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PubMed1975

Acetylene reduction assays for nitrogen fixation in freshwaters: a note of caution.

Lake water samples were observed to transform [14-C]ethylene into water-soluble compounds that were undetectable by conventional acetylene reduction assay procedures. Methane oxidizing bacteria, which are known to be common in freshwaters, appeared to be responsible for this activity. As much as 28 percent of added ethylene has been observed to be transformed and this figure is probably an underestimate. It is suggested that acetylene reduction assays may not be accurately applied to samples containing methane oxidizing bacteria.

Acetylene↗

PubMed1969

Regulation of nitrogen fixation in Azotobacter vinelandii OP: the role of nitrate reductase.

A number of chlorate-resistant mutants were selected, and one of these, clr68-5, was studied in detail. This mutant cannot utilize nitrate in vivo to overcome the effect of nonmetabolizable repressors of nitrogenase. The reason for this inability was that strain clr68-5 lacked nitrate reductase. Nitrate inhibited the activity of nitrogenase but did not act as a corepressor of nitrogenase in strain clr68-5 as it does in the wild type. Ammonia seemed to act as corepressor of nitrogenase in both strains.

Ammonia↗

PubMed1997

Nitrogenase phylogeny and the molybdenum dependence of nitrogen fixation in Methanococcus maripaludis.

We studied the effects of molybdenum, vanadium, and tungsten on the diazotrophic growth of Methanococcus maripaludis. Mo stimulated growth, with a maximal response at 4.0 microM, while V had no effect at any concentration tested. W specifically inhibited diazotrophic growth in the presence of Mo. Coupling the results of our analysis and other known metal requirements with phylogenies derived from nifD and nifK genes revealed distinct clusters for Mo-, V-, and Fe-dinitrogenases and suggested that most methanogens also have molybdenum-type nitrogenases.

Base Sequence↗

PubMed2006

In situ analysis of nitrogen fixation and metabolic switching in unicellular thermophilic cyanobacteria inhabiting hot spring microbial mats.

Genome sequences of two Synechococcus ecotypes inhabiting the Octopus Spring microbial mat in Yellowstone National Park revealed the presence of all genes required for nitrogenase biosynthesis. We demonstrate that nif genes of the Synechococcus ecotypes are expressed in situ in a region of the mat that varies in temperature from 53.5 degrees C to 63.4 degrees C (average 60 degrees C); transcripts are only detected at the end of the day when the mat becomes anoxic. Nitrogenase activity in mat samples was also detected in the evening. Hitherto, N2 fixation in hot spring mats was attributed either to filamentous cyanobacteria (not present at >50 degrees C in these mats) or to heterotrophic bacteria. To explore how energy-generating processes of the Synechococcus ecotypes track natural light and O2 conditions, we evaluated accumulation of transcripts encoding proteins involved in photosynthesis, respiration, and fermentation. Transcripts from photosynthesis (cpcF, cpcE, psaB, and psbB) and respiration (coxA and cydA) genes declined in the evening. In contrast, transcripts encoding enzymes that may participate in fermentation fell into two categories; some (ldh, pdhB, ald, and ackA) decreased in the evening, whereas others (pflB, pflA, adhE, and acs) increased at the end of the day and remained high into the night. Energy required for N2 fixation during the night may be derived from fermentation pathways that become prominent as the mat becomes anoxic. In a broader context, our data suggest that there are critical regulatory switches in situ that are linked to the diel cycle and that these switches alter many metabolic processes within the microbial mat.

Energy Metabolism↗

PubMed1999

Effect of salt stress on nodulation and nitrogen fixation in legumes.

It is now well established that almost all phases of root nodule development in legumes are adversely affected by saline conditions in the rooting medium. There is also a general agreement that the rhizobia are more tolerant to salt stress than the host plant, but they show considerable strain variability in growth and survival under saline conditions. Inhibitory effect of salinity on nodulation has been attributed to decrease in rhizobial colonisation and shrinkage and lack of root hair formation. Salt stress also induces premature senescence of already formed nodules. Both N2-fixation activity and nodule respiration are inhibited sharply on exposure of plants to saline conditions. The decrease in N2-fixation has been ascribed to direct effect on nitrogenase activity or an indirect effect through decrease in leghemoglobin content, respiratory rate, malate concentrations in nodules and photosynthate availability. Salinity increases oxygen diffusion resistance in the nodules and alters their ultrastructure. Decrease in N2-fixation in nodules under salinity is also accompanied by parallel decrease in the activity of H2O2-scavenging enzymes like catalase, ascorbate peroxidase and the level of antioxidants like ascorbic acid. Nodules appear to undergo osmoregulation under saline conditions by accumulating physiologically compatible solutes like proline, sugars (pinnitol) and lactic acid. The intensity of the adverse effects of salinity on nodule functioning depends on plant species, rhizobial strain, duration of exposure to saline conditions, nature, concentration and mode of salt application.

