NH4+-Excreting Azospirillum brasilense Mutants Enhance the Nitrogen Supply of a Wheat Host

Spontaneous ethylenediamine-resistant mutants of Azospirillum brasilense were selected on the basis of their excretion of NH₄⁺. Two mutants exhibited no repression of their nitrogenase enzyme systems in the presence of high (20 mM) concentrations of NH₄⁺. The nitrogenase activities of these mutants on nitrogen-free minimal medium were two to three times higher than the nitrogenase activity of the wild type. The mutants excreted substantial amounts of ammonia when they were grown either under oxygen-limiting conditions (1 kPa of O₂) or aerobically on nitrate or glutamate. The mutants grew well on glutamate as a sole nitrogen source but only poorly on NH₄Cl. Both mutants failed to incorporate [¹⁴C]methylamine. We demonstrated that nitrite ammonification occurs in the mutants. Wild-type A. brasilense, as well as the mutants, became established in the rhizospheres of axenically grown wheat plants at levels of > 10⁷ cells per g of root. The rhizosphere acetylene reduction activity was highest in the preparations containing the mutants. When plants were grown on a nitrogen-free nutritional medium, both mutants were responsible for significant increases in root and shoot dry matter compared with wild-type-treated plants or with noninoculated controls. Total plant nitrogen accumulation increased as well. When they were exposed to a ¹⁵N₂-enriched atmosphere, both A. brasilense mutants incorporated significantly higher amounts of ¹⁵N inside root and shoot material than the wild type did. The results of our nitrogen balance and ¹⁵N enrichment studies indicated that NH₄⁺-excreting A. brasilense strains potentially support the nitrogen supply of the host plants.     

Energy status,growth and nitrogenase activity in continuous cultures of Rhizobium sp. strain CB756 supplied with NH+4 and various rates of aeration

Continuous cultures of the cowpea-type Rhizobium sp., strain CB756, were grown in the presence of NH⁺₄ at automatically controlled concentrations of dissolved O₂ and rates of aeration. Nitrogenase activity of steady-state cultures was only detected under microaeration conditions (dissolved O₂ typically <0.03 μM; aeration rate typically 0.6 μmol O₂/ml per h), when the cellular ATP pool size was 0.8–1.8 nmol/mg dry wt., (optimum 1.1) and the energy charge 0.6–0.7. At twice this aeration rate and dissolved O₂ concentration of about 0.15 μM, the yield of bacteria doubled, the ATP pool increased and energy charge increased to 0.8. With similar rates of O₂ supply but high concentration of dissolved O₂ (approx. 150 μM), cultures were NH⁺₄-limited and the ATP pool and energy charge were slightly reduced. Amongst all of these O₂ supply conditions the total pool of adenosine phosphates was not significantly different (2.6 S.D. 0.7 nmol/mg dry wt.). In steady-state, O₂-limited cultures, concentrations of cyclic GMP were higher when nitrogenase was present. When rates of O₂ supply to steady-state cultures were changed, oscillations in bacterial energy status and growth rate were induced decreasing in amplitude until a new steady state was reached. This made it difficult to discern precisely the energy status in which nitrogenase activity was derepressed or repressed. However, generally, increases in nitrogenase activity followed decreases in ATP and energy charge and decreased nitrogenase activity accompanied increases in these energy parameters. These results are discussed in relation to the possible involvement of adenylation or deadenylation of glutamine synthetase and to the control of nitrogenase synthesis in the presence of NH⁺₄. It is concluded that the small ATP pool size is responsible for failure of adenylylation of glutamine synthetase and is related to nitrogenase synthesis at microaeration rates.     

