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 concentrations.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.     

Atmospheric nitrogen fixation by hydrocarbon-oxidizing bacteria

Microorganisms have been found which concomitantly convert hydrocarbons, selected naphthenic acids, and atmospheric nitrogen into cellular substance. Bacteria are included in the genera Pseudomonas, Mycobacterium, and Azotobacter. Carbon sources utilized include the hydrocarbons methane, n-butane, n-tetradecane, toluene, and a naphthenic acid, cyclohexane-carboxylate. Uptake of isotopic nitrogen was employed as a criterion of nitrogen fixation. The results indicate a rather wide prevalence in nature of hydrocarbon-oxidizing bacteria capable of fixing atmospheric nitrogen. Their occurrence helps explain the high concentration of organic nitrogen commonly found in soils exposed to gas leakage from pipelines or natural-gas seeps, and suggests further consideration of the possibility of applying selected petroleum residua to soils in order to increase the agricultural potential by nitrogen-fixing processes.     

Seasonal variation in rates of heterotrophic nitrogen fixation (acetylene reduction) in Zostera noltii meadows and uncolonised sediments of the Bassin d'Arcachon, south-west France

Nitrogen fixation (acetylene reduction) rates were measured over an annual cycle in meadows of the seagrass Z. noltii and uncolonised sediments of the Bassin d'Arcachon, south-west France, using both slurry and whole core techniques. Measured rates using the slurry technique in Z. noltii colonised sediments were consistently higher than those determined in isolated cores. This was probably due to the release of labile organic carbon sources during preparation of the slurries. Thus, in colonised sediments the whole core technique may provide a more accurate estimate of in situ activity. Acetylene reduction rates measured by the whole core technique in colonised sediments were 1.8 to 4-fold greater, dependent upon the season, in the light compared with those measured in the dark, indicating that organic carbon released by the plant roots during photosynthesis was an important factor regulating nitrogen fixation. In contrast acetylene reduction rates in uncolonised sediments were independent of light.Addition of sodium molybdate, a specific inhibitor of sulphate reduction inhibited acetylene reduction activity in Z. noltii colonised sediments by > 80% as measured by both slurry and whole core techniques irrespective of the light regime, throughout the year inferring that sulphate reducing bacteria (SRB) were the dominant component of the nitrogen fixing microflora. A mutualistic relationship between Z. noltii and nitrogen fixing SRB in the rhizosphere, based on the exchange of organic carbon and fixed nitrogen is proposed. In uncolonised sediments sodium molybdate initially severely inhibited acetylene reduction rates, but the level of this inhibition declined over the course of the year. These data indicate that the nitrogen fixing SRB associated with the Zostera roots and rhizomes were progressively replaced by an aerobic population of nitrogen fixers associated with the decomposition of this recalcitrant high C:N ratio organic matter.Acetylene and sulphate reduction rates in the seagrass beds showed distinct summer maxima which correlated with a reduced availability of NH ₄ ⁺ in the sediment and the growth cycle of Z. noltii in the Bassin. Overall, these data indicate that acetylene reduction (nitrogen fixation) activity in the rhizosphere of Z. noltii was regulated both by release of organic carbon from the plant roots and maintenance of low ammonium concentrations in the root zone due to efficient ammonium assimilation.Nitrogen fixation rates determined from acetylene reduction rates measured by the whole core technique ranged from 0.1 to 7.3 mg N m^–2 d^–1 in the Z. noltii beds and between 0.02 and 3.7 mg N m^–2 d^–1 in uncolonised sediments, dependent upon the season. Nitrogen fixation in the rhizosphere of Z. noltii was calculated to contribute between 0.4 and 1.1 g N m^–2 y^–1 or between 6.3 and 12% of the annual fixed nitrogen requirement of the plants. Heterotrophic nitrogen fixation therefore represents a substantial local input of fixed nitrogen to the sediments of this shallow coastal lagoon and contributes to the overall productivity of Z. noltii in this ecosystem.     

