Nitrogenase activity of the antarctic cyanobacteriumNostoc commune: Influence of temperature

Nitrogenase activity (C₂H₂ reduction) ofNostoc commune isolated from Schirmacher Oasis (Antarctica) was compared withNostoc muscorum, N. calcicola, Anabaena doliolum andGloeocapsa sp. The temperature profile of acetylene reduction (5–30 °C) forN. commune revealed (a) that the highest rate of nitrogenase activity was at 25±1 °C, (b) that it was low (69 %) in comparison withN. muscorum, and (c) that nitrogenase activity continued at lower temperatures, which was not evident for other cyanobacteria. The results suggest thatN. commune is adapted to lower temperatures in terms of nitrogen fixation.     

Combination and positional distribution of fatty acids in lipids from blue-green algae

The main glycerolipids (monogalactosyl-, digalactosyl-, sulphoquinovosyl diacylglycerol, phosphatidylglycerol) from five blue-green algae (Microcystis, Anabaena, Nostoc, Oscillatoria, Tolypothrix) were analyzed for fatty acid composition, occurrence of diglyceride species and positional distribution of fatty acids between thesn-1- andsn-2-position of glycerol. In contrast to eucaryotic plants biosynthetically closely related lipids (monogalactosyl-, digalactosyl-, trigalactosyl diacylglycerol) show nearly identical diglyceride moieties, whereas sulphoquinovosyl diacylglycerol and phosphatidylglycerol are separated from galactolipids by composition as well as occurrence of fatty acids. On the other hand the positional distribution of fatty acids in all lipids is controlled exclusively by chain length and not by degree of unsaturation with C₁₈-fatty acids at thesn-1- and C₁₆-fatty acids at thesn-2-position. These results show that in procaryotic organisms the diversity in diglyceride portions of lipids is reduced as compared to eucaryotic organisms, but nevertheless does exist.Abbreviations MGD, DGD, TGD, SQD monogalactosyl-, digalactosyl-, trigalactosyl-, sulphoquinovosyl diacylglycerol - PG phosphatidyl glycerol     

Rewetting of drought-resistant blue-green algae: Time course of water uptake and reappearance of respiration,photosynthesis, and nitrogen fixation

Summary The response of the terrestrial blue-green algae Nostoc flagelliforme, Nostoc commune, and Nostoc spec. to water uptake has been investigated after a drought period of approximately 2 years. Rapid half-times of rewetting (0.6, 3.3, and 15.5 min, respectively) are found. The surfaceto-mass ratio of the three species is inversely correlated to the speed of water uptake and loss. The ecological relevance of these different time courses is discussed.Respiration starts immediately after a 30-min rewetting period, whereas photosynthetic oxygen evolution reaches its maximum activity after 6 and 8 h with N. commune and N. flagelliforme, respectively. In the dark, recovery of oxygen uptake by N. commune is somewhat impaired, while slightly stimulated with N. flagelliforme. With both species, recovery of photosynthesis is inhibited by darkness.Using colonies kept dry for two years, nitrogenase activity of N. commune attains its maximum 120 to 150 h after rewetting, while only 50 h were needed with algal mats kept dry for two days.Thus, after a 2-year drought period, the physiological sequence of reactivation is respiration—photosynthesis—nitrogen fixation. Respiration and photosynthesis precede growth and are exhibited by existing vegetative cells, whereas recovery of nitrogen fixation is dependent on newly differentiated heterocysts.     

Determination of oxidized scytonemin in Nostoc commune Vauch cultured on different conditions by high performance liquid chromatography coupled with triple quadrupole mass spectrometry

A facile method based on high performance liquid chromatography coupled with electrospray ionization tandem triple quadrupole mass spectrometry (HPLC-ESI-QqQ-MS) has been established to investigate the production of the oxidized scytonemin by Nostoc commune Vauch in different environmental conditions. In optimized HPLC-ESI-QqQ-MS conditions, the oxidized scytonemin can be effectively detected in selected reaction monitoring (SRM) mode. After regression, high linearity of the calibration curve was achieved with a correlation coefficient (r ²) at 0.99. The limit of detection and limit of quantification were 0.03 and 0.10 ng mL^?1, respectively. The recovery of the oxidized scytonemin was at 76.90 %, and the relative standard deviations of inter- and intraday precisions were lower than 8.95 % (n?=?4). Subsequently, the production of the oxidized scytonemin from N. commune has been investigated in different culture conditions. High culture temperature, strong illumination intensity, and light–dark cycle (12:12 h) were found to be good for producing the oxidized scytonemin by N. commune. In short, the HPLC-QqQ-SRM-MS method is a powerful tool for comprehensive analysis of the oxidized scytonemin from N. commune, owing to its excellent selectivity and sensitivity.     