A changed nitrogenase activity in Rhodospirillum rubrum after substitution of tungsten for molybdenum

The nitrogenase activity in Rhodospirillum rubrum was changed when the cells were made either Mo-deficient or when Mo was replaced by tungsten (W) as trace element in the growth medium: In the absence of N₂, normal Mo cells evolved H₂ (via nitrogenase) from added malate in the light faster than W cells, which in turn evolved H₂ faster than Mo-deficient cells. In the presence of N₂, on the other hand, nitrogen fixation rate in W cells was very close to the low rate found with Mo-deficient cells. Incubation after harvesting of Mo-deficient cells with 2×10^-5 M molybdate or tungstate stimulated the H₂ evolution (similarly with both trace elements) as well as the N₂ fixation (Mo was more effective than W). This indicates that the nitrogenase activity of W cells was truly caused by W and not merely by remaining traces of Mo. The ATP consumption is apparently higher with a W-containing nitrogenase than with the normal Mo-nitrogenase. Further, the affinity to N₂ of the W cells seems to be lower than with the Mo cells.     

The effect of waterlogging on the synthesis of the nitrogenase components in bacteroids of Rhizobium leguminosarum in root nodules of Pisum sativum

The effect of waterlogging of root nodules on nitrogenase activity and synthesis was studied in Pisum sativum inoculated with Rhizobium leguminosarum (strain PRE). It was shown that: 1. nitrogenase activity of intact pea plants was decreased by waterlogging, 2. this decrease was paralleled by a decline of the amount of active nitrogenase determined in toluene EDTA treated bacteroids, 3. SDS-polyacrylamide gel electrophoresis revealed that the amount of nitrogenase component II (CII) decreased by waterlogging while the amount of component I (CI) was not markedly affected, and 4. analysis of bacteroid proteins after ³⁵SO₄ labeling of pea plants showed that CII synthesis was repressed while CI synthesis continued indicating that the synthesis of CI and CII is regulated by independent mechanisms.     

Properties and regulation of synthesis of two ferredoxins from Rhodopseudomonas capsulata

Two ferredoxins from nitrogen-fixing cells of the phototrophic bacterium Rhodopseudomonas capsulata, strain B10, are purified to a homogeneous state and characterized. The molecular mass of ferredoxin I is about 12 kDa and that of ferredoxin II, 18 kDa. Ferredoxin I contains 8 Fe²⁺ and 8 S^2?; ferredoxin II has 4 Fe²⁺ and 4 S^2? per molecule. The redox potential of ferredoxin I is about ?270 mV and that of ferredoxin II ?419 mV. Ferredoxin I is more labile to the action of O₂, O^?₂, H₂O₂ and heating. The ferredoxins are also different in their absorption and EPR spectra, amino acid composition and electron-transfer activity to Rps. capsulata nitrogenase: both C₂H₂ reduction and H₂ evolution by Rps. capsulata nitrogenase proceed faster in the presence of ferredoxin I than in case of ferredoxin II. Synthesis of ferredoxin I takes place only in Rps. capsulata nitrogen-fixing cells grown in light under anaerobic conditions whereas ferredoxin II formation does not depend on the source of nitrogen or the growth medium, though the amount of ferredoxin II varies with the growth conditions. Its highest level has been found in the cells grown in lactate-limited medium in the presence of CO₂ and light or in the presence of glutamate in darkness under anaerobic conditions.     

Catechol Formation and Melanization by Na+ -Dependent Azotobacter chroococcum: a Protective Mechanism for Aeroadaptation?

