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PubMed1984

Reiteration of genes involved in symbiotic nitrogen fixation by fast-growing Rhizobium japonicum.

By using cloned Rhizobium meliloti nodulation (nod) genes and nitrogen fixation (nif) genes, we found that the genes for both nodulation and nitrogen fixation were on a plasmid present in fast-growing Rhizobium japonicum strains. Two EcoRI restriction fragments from a plasmid of fast-growing R. japonicum hybridized with nif structural genes of R. meliloti, and three EcoRI restriction fragments hybridized with the nod clone of R. meliloti. Cross-hybridization between the hybridizing fragments revealed a reiteration of nod and nif DNA sequences in fast-growing R. japonicum. Both nif structural genes D and H were present on 4.2- and 4.9-kilobase EcoRI fragments, whereas nifK was present only on the 4.2-kilobase EcoR2 fragment. These results suggest that the nif gene organizations in fast-growing and in slow-growing R. japonicum strains are different.

Base Sequence↗
PubMed1969

Nitrogen fixation by Rhodospirillum rubrum grown in nitrogen-limited continuous culture.

Cell-free extracts of the photosynthetic bacterium Rhodospirillum rubrum were inconsistent in reducing N(2). An internally illuminated fermentor, designed for the continuous culture of this organism on N(2) under nitrogen-limited conditions, produced cells which yielded cell extracts with consistent activity for cell-free N(2) fixation. A nitrogen-limited continuous culture, supplied ammonia rather than N(2), gave cell-free extracts with even more active N(2) fixation. Extracts of cells grown in the fermentor with glutamate nitrogen as the limiting nutrient in continuous culture did not reduce N(2), but whole cells fixed (15)N-enriched N(2). The discovery that cells from ammonia and glutamate nitrogen-limited continuous cultures are capable of N(2) reduction suggests that R. rubrum cells produce the N(2)-reducing enzymes in response to conditions of nitrogen deficiency rather than in response to the presence of N(2). Examination of the effect of the pN(2) on N(2) reduction by cell-free preparations of R. rubrum indicated that the K(N(2)) is approximately 0.071 atm. Cell-free extracts from R. rubrum were tested for their ability to reduce substrates other than N(2).

Ammonia↗
PubMed1965

NITROGEN FIXATION BY MEMBERS OF THE TRIBE KLEBSIELLEAE.

Mahl, M. C. (University of Wisconsin, Madison), P. W. Wilson, M. A. Fife, and W. H. Ewing. Nitrogen fixation by members of the tribe Klebsielleae. J. Bacteriol. 89:1482-1487. 1965.-Strains of species of the tribe Klebsielleae were tested for nitrogen fixation by inoculating actively growing cultures into side-arm flasks containing a medium with a growth-limiting quantity of combined nitrogen (25 mug of nitrogen per ml as ammonium sulfate). The flasks were evacuated, filled with pure N(2), sealed, and placed on a shaker at 30 C. Growth was followed by optical-density measurements; maximal growth was obtained in 9 to 10 hr. Yeast extract was then added as a source of amino acids to shorten the induction time for the nitrogen-fixing enzymes. Fixation was determined either by estimating total nitrogen with a semimicro Kjeldahl technique or by exposing 20- to 22-hr-old samples to an atmosphere of N(2) (15) and helium for 5 hr and then analyzing the digested sample for N(15) in a mass spectrometer. None of the 22 strains of the two Enterobacter (formerly Aerobacter) species fixed nitrogen; neither did any of eight strains of Serratia species that were tested. Of 31 strains of Klebsiella pneumoniae, 13 incorporated atmospheric nitrogen. Net nitrogen fixed ranged from 17 to 65 mug/ml. It is concluded that these facultative anaerobic, gram-negative, nitrogen-fixing rods should be placed in the genus Klebsiella pneumoniae. The nitrogen-fixing organism tentatively classified as Achromobacter N-4 should also be changed to K. pneumoniae strain N-4, because it has been found to be an anaerogenic strain of K. pneumoniae.

Achromobacter↗
PubMed1999

Increase in alfalfa nodulation, nitrogen fixation, and plant growth by specific DNA amplification in Sinorhizobium meliloti.

