identified a novel modular domain in these NifU proteins. These findings support the hypothesis that NifU is indeed a modular protein. The high degree of sequence similarity between NifU-like proteins from species as divergent as humans and H. influenzae suggests that these proteins perform some basic cellular function and may be among the most highly conserved proteins.

Amino Acid Sequence↗
PubMed2005

History on the biological nitrogen fixation research in graminaceous plants: special emphasis on the Brazilian experience.

This review covers the history on Biological Nitrogen Fixation (BNF) in Graminaceous plants grown in Brazil, and describes research progress made over the last 40 years, most of which was coordinated by Johanna Döbereiner. One notable accomplishment during this period was the discovery of several nitrogen-fixing bacteria such as the rhizospheric (Beijerinckia fluminensis and Azotobacter paspali), associative (Azospirillum lipoferum, A. brasilense, A. amazonense) and the endophytic (Herbaspirillum seropedicae, H. rubrisubalbicans, Gluconacetobacter diazotrophicus, Burkholderia brasilensis and B. tropica). The role of these diazotrophs in association with grasses, mainly with cereal plants, has been studied and a lot of progress has been achieved in the ecological, physiological, biochemical, and genetic aspects. The mechanisms of colonization and infection of the plant tissues are better understood, and the BNF contribution to the soil/plant system has been determined. Inoculation studies with diazotrophs showed that endophytic bacteria have a much higher BNF contribution potential than associative diazotrophs. In addition, it was found that the plant genotype influences the plant/bacteria association. Recent data suggest that more studies should be conducted on the endophytic association to strengthen the BNF potential. The ongoing genome sequencing programs: RIOGENE (Gluconacetobacter diazotrophicus) and GENOPAR (Herbaspirillum seropedicae) reflect the commitment to the BNF study in Brazil and should allow the country to continue in the forefront of research related to the BNF process in Graminaceous plants.

Brazil↗
PubMed1977

Energetics of biological nitrogen fixation: determination of the ratio of formation of H2 to NH4+ catalysed by nitrogenase of Klebsiella pneumoniae in vivo.

Nitrogen fixation (Nif)-derepressed mutants of Klebsiella pneumoniae consumed, under optimum conditions, 7.5 to 8.5 mol glucose per mol N2 fixed. The nitrogenase system of these mutants catalysed the production of about 1.3 mol H2 per mol N2 reduced. Almost one-third of the energy as ATP and reductant used by nitrogenase in vivo may be lost in H2 production, since an ATP/2e ratio of approximately 4 was obtained. Nitrogenase-catalysed H2 production was not substantially suppressed by increasing the partial pressure of N2 from 0.2 atm (20 kPa) to 1 atm (101 kPa). In the absence of N2, H2 production catalysed by nitrogenase increased about threefold. It is concluded that nitrogenase-catalysed H2 production is of major importance in the overall efficiency of biological N2 fixation in vivo.

Catalysis↗
PubMed2006

Cloning and characterization of a novel gene involved in nitrogen fixation in Heliobacterium chlorum: a possible regulatory gene.

In the present study, the transcriptional properties of the nitrogen fixation gene cluster of Hbt. chlorum, a strictly anaerobic, gram-positive, phototrophic bacterium, were explored. The cluster consisted of eleven genes in the same orientation in the order nifI ( 1 ) , nifI ( 2 ) , nifH, nifD, nifK, nifE, nifN, nifX, fdx, nifB, and nifV as detected previously. An open reading frame (orf1) preceding these genes was revealed by further cloning. The orf1 was co-transcribed with downstream nif genes in a single polycistronic transcript, the transcription start site (TSS) was located upstream of the orf1, and a putative promoter was identified 10 bp preceding the TSS. Unlike most diazotrophs which have a sigma(54)-dependent -24/-12 promoter, the promoter was similar to the -35/-10 E. coli promoter. The orf1 had no nif homolog in DNA databases, and the highest level of identity (27% at amino acid level) was found with hutP, a positive regulatory gene of the histidine utilization (hut) operon in B. subtilis. Analogous to the regulatory mechanism of the hut operon in B. subtilis, it is conceivable that the orf1 product interacts with the terminator-like structure located downstream of the orf1 during N-deficient condition and prevents transcription termination; thus, the transcription continues into the nif structural genes.

Amino Acid Sequence↗
PubMed2002

Novel aspects of symbiotic nitrogen fixation uncovered by transcript profiling with cDNA arrays.

An array of 2,304 cDNA clones derived from nitrogen-fixing nodules of Lotus japonicus was produced and used to detect differences in relative gene transcript abundance between nodules and uninfected roots. Transcripts of 83 different genes were found to be more abundant in nodules than in roots. More than 50 of these have never before been identified as nodule-induced in any species. Expression of 36 genes was detected in nodules but not in roots. Several known nodulin genes were included among the nodule-induced genes. Also included were genes involved in sucrose breakdown and glycolysis, CO2 recycling, and amino acid synthesis, processes that are known to be accelerated in nodules compared with roots. Genes involved in membrane transport, hormone metabolism, cell wall and protein synthesis, and signal transduction and regulation of transcription were also induced in nodules. Genes that may subvert normal plant defense responses, including two encoding enzymes involved in detoxification of active oxygen species and one that may prohibit phytoalexin synthesis, were also identified. The data represent a rich source of information for hypothesis building and future exploration of symbiotic nitrogen fixation.

Cloning, Molecular↗
PubMedDate not supplied

[Characteristics of nitrogen fixation and denitrification in Termites Neotermes castaneus, Zootermopsis angusticollis, and Reticulitermes lucifugus].

We studied specific features of microbial nitrogen fixation and denitrification in laboratory cultures of the termites Neotermes castaneus, Zootermopsis angusticollis, and Reticulitermes lucifugus, as well as in their nest materials. The nitrogenase activity in the termites was much higher than in the materials of termitarium. Denitrification was found only in the nest materials of termitarium. Studies of the bacterial community of gut nitrogen fixers Neotermes castaneus have shown the predominance of anaerobic and facultatively anaerobic bacteria that amount to up to 60% of the total number of "gut" bacteria. In the materials of termitarium, aerobic cellulose-destroying myxobacteria predominated, which are typical inhabitants of plant substrates, a food for the termite.

Animals↗