Nitrogen fixation associated with sago (Metroxylon sagu) and some implications

Aims: To determine the presence and contribution of diazotrophic bacteria to nitrogen concentrations in edible starch derived from the sago palm (Metroxylon sagu). Methods and Results: Isolation of diazotrophic bacteria and analysis of nitrogen fixation were conducted on pith, root and sago starch samples. Acetylene reduction showed that five of ten starch samples were fixing nitrogen. Two presumptive nitrogen‐fixing bacteria from starch fixed nitrogen in pure culture and five isolates were positive for the nif H gene. Nitrogen concentrations in 51 starch samples were low (37 samples <0·2 g kg^?1; 14 ranging from 0·2 to 2·0 g kg^?1). Conclusions: Nitrogen fixation occurs in sago starch, which undoubtedly plays a role in fermentation ecology. Nitrogen levels are considered too low to be of nutritional benefit and to protect against nutritional‐associated illnesses. Significance and Impact of the Study: Sago starch does not add significantly to the protein calorie intake and may be associated with susceptibility to nutritional‐associated illness.     

Molecular identification of diazotroph microbial consortia in mountain soil

Nitrogen fixing microbial consortia from soil samples taken from five altitudinal vegetation zones (alpine, subalpine, coniferous, beech, Maleia flood plain) of Parang Massif, Romania, were isolated and identified. Molecular characterisation of nitrogen fixing consortia was carried out by PCR and nested PCR with 7 primer sets specific to nifH genes. All nifH genes are specific to nitrogen fixation and are found within phylogenetically related organisms which have the nitrogenase enzyme complex. These molecular studies allowed the assessment of nifH gene diversity within these nitrogen fixing microbial consortia from different type of soils. At high altitude, a consortium of nitrogen fixing bacteria dominated by Azotobacter chroococcum and Azospirillum brasilense was found. Clostridium, Rhizobiales, Herbaspirillum, Frankia species were also found in different rations depending on the altitudinal vegetation zone.     

MALDI mass spectrometry‐assisted molecular imaging of metabolites during nitrogen fixation in the Medicago truncatula–Sinorhizobium meliloti symbiosis

Symbiotic associations between leguminous plants and nitrogen‐fixing rhizobia culminate in the formation of specialized organs called root nodules, in which the rhizobia fix atmospheric nitrogen and transfer it to the plant. Efficient biological nitrogen fixation depends on metabolites produced by and exchanged between both partners. The Medicago truncatula–Sinorhizobium meliloti association is an excellent model for dissecting this nitrogen‐fixing symbiosis because of the availability of genetic information for both symbiotic partners. Here, we employed a powerful imaging technique – matrix‐assisted laser desorption/ionization (MALDI)/mass spectrometric imaging (MSI) – to study metabolite distribution in roots and root nodules of M. truncatula during nitrogen fixation. The combination of an efficient, novel MALDI matrix [1,8–bis(dimethyl‐amino) naphthalene, DMAN] with a conventional matrix 2,5–dihydroxybenzoic acid (DHB) allowed detection of a large array of organic acids, amino acids, sugars, lipids, flavonoids and their conjugates with improved coverage. Ion density maps of representative metabolites are presented and correlated with the nitrogen fixation process. We demonstrate differences in metabolite distribution between roots and nodules, and also between fixing and non‐fixing nodules produced by plant and bacterial mutants. Our study highlights the benefits of using MSI for detecting differences in metabolite distributions in plant biology.     