PHYSIOLOGICAL ECOLOGY OF NITROGEN FIXING BLUE-GREEN ALGAL CRUSTS IN THE UPPER-SUBALPINE LIFE ZONE1

Blue-green algal (cyanobacterial) crusts composed of nitrogen fixing Nostoc commune Voucher ex Born. et Flah. and Tolypothrix conglutinata var. colorata Ghose were studied in the upper-subalpine life zone, Mission Mountain Wilderness, Montana. Rates of ethylene production were highest in the submerged shoreline crusts, lower for exposed crusts pioneering rocky shorelines and lowest in the Carex meadow. Nitrogenase activity (acetylene reduction technique) was constant between 200–285% crust moisture content (wet/dry weight) and then rapidly declined to 0 between 200–140%. Optimal temperatures for ethylene production by illuminated cells was between 20–30° C for T. conglutinata, 20° C for N. commune and about 25° C in darkness for both species. Nitrogenase activity by T. conglutinata in culture was unaffected by repeated freeze-thaw treatments whereas N. commune was severely inhibited. In contrast, N₂-ase activity of these two species in an intact crust was unaffected by repeated freeze-thaw treatments. Application of nitrogen-free growth medium to intact crusts increased nitrogenase activity by 3.7 times implying that these were mineral deficient under field conditions. Photosynthesis was light saturated at 125 μmol-m^?2.s^?1 whereas nitrogenase activity was light independent for cells with carbohydrate reserves. When carbohydrate reserves were reduced by 8 h incubation in darkness, between 1–3 h of illumination were required to restore nitrogenase activity to 80% of the maximum rate. Biochemical pathway inhibitor studies employing DCMU, MFA, and CCCP showed that oxidative metabolism was the source of reductant for acetylene reduction. Tetrazolium precipitation in heterocysts paralleled acetylene reduction activity in the inhibitor treated cells.     

Algal nitrogen fixation on the lava field of Heimaey,Iceland

Summary Nitrogen fixation (C₂H₂ reduction) by blue-green algae occurring on the juvenile lava field of Heimaey, Iceland was examined both in the laboratory (potential at 20° C and 39° C) and in the field, three and a half years after the volcanic eruption.Already at this early stage of colonization representatives of unicellular and filamentous heterocystous and non-heterocystous blue-green algae were commonly observed. The predominating algae were Nostoc sp. (20° C) and Schizothrix sp. — Microcoleus chthonoplastes, (39° C), the former often in association with the protonemata-rhizoids of moss plants.The potential for nitrogen fixation was recorded at an average rate of 109.2 (20° C) and 138.1 (39° C) ng N g^-1 h^-1 in soil collected from localities randomly distributed over the lava field.Tests for nitrogen fixation performed in situ revealed significant fixation activities in all the eleven localities subject to examination. The activities ranged from 2.8 to 63.4 (mean 21.5) ng N g^-1 h^-1 and 1.9 to 17.7 (mean 7.9) ng N cm^-2 h^-1.All the nitrogen fixation data noted imply that blue-green algae contribute a substantial part of the nitrogen input to the lava. Further, it was found that material incubated under micro-aerophilic conditions exhibited considerably enhanced nitrogenase activity.The role of nitrogen-fixing blue-green algae in general and Nostoc muscorum in particular in being suitable as pioneering organisms preparing the bare lava for ingress of other plants is also discussed.     

Ca requirement for aerobic nitrogen fixation by heterocystous blue-green algae

The requirement of Ca²⁺ for growth and nitrogen fixation has been investigated in two strains of heterocystous blue-green algae (Anabaena sp. and Anabaena ATCC 33047). With combined nitrogen (nitrate or ammonium) or with N₂ under microaerobic conditions, Ca²⁺ was not required for growth, at least in concentrations greater than traces. In contrast, Ca²⁺ was required as a macronutrient for growth and nitrogen fixation with air as the nitrogen source. Addition of Ca²⁺ to an aerobic culture without Ca²⁺ promoted, after a lag of several hours, development of nitrogenase activity and cell growth. Provision of air to a microaerobic culture in the absence of Ca²⁺ promoted a drastic drop in nitrogenase activity, which rapidly recovered its initial level upon restoration of microaerobic conditions. Development of nitrogenase activity in response to either Ca²⁺ or low oxygen tension was dependent on de novo protein synthesis. The role of Ca²⁺ seems to be related to protection of nitrogenase from inactivation, by conferring heterocysts resistance to oxygen.     