Fabaceae↗

PubMedDate not supplied

Genetic analysis of the nitrogen fixation system in Klebsiella pneumoniae.

Fine structure mapping of nif mutations of Klebsiella pneumoniae was accomplished by means of Pl-transductional crosses and the plasmid R144 drd mediated conjugations. The physical distance between nif mutations based on the percentage of co-transduction with hisD of the nif mutations was estimated. The maximal distance between two mutations was calculated about 3 Kb, and the average distance between different nif mutations was about 1 to 2 Kb. So no "silent region" was shown within the nif cluster nearby the histidine operon. Several hisD-unlinked nif mutants were isolated and investigated genetically and biochemically. They all differed from the glutamineless mutants, one of these mutants was tentatively assigned as a sort of N-assimilation mutant with little activity of glutamate synthetase. It differed from the known N-assimilation mutants in its absence of nitrogenase activity. Since the wild type hisD-linked nif genes carried by the plasmid RP4 failed to complement the defects of the hisD-unlinked nif genes in the recipient cells but they were effective to facilitate E. coli in acquiring the ability to fix nitrogen, which indicates that the hisD-unlinked nif genes necessary for the functioning of the hisD-linked nif genes are present in E. coli.

Chromosome Mapping↗

PubMed2004

Successional changes in soil nitrogen availability, non-symbiotic nitrogen fixation and carbon/nitrogen ratios in southern Chilean forest ecosystems.

Vast areas of southern Chile are now covered by second-growth forests because of fire and logging. To study successional patterns after moderate-intensity, anthropogenic fire disturbance, we assessed differences in soil properties and N fluxes across a chronosequence of seven successional stands (2-130 years old). We examined current predictions of successional theory concerning changes in the N cycle in forest ecosystems. Seasonal fluctuations of net N mineralization (N(min)) in surface soil and N availability (N(a); N(a)=NH4+-N+NO3--N) in upper and deep soil horizons were positively correlated with monthly precipitation. In accordance with theoretical predictions, stand age was positively, but weakly related to both N(a) ( r(2)=0.282, P<0.001) and total N (N(tot); r(2)=0.192, P<0.01), and negatively related to soil C/N ratios ( r(2)=0.187, P<0.01) in surface soils. A weak linear increase in soil N(min) (upper plus deep soil horizons) was found across the chronosequence ( r(2)=0.124, P<0.022). N(min) occurred at modest rates in early successional stands, suggesting that soil disturbance did not impair microbial processes. The relationship between N fixation (N(fix)) in the litter layer and stand age best fitted a quadratic model ( r(2)=0.228, P<0.01). In contrast to documented successional trends for most temperate, tropical and Mediterranean forests, non-symbiotic N(fix) in the litter layer is a steady N input to unpolluted southern temperate forests during mid and late succession, which may compensate for hydrological losses of organic N from old-growth ecosystems.

Age Factors↗

PubMed1984

Repression of nitrogen fixation in Klebsiella pneumoniae at high temperature.

The effect of elevated growth temperature on nif mRNA synthesis, nif protein synthesis, and nitrogenase activity was investigated in wild-type and NifL- strains of Klebsiella pneumoniae. Nitrogenase activity is not affected at 41 degrees C; however, nitrogenase is not synthesized at that temperature. Transcription of three nif operons studied is repressed at 41 degrees C. We show that the nifL protein is not required for repression by high temperature and propose that repression of nif at 41 degrees C results from a reversible inactivation of the nifA gene product.

Acetylene↗

PubMed1981

Regulation of nitrogen fixation in Klebsiella pneumoniae: isolation and characterization of strains with nif-lac fusions.