Isolation,partial characterization,and the effect of plant growth-promoting bacteria (PGPB) on micro-propagated sugarcane in vitro

We report the isolation of nitrogen fixing, phytohormone producing bacteria from sugarcane and their beneficial effects on the growth of micropropagated sugarcane plantlets. Detection of the nitrogen fixing bacteria by ARA-based MPN (acetylene reduction assay-based most probable number) method indicated the presence of up to 10⁶ bacteria per gram dry weight of stem and 10⁷ bacteria per gram dry weight of root of field-grown sugarcane. Two nitrogen fixing bacterial isolates were obtained from stem (SC11, SC20) and two from the roots (SR12, SR13) of field-grown plants. These isolates were identified as Enterobacter sp. strains on the basis of their morphological characteristics and biochemical tests. The isolate SC20 was further characterized by 16S rRNA sequence analysis, which showed high sequence similarity to the sequence of Enterobacter cloacae and Klebsiella oxytoca. All the isolates produced the phytohormone indoleacetic acid (IAA) in pure culture and this IAA production was enhanced in growth medium containing tryptophan. The bacterial isolates were used to inoculate micro-propagated sugarcane in vitro where maximum increase in the root and shoot weight over control was observed in the plantlets inoculated with strain SC20. By using the¹⁵N isotope dilution technique, maximum nitrogen fixation contribution (28% of total plant nitrogen) was detected in plantlets inoculated with isolate SC20.     

Organic matter and nitrogen accumulation and nitrogen fixation during early ecosystem development in Hawaii

We used a chronosequence comprised of 10 y, 52 y and 142 yold `a'a lava flows on Mauna Loa, Hawaii, to determine theaccumulation of organic matter and nitrogen and rates of nitrogenfixation through time. The mass of organic matter (live and deadbiomass and soil organic matter) on the 1984, 1942 and 1852 lavaflows was 0.6, 2.2 and 7.6 kg m^{– 2}, respectively, while total N was 4.8, 10.9 and 85.7 g m^{– 2}.We estimated the total rates of nitrogen fixation for thethree different aged ecosystems using an acetylene reduction assaycalibrated with ¹⁵N incubations. While mean rates of total N fixation remained largely constant across the three sites – between2.0 and 3.1 kg ha^{– 1} y^{– 1} – the most important sources of N fixation changed. On the 10 y flow, the most important fixer was the pioneering cyanolichen, Stereocaulon vulcani. After 52 years ofecosystem development, the most important N fixer was a cyanoalga,while after 142 years, the predominant N fixers were heterotrophicbacteria associated with leaf litter, twigs and detritus. The totalamount of N accumulated after 52 years of ecosystem development wasequivalent to cumulative inputs through biological N fixation. After 142 years, however, cumulative inputs from N fixation couldonly account for between 27–59% of the total nitrogen accrued.We used fertilizer additions of all essential nutrients otherthan N to test whether the availability of lithophilic nutrientsregulated rates of N fixation in early ecosystem development. Ratesof nitrogen fixation by the lichen, S. vulcani, approximately doubled when fertilized on the 1984 and 1942 flows. Rates of N-fixation by heterotrophic nitrogen fixing bacteria on leaf litter ofMetrosideros polymorpha also increased significantly when fertilized with lithophilic nutrients. These findings suggest that weathering rates of lava in part regulate rates of nitrogen fixation in these young ecosystems.     

Insights into the history of the legume‐betaproteobacterial symbiosis

The interaction between legumes and rhizobia has been well studied in the context of a mutualistic, nitrogen‐fixing symbiosis. The fitness of legumes, including important agricultural crops, is enhanced by the plants’ ability to develop symbiotic associations with certain soil bacteria that fix atmospheric nitrogen into a utilizable form, namely, ammonia, via a chemical reaction that only bacteria and archaea can perform. Of the bacteria, members of the alpha subclass of the protebacteria are the best‐known nitrogen‐fixing symbionts of legumes. Recently, members of the beta subclass of the proteobacteria that induce nitrogen‐fixing nodules on legume roots in a species‐specific manner have been identified. In this issue, Bontemps et al. reveal that not only are these newly identified rhizobia novel in shifting the paradigm of our understanding of legume symbiosis, but also, based on symbiotic gene phylogenies, have a history that is both ancient and stable. Expanding our understanding of novel plant growth promoting rhizobia will be a valuable resource for incorporating alternative strategies of nitrogen fixation for enhancing plant growth.     

