Aerobic Nitrogen Fixation Research Articles

Oxygen protection of nitrogenase in the aerobically nitrogen fixing, non-heterocystous cyanobacterium Oscillatoria sp.

Oscillatoria sp. strain 23 is a filamentous, non-heterocystous cyanobacterium that fixes nitrogen aerobically. Although, in this organism nitrogenase is inactivated by oxygen a high tolerance is observed. Up to a pO2 of 0.15 atm, oxygen does not have any measurable effects on acetylene reduction. Higher concentrations of oxygen inhibited the activity to a relatively high degree. Evidence for two mechanisms of oxygen protection of nitrogenase in this cyanobacterium was obtained. A high rate of synthesis of nitrogenase may allow the organism to maintain a certain amount of active enzyme under aerobic conditions. Secondly, a switch off/on mechanism may reversibly convert the active enzyme into a non-active form which is insensitive to oxygen inactivation after a sudden and short-term exposure to high oxygen concentrations. It is conceived that these mechanisms in addition to a temporal separation of nitrogen fixation from oxygenic photosynthesis sufficiently explain the regulation process of aerobic nitrogen fixation in this organism.

Isolation and Properties of Mutants of Azotobacter chroococcum Defective in Aerobic Nitrogen Fixation

Mutants of Azotobacter chroococcum which showed unusually O2-sensitive N2 fixation when grown on sugars were isolated following mutagenesis with nitrosoguanidine or the transposon Tm1. These mutants we called Fos– (inability to fix N2 on sugars). Aerotolerant growth of Fos– mutants was, in general, restored by carboxylic acids, Ca2+ or a combined-N source. Two main groups of mutants were distinguished: (1) regulatory mutants which failed to synthesize nitrogenase under aerobic conditions and which were complemented by the Klebsiella pneumoniae nifA gene product; (2) mutants affected at different steps in intermediary metabolism. The latter strains exhibited low respiration rates or high apparent K s(O2) values (when grown on sucrose) compared with the parent strain. They seemed to be defective in respiratory protection of nitrogenase. The restoration of aerotolerant N2-dependent growth of Fos– mutants by carboxylic acids was correlated with their ability to induce a decrease in the apparent K s(O2) value; however, in both the mutant and the parent strains maximum respiration rates did not change significantly. The Ca2+ requirement for diazotrophic growth in A. chroococcum seems to be related to the capability of these organisms to fix N2 in air. Ca2+ might be required for high fluxes in the tricarboxylic acid cycle and might act at the level of phosphoenolpyruvate carboxylase, which is involved in the replenishment of the tricarboxylic acid cycle and/or at the level of tricarboxylic acid cycle enzymes per se. It is suggested that respiratory protection requires the maintenance of high respiratory rates even at low O2 tension.

Lesions in citrate synthase that affect aerobic nitrogen fixation by Azotobacter chroococcum.

A class of Azotobacter chroococcum mutants induced by Tn1 that were defective in normal aerobic nitrogen fixation when grown on sugars (Fos-) were corrected by provision of alpha-ketoglutarate or glutamate. In a representative mutant, Fos252, rates of evolution of 14CO2 from [14C]acetate or [14C]glucose were 5% of the parental values, although uptake and incorporation were normal for both substrates. The results suggest that a lesion affects the entry of substrates into the tricarboxylic acid cycle. The activity of citrate synthase in Fos252 in vitro was 5% that of the parents. The citrate synthase (gltA) gene from Escherichia coli was cloned into broad-host-range vectors and mobilized into Fos252. The plasmids restored parental citrate synthase activities to Fos252 and complemented the inability to fix N2 in air. The data indicate that a mutation causing an intrinsic limitation in respiratory capacity abolishes normal aerobic N2 fixation, which is consistent with the hypothesis of respiratory protection for nitrogenase in Azotobacter species.