Aeroadaptive microaerophilic Azotobacter chroococcum 184 produced a cell-associated black pigment when grown at high aeration rates under nitrogen-fixing conditions. This pigment was shown to be a catechol melanin. Polyphenol oxidase activity was detected in cell extracts of cells grown for 72 h. Melanin formation was optimal in the later stages of growth, and there was no correlation between nitrogenase activity and melanization. Nitrogenase activity in strain 184 was optimal at 10% O₂, and melanin formation was suppressed by O₂ limitation. In the presence of charcoal, an adsorbent of toxic oxygen intermediates, and benzoic acid, a scavenger of hydroxyl radicals, melanization was inhibited. However, in the presence of copper, the intensity of pigment color increased and melanization was accelerated. Copper also eliminated catalase and peroxidase activities of the organism but still permitted aerobic growth. In the presence of low levels of iron, melanization was accelerated under high aeration rates, and under low rates of aeration, melanization was observed only at higher levels of iron. Hydroxamate-siderophore production was detectable in the presence of soluble iron under high rates of aeration but was repressed by the same levels of iron under low aeration rates. Unlike melanization and hydroxamate formation, catechol formation was observed under both low and high rates of aeration under nitrogen-fixing conditions. Catechol formation and melanization were repressed by 14 mM NH₄⁺, at which level nitrogenase activity was also repressed. Copper reversed the repressive effect of NH₄⁺. A role for catechol formation and melanization in aeroadaptation is proposed.     

Nitrogen-Fixing Activity in Peat Soils from a Raised Bog

The nitrogenase (acetylene reductase) activity in monolithic and minced peat samples was found to be low, no more than 0.014–0.022 mg N/(kg h). Incorporation of the ¹⁵N₂ isotope into organic compounds of peat soil was 2.71–8.13 mg N/kg over 15 days. The nitrogen-fixing activity was the highest in a 10- to 20-cm layer of soil and much lower in the upper (under green moss) and deeper (20- to 30-cm) layers. The addition of glucose to soil samples stimulated nitrogen fixation considerably after 18–26 h. The maximum nitrogenase activity (3.5–3.8 mg N/(kg h)), observed after 60–70 h, coincided with the peak of respiratory activity. A repeated addition of glucose after its exhaustion increased nitrogenase activity, without a lag period, to 8.5 mg N/(kg h). Investigation of the effect of environmental factors (temperature, pH, aeration, and light intensity) on potential nitrogen-fixing activity in peat samples revealed that nitrogen fixation could proceed in a wide range of pH values (from 3.0 to 7.5) and temperatures (from 5 to 35°C). The nitrogen-fixing bacteria belonging to different trophic groups were enumerated by using nitrogen-free media with pH values and mineralization levels close to those in situ. In samples of peat soil, diazotrophic methanol-utilizing bacteria prevailed (2.0–2.5 × 10⁶ cells/g); the second largest group was facultatively anaerobic bacteria of the family Enterobacteriaceae.     

Comparative physiology of nitrogenase activity and vesicle development for Frankia strains CpI1, ACN1 ag,EAN1pec and EUN1f

The relationship between nitrogen fixation and development of a specialized cell structure, called the vesicle, was studied using four Frankia isolates. Nitrogenase activity was repressed in all four strains during growth with ammonia. Strain CpI1 formed no vesicles during NH₄ growth. Strains ACN1 ^ag , EAN1_pec and EUN1_f produced low numbers of vesicles in the presence of ammonia. Following transfer to nitrogen-free media, a parallel increase in nitrogenase activity and vesicle numbers occurred with all four isolates. Appearance of nitrogenase activity was more rapid in those strains that possessed some vesicles at the time of shift to N₂ as a nitrogen source. The ratio of vesicle numbers to level of nitrogenase activity varied widely among the four strains and in response to different growth conditions and culture age of the individual strains. Optimum conditions of temperature, carbon and energy source, nitrogen source and availability of iron and molybdenum were different for each of the four strains. Those conditions that significantly reduced nitrogenase activity were always associated with decreased numbers of vesicles.     

Azolla filiculoides Nitrogenase Activity Decrease Induced by Inoculation with Chlamydomonas sp.

