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PubMed1993

Growth, nitrogen fixation, and spectral attenuation in cultivated trichodesmium species.

Physiological studies of Trichodesmium species have been hindered by difficulties in maintaining actively growing, nitrogen-fixing cultures. Previous cultivation successes have not been widely duplicated. We present here a simple modified seawater medium and handling techniques which have been used to maintain actively growing Trichodesmium thiebautii in laboratory culture for over 1 year. The cultured population, isolated from coastal Atlantic waters, has a growth rate of 0.23 division day and exhibits light-dependent nitrogen fixation during exponential growth. Various morphologies, including solitary trichomes, and aggregates (spherical puffs and fusiform tufts) are present during growth. Spectral and scalar irradiance were measured within naturally occurring (coastal Atlantic) aggregates with small (diameter, 50 to 70 mum) spherical fiber-optic sensors. In contrast to naturally occurring puffs, cultivated Trichodesmium aggregates exhibited spectral properties consistent with low-light adaptation. Cultivated puff-type aggregates were also examined by using oxygen microelectrodes. The simple medium and approach used for cultivation should be easily reproducible and amenable to further manipulations and modifications useful for physiological studies of Trichodesmium spp. in culture.

Journal Article↗
PubMed1982

Directed transposon Tn5 mutagenesis and complementation analysis of Rhizobium meliloti symbiotic nitrogen fixation genes.

An 18 kb region adjacent to and surrounding the genes for nitrogenase (nif) was cloned from the genome of the symbiotic nitrogen-fixing species Rhizobium meliloti. A total of 31 Tn5 insertions in the nif region were constructed and assayed for their effect on symbiotic nitrogen fixation (Fix phenotype). Fix- insertions were found in two clusters, one 6.3 kb region not containing essential symbiotic genes. The locations of at least three transcription units containing Fix genes were deduced from complementation analysis between genomic nif::Tn5 insertions and nif::Tn5 insertions on mobilizable cloning vectors. The locations of R. meliloti genes nifH, nifD and nifK, which code for the single subunit of the nitrogenase Fe protein and for the two subunits of the nitrogenase MoFe protein respectively, were determined by DNA hybridization to cloned Klebsiella pneumoniae nif genes and by comparison of partial R. meliloti DNA sequences with K. pneumoniae nif gene sequences. R. meliloti nifH, D and K are located in the 6.3 kb fix-::Tn5 cluster and are transcribed in the order nifH, nifD, nifK, which is the same order as in K. pneumoniae.

Base Sequence↗
PubMed1975

Limitation of acetylene reduction (nitrogen fixation) by photosynthesis in soybean having low water potentials.

The role of photosynthesis and transpiration in the desiccation-induced inhibition of acetylene reduction (nitrogen fixation) was investigated in soybean (Glycine max [L.] Merr. var. Beeson) using an apparatus that permitted simultaneous measurements of acetylene reduction, net photosynthesis, and transpiration. The inhibition of acetylene reduction caused by low water potentials and their aftereffects could be reproduced by depriving shoots of atmospheric CO(2) even though the soil remained at water potentials that should have favored rapid acetylene reduction. The inhibition of acetylene reduction at low water potentials could be partially reversed by exposing the shoots to high CO(2) concentrations. When transpiration was varied independently of photosynthesis and dark respiration in plants having high water potentials, no effects on acetylene reduction could be observed. There was no correlation between transpiration and acetylene reduction in the CO(2) experiments. Therefore, the correlation that was observed between transpiration and acetylene reduction during desiccation was fortuitous. We conclude that the inhibition of shoot photosynthesis accounted for the inhibition of nodule acetylene reduction at low water potentials.

Journal Article↗
PubMed1981

Nitrogen Fixation (Acetylene Reduction) Associated with Decaying Leaves of Pond Cypress (Taxodium distichum var. nutans) in a Natural and a Sewage-Enriched Cypress Dome.