Rhizosphere mediated growth enhancement using phosphate solubilizing rhizobacteria and their tri-calcium phosphate solubilization activity under pot culture assays in Rice (Oryza sativa.)

Phosphate solubilizing rhizobacteria are considered as an important alternative to increase the availability of accumulated phosphates through solubilization. These increase the growth of plant by enhancing the efficiency of fixing biological nitrogen. This was studied through a pot experiment involving two Phosphate Solubilizing Rhizobacteria (PSRB) isolates, Pseudomonas aeruginosa and Bacillus subtilis along with Tri-calcium phosphate (TCP) on availibity of nutrients, biological composition of soil and yield attributes of rice crop at its growth stages. Experiment was laid in factorial completely randomized design (CRD) comprising of eight treatments replicated thrice with two factors viz. factor 1 with or without TCP (1 g^?1soil) and factor 2 with single or combined inoculation of PSRB isolates. Considerable enhancement in available content of potassium (K), phosphorous (P), nitrogen (N) in soil was found with TCP 1 g^?1soil (P₁) and consortium of Pseudomonas aeruginosa and Bacillus subtilis broth culture at crop growth stages. Highest increase in available N (17.13% and 19.1%), available P (232% and 265%), available K (19.6% and 29.2%) over control were recorded in B₃ (consortium of Pseudomonas aeruginosa and Bacillus subtilis broth culture). Similarly, maximum nutrient uptake N (6.4%), P (15.8%) and K (8.9%) were recorded with same treatment. A considerable growth in soil microbial biomass carbon and dehydrogenase activity at crop growth stages was recorded on application of TCP 1 g^?1soil (P₁) and consortium of PSRB isolates' Pseudomonas aeruginosa and Bacillus subtilis (B₃). Highest increase in microbial biomass carbon (16.4% and 16.5%) and dehydrogenase activity 34.7% and 43.8% over control were recorded in B₃ (consortium of PSRB isolates Pseudomonas aeruginosa and Bacillus subtilis) and was found best among all treatments in terms of yield (63.2%) and yield attributes; number of panicles^?1plant (54.8%), number of grains^?1panicle (156%) and average panicle length (63.9%).     

Regulation of nitrate reductase in cyanobacteria. Repression-derepression control of nitrate reductase apoprotein in the cyanobacterium Nostoc muscorum

The repression-derepression control of Nostoc muscorum nitrate reductase was studied with regard to the Mo-cofactor and apoprotein levels. It was found that the synthesis of Mo-cofactor is constitutive but the apoprotein is subject to the repression-derepression control. In NH₄⁺ medium apoprotein synthesis was repressed and in N₂ and NO₃^? media apoprotein synthesis was derepressed. The apoprotein levels were similar in NO₃^? and N₂ media; however, the nitrate reductase activity was lower in N₂ medium due to lower Mo-cofactor activity. The lower Mo-cofactor activity in N₂-fixing conditions as compared to that in non-N₂-fixing conditions was consistent with the earlier view that the Mo-cofactor of nitrate reductase may be a precursor for FeMo-cofactor of nitrogenase.     

The toxicity of phenolic compounds to soil algal population and toChlorella vulgaris andNostoc linckia

Summary p-Nitrophenol (PNP),m-nitrophenol (MNP), 2,4-dinitrophenol (DNP) and catechol were tested for their effects on algal population in a soil and on pure cultures of two algae isolated from soil. Both PNP and MNP, even at 0.5 kg ha⁻¹ level were toxic to the soil algae; high doses effected increase in toxicity. Inhibition of algae was relatively more with PNP compared to the other two nitrophenols. Catechol treatment up to 1.0 kg ha⁻¹ led to a significant initial enhancement of algae with a subsequent far less toxic effect. The toxicity of the phenolic compounds towardChlorella vulgaris, a green alga andNostoc linckia, a blue-green alga, decreased in the order: MNP≧PNP>DNP>Catechol. However, algicidal or algistatic effect of the test chemicals was fairly more againstC. vulgaris, suggesting that the eukaryotic alga is highly sensitive to such soil pollutants compared to the prokaryotic alga.     