To improve symbiotic nitrogen fixation on alfalfa plants, Sinorhizobium meliloti strains containing different average copy numbers of a symbiotic DNA region were constructed by specific DNA amplification (SDA). A DNA fragment containing a regulatory gene (nodD1), the common nodulation genes (nodABC), and an operon essential for nitrogen fixation (nifN) from the nod regulon region of the symbiotic plasmid pSyma of S. meliloti was cloned into a plasmid unable to replicate in this organism. The plasmid then was integrated into the homologous DNA region of S. meliloti strains 41 and 1021, which resulted in a duplication of the symbiotic region. Sinorhizobium derivatives carrying further amplification were selected by growing the bacteria in increased concentrations of an antibiotic marker present in the integrated vector. Derivatives of strain 41 containing averages of 3 and 6 copies and a derivative of strain 1021 containing an average of 2.5 copies of the symbiotic region were obtained. In addition, the same region was introduced into both strains as a multicopy plasmid, yielding derivatives with an average of seven copies per cell. Nodulation, nitrogenase activity, plant nitrogen content, and plant growth were analyzed in alfalfa plants inoculated with the different strains. The copy number of the symbiotic region was critical in determining the plant phenotype. In the case of the strains with a moderate increase in copy number, symbiotic properties were improved significantly. The inoculation of alfalfa with these strains resulted in an enhancement of plant growth.

Blotting, Southern↗
PubMed1977

The effect of 2,4-dichlorophenoxyacetic acid on growth and nitrogen-fixation of blue-green alga Anabaenopsis raciborskii.

Sodium salt of 2,4-dichlorophenoxyacetic acid (80% active ingredient), commonly applied for the control of aquatic weeds, was used to observe its effect on the growth and nitrogen fixation of a heterocystous bloom forming blue-green alga Anabaenopsis raciborskii. A concentration of 10 microgram per ml of 2,4-D showed stimulation of growth and nitrogen fixation and these were almost unaffected in presence of its 100 microgram per ml in the medium. The alga could tolerate up to 800 microgram per ml in liquid culture media with and without nitrate nitrogen and up to 90 microgram per ml on to agar plates. Nitrogen fixation was inhibited in presence of its higher concentrations.

2,4-Dichlorophenoxyacetic Acid↗
PubMed2001

Segregation of nitrogen fixation and oxygenic photosynthesis in the marine cyanobacterium Trichodesmium.

In the modern ocean, a significant amount of nitrogen fixation is attributed to filamentous, nonheterocystous cyanobacteria of the genus Trichodesmium. In these organisms, nitrogen fixation is confined to the photoperiod and occurs simultaneously with oxygenic photosynthesis. Nitrogenase, the enzyme responsible for biological N2 fixation, is irreversibly inhibited by oxygen in vitro. How nitrogenase is protected from damage by photosynthetically produced O2 was once an enigma. Using fast repetition rate fluorometry and fluorescence kinetic microscopy, we show that there is both temporal and spatial segregation of N2 fixation and photosynthesis within the photoperiod. Linear photosynthetic electron transport protects nitrogenase by reducing photosynthetically evolved O2 in photosystem I (PSI). We postulate that in the early evolutionary phase of oxygenic photosynthesis, nitrogenase served as an electron acceptor for anaerobic heterotrophic metabolism and that PSI was favored by selection because it provided a micro-anaerobic environment for N2 fixation in cyanobacteria.

Aerobiosis↗
PubMed2006

Effect of composted textile sludge on growth, nodulation and nitrogen fixation of soybean and cowpea.

The effect of composted textile sludge on growth, nodulation and nitrogen fixation of soybean and cowpea was evaluated in a greenhouse experiment. The compost was incorporated into soil at 0, 9.5, 19 and 38 t ha(-1) (bases upon the N requirement of the crops, i.e., 0, 50, 100 and 200 kg available N ha(-1)). Growth, nodulation and shoot accumulation of nitrogen were evaluated 36 and 63 days after plant emergence. Nodule glutamine synthetase (GS) activity and leghemoglobin content were evaluated 63 days after emergence. Composted textile sludge did not show negative effects on nodule number and weight, nodule GS activity and leghemoglobin content. Nitrogen accumulation in shoot dry matter in soybean and cowpea was higher than other treatments with application of 19 t ha(-1) of compost. Composting can be an alternate technology for the management of solid textile mill sludge. This study verifies that the composted textile sludge was not harmful to growth, nodulation and nitrogen fixation of soybean and cowpea.

Fabaceae↗
PubMed1982

Nodule and Leaf Nitrate Reductases and Nitrogen Fixation in Medicago sativa L. under Water Stress.

The effect of water stress on patterns of nitrate reductase activity in the leaves and nodules and on nitrogen fixation were investigated in Medicago sativa L. plants watered 1 week before drought with or without NO(3) (-). Nitrogen fixation was decreased by water stress and also inhibited strongly by the presence of NO(3) (-). During drought, leaf nitrate reductase activity (NRA) decreased significantly particularly in plants watered with NO(3) (-), while with rewatering, leaf NRA recovery was quite important especially in the NO(3) (-)-watered plants. As water stress progressed, the nodular NRA increased both in plants watered with NO(3) (-) and in those without NO(3) (-) contrary to the behavior of the leaves. Beyond -15.10(5) pascal, nodular NRA began to decrease in plants watered with NO(3) (-). This phenomenon was not observed in nodules of plants given water only.Upon rewatering, it was observed that in plants watered with NO(3) (-) the nodular NRA increased again, while in plants watered but not given NO(3) (-), such activity began to decrease. Nitrogen fixation increased only in plants without NO(3) (-).

