Free-living Nitrogen Fixing Research Articles

Purification and properties of glutamine synthetase from Bacillus polymyxa

Glutamine synthetase (EC 6.3.1.2) was purified to homogeneity from a free-living nitrogen fixing bacteria, Bacillus polymyxa. The holoenzyme, relative molecular mass (Mr) of 600 000 is composed of monomeric sub-units of 60 000 (Mr). The isoelectric point of the sub-units was 5.2. The pH optimum for the biosynthetic and transferase enzyme activity was 8.2 and 7.8, respectively. The apparent Km values (Kmapp) in the biosynthetic reaction for glutamate, NH4Cl and ATP were 3.2, 0.22 and 1 mM, respectively. In the transferase reaction the Km values for glutamine, hydroxylamine and ADP were 6.5, 3.5 and 8×10-4 mM respectively. L-Methionine-D-L-sulfoximine was a very potent inhibitor in both biosynthetic and transferase reactions. Similar to most Gram positive bacteria there was no evidence of in vivo adenylylation and the enzyme seemed to be mainly regulated by feed-back mechanism.

Regulation of Nitrogen Fixation (nif) Genes of Azorbizobium caulinodans ORS571 in Culture and in planta

Summary Translational fusions of nitrogen fixation ( nif ) genes to the β-galactosidase ( lacZ ) gene are used to investigate nif gene regulation, in response to different nitrogen sources and oxygen concentrations, to free living nitrogen fixing cultures of Azorhizobium caulinodans ORS571. Derepression of nif gene promoters is also examined in nitrogen fixing stem- and root nodules induced on Sesbania rostrata . The effect of increasing nitrate concentrations on nodulation and nitrogen fixation is determined. These results are reviewed in the light of recent progress in understanding the structure, function and regulation of nitrogen fixation and assimilation genes of A. caulinodans ORS571.

Characterization of Azorhizobium caulinodans gen. nov., sp. nov., a Stem-Nodulating Nitrogen-Fixing Bacterium Isolated from Sesbania rostrata

Twenty stem- and root-nodulating bacterial strains isolated from stem nodules of Sesbania rostrata were compared by numerical analysis of 221 phenotypic features with nine strains which effectively nodulate only the roots of this plant and with representative strains from the genera Rhizobium and Bradyrhizobium. Representative organisms from the different clusters were investigated further, together with possibly related organisms, by performing comparative gel electrophoresis of whole-cell proteins and by performing deoxyribonucleic acid (DNA)-DNA and DNA-ribosomal ribonucleic acid (rRNA) hybridizations. 3H-labeled rRNA was prepared from Sesbania stem- and root-nodulating bacterial strain ORS 571T (T = type strain); [14C]rRNA from Bradyrhizobium japonicum NZP 5549T was also used. The following conclusions were drawn: (i) the Sesbania root-nodulating bacterial strains are genuine rhizobia; (ii) the Sesbania stem- and root-nodulating strains are quite different from Rhizobium and Bradyrhizobium, and thus they constitute a separate rRNA subbranch on the Rhodopseudomonas palustris rRNA branch in rRNA superfamily IV; and (iii) the closest relative of these organisms is Xanthobacter, but they are phenotypically and genotypically sufficiently different from the latter genus to deserve a separate generic rank. Because the feature of free-living nitrogen fixation is quite discriminative, a new genus, Azorhizobium, is proposed, with one species, Azorhizobium caulinodans. The type strain is strain ORS 571 (= LMG 6465).

Rates of Free-Living Nitrogen Fixation in Some Piedmont Forest Types

Abstract We examined rates of free-living (nonsymbiotic) nitrogen fixation in a successional sequence of forest types in the Piedmont of North Carolina. Annual nitrogen input through this process did not differ among ecosystems, averaging roughly 0.002 kg/ha annually. The rate in a 20- to 30-year-old stand did not increase with irrigation or additions of nitrogen, phosphorus, or molybdenum. Free-living nitrogen fixation in these forests should not be expected to compensate for nitrogen losses through natural processes or management activities. For. Sci. 33(2):548-551.