Hydrogen oxidation and efficiency of nitrogen fixation in succinate-limited chemostat cultures ofRhizobium ORS 571

Rhizobium ORS 571, isolated from stem nodules of the tropical legumeSesbania rostrata is able to grow in the chemostat with molecular nitrogen as sole nitrogen source at a specific growth rate of 0.1 h-1. Samples from nitrogenfixing cultures showed high acetylene reduction activities: 1,500 nmol ethylene formed per milligram dry weight per hour. Under nitrogen-fixing conditions an uptake hydrogenase is induced. Ammonia-assimilating cultures, without additional hydrogen, did not induce hydrogenase. The addition of hydrogen to succinate-limited nitrogen-fixing cultures resulted in an increase in the molar growth yield on succinate (Ysuccinate) from 27 to 35 and a slight decrease in the molar growth yield on oxygen (\(Y_{O_2 }\)), showing that hydrogen oxidation is less energy-yielding than the oxidation of endogenous substrates. Respiration-driven proton translocation measured with starved cells indicated the functioning of site 1 and 2 of oxidative phosphorylation. Cytochrome spectra showed that cytochromea600, present at high dissolved oxygen tension (d.o.t.) almost completely disappeared at low d.o.t. In flash-photolysis spectra only thea-type cytochrome could be detected as an oxidase in cells both grown at high and low d.o.t. Growth yields in ammonia-assimilating cultures were higher than those measured in nitrogen-fixing cultures. Assuming two sites of oxidative phosphorylation, a molar growth yield on ATP (YATP) of about 3 and 6 was calculated for respecticely nitrogen-fixing and ammonia-assimilating cultures. TheYATP under nitrogen-fixing conditions is dependent on the amount of H2 formed per mol N2 fixed (H2/N2 ratio). A method has been described to calculate the total amount of ATP use by nitrogenase during the fixation of 1 mol N2 (ATP/N2 ratio) and H2/N2 ratios in aerobic nitrogen fixing organisms. This calculation yielded that nitrogen fixation inRhizobium ORS 571 is a high ATP-consuming process. The calculated ATP/N2 and H2/N2 ratios were respectively 42 and 7.5.

Construction of shuttle vectors capable of conjugative transfer from Escherichia coli to nitrogen-fixing filamentous cyanobacteria.

Wild-type cyanobacteria of the genus Anabaena are capable of oxygenic photosynthesis, differentiation of cells called heterocysts at semiregular intervals along the cyanobacterial filaments, and aerobic nitrogen fixation by the heterocysts. To foster analysis of the physiological processes characteristic of these cyanobacteria, we have constructed a family of shuttle vectors capable of replication and selection in Escherichia coli and, in unaltered form, in several strains of Anabaena. Highly efficient conjugative transfer of these vectors from E. coli to Anabaena is dependent upon the presence of broad host-range plasmid RP-4 and of helper plasmids. The shuttle vectors contain portions of plasmid pBR322 required for replication and mobilization, with sites for Anabaena restriction enzymes deleted; cyanobacterial replicon pDU1, which lacks such sites; and determinants for resistance to chloramphenicol, streptomycin, neomycin, and erythromycin.

Microbial and biochemical changes in soil and rhizosphere of tomato and common bean plants

Summary Total microbial flora, as well as some of the important microbial groups were determined in soil and rhizosphere of both common bean and tomato plants at different stages of growth. The accumulation of some important biological substances in root zone was also interpreted. The total microbial flora in general decreased slightly during the growth season in both soil and rhizosphere region. The R/S ratios were positive and ranged from 5 to 20 depending on plant species and ages. There was a distinct relation between the stages of plant development and the densities of total microbial flora, actinomycetes, nitrogen fixing Clostridia, sporeformers, aerobic and anaerobic cellulose decomposers. Densities of aerobic nitrogen fixers (Azotobacter) and fungi seemed to be constant in their low count in the rhizosphere regions during the growth season. The rhizosphere soil was characterised by a higher quantitative content of organic acids and amino acids as compared with the non-rhizosphere soil.