Journal Article↗
PubMed1980

Nitrate Effect on Nitrogen Fixation (Acetylene Reduction): ACTIVITIES OF LEGUME ROOT NODULES INDUCED BY RHIZOBIA WITH VARIED NITRATE REDUCTASE ACTIVITIES.

The effect of nitrate on symbiotic nitrogen fixation by root nodules of cowpea (Vigna unguiculata L., Walp., cv. California Blackeye) and lupine (Lupinus augustifolius L., cv. Frost) plants inoculated with nitrate reductase-expressing and nitrate reductase-nonexpressing Rhizobium strains were examined. Nitrate reductase of Rhizobium bacteroids in the nodules of cowpea and lupine reduced nitrate to nitrite. Both cowpea and lupine nodules accumulated nitrite when grown in the presence of 15 millimolar nitrate and induced by Rhizobium strains which express nitrate reductase activity (Rhizobium sp. 32H1 and 127E15). The nitrogen fixation (acetylene reduction) activities of cowpea and lupine nodules were inhibited by nitrate whether the nodules were induced by Rhizobium strains that express (Rhizobium sp. 32H1 and 127E15) or do not express (Rhizobium sp. 127E14 and R. lupini ATCC 10318) nitrate reductase activity. These findings indicate that nitrite, the product of bacteroid nitrate reductase, may not play a role in the inhibitory effect of nitrate on nitrogen fixation activities of legume root nodules. However, the degree of inhibition on the fixation activity by nitrate varied in different legume-Rhizobium combinations.

Journal Article↗
PubMed1991

Involvement of fixLJ in the regulation of nitrogen fixation in Azorhizobium caulinodans.

A gene bank of Azorhizobium caulinodans DNA constructed in the bacteriophage lambda GEM11 was screened with Rhizobium meliloti fixL and fixJ genes as probes. One positive recombinant phage, ORS lambda L, was isolated. The nucleotide sequence of a 3.7 kb fragment was established. Two open reading frames of 1512bp and 613bp were identified as fixL and fixJ. Kanamycin cartridges were inserted into the cloned fixL and fixJ genes and recombined into the host genome. The resulting mutants were Nif- Fix-, suggesting that the two genes were required for symbiotic nitrogen fixation and for nitrogen fixation in the free-living state. Using pnifH-lacZ and pnifA-lacZ fusions, it was shown that the FixLJ products controlled the expression of nifH and nifA in bacteria grown in the free-living state.

Amino Acid Sequence↗
PubMed1990

Ca requirement for aerobic nitrogen fixation by heterocystous blue-green algae.

The requirement of Ca(2+) for growth and nitrogen fixation has been investigated in two strains of heterocystous blue-green algae (Anabaena sp. and Anabaena ATCC 33047). With combined nitrogen (nitrate or ammonium) or with N(2) under microaerobic conditions, Ca(2+) was not required for growth, at least in concentrations greater than traces. In contrast, Ca(2+) was required as a macronutrient for growth and nitrogen fixation with air as the nitrogen source. Addition of Ca(2+) to an aerobic culture without Ca(2+) promoted, after a lag of several hours, development of nitrogenase activity and cell growth. Provision of air to a microaerobic culture in the absence of Ca(2+) promoted a drastic drop in nitrogenase activity, which rapidly recovered its initial level upon restoration of microaerobic conditions. Development of nitrogenase activity in response to either Ca(2+) or low oxygen tension was dependent on de novo protein synthesis. The role of Ca(2+) seems to be related to protection of nitrogenase from inactivation, by conferring heterocysts resistance to oxygen.

Journal Article↗
PubMed1995

Genetic evidence of a major role for glucose-6-phosphate dehydrogenase in nitrogen fixation and dark growth of the cyanobacterium Nostoc sp. strain ATCC 29133.