Experiments were conducted to determine the influence of Chlamydomonas sp. on nitrogen fixation (C₂H₂ → C₂H₄) in Azolla filiculoides and on the nitrogen fixation and growth of free-living Anabaena azollae 2B organisms. Inoculation of azolla medium with Chlamydomonas sp. was associated with decreased nitrogenase activity in A. filiculoides and with increases in the density of a fungal population identified as Acremonium sp. Subsequent inoculation of azolla medium with this fungus was also accompanied by a significant decrease in nitrogenase activity of A. filiculoides. However, the extent of depression of nitrogenase activity was significantly higher when azolla medium was inoculated with Chlamydomonas sp. than when it was inoculated with Acremonium sp. Inoculation of nitrogen-free Stanier medium with either Acremonium sp. or Chlamydomonas sp. did not adversely affect the growth or nitrogenase activity of free-living A. azollae. Decreased nitrogenase activity in A. filiculoides is apparently related to the adverse influence of the green alga and the fungus on the macrosymbiont. The mechanisms that might be involved are discussed.     

Whole-Cell Immunolocalization of Nitrogenase in Marine Diazotrophic Cyanobacteria, Trichodesmium spp.

The mechanism by which planktonic marine cyanobacteria of the genus Trichodesmium fix N₂ aerobically during photosynthesis without heterocysts is unknown. As an aid in understanding how these species protect nitrogenase, we have developed an immunofluorescence technique coupled to light microscopy (IF-LM) with which intact cyanobacteria can be immunolabeled and the distribution patterns of nitrogenase and other proteins can be described and semiquantified. Chilled ethanol was used to fix the cells, which were subsequently made permeable to antibodies by using dimethyl sulfoxide. Use of this technique demonstrated that about 3 to 20 cells (mean ± standard deviation, 9 ± 4) consecutively arranged in a Trichodesmium trichome were labeled with the nitrogenase antibody. The nitrogenase-containing cells were distributed more frequently around the center of the trichome and were rarely found at the ends. On average 15% of over 300 randomly encountered cells examined contained nitrogenase. The percentage of nitrogenase-containing cells (nitrogenase index [NI]) in an exponential culture was higher early in the light period than during the rest of the light-dark cycle, while that for a stationary culture was somewhat constant at a lower level throughout the light-dark cycle. The NI was not affected by treatment of the cultures with the photosynthetic inhibitor dichloro 1,3′-dimethyl urea or with low concentrations of ammonium (NH₄Cl). However, incubation of cultures with 0.5 μM NH₄Cl over 2 days reduced the NI. The IF technique combined with ¹⁴C autoradiography showed that the CO₂ fixation rate was lower in nitrogenase-containing cells. The results of the present study suggest that (i) the IF-LM technique may be a useful tool for in situ protein localization in cyanobacteria, (ii) cell differentiation occurs in Trichodesmium and only a small fraction of cells in a colony have the potential to fix nitrogen, (iii) the photosynthetic activity (CO₂ uptake) is reduced if not absent in N₂-fixing cells, and (iv) variation in the NI may be a modulator of nitrogen-fixing activity.     

The program of protein synthesis during heterocyst differentiation in nitrogen-fixing blue-green algae

The program of protein synthesis that accompanies cellular differentiation following transfer of the blue-green alga Nostoc muscorum from nitrogen-containing to nitrogen-free medium has been determined by polyacrylamide gel electrophoresis of whole cell proteins labeled with ³⁵SO₄⁼ during successive intervals of the differentiation. Differentiating cells (proheterocysts, which become heterocysts) are distinguished from vegetative cells on the basis of the latter's susceptibility to lysis with lysozyme.At least ten sets of proteins can be distinguished on the basis of the time at which they are synthesized or the type of cell in which they are located. Regulation of most of these sets can be accounted for by classical induction or repression involving NH₄⁺ or a simple derivative of NH₄⁺. An additional mechanism is required to explain how the synthesis of several sets of proteins is initiated in all cells following transfer to nitrogen-free medium, but is permitted to continue only in developing proheterocysts. The structural polypeptides of the nitrogenase enzyme complex are members of the latter set.In differentiated filaments, very few proteins are synthesized in both vegetative cells and heterocysts. The qualitatively different pattern of protein synthesis is established very early, within the first 9 hr after transfer. Moreover, the proteins present in proheterocysts at that time are already qualitatively different from those of vegetative cells. Rapid turnover of vegetative cell proteins appears to be a characteristic of the early development of proheterocysts.     