Loss of aerobic nitrogen fixation associated with kasugamycin resistance in the heterocystous nitrogen-fixing, streptomycin resistant blue-green alga Nostoc muscorum

Heterocystous N2-fixing, Streptomycin resistant (Het (+)Nif(+)Str-R) Nostoc muscorum produces two groups of Kasugamycin resistant (Kas-R) phenotypes: one group showing normal heterocyst frequency and normal aerobic growth with N2 as a nitrogen source, the other group showing loss of growth with N2 as nitrogen source under aerobic growth conditions. Examination of strains showing Kas-R associated loss of aerobic N2 growth shows them to comprise three classes: Class A showing loss of both heterocyst and nitrogenase activity, class B showing formation of heterocyst but no nitrogenase activity and class C lacking heterocyst but containing microaerobic nitrogenase activity. The three classes of mutants revert to aerobic prototrophy with almost similar frequency of about 10(-6) suggesting them to have arisen as a result of single mutational events. Their reversion to prototrophy is simultaneously accompanied by the loss of Kas-R phenotype. All the phenotypic revertants in general appear more or less similar to the parental strain in respect to heterocyst formation, aerobic N2 growth and Kasugamycin sensitivity (Kas-S).

Energy Requirement for Diazotrophic Growth of the Cyanobacterium Anabaena variabilis Determined from Growth Yields in the Dark

SUMMARY: The cyanobacterium Anabaena variabilis CCAP 1403/13a (UTEX 1444, ATCC 29413) was grown diazotrophically in the dark in the presence of fructose with a doubling time of 25 h at 35 °C. This was 40% of the maximum rate observed in the light. Growth rates in the dark were similar with nitrate or ammonium as nitrogen sources or under diazotrophic conditions. Dark-grown cyanobacteria reduced acetylene in the dark at 25% of the rate observed for photoautotrophic cultures in the light. Heterotrophic growth yields for dark growth with different nitrogen sources were estimated from the extent of fructose-dependent growth in batch cultures. The ratio between cell carbon and fructose carbon was 0.38 in diazotrophically grown cells, 0.38 with nitrate and 0.52 with ammonia. Thus the energy requirement for growth on molecular nitrogen is similar to the energy requirement for growth on nitrate, but greater than that for growth on ammonia. Since the diazotrophic culture always had a dissolved O2 concentration of at least 80% of air saturation, this demonstrates that there are no detectable extra energy requirements for aerobic nitrogen fixation compared to nitrate reduction, such as would result from a need for respiratory protection of nitrogenase.

The structural nif genes of the cyanobacteria Gloeothece sp. and Calothrix sp. share homology with those of Anabaena sp., but the Gloeothece genes have a different arrangement.

Probes carrying the Anabaena sp. strain PCC 7120 nitrogenase reductase (nifH) and nitrogenase (nifK and nifD) genes were hybridized to Southern blots of DNA from the unicellular, aerobic nitrogen-fixing cyanobacterium Gloeothece sp. strain PCC 6909 and from the filamentous cyanobacterium Calothrix sp. strain PCC 7601. These data suggest that the Gloeothece sp. nif structural proteins must be similar to those of other diazotrophs and that the ability for aerobic nitrogen fixation does not reside in the nif protein complex. We also found that the nif structural genes of Gloeothece sp. are clustered, whereas those of Calothrix sp. are arranged more like those of Anabaena sp.

Chlorflurenol-methyl in soil: degradation, leaching, and effects on microbiological processes.

Tests were conducted with the synthetic growth regulator chlorflurenol-methyl to investigate its rate of degradation in soil, leaching behavior, and possible side-effects on the soil microflora and on soil physiological processes. With two sandy soils (Ct = 1.0 and 2.58%) which were treated with 11.35 mg kg-1 chlorflurenol-methyl (congruent to 2.8 kg a.i. ha-1), over 90% of the compound disappeared within 4 to 8 days. The degradation products were 2-chloro-9-hydroxyfluorene-9-carboxylic acid and 2-chlorofluorenone, which undergo further decomposition. In leaching tests with three sandy soils (Ct = 0.69, 1.0 and 2.58%), chlorflurenol-methyl was not washed from the soil; however, with one soil (0.69% C), very small residues were observed in the effluent identified as 2-chlorofluorenone. In side-effects experiments with a parabrown (Ct = 1.26%) and a chernozem soil (Ct = 2.3%), which were treated with 1 and 10 mg kg-1 chlorflurenolmethyl, no persistent inhibition of anaerobic or aerobic nitrogen fixation (C2H2-reduction) was detected. Ammonification, nitrification, and mineralization of soluble starch were also not influenced. The mineralization of cellulose in compost soil (Ct = 13.59%) was temporarily delayed; however, this delay was later compensated for by a higher mineralization rate. The colonization density of fungi on soil particles and the numbers of bacteria, actinomycetes, and fungi were not negatively influenced. Chlorflurenol-methyl does not significantly influence these microbiological processes and populations in the soil.