Preliminary results of a study on short term effects of prescribed fire in pine stands in NW Spain

Short term effects cf prescribed burning experiments carried out in P. pinaster stands in the N.W. of Spain on fuels ; soil nutrients , soil microflora and forest floor meso fauna were studied. Fire intensities range from 21 to 420 Kw.m-1. The mean maximum temperature recorded on the litter duff interface was 408 °C ; the temperature was greater than 60°C for about 5 minutes (mean value). On the duff-soil interface, 59° was reached and 21 °C was measured 2,5 cm below the top of the mineral soil. Fires consumed 32 per cent of the total fuel and 55 per cent of the available fuel. Two years after the fires, the total fuel was practically equal to the prebum load but the available fuel represented 64 per cent of its initial load. Changes in soil (0-5cm layers) nutrients were very small. Soon after the fires a little increment for the organic matter content, a small drop in pH and an increment in K, ca, Mg and P were detected. Nitrogen content was practically equal to pre-fire level. Two years later, all the nutrients remained at the same initial level, except for Ca which was 65 per cent higher than before burning. The organic matter per cent was a little smaller than before fire but pH was higher. Some groups of the soil microflora, especially aerobic free living nitrogen fixers, were enhanced by the fire. The others remained at the same level as in unbumt areas. Forest floor mesofauna populations were affected by fire and depending on groups, duff heating and duff consumption, their recovery lasted from six months to three and a half years.

Bradyrhizobium japonicum mutants defective in root-nodule bacteroid development and nitrogen fixation

The genome of the slow-growing Bradyrhizobium japonicum (strain 110) was mutagenized with transposon Tn5. A total of 1623 kanamycin/streptomycin resistant derivatives were screened in soybean infection tests for nodulation (Nod) and symbiotic nitrogen fixation (Fix). In this report we describe 14 strains possessing a stable, reproducible Nod+Fix- phenotype. These strains were also grown under microaerobic culture conditions to test them for free-living nitrogen fixation activity (Nif). In addition to strains having reduced Fix and Nif activities, there were also strains that had reduced symbiotic Fix activity but were Nif+ex planta.

Rhizobium free-living nitrogen fixation occurs in specialized nongrowing cells

A model for free-living N(2) fixation by Rhizobium sp. RC3200 is presented that asserts that this process occurs in nongrowing cells. Cultures containing mixed populations of cell types, N(2)-fixing and vegetative, grow cooperatively. In nitrogen-limited liquid suspension cultures, cooperative growth occurs by means of ammonium that is produced and exported by nongrowing, N(2)-fixing cells and transported to vegetative cells. This model implies prokaryotic differentiation: the creation of metabolically specialized cells, terminally nonviable, that functionally cooperate in a higher cell order. Here, the switch to a Rhizobium N(2)-fixing cell state is regulated by both O(2) and utilizable nitrogen.

Free-living and symbiotic nitrogen fixing ability ofRhizobium japonicumis unaffected by rifampicin resistance mutations

The hypothesis that rifampicin resistance mutations (possibly leading to altered RNA polymerases) have a pleiotropic effect on symbiotic nitrogen fixation was tested using the Rhizobium japonicum-soybean symbiosis. A total of 20 spontaneous rifampicin-resistant mutants of R. japonicum strain 110 were analyzed biochemically. RNA polymerase assays revealed that the enzyme from 15 mutants was indeed rifampicin-insensitive. Two of these mutants were found to possess an enzyme with an electrophoretically altered β subunit. All rifampicin-resistant mutants were able to form nodules on soybeans and fix nitrogen symbiotically; free-living nitrogen fixation under microaerophilic culture conditions was also unaffected.

Free-living and symbiotic nitrogen fixing ability of Rhizobium japonicum is unaffected by rifampicin resistance mutations

Free-living and symbiotic nitrogen fixing ability of Rhizobium japonicum is unaffected by rifampicin resistance mutations

Effect of Growth Stages of Soybean and Broadbean on Phyllosphere Densities of Free-Living Nitrogen Fixers