Sodium-Dependent Azotobacter chroococcum Strains Are Aeroadaptive, Microaerophilic, Nitrogen-Fixing Bacteria

Na⁺ -dependent strains of Azotobacter chroococcum were observed to have very low reactivities with the H₂O₂ spot test for catalase. The cell extract of the representative Na⁺ -dependent strain 184 contained a catalase specific activity that was 10-to 600-fold lower than those found in Na⁺ -independent strains of A. chroococcum. Peroxidase and superoxide dismutase activities existed in all strains, although only certain Na⁺ -dependent strains contained a peroxidase reactive with p-phenylenediamine. The activities of catalase and peroxidase in the Na⁺ -dependent strain 184 were dependent on iron availability, which helped to explain the iron-dependent growth characteristic of this strain. The activities of these enzymes were not increased by subjecting the cells to increased aeration, nitrogen-fixing conditions, or paraquat. Strain 184 was found to be very sensitive to H₂O₂ or paraquat, even under iron-sufficient conditions, and was difficult to recover quantitatively on solid plating media. Strain 184 was more susceptible to H₂O₂ when grown under low-aeration, nitrogen-fixing conditions than when it was grown in the presence of NH₄⁺. Low population densities of strain 184 grew in nitrogen-free medium under microaerophilic conditions, while more dense populations were able to fix nitrogen under aerobic conditions. Therefore, these bacteria appeared to be aeroadaptive, microaerophilic, nitrogen-fixing bacteria.     

The effect of ammonium nitrate on the synthesis of nitrogenase and the concentration of leghemoglobin in pea root nodules induced by Rhizobium leguminosarum

The effects of NH₄NO₃ on the development of root nodules of Pisum sativum after infection with Rhizobium leguminosarum (strain PRE) and on the nitrogenase activity of the bacteriods in the nodule tissue were studied. The addition of NH₄NO₃ decreased the nitrogenase activity measured on intact nodules. This reduction of nitrogen fixation did not result from a reduced number of bacteroids or a decreased amount of bacteroid proteins per gram of nodule. The synthesis of nitrogenase, measured as the relative amount of incorporation of [³⁵S]sulfate into the components I and II of nitrogenase was similarly not affected.The addition of NH₄NO₃ decreased the amount of leghemoglobin in the nodules and there was a quantitative correlation between the leghemoglobin content and the nitrogen-fixing capacity of the nodules. The conclusion is that the decrease of nitrogen-fixing capacity is caused by a decrease of the leghemoglobin content of the root nodules and not by repression of the nitrogenase synthesis.     

Temperature control of nitrogen fixation in Klebsiella pneumoniae

At growth temperatures above 37°C, Klebsiella pneumoniae does not grow in a medium containing N₂ or NO ₃ ^- as nitrogen sources. However, both the growth in the presence of other nitrogen sources as well as the in vitro nitrogenase activity are not affected at this temperature. The inability to fix N₂ at high temperature is due to the failure of the cells to synthesize nitrogenase and other nitrogen fixation (nif) gene encoded proteins. When cells grown under nitrogen fixing conditions at 30°C were shifted to 39°C, there was a rapid decrease of the rate of de novo biosynthesis of nitrogenase (component 1), nitrogenase reductase (component 2), and the nifJ gene product. There was no degradation of nitrogenase at the elevated temperature since preformed enzyme remained stable over a period of at least 3 h at 39°C. Thus, temperature seems to represent a third control system, besides NH ₄ ⁺ and O₂, governing the expression of nif genes of K. pneumoniae.     