Aerobic nitrogen fixation by the marine non-heterocystous cyanobacterium Trichodesmium (Oscillatoria) spp.: Its protective mechanism against oxygen

Nitrogen fixation (acetylene reduction) by the marine non-heterocystous cyanobacteria, Trichodesmium thiebautii and T. erythraeum, is sensitive to oxygen. Its sensitivity to oxygen was intensified when the colonies of T. thiebautii were disintegrated, but the separate trichomes yielded still retained the capacity for light dependent acetylene reduction. Trichodesmium colonies evolved hydrogen under argon in the light. The addition of carbon monoxide with DCMU [3-(3,4-dichlorophenyl)-1,1-dimethylurea] enhanced the rate of hydrogen evolution to approximately the same level as that of the maximum acetylene reduction on an electron basis. This probably results from the inhibition of the uptake hydrogenase. We propose that the uptake hydrogenase functions to protect nitrogenase from damage by oxygen.

Occurrence of aerobic nitrogen fixing bacteria in wetland and dryland plants

Abstract We recently suggested that N2-fixing bacteria other than Azospirillum are present in greater abundance in the roots of wetland rice (2). Using a low concentration of tryptic soy agar, we isolated aerobic heterotrophic bacteria that required low levels of combined nitrogen for nitrogenase activity from wetland rice roots (1). In a liquid medium under batch culture conditions, the bacteria were unable to grow without combined nitrogen under either aerobic or anaerobic conditions. Neither could they grow nor exhibit nitrogenase activity in a semi-solid N-free medium unless the medium was supplemented with a small amount of yeast extract, casamino acids or any amino acid. Among the nitrogenase positive aerobic heterotrophic isolates from the inner tissue of rice roots that accounted for 80% of the isolated heterotrophs, about 90% were found to be Pseudomonas-like, which require a low level of combined nitrogen. We further report here the occurrence of aerobic nitrogen fixing bacteria in association w...

The developmental biology of heterocyst and akinete formation in cyanobacteria.

We will be concerned with the two major differentiated cell types of filamentous cyanobacteria--the heterocyst and the akinete. The former is generally accepted to be the site of aerobic nitrogen fixation in heterocystous cyanobacteria. The latter is a spore-like cell capable of withstanding certain environmental extremes and of germination. A short general introduction to cyanobacteria and their cell types will be included. The remainder of the review will fall into four main sections. The first will deal with the metabolism of the heterocyst and akinete, with particular reference to nitrogen fixation in the former. The next will be concerned with a description of the metabolic and ultrastructural changes associated with heterocyst and a kinete development. A third section will describe the special arrangement of the heterocyst and akinete (one of the features which makes this group of prokaryotes unique), the relationship between the cell types, and methods of altering this normal regular spatial pattern. The final section will describe in detail present theories of pattern control in cyanobacteria and the mechanisms by which the process of differentiation itself is regulated.

Control of phycobiliprotein proteolysis and heterocyst differentiation in Anabaena.

Phycobiliprotein degradation can be initiated in cultures of the cyanobacterium Anabaena by removal of combined nitrogen from the medium. Certain strains of Anabaena differentiate cells specialized for aerobic nitrogen fixation (heterocysts) under such conditions. We describe here a procedure for the preparation of extracts from heterocysts or vegetative cells that contain an activity capable of degrading only the phycobiliproteins in a mixture of soluble Anabaena proteins in vitro. This activity increased under nitrogen starvation conditions or in ammonia-replete cultures treated with the glutamine synthetase inhibitor methionine sulfoximine. The increase in activity induced by nitrogen starvation was prevented by chloramphenicol or by carbon starvation. Under all these conditions, phycobiliprotein degradative activity assayed in vitro was correlated with the loss of phycobiliprotein absorbance in vivo. Finally, starvation of a met auxotroph of Anabaena for methionine (in the presence of ammonia) did not induce phycobiliprotein degradation in vivo or the increase in proteinase activity. Together with direct measurements of ppGpp, these results indicate that proteolysis in Anabaena is not controlled by compounds associated with the stringent response in Escherichia coli. Since the increase in proteinase activity appears to be regulated by the same variables that control heterocyst differentiation, the activity should provide a useful biochemical marker for the early events of differentiation.

Identification of blue-green algal nitrogen fixation genes by using heterologous DNA hybridization probes

In the filamentous blue-green alga Anabaena 7120, aerobic nitrogen fixation is linked to the differentiation of specialized cells called heterocysts. In order to study control of heterocyst development and nitrogen fixation in Anabaena, we have used cloned fragments of the Klebsiella pneumoniae nitrogen fixation (nif) genes as probes in DNA.DNA hybridizations with restriction endonuclease fragments of Anabaena DNA. Using this technique, we were able to identify and clone Anabaena nif genes, demonstrating the feasibility of using heterologous probes to identify genes for which no traditional genetic selection exists. From the patterns of hybridization observed, we deduced that although DNA sequence homology has been retained between some of the nif genes of these divergent organisms, the nif gene order has been rearranged.

Hydrogen metabolism in blue-green algae

This manuscript reviews the literature on hydrogen metabolism in blue-green algae and reports some new data from this laboratory. H2-formation by intact cells is found to be catalyzed exclusively by nitrogenase. Its rate appears to be variable from strain to strain used byt is--in our hands--very small. Therefore, blue-green algae are presumably of limited value in projects of solar energy conversion to form molecular hydrogen. These organisms are also able to consume the gas in a reaction catalysed by hydrogenase. Hydrogen is mainly consumed in an oxygen dependent reaction, as in aerobic nitrogen fixing bacteria. It can also serve as an electron donor for nitrogen fixation under certain physiological conditions. In experiments with a cell-free preparation, hydrogenase is found to be membrane-bound. The enzyme is characterized with respect to its specifity towards electron donors and acceptors.

Influence of some environmental factors on nitrogen fixation in the rhizosphere of rice

Nitrogen fixers make up a large percentage of the total microflora in the rhizosphere of lowland rice. There are more aerobic nitrogen fixers than there are anaerobic ones.

A quantitative study of the bacteria of a tempory pond.

Surmmary To examine the role of bacteria in the life history of a temporary pond, nine physiological groups of bacteria were determined by plate counts and enrichment culture techniques in pond water, in soil from the pond basin, and in soil from the pinewoods area surrounding the basin. Sampling was begun when the pond was dry and continued through the period when it contained water, into the next dry period. Low counts were obtained for sulphur-, ammonia-, and nitrite-oxidizing autotrophs; iron-oxidizing autotrophs were not detected. This indicates that bacteria did not make a major contribution to the ecosystem as primary producers. Soil samples contained millions to hundreds of millions (106-108) of aerobic nitrogen-fixers and millions to tens of millions (106-107) of urea-utilizing organisms per gramme, suggesting that they may be of significance in the nitrogen cycle in the ecosystem. Hundreds of thousands to tens of millions (105-107) of cellulose-decomposers, and tens of millions to hundreds of millions (107-108) of aerobic and of anaerobic heterotrophs were present per gramme in soil samples. The several heterotrophic types were also present in pond water in maximum numbers ranging from tens of thousands per millitre for nitrogen-fixers to millions per millilitre for anaerobic heterotrophs. As the pond dried the numbers of bacteria in its water decreased. These data suggest that bacteria in the pond ecosystem play a role in the nitrogen, carbon and energy cycles as decomposers and transformers, as a source of nutrilites and as members of the food chain.

NON-SYMBIOTIC NITROGEN FIXATION IN SOME QUEBEC SOILS

The occurrence of free-living nitrogen fixers, the potential for nitrogen fixation, and the correlation between the nitrogen-fixing capacities of the soils and bacterial counts were studied using representative Quebec soils.Clostridium occurred more frequently than did Azotobacter. Studies with N15showed that nitrogen fixation was more frequent under anaerobic than under aerobic conditions in all the soil types studied in their unamended state. The addition of glucose stimulated nitrogen fixation. During anaerobic incubation, nitrogen fixation was found to be correlated significantly with the increase in numbers of both total aerobes and Clostridia. The results suggested that facultatively anaerobic nitrogen fixers, and aerobic nitrogen fixers other than Azotobacter, were present.