The distribution of oligonitrophilic bacteria and Azotobacter on soybean and broadbean leaves was studied during the growth season. The results revealed that the densities of oligonitrophilic bacteria were generally higher than those of Azotobacter in the phyllosphere during the growth season of both soybean and broadbean. The densities of these groups of nitrogen fixers were often higher on the upper surfaces of the leaves than those on lower ones during the growth season. The data also indicate that the densities of nitrogen-fixing free-living micro-organisms were generally higher in the phyllosphere of broadbean than in that of soybean. Es wurde die Verteilung von oligonitrophilen Bakterien und Azotobacter auf Sojabohnen-und Ackerbohnenblättern während der Entwicklung der Pflanzen studiert. Die Keimzahl der oligonitrophilen Bakterien in der Phyllosphäre war während des Wachstums im allgemeinen höher als die von Azotobacter, sowohl bei Sojabohnen als auch bei Ackerbohnen. Die Keimzahl der geprüften Stickstoffbinder war auf den oberen Blättern häufig höher als auf den unteren und die Zahl der freilebenden N-Binder in der Phyllosphäre von Ackerbohnen im allgemeinen höher als in der der Sojabohne.

Gnotobiotic cultures of rice plants up to ear stage in the absence of combined nitrogen source but in the presence of free living nitrogen fixing bacteriaAzotobacter vinelandii andRhodopseudomonas capsulata

An all glass tight growth chamber, entirely sterilizable, has been constructed to carry out axenic and gnotobiotic cultures of rice plants (Oryza sativa L.). When grown in liquid medium and in the absence of combined nitrogen but in the presence of the diazotrophsAzotobacter vinelandii andRhodopseudomonas capsulata, rice plants exhibited a complete biological cycle from germination up to ear stage, during a period of time similar to the one encountered in french paddy soil of Camargue. In one experiment, mannitol was given to rice culture medium together withAzotobacter vinelandii andRhodopseudomonas capsulata. In another experiment, mannitol was not given together with Rhodopseudomonas, and still positive nitrogen gain was obtained, although it was less than culture with mannitol. When15N labeled cells of Rhodopseudomonas were added in rice culture medium,15N was partly transferred to rice plant. Among the nitrogen substances excreted from the bacteria in the rhizosphere medium, large organic molecules were shown to be the most abundant in our experimental conditions. Moreover, the concentration of free ammonia or aminoacids present in the rice rhizosphere were always compatible with a bacterial nitrogenase activity.

Further examination of presumptive Rhizobium trifolii mutants that nodulate Glycine max

Two recent reports described the isolation of derivatives of a Rhizobium trifolii strain that had gained the ability to nodulate Glycine max and Vigna radiata and that had demonstrated altered patterns of carbon source utilization, free-living nitrogen fixation, and hydrogen uptake. More extensive characterization of these strains now supports the conclusion that these strains are R. japonicum and are not derived from the putative parent R. trifolii.

Why Do Aphids Synthesize Melezitose?

Aphids feed by removing sugars and amino acids in solution from the phloem of plants. Most of the sugar taken up is excreted as honeydew, a high proportion of which (over 40% in some species) is in the form of the trisaccharide, melezitose, which apparently does not occur in plants, and which must therefore by synthesized by aphids. It is predicted that melezitose provides a carbon source for free-living nitrogen fixing bacteria, and that the plant from which the honeydew came in the first place benefits from this arrangement, particularly if the supply of nitrogen is a limiting factor to plant growth. Throughout most of the season aphids are parthenogenetic and by making use of the concept of the evolutionary individual it is further suggested that the aphids themselves benefit from melezitose synthesis. Experiments are needed to confirm or refute the hypothesis.

Regulation of symbiotic nitrogen fixation in root nodules of alfalfa (Medicago sativa) infected with Rhizobium meliloti.

Symbiotic nitrogen fixation of Rhizobium meliloti bacteroids in Medicago sativa root nodules was suppressed by several inorganic nitrogen sources. Amino acids like glutamine, glutamic acid and aspartic acid, which can serve as sole nitrogen sources for the unnodulated plant did not influence nitrogenase activity of effective nodules, even at high concetrations. Ammonia and nitrate suppressed symbiotic nitrogen fixation in vivo only at concentrations much higher than those needed for suppression of nitrogenase activity in free living nitrogen fixing bacteria. The kinetics of suppression were slow compared with that of free living nitrogen fixing bacteria. On the other hand, nitrite, which acts as a direct inhibitor of nitrogenase, suppressed very quickly and at low concentrations. Glutamic acid and glutamine enhanced the effect of ammonia dramatically, while the suppression by nitrate was enhanced only slightly.