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.     

Contribution of nitrogen fixation to first year Miscanthus × giganteus

Many characteristics make Miscanthus × giganteus an appealing bioenergy feedstock in temperate North America, but the degree to which this plant species interacts with nitrogen‐fixing bacteria remains understudied. Demonstration of associative nitrogen fixation in Miscanthus would support management with minimal fertilizer inputs that is demanded of long‐term biofuel sustainability. As a first step, we investigate the role of biological nitrogen fixation in nutrition of immature Miscanthus and temporal dynamics of plant‐associated nitrogen fixers. The contribution of biological nitrogen fixation to plant nitrogen acquisition in first year Miscanthus × giganteus was estimated using a yield‐dependent ¹⁵N isotope dilution model. Temporal changes in plant‐associated diazotroph relative abundance and community composition were analyzed with quantitative PCR and terminal restriction fragment length polymorphism of the nifH gene in rhizome and rhizosphere DNA extracts. We estimate 16% of new plant nitrogen was derived by nitrogen fixation during the growing season, despite non‐limiting soil nitrogen. Diazotroph communities from rhizome and rhizosphere changed with plant development and endophytic nitrogen fixers had significantly higher relative abundance and altered community composition at sampling dates in July and August. This study provides evidence for a small, but measurable, benefit of associative nitrogen fixation to first year Miscanthus × giganteus that underscores the potential and need for selection of breeding lines that maximize this trait.     

Carbon availability controls the growth of detritivores (Lumbricidae) and their effect on nitrogen mineralization

Activity of soil decomposer microorganisms is generally limited by carbon availability, but factors controlling saprophagous soil animals remain largely unknown. In contrast to microorganisms, animals are unable to exploit mineral nutrient pools. Therefore, it has been suggested that soil animals, and earthworms in particular, are limited by the availability of nitrogen. In contrast to this view, a strong increase in density and biomass of endogeic earthworms in response to labile organic carbon addition has been documented in field experiments. The hypothesis that the growth of endogeic earthworms is primarily limited by carbon availability was tested in a laboratory experiment lasting for 10 weeks. In addition, it was investigated whether the effects of earthworms on microbial activity and nutrient mineralization depend on the availability of carbon resources. We manipulated food availability to the endogeic earthworm species Octolasion tyrtaeum by using two soils with different organic matter content, providing access to different amounts of soil, and adding labile organic carbon (glucose) enriched in ¹³C.Glucose addition strongly increased the growth of O. tyrtaeum. From 8 to 17% of the total C in earthworm tissue was assimilated from the glucose added. Soil microbial biomass was not strongly affected by the addition of glucose, though basal respiration was significantly increased and up to 50% of the carbon added as glucose was incorporated into soil organic matter. The impact of earthworms on the mineralization and leaching of nitrogen depended on C availability. As expected, in C-limited soil, the presence of earthworms strongly increased nitrogen leaching. However, when C availability was increased by the addition of glucose, this pattern was reversed, i.e. the presence of O. tyrtaeum decreased nitrogen leaching and its availability to soil microflora. We conclude that irrespective of the total carbon content of soils, O. tyrtaeum was primarily limited by carbon, and that increased carbon availability allowed earthworms to be more effective in mobilizing N. The presence of earthworms increases C limitation of soil microorganisms, due to increased availability of N and P in earthworm casts or a direct depletion of easily available carbon resources by earthworms.     