Heterocysts, sites of nitrogen fixation in certain filamentous cyanobacteria, are limited to a heterotrophic metabolism, rather than the photoautotrophic metabolism characteristic of cyanobacterial vegetative cells. The metabolic route of carbon catabolism in the supply of reductant to nitrogenase and for respiratory electron transport in heterocysts is unresolved. The gene (zwf) encoding glucose-6-phosphate dehydrogenase (G6PD), the initial enzyme of the oxidative pentose phosphate pathway, was inactivated in the heterocyst-forming, facultatively heterotrophic cyanobacterium, Nostoc sp. strain ATCC 29133. The zwf mutant strain had less than 5% of the wild-type apparent G6PD activity, while retaining wild-type rates of photoautotrophic growth with NH4+ and of dark O2 uptake, but it failed to grow either under N2-fixing conditions or in the dark with organic carbon sources. A wild-type copy of zwf in trans in the zwf mutant strain restored only 25% of the G6PD specific activity, but the defective N2 fixation and dark growth phenotypes were nearly completely complemented. Transcript analysis established that zwf is in an operon also containing genes encoding two other enzymes of the oxidative pentose phosphate cycle, fructose-1,6-bisphosphatase and transaldolase, as well as a previously undescribed gene (designated opcA) that is cotranscribed with zwf. Inactivation of opcA yielded a growth phenotype identical to that of the zwf mutant, including a 98% decrease, relative to the wild type, in apparent G6PD specific activity. The growth phenotype and lesion of G6PD activity in the opcA mutant were complemented in trans with a wild-type copy of opcA. In addition, placement in trans of a multicopy plasmid containing the wild-type copies of both zwf and opcA in the zwf mutant resulted in an approximately 20-fold stimulation of G6PD activity, relative to the wild type, complete restoration of nitrogenase activity, and a slight stimulation of N2-dependent photoautotrophic growth and fructose-supported dark growth. These results unequivocally establish that G6PD, and most likely the oxidative pentose phosphate pathway, represents the essential catabolic route for providing reductant for nitrogen fixation and respiration in differentiated heterocysts and for dark growth of vegetative cells. Moreover, the opcA gene product is involved by an as yet unknown mechanism in G6PD synthesis or catalytic activity.

Cell Division↗
PubMedDate not supplied

[Nitrogen fixation in combined cultures of Lipomyces and bacteria].

Nitrogen fixation by pure cultures of soil yeasts belonging to the genus Lipomyces Lodder et Kreger van Rij was not found by the acetylene method on a medium containing microelements and yeast autolysate. In a binary culture of L. lipofer 133 and Pseudomonas sp. 5, a nitrogen fixing bacterium, the content of bound nitrogen was 3.6 times higher in aerobic conditions and 15 times higher in anaerobic conditions than its accumulation by individual bacterial culture.

Aerobiosis↗
PubMed1993

The ORF encoding a putative ferredoxin-like protein downstream of the vnfH gene in Azotobacter vinelandii is involved in the vanadium-dependent alternative pathway of nitrogen fixation.

An open reading frame (ORF) in the same operon as, but downstream of, vnfH in Azotobacter vinelandii can code for a ferredoxin-like protein. The role this ORF may play in the vnf (vanadium-dependent alternative) pathway of nitrogen fixation was investigated. Site-directed mutagenesis was used to alter one base in each of the codons specifying amino acids 18 and 19 generating a unique Bg/II site. A kanamycin resistance cartridge was cloned into the Bg/II site. This construct was mobilized into A. vinelandii CA12 (delta nifHDK) strain by conjugation and the mutation was introduced into the genome by marker exchange. The resulting mutant was unable to fix nitrogen under conditions in which the vnf pathway of nitrogen fixation operates. This suggests that this ORF is functional and is essential for the vanadium-dependent alternative pathway of nitrogen fixation in A. vinelandii.

Amino Acid Sequence↗
PubMed1997

Structural analysis of the Trichodesmium nitrogenase iron protein: implications for aerobic nitrogen fixation activity.

Trichodesmium spp. are marine filamentous nitrogen-fixing cyanobacteria which play an important role in the nitrogen budget of the open ocean. Trichodesmium is unique in that it is nonheterocystous and fixes nitrogen during the day, while evolving oxygen through photosynthesis, even though nitrogenase is sensitive to oxygen inactivation. The sequence of the gene encoding the Fe protein component of nitrogenase from the recently cultivated isolate Trichodesmium sp. IMS 101 was used to construct a 3-dimensional model of the Fe protein, by comparison to the X-ray crystallographic structure of the Fe protein of the gamma-proteobacterium Azotobacter vinelandii. The primary differences in amino acid sequences of the Fe protein from diverse organisms do not impact the critical structural features of the Fe protein. It can be concluded that aerobic nitrogen fixation in Trichodesmium spp. is not facilitated by unique structural features of Trichodesmium Fe protein.

Aerobiosis↗
PubMed1999

Oligotrophy and nitrogen fixation during eastern mediterranean sapropel events

Nitrogen isotopic measurements in fossil chlorophyll from late Pleistocene organic-rich sediments (sapropels) in the eastern Mediterranean Sea provide geochemical evidence for stratified, nutrient-depleted surface water and extensive nitrogen fixation. This evidence is reconciled with previous indications of high productivity by invoking a model of sapropel formation in which increased river discharge facilitates development of a specialized phytoplankton population whose annual mass sinking provides the organic flux to generate sapropels. This interpretation is consistent with the widespread occurrence of mat-forming diatoms that thrive in stratified water and can harbor diazotrophic bacterial symbionts, but does not support eutrophication of surface waters by enhanced river runoff or a circulation reversal.

Journal Article↗