Surface litter from a natural and a sewage-enriched cypress dome in north-central Florida showed a pronounced seasonal pattern of nitrogenase (acetylene reduction) activity associated with seasonal leaf fall from deciduous trees in the domes. Samples of peat from cores indicated negligible nitrogenase activity below the surface layer. Integrating the monthly rates of nitrogen fixation (based on the theoretical molar ratio of 3:2 for C(2)H(4)/NH(3)) yielded 0.39 and 0.12 g of N/m per year fixed in the litter of the natural and sewage-enriched domes, respectively. The nitrogen fixed in the first 3 months after leaf fall in the natural dome represented about 14% of the nitrogen increment in the decomposing cypress leaves, but fixation contributed a negligible amount of nitrogen (<1%) to decomposing litter in the sewage-enriched dome.

Journal Article↗
PubMed2003

Exploring symbiotic nitrogen fixation and assimilation in pea root nodules by in vivo 15N nuclear magnetic resonance spectroscopy and liquid chromatography-mass spectrometry.

Nitrogen (N) fixation and assimilation in pea (Pisum sativum) root nodules were studied by in vivo (15)N nuclear magnetic resonance (NMR) by exposing detached nodules to (15)N(2) via a perfusion medium, while recording a time course of spectra. In vivo (31)P NMR spectroscopy was used to monitor the physiological state of the metabolically active nodules. The nodules were extracted after the NMR studies and analyzed for total soluble amino acid pools and (15)N labeling of individual amino acids by liquid chromatography-mass spectrometry. A substantial pool of free ammonium was observed by (15)N NMR to be present in metabolically active, intact nodules. The ammonium ions were located in an intracellular environment that caused a remarkable change in the in vivo (15)N chemical shift. Alkalinity of the ammonium-containing compartment may explain the unusual chemical shift; thus, the observations could indicate that ammonium is located in the bacteroids. The observed (15)N-labeled amino acids, glutamine/glutamate and asparagine (Asn), apparently reside in a different compartment, presumably the plant cytoplasm, because no changes in the expected in vivo (15)N chemical shifts were observed. Extensive (15)N labeling of Asn was observed by liquid chromatography-mass spectrometry, which is consistent with the generally accepted role of Asn as the end product of primary N assimilation in pea nodules. However, the Asn (15)N amino signal was absent in in vivo (15)N NMR spectra, which could be because of an unfavorable nuclear Overhauser effect. gamma-Aminobutyric acid accumulated in the nodules during incubation, but newly synthesized (15)N gamma-aminobutyric acid seemed to be immobilized in metabolically active pea nodules, which made it NMR invisible.

Amino Acids↗
PubMed1980

Nodulation and Nitrogen Fixation in Cliffrose (Cowania mexicana var. stansburiana (Torr.) Jeps.).

Nodulation and acetylene reduction were demonstrated for cliffrose (Cowania mexicana var. stansburiana [Torr.] Jeps.) in a greenhouse trial. Nitrogen fixation was also verified with (15)N. Seedlings were grown in a soil known to nodulate both bitterbrush species (Purshia tridentata (Pursh) D.C. and P. glandulosa [Curran]) to which a suspension of crushed bitterbrush nodules had been added. Bitterbrush seedlings grew faster and reduced more acetylene than cliffrose seedlings planted in the same pots, but the specific activity of the nodules and the nodule mass to root mass ratios were approximately the same.

Journal Article↗
PubMed1999

Enhanced nitrogen fixation in a rhizobium etli ntrC mutant that overproduces the bradyrhizobium japonicum symbiotic terminal oxidase cbb3

The ntrC gene codes for a transcriptional activator protein that modulates gene expression in response to nitrogen. The cytochrome production pattern of a Rhizobium etli ntrC mutant (CFN2012) was studied. CO difference spectral analysis of membranes showed that CFN2012 produced a terminal oxidase similar to the symbiotic terminal oxidase of bacteroids in free-living cells under aerobic conditions, with a characteristic trough at 553 nm. CFN2012 produced two c-type cytochromes with molecular masses of 27 and 32 kDa, in contrast with the wild-type strain, which produced only a 32-kDa c-type cytochrome. The expression levels of the R. etli fixNOQP operon, which codes for terminal oxidase cbb3, were not affected by the ntrC mutation. However, the production levels of the two c-type cytochromes (27 and 32 kDa) were enhanced at least eightfold when the Bradyrhizobium japonicum fixNOQP operon was expressed in CFN2012 from the nptII promoter (pMSfixc), suggesting that these proteins are subunits FixO (27 kDa) and FixP (32 kDa) of cbb3 and that CFN2012/pMSfixc overproduced this terminal oxidase. CFN2012/pMSfixc showed a significant increase in its symbiotic performance as judged by the determination of nitrogenase activities of plants inoculated with this strain, suggesting that the overproduction of cbb3 terminal oxidase correlates with an enhancement in symbiotic nitrogen fixation.

Journal Article↗
PubMed2005

Legume species identity and soil nitrogen supply determine symbiotic nitrogen-fixation responses to elevated atmospheric [CO2].

In nitrogen (N)-limited systems, the response of symbiotic N fixation to elevated atmospheric [CO2] may be an important determinant of ecosystem responses to this global change. Experimental tests of the effects of elevated [CO2] have not been consistent. Although rarely tested, differences among legume species and N supply may be important. In a field free-air CO2 enrichment (FACE) experiment, we determined, for four legume species, whether the effects of elevated atmospheric [CO2] on symbiotic N fixation depended on soil N availability or species identity. Natural abundance and pool-dilution 15N methods were used to estimate N fixation. Although N addition did, in general, decrease N fixation, contrary to theoretical predictions, elevated [CO2] did not universally increase N fixation. Rather, the effect of elevated [CO2] on N fixation was positive, neutral or negative, depending on the species and N addition. Our results suggest that legume species identity and N supply are critical factors in determining symbiotic N-fixation responses to increased atmospheric [CO2].

Atmosphere↗
PubMed1985

Nitrogen fixation specific regulatory genes of Klebsiella pneumoniae and Rhizobium meliloti share homology with the general nitrogen regulatory gene ntrC of K. pneumoniae.

We have determined the complete nucleotide sequences of three functionally related nitrogen assimilation regulatory genes from Klebsiella pneumoniae and Rhizobium meliloti. These genes are: 1) The K. pneumoniae general nitrogen assimilation regulatory gene ntrC (formerly called glnG), 2) the K. pneumoniae nif-specific regulatory gene nifA, and 3) an R. meliloti nif-specific regulatory gene that appears to be functionally analogous to the K. pneumoniae nifA gene. In addition to the DNA sequence data, gel-purified K. pneumoniae nifA protein was used to determine the amino acid composition of the nifA protein. The K. pneumoniae ntrC and nifA genes code for proteins of 52,259 and 53,319 d respectively. The R. meliloti nifA gene codes for a 59,968 d protein. A central region within each polypeptide, consisting of approximately 200 amino acids, is between 52% and 58% conserved among the three proteins. Neither the amino termini nor the carboxy termini show any conserved sequences. Together with data that shows that the three regulatory proteins activate promoters that share a common consensus sequence in the -10 (5'-TTGCA-3') and -23 (5'-CTGG-3') regions, the sequence data presented here suggest a common evolutionary origin for the three regulatory genes.

Amino Acid Sequence↗
PubMed1979

Nitrogen fixation by Rhizobium sp. 32H1. A morphological and ultrastructural comparison of asymbiotic and symbiotic nitrogen-fixing forms.

The induction of nitrogenase (C2H2) activity in asymbiotically cultured Rhizobium sp. 32H1 was found to be associated with morphological changes in the cells which were more pronounced than those seen in bacteroids. Polyphosphate granules were found in both bacteroids and cultured cells, but poly-beta-hydroxybutyrate vesicles were almost absent in bacteroids but were present in cultured cells. Freeze-etching techniques revealed no differences between the asymbiotically cultured nitrogen-fixing forms and bacteroids in that both the cell wall and cytoplasmic membrane cleavage planes were normal for gram-negative bacteria.

Cytoplasmic Granules↗