Different cytokinin histidine kinase receptors regulate nodule initiation as well as later nodule developmental stages in Medicago truncatula

Legume plants adapt to low nitrogen by developing an endosymbiosis with nitrogen‐fixing soil bacteria to form a new specific organ: the nitrogen‐fixing nodule. In the Medicago truncatula model legume, the MtCRE1 cytokinin receptor is essential for this symbiotic interaction. As three other putative CHASE‐domain containing histidine kinase (CHK) cytokinin receptors exist in M. truncatula, we determined their potential contribution to this symbiotic interaction. The four CHKs have extensive redundant expression patterns at early nodulation stages but diverge in differentiated nodules, even though MtCHK1/MtCRE1 has the strongest expression at all stages. Mutant and knock‐down analyses revealed that other CHKs than MtCHK1/CRE1 are positively involved in nodule initiation, which explains the delayed nodulation phenotype of the chk1/cre1 mutant. In addition, cre1 nodules exhibit an increased growth, whereas other chk mutants have no detectable phenotype, and the maintained nitrogen fixation capacity in cre1 requires other CHK genes. Interestingly, an AHK4/CRE1 genomic locus from the aposymbiotic Arabidopsis plant rescues nodule initiation but not the nitrogen fixation capacity. This indicates that different CHK cytokinin signalling pathways regulate not only nodule initiation but also later developmental stages, and that legume‐specific determinants encoded by the MtCRE1 gene are required for later nodulation stages than initiation.     

Bacteriophage in relation to nitrogen fixation by red clover

Summary Cultures of Rhizobium trifolii resistant to the action of a given bacteriophage, may vary appreciably in their nitrogen fixing ability in association with the proper host plant. Likewise, cultures of Rh. trifolii, sensitive to bacteriophage may or may not benefit the host plant as judged by nitrogen fixation.Since sensitive and resistant cultures may be comparable in their nitrogen fixing capacity, it appears that the behavior of a culture of Rh. trifolii toward the lytic action of a specific bacteriophage in vitro cannot be correlated with its nitrogen fixing ability in association with the host plant.If a bacteriophage is added to a sensitive strain of Rh. trifolii used for inoculation of red clover, the cultures recovered from the nudules are often only of the resistant type. When this occurs the nitrogen fixed through association of plant and bacteria is decreased in the case of a good strain, but is unaffected if the strain is of the poor type. The addition of phage to a resistant strain of Rh. trifolii used for inoculation of red clover plants does not change the resistant type of culture recovered from the nodules, nor is the fixation of nitrogen by plant and bacteria affected.     

Nitrogen Fixation by Free-living Bacteria in the Soil and in the Canopy of Douglas Fir

Nitrogen fixing bacteria have been isolated from the soil andthe leaves of Douglas fir. Measurements, taken at monthly intervalsusing ¹⁵N, have shown that small quantities of nitrogen arefixed on the leaves and in the various soil layers and thatthe highest rates of fixation occur in the spring. Further studieswith ¹⁵N have shown that the products of nitrogen fixation inDouglas fir soils are available for growth of seedlings andfor denitrification.     

Chemical and microbiological changes during vermicomposting of coffee pulp using exotic (<Emphasis Type="Italic">Eudrilus eugeniae</Emphasis>) and native earthworm (<Emphasis Type="Italic">Perionyx ceylanesis</Emphasis>) species

Coffee pulp is the main solid residue from the wet processing of coffee berries. Due to presence of anti-physiological and anti-nutritional factors, coffee pulp is not considered as adequate substrate for bioconversion process by coffee farmers. Recent stringent measures by Pollution Control authorities, made it mandatory to treat all the solid and liquid waste emanating from the coffee farms. A study was conducted to evaluate the efficiency of an exotic (Eudrilus eugeniae) and a native earthworm (Perionyx ceylanesis) from coffee farm for decomposition of coffee pulp into valuable vermicompost. Exotic earthworms were found to degrade the coffee pulp faster (112 days) as compared to the native worms (165 days) and the vermicomposting efficiency (77.9%) and vermicompost yield (389 kg) were found to significantly higher with native worms. The multiplication rate of earthworms (280%) and worm yield (3.78 kg) recorded significantly higher with the exotic earthworms. The percentage of nitrogen, phosphorous, potassium, calcium and magnesium in vermicompost was found to increase while C:N ratio, pH and total organic carbon declined as a function of the vermicomposting. The plant nutrients, nitrogen (80.6%), phosphorus (292%) and potassium (550%) content found to increase significantly in the vermicompost produced using native earthworms as compared to the initial values, while the calcium (85.7%) and magnesium (210%) content found to increase significantly in compost produced utilizing exotic worms. Vermicompost and vermicasts from native earthworms recorded significantly higher functional microbial group’s population as compared to the exotic worms. The study reveals that coffee pulp can be very well used as substrate for vermicomposting using exotic (Eudrilus eugeniae) and native earthworm (Perionyx ceylanesis).     

Eisenia fetida (Oligochaeta, Lumbricidae) Activates Fungal Growth, Triggering Cellulose Decomposition During Vermicomposting

Cellulose is the most abundant polymer in nature and constitutes a large pool of carbon for microorganisms, the main agents responsible for soil organic matter decomposition. Cellulolysis occurs as the result of the combined action of fungi and bacteria with different requirements. Earthworms influence decomposition indirectly by affecting microbial population structure and dynamics and also directly because the guts of some species possess cellulolytic activity. Here we assess whether the earthworm Eisenia fetida (Savigny 1826) digests cellulose directly (i.e., with its associated gut microbiota) and also whether the effects of E. fetida on microbial biomass and activity lead to a change in the equilibrium between fungi and bacteria. By enhancing fungal communities, E. fetida would presumably trigger more efficient cellulose decomposition. To evaluate the role of E. fetida in cellulose decomposition, we carried out an experiment in which pig slurry, a microbial-rich substrate, was treated in small-scale vermireactors with and without earthworms. The presence of earthworms in vermireactors significantly increased the rate of cellulose decomposition (0.43 and 0.26% cellulose loss day⁻¹, with and without earthworms, respectively). However, the direct contribution of E. fetida to degradation of cellulose was not significant, although its presence increased microbial biomass (C_mic) and enzyme activity (cellulase and β-glucosidase). Surprisingly, as fungi may be part of the diet of earthworms, the activity of E. fetida triggered fungal growth during vermicomposting. We suggest that this activation is a key step leading to more intense and efficient cellulolysis during vermicomposting of organic wastes.     

Loss‐of‐function of ASPARTIC PEPTIDASE NODULE‐INDUCED 1 (APN1) in Lotus japonicus restricts efficient nitrogen‐fixing symbiosis with specific Mesorhizobium loti strains

The nitrogen‐fixing symbiosis of legumes and Rhizobium bacteria is established by complex interactions between the two symbiotic partners. Legume Fix^– mutants form apparently normal nodules with endosymbiotic rhizobia but fail to induce rhizobial nitrogen fixation. These mutants are useful for identifying the legume genes involved in the interactions essential for symbiotic nitrogen fixation. We describe here a Fix^– mutant of Lotus japonicus, apn1, which showed a very specific symbiotic phenotype. It formed ineffective nodules when inoculated with the Mesorhizobium loti strain TONO. In these nodules, infected cells disintegrated and successively became necrotic, indicating premature senescence typical of Fix^– mutants. However, it formed effective nodules when inoculated with the M. loti strain MAFF303099. Among nine different M. loti strains tested, four formed ineffective nodules and five formed effective nodules on apn1 roots. The identified causal gene, ASPARTIC PEPTIDASE NODULE‐INDUCED 1 (LjAPN1), encodes a nepenthesin‐type aspartic peptidase. The well characterized Arabidopsis aspartic peptidase CDR1 could complement the strain‐specific Fix^– phenotype of apn1. LjAPN1 is a typical late nodulin; its gene expression was exclusively induced during nodule development. LjAPN1 was most abundantly expressed in the infected cells in the nodules. Our findings indicate that LjAPN1 is required for the development and persistence of functional (nitrogen‐fixing) symbiosis in a rhizobial strain‐dependent manner, and thus determines compatibility between M. loti and L. japonicus at the level of nitrogen fixation.     

Ammonium sensing in nitrogen fixing bacteria: Functions of theglnB andglnD gene products

A plentiful supply of fixed nitrogen as ammonium (or other compounds such as nitrate or amino acids) inhibits nitrogen fixation in free-living bacteria by preventing nitrogenase synthesis and/or activity. Ammonium and nitrate have variable effects on the ability ofRhizobiaceae (Rhizobium, Bradyrhizobium andAzorhizobium) species to nodulate legume hosts and on nitrogen fixation capacity in bacteroid cells contained in nodules or in plant-free bacterial cultures. In addition to effects on nitrogen fixation, excess ammonium can inhibit activity or expression of other pathways for utilization of nitrogenous compounds such as nitrate (through nitrate and nitrite reductase), or glutamine synthetase (GS) for assimilation of ammonium. This paper describes the roles of two key genesglnB andglnD, whose gene products sense levels of fixed nitrogen and initiate a cascade of reactions in response to nitrogen status. While work onEscherichia coli and other enteric bacteria provides the model system,glnB and, to a lesser extent,glnD have been studied in several nitrogen fixing bacteria. Such reports will be reviewed here. Recent results on the identity and function of theglnB andglnD gene products inAzotobacter vinelandii (a free-living soil diazotroph) and inRhizobium leguminosarum biovarviciae, hereinafter designatedR.l. viciae will be presented. New data suggests thatAzotobacter vinelandii probably contains aglnB-like gene and this organism may have twoglnD-like genes (one of which was recently identified and namednfrX). In addition, evidence for uridylylation of theglnB gene product (the PII protein) ofR. l. viciae in response to fixed nitrogen deficiency is presented. Also, aglnB mutant ofR. l. viciae has been isolated; its characteristics with respect to expression of nitrogen regulated genes is described.     

Isolation of Nitrogen Fixing Bacteria from Fungus Termites

Biological nitrogen fixation by the microorganisms in the gut of termites is one of the singularly important symbiotic processes, since termites invariably thrive on nitrogen poor diet. Two isolates of free living aerobic and facultative anaerobic N fixing bacteria were obtained from the guts of fungus cultivating termite, Macrotermes sp. Among the total bacterial isolates from termite gut, the per cents of N fixing aerobes viz., Azotobacter and Beijerinckia spp were 49% and 37% from the salivary gland while facultative N fixing anaerobe viz., Klebsiella and Clostridium contributed (51% and 93%). The free living aerobic bacteria were identified as Azotobacter spp (19 x 10⁴ CFU mL^‐1) and Beijerinckia (13.2 x 10⁴ CFU mL^‐1) from the salivary gland of the termite; interestingly, foregut, mid gut and hind gut registered a low population of these bacteria. The isolates of Azotobacter were smooth, glistening, vicid in nature, rods, gram negative and cyst forming. Isolates of Beijerinckia sp. produced copious slime, tenacious, rods, gram negative with no cyst formations. Both the isolates emitted green fluorescence and produced acid. Facultative N fixing anaerobes were harbored in the hind gut. The isolates were identified as Klebsiella (20 x 10⁴ CFU mL^‐1) and Clostridium pasteurianum 39.1 x 10⁴ CFU mL^‐1. Klebsiella were straight rods arranged singly or in pairs, non‐motile, gram negative, whereas Clostridium pasteurianum was viscoid, motile with terminal spores. A positive correlation was observed between the extractable polysaccharides of these isolates and soil aggregation. The aggregates formed by the isolates increased soil aeration, porosity, water holding capacity and helped in better plant growth. Thus, the gut microflora of termite, apart from harnessing nitrogen from the atmosphere, also helps improving soil fertility.