Contemporaneous N2 Fixation and Oxygenic Photosynthesis in the Nonheterocystous Mat-Forming Cyanobacterium Lyngbya aestuarii

The nonheterocystous filamentous cyanobacterial genus Lyngbya is a widespread and frequently dominant component of marine microbial mats. It is suspected of contributing to relatively high rates of N₂ fixation associated with mats. The ability to contemporaneously conduct O₂-sensitive N₂ fixation and oxygenic photosynthesis was investigated in Lyngbya aestuarii isolates from a North Carolina intertidal mat. Short-term (<4-h) additions of the photosystem II (O₂ evolution) inhibitor 3(3,4-dichlorophenyl)-1,1-dimethylurea stimulated light-mediated N₂ fixation (nitrogenase activity), indicating potential inhibition of N₂ fixation by O₂ production. However, some degree of light-mediated N₂ fixation in the absence of 3(3,4-dichlorophenyl)-1,1-dimethylurea was observed. Electron microscopic immunocytochemical localization of nitrogenase, coupled to microautoradiographic studies of ¹⁴CO₂ fixation and cellular deposition of the tetrazolium salt 2,4,5-triphenyltetrazolium chloride, revealed that (i) nitrogenase was widely distributed throughout individual filaments during illuminated and dark periods, (ii) ¹⁴CO₂ fixation was most active in intercalary regions, and (iii) daylight 2,4,5-triphenyltetrazolium chloride reduction (formazan deposition) was most intense in terminal regions. Results suggest lateral partitioning of photosynthesis and N₂ fixation during illumination, with N₂ fixation being confined to terminal regions. During darkness, a larger share of the filament appears capable of N₂ fixation.     

Effects of in vivo treatments on the activity of nitrogenase isolated from Rhodospirillum rubrum

Nitrogenase activities of partially purified extracts of Rhodospirillum rubrum grown on different nitrogen sources were examined. Most of the nitrogenase from cells grown on N₂ or glutamate was in the inactive form. This form was also predominant in extracts from cells grown on limiting N₂ or glutamate plus N₂. The enzyme from cells grown with limiting NH⁺₄ was fully active. Nitrogenase displayed varying degrees of sensitivity to in vivo inhibition by NH⁺₄, depending on the culture conditions. However, addition of NH⁺₄ to the cultures prior to harvest did not change the proportion of the active form of the enzyme in extracts from that found in control samples. Several of these observations are inconsistent with the three component model of nitrogenase regulation of Yoch and Cantu (Yoch, D.C. and Cantu, M. (1980) J. Bacteriol, 142, 899–907). A regulatory system controlled by products of NH⁺₄ assimilation is suggested.     

Diazotrophy and Nitrogenase Activity in the Archaebacterium Methanosarcina barkeri 227

Nitrogen fixation (diazotrophy) has recently been demonstrated in several methanogenic archaebacteria. To compare the process in an archaebacterium with that in eubacteria, we examined the properties of diazotrophic growth and nitrogenase activity in Methanosarcina barkeri 227. Growth yields with methanol or acetate as a growth substrate were significantly lower in N₂-grown cultures than in NH₄⁺-grown cultures, and the culture doubling times were increased, indicating that diazotrophy was energetically costly, as it is in eubacteria. Growth of nitrogen-fixing cells was inhibited when molybdenum was omitted from the medium; addition of 10 nM molybdate stimulated growth, while 1 μM molybdate restored maximum diazotrophic growth. Omission of molybdenum did not inhibit growth of ammonia-grown cells. Tungstate (100 μM) strongly inhibited growth of molybdenum-deficient diazotrophic cells, while ammonia-grown cells were unaffected. The addition of 100 nM vanadate or chromate did not stimulate diazotrophic growth of molybdenum-starved cells. These results are consistent with the presence of a molybdenum-containing nitrogenase in M. barkeri. Acetylene, the usual substrate for assaying nitrogenase activity, inhibited methanogenesis by M. barkeri and consequently needed to be used at a low partial pressure (0.3% of the headspace) when acetylene reduction by whole cells was assayed. Whole cells reduced 0.3% acetylene to ethylene at a very low rate (1 to 2 nmol h⁻¹ mg of protein⁻¹), and they “switched off” acetylene reduction in response to added ammonia or glutamine. Crude extracts from diazotrophic cells reduced 10% acetylene at a rate of 4 to 5 nmol of C₂H₄ formed h⁻¹ mg of protein⁻¹ when supplied with ATP and reducing power, while extracts of Klebsiella pneumoniae prepared by the same procedures had rates 100-fold higher. Acetylene reduction by extracts required ATP and was completely inhibited by 1 mM ADP in the presence of 5 mM ATP. The low rates of C₂H₂ reduction could be due to improper assay conditions, to switched-off enzyme, or to the nitrogenase's having lower activity towards acetylene than towards dinitrogen.     