Increase of Nitrogenase Activity in the Blue-Green Alga Nostoc muscorum (Cyanobacterium)

Preincubation of the blue-green alga (cyanobacterium) Nostoc muscorum under hydrogen or argon (nongrowing conditions, neither CO₂ nor N₂ or bound nitrogen present) in the light resulted in a two- to fourfold increase of light-induced hydrogen evolution and a 30% increase of acetylene reduction. Preincubation under the same gases in the dark led to a decrease of both activities. Cultivation of algae under a hydrogen-containing atmosphere (N₂, H₂, CO₂) increased neither hydrogen nor ethylene evolution by the cells. Formation of both ethylene and hydrogen is due to nitrogenase activity, which apparently was induced by the absence of N₂ or bound nitrogen and not by the presence of hydrogen. Inhibitors of protein biosynthesis prevented the increase of nitrogenase activity. Hydrogen uptake by the cells was almost unaffected under all of these conditions. With either ammonia or chloramphenicol present, nitrogenase activity decreased under growing conditions (i.e., an atmosphere of N₂ and CO₂). The kinetics of decrease were the same with ammonia or chloramphenicol, which was interpreted as being due to rapid protein breakdown with a half-life of approximately 4 h. The decay of nitrogenase activity caused by chloramphenicol could be counteracted by nitrogenase-inducing conditions, i.e., by the absence of N₂ or bound nitrogen. A cell-free system from preconditioned algae with an adenosine 5′-triphosphate-generating system exhibited the same increase or decrease of nitrogenase activity as the intact cell filaments, indicating that this effect resided in the nitrogenase complex only. We tentatively assume that not the whole nitrogenase complex, but merely a subunit or a special protein with regulatory function, is susceptible to fast turnover.     

In vitro translation of mRNA from Nostoc commune (Cyanobacteria)

RNA pools were extracted from cells of Nostoc commune UTEX 584 in exponential growth (liquid cultures) and from cells which had been immobilized and dried rapidly at -99.5 MPa. Levels of incorporation of ³⁵S-methionine, five- to sixfold higher than the endogenous level, were obtained after in vitro translation of the RNA preparations in a heterologous S30 cell-free system purified from Escherichia coli Q13. The levels of incorporation, obtained with a homologous N. commune UTEX 584 S30 system, were much lower. The requirement for magnesium in the heterologous system was 15–21 mM, translation of N. commune UTEX 584 RNA was inhibited when the RNA concentration was greater than 0.3 mg ml^–1, and translation was stimulated significantly by the presence of ammonium chloride. Few qualitative differences were observed between the pattern of proteins (SDS-PAGE) obtained after translation of the RNA pools from cells in exponential growth, and from those cells subjected to immobilization and rapid drying. The data suggest that short-term desiccation of N. commune UTEX 584 does not have a marked selective effect on the composition of the mRNA pool. In contrast, preparations of RNA from field materials of Nostoc commune HUN (desiccated for 5 years) were unable to drive high rates of translation in any of the systems tested and optimized for use in this study.     

DEPRESSION OF MASTIGOCLADUS LAMINOSUS (CYANOPHYTA) NITROGENASE ACTIVITY UNDER NORMAL SUNLIGHT INTENSITIES1

The effect of light intensity (PAR) on the nitrogenase activity of Mastigocladus laminosus Cohn was studied by the acetylene reduction technique. Benthic mat from a thermal stream, Hot River, in Yellowstone National Park was used in both experimental and in situ incubations. This hot spring maintained a mean pH of 7.0, was essentially isothermal (ca. 50°C), and had virtually no upstream to downstream physicochemical gradients (P > 0.05). Two surveys of the stream showed that nitrogenase of the M. laminosus mat was significantly more active (P > 0.02) under low light intensities than under high intensities, 252 and 712 μE · m^?2· s^?1, respectively. Maximum activity of Hot River Mastigocladus (268 nmol C₂H₄· mg Chl a^?1· h^?1) occurred at 50% full midday light intensities; the rates at low light (mean = 247 nmol C₂H₄· mg Chl a^?1· h^?1) were significantly (P > 0.