The acetylene reduction technique as an assay for nitrogenase activity in the methane oxidizing bacterium Methylococcus capsulatus strain bath

The use of acetylene as a convenient assay substrate for nitrogenase in methane oxidising bacteria is complicated by the observation that it is a potent inhibitor of the methane monooxygenase enzyme in both whole cells and cell-free extracts. If the cells were provided with alternative oxidisable carbon substrates other than methane then nitrogen fixing cells would reduce acetylene to ethylene. Hydrogen gas also served as an oxidisable substrate in the assay. Nitrous oxide, which is reduced by nitrogenase to N2 and H2O, was not an inhibitor of methane monooxygenase function and could be used as a convenient assay substrate for nitrogenase. Reduction of both substrates by whole cells showed similar response to oxygen in the assay system and in this respect Methylococcus resembles other free living nitrogen fixing aerobes.

Rhizobium as a free-living nitrogen fixer

Rhizobium as a free-living nitrogen fixer

ALLELOPATHIC EFFECTS OF ANDROPOGON VIRGINICUS AND ITS PERSISTENCE IN OLD FIELDS

Aqueous extracts of fresh roots and shoots of Andropogon virginicus (broomsedge) were found to be inhibitory to the growth of seedlings of Amaranthus palmeri, Bromus japonicus, Aristida oligantha, and Andropogon scoparius. The first two species are often important in the pioneer stage of old‐field succession in eastern Oklahoma, Aristida is prominent in the second stage, and Andropogon scoparius is important later in succession including the climax Quercus stellata–Quercus marilandica savanna. Sterile dilute extracts of roots and shoots of broomsedge were inhibitory to two test species of Azotobacter, a free living nitrogen fixer, and to two species of Rhizobium, a symbiotic nitrogen fixer. Small amounts of decaying shoots of broomsedge (1 g per 454 g of soil) were very inhibitory to the growth of the four test species listed above and to Amaranthus retroflexus, another species often important in the first stage of succession. Similar amounts of decaying material in soil also significantly inhibited growth and nodulation of the two most important species of legumes in old‐field succession in eastern Oklahoma, Lespedeza stipulacea and Trifolium repens. Broomsedge is known to compete vigorously and grow well on soils of low fertility, so the inhibition of nodulation of legumes could help keep the nitrogen supply low and give broomsedge a selective advantage in competition over species that have higher nitrogen requirements. The combined interference of broomsedge against other species resulting from competition and allelopathy could help explain why it invades old fields in 3–5 yr after abandonment from cultivation and remains so long in almost pure stands.

The response to inoculation of the three chromosome races of Trifolium ambiguum sown with and without a companion grass. II. The effect of the method of inoculation on the clover and grass

The inoculation of legumes is often carried out using skim-milk as the carrier for the nodule bacteria. Increased germination due to inoculation has often been reported but in this investigation the effects of the skim-milk and the bacteria have been separated. The results indicate that the early establishment of T. ambiguum was stimulated by the skim milk rather than the nodule bacteria. The mechanism of this effect is thought to be due to extra N present in the milk and possibly to an enhanced nitrogen-cycle in the inoculated soil and evidence is produced of increases in certain freeliving nitrogen fixing and nitrifying microorganisms.

Utilization of phosphates byRhizobium spp.in relation to their efficiencies

Abstract The relationship between the amount of phosphorous supplied and nitrogen fixation by free living nitrogen fixing organisms like Azotobacter and Clostridium has been established among others by Green (2). A quantitative relationship between the availability of phosphates and nitrogen fixation by Azotobacter has been expressed by several workers (Stoklassa (6) ; Waksman and Karunakar (7) ; Singh and Shrikhande (4); Sen et. al. (5)). In the case of a symbiotic organism like Rhizobium, phosphorous has been considered essential for the normai nodule growth and increased race of infection by Anderson and Thomas (1). The intimate relationship observed between phosphate supply and nitrogen fixation in the case of free living organism might cause one to suspect that the more efficient strains of Rhizobium spp. might have also higher capacities of phosphate utilization.

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.