N(2)-Fixation by Freshly Isolated Nostoc from Coralloid Roots of the Cycad Macrozamia riedlei (Fisch. ex Gaud.) Gardn

Nitrogenase (EC 1.7.99.2) activity (acetylene reduction) and nitrogen fixation (¹⁵N₂ fixation) were measured in cyanobacteria freshly isolated from the coralloid roots of Macrozamia riedlei (Fisch. ex Gaud.) Gardn. Light and gas phase oxygen concentration had marked interactive effects on activity, with higher (up to 100-fold) rates of acetylene reduction and ¹⁵N₂ fixation in light. The relationship between ethylene formation and N₂-fixation varied in the freshly isolated cyanobacteria from 4 to 7 nanomoles of C₂H₄ per nanomole ¹⁵N₂. Intact coralloid roots, incubated in darkness and ambient air, showed a value of 4.3. Maximum rates of nitrogenase activity occurred at about 0.6% O₂ in light, while in darkness there was a broad optimum around 5 to 8% O₂. Inhibition of nitrogenase, in light, by pO₂ above 0.6% was irreversible. Measurements of light-dependent O₂ evolution and ¹⁴CO₂ fixation indicated negligible photosynthetic electron transport involving photosystem II and, on the basis of inhibitor studies, the stimulatory effect of light was attributed to cyclic photophos-phorylation. Nitrogenase activity of free-living culture of an isolate from Macrozamia (Nostoc PCC 73102) was only slightly inhibited by O₂ levels above 6% O₂ and the inhibition was reversible. These cells showed rates of light-dependent O₂ evolution and ¹⁴CO₂ fixation which were 100- to 200-fold higher than those by the freshly isolated symbiont. Furthermore, nitrogenase activity was dependent on both photosynthetic electron transport and photophosphorylation. These data indicate that cyanobacteria within cycad coralloid roots are differentiated specifically for symbiotic functioning in a microaerobic environment. Specializations include a high heterocyst frequency, enhanced permeability to O₂, and a direct dependence on the cycad for substrates to support nitrogenase activity.     

Occurrence of effective nitrogen-scavenging bacteria in the rhizosphere of kallar grass

Bacteria occurring in high numbers on the rhizoplane of kallar grass grown at a natural site in Pakistan were effective scavengers of traces of combined nitrogen from the atmosphere. Bacteria grew under appropriate conditions in nitrogen-free semi-solid malate medium in the form of a typical subsurface pellicle which resulted in a significant nitrogen gain in the medium within 3 to 4 days of incubation; this could be also measured by¹⁵N-dilution. Bacteria grew and incorporated nitrogen under an atmosphere containing NH₃ and N₂O. A rapid and strong binding of strain W1 to roots of kallar grass grown in hydroponic culture was found by using a³²P-tracer technique. We obtained no evidence for diazotrophy of our strains because they failed to grow on nitrogen-free media when gases of high purity were used. No¹⁵N₂ was incorporated when bacteria were grown on¹⁵N₂ although a nitrogen gain was found, no acetylene reduction was observed and no homology with DNA containing sequences ofnifHDK structural genes for the nitrogenase components fromKlebsiella pneumoniae were detected. Owing to close contact of these bacteria with roots of kallar grass, utilization of scavenged nitrogen by the plant may have to be taken into account.