Strains with lac fused to each of the seven nif operons were isolated by two different methods. Repressing conditions prevented expression of all nif operons, whereas derepressing conditions led to the expression of all nif operons. No differences in Nif regulation were observed between Escherichia coli and Klebsiella pneumoniae with the same nif-lac fusions. Most derivatives of nif-lac fusion strains selected on lactose and NH4+ contained nif operator mutations. Some derivative contained deletions, which establishes that the direction of transcription of all seven nif operons is toward his

DNA, Recombinant↗

PubMed1983

Adenine nucleotide levels in and nitrogen fixation by the cyanobacterium Anabaena sp. strain 7120.

Adenine nucleotide levels were determined in whole filaments of Anabaena sp. 7120 grown under different N2-fixing or non-N2-fixing conditions. These were compared with levels in isolated heterocysts, Rhodospirillum rubrum, and Azotobacter vinelandii. Adenine nucleotides in whole filaments of Anabaena sp. do not reflect the energetic expense of N2 fixation as they do in R. rubrum and A. vinelandii. However, adenine nucleotide levels in heterocysts were similar to the levels found in N2-fixing R. rubrum, i.e., an ATP:ADP ratio near 1 and an energy charge between 0.5 and 0.7. Nitrogenase activity was only 50% of optimal in permeabilized heterocysts at an exogenous ATP:ADP ratio of 3.33. Hydrogen, which increases acetylene reduction activity, also causes a transient increase (2 to 5 min) in the ATP:ADP ratio. Hydrogen has little effect on energy charge.

Adenine Nucleotides↗

PubMed1983

High-frequency induction of nodulation and nitrogen fixation mutants of Rhizobium japonicum.

More than 50 symbiotic mutants of Rhizobium japonicum were isolated by purported plasmid-curing techniques. Wild-type R. japonicum strains were grown in liquid culture at 28 or 36 degrees C in different concentrations of acridine orange, ethidium bromide, or sodium dodecyl sulfate for selection of mutants. The symbiotic traits of 133 isolates from nine treatment groups were determined. Forty-two isolates were Nod- Nif+, seven were Nod+ Nif-, and two were Nod- Nif-. The nifDH genes were deleted in three mutants and consequently showed no hybridization to a nifDH probe. None of these mutants showed any detectable loss of plasmid DNA.

Acridine Orange↗

PubMed1980

Evolutionary considerations on the thermodynamics of nitrogen fixation.

An evolutionary explanation is sought for the fact that ATP is needed for N2 fixation in spite of the exergonicity of the process. After a survey of the state of knowledge about the thermodynamics of N2 fixation in fermenters, photosynthesizers and respirers it is suggested that nitrogenase, which still shows ATP-dependent hydrogenase activity, evolved from an ATP-requiring hydrogenase that lacked nitrogenase activity. The hydrogenase action in the Archaean, reducing, biosphere may have needed ATP to ensure expulsion of H2. Extant non-nitrogenase hydrogenases have lost the dependence on ATP. Because of its complexity, nitrogenase could not rid itself of the ATP dependence or of hydrogenase activity, both wasteful. Presumably all hydrogenases evoled from ferredoxin-like Fe-S proteins.

Bacteria↗

PubMed1998

The Bradyrhizobium japonicum phoB gene is required for phosphate-limited growth but not for symbiotic nitrogen fixation.

We identified by cloning and DNA sequence analysis the phosphate regulatory gene phoB of Bradyrhizobium japonicum. The deduced gene product displayed pronounced similarity to the PhoB protein of Sinorhizobium meliloti (71.4% identical amino acids). Escherichia coli (50.2%) and other bacterial species. Insertion of a kanamycin resistance cassette into phoB led to impaired growth of the B. japonicum mutant in media containing approximately 25 microM phosphate or less. A standard plant infection test using wild-type and phoB-defective B. japonicum strains showed that the phoB mutation had no effect on the symbiotic properties of B. japonicum with its soybean host plant.

Amino Acid Sequence↗

PubMed1982

Cloning of a nitrogen fixation (nif) gene cluster of Azospirillum brasilense.

Homology was detected between the structural genes for the nitrogenase complex of K. pneumoniae (nifHDK genes) and the total DNA of several Azospirillum strains. Bacteriophage lambda gt 7-ara6 was used to construct a gene bank of A. brasilense strain 7000 DNA and a recombinant phage carrying a 6.7 kb Eco RI fragment, termed AbRI, was selected by hybridization with the K. pneumoniae nif probe. Using heteroduplex analysis the extent of the homology of the AbRI fragment and the K. pneumoniae nif genes was found to be approximately 5 kb. Proteins encoded by the AbRI fragment were examined after infection of E. coli minicells.

Cloning, Molecular↗

PubMed1976

Derivation and properties of F-prime factors in Escherichia coli carrying nitrogen fixation genes from Klebsiella pneumoniae.

A His+ Nif+ Escherichia coli K12, Hfr strain (UNF43) was constructed by an intergeneric mating between a Klebsiella pneumoniae donor strain (HF3) and a his-HFR E. coli strain (SBI824) which transfers his as an early marker. An F-prime nif plasmid, FN39, carrying genes which correspond to the E. coli chromosomal region, metG gnd his shiA, but excluding purF and aroD, was isolated from UNF43. Translocation of carbenicillin resistance genes from a P-type R-factor, R68, to FN39 increased the stability of his and nif on the derivative F-prime, FN68. Sedimentation analysis of both F-primes in sucrose gradients revealed our covalently closed circular(CCC) DNA species of molecular weights 279 +/- 9, 136 +/- 3, 90 +/- 1 and 44 +/- 1 megadaltons. It is suggested that the two smallest CCC-DNA species are component replicons of the composite F-primes of molecular weight 136 +/- 3 megadaltons, and that the molecules of 279 +/- 9 megadaltons are CCC-dimers. FN68 was transferable in intergeneric matings to Klebsiella aerogenes, K. pneumoniae and Salmonella typhimurium but not to Proteus mirabilis; only carbenicillin resistance and sex factor activity were transferred to Erwinia herbicola. nif genes on FN68 were expressed in a Nif- mutant of K. pneumoniae and also in S. typhimurium, which in conventional tests is naturally non-nitrogen-fixing; expression of the his determinant of FN68 became temperature-sensitive in S. typhimurium.

Carbenicillin↗

PubMed1992

Regulation of nitrogen fixation in Azospirillum brasilense Sp7: involvement of nifA, glnA and glnB gene products.

The expression of nifA-, niH- and nifB-lacZ fusions was examined in different mutants of Azospirillum brasilense. Mutations in nifA, glnA and glnB severely impaired the expression of nifH- and nifB-lacZ fusions. By contrast, a nifA-lacZ fusion was not affected in a nifA or a glnB background and was only partially impaired in glnA mutants. It is proposed that in A. brasilense, the PII protein and glutamine synthetase are involved in a post-translational modification of NifA.

Amino Acid Sequence↗

PubMed1990

[Transposon mutagenesis, elimination and mobilization of plasmids in nitrogen-fixating bacterium Azospirillum brasilense Sp245].

The expressed difference in the plasmid profile of A. brasilense Sp245 is registered as a result of Tn5-Mob-mutability. Integration of the vector pSUP5011 into one of the A. brasilense Sp245 plasmid and using of the Tn5-Mob transposon to mobilize the 85Md cryptic plasmid are reported. The properties of A. brasilense Sp245 with the mutant plasmids composition (surface structure, acetylene and nitrate reduction, ability to a number of carbohydrates utilization, formation of melanin, antibiotics resistance specter) have been analyzed. The transposon Tn5-Mob insertion into the 85Md plasmid resulted in isolation of a mutant excreting a melanin-like pigment into the medium. The results suppose 85Md plasmid participation in melaninogenesis.

Azospirillum brasilense↗

PubMed1997

Effect of nitrate on nitrogen fixation and nitrate assimilation in Azorhizobium-Sesbania rostrata symbiotic system.

Root nodule formation was inhibited by 30% and 50% respectively at low concentration of 1 mM and 2 mM nitrate, while stem nodule formation was enhanced by 50% only at 1 mM nitrate. The nodule specific nitrogenase activity decreased with the increasing concentration of nitrate. At 1 mM nitrate nitrogenase activity per plant stem nodule was not affected, but it was less than 50% in the root nodules as compared to control. Increasing concentration of nitrate increased in vivo activity of nitrate reductase (NR) significantly in stem, root nodules and leaves. Nodule cytosolic NR utilized both NADH and succinate as electron donor, but not reduced MV. However bacteroidal NR utilised reduced MV as reductant more efficiently than succinate.

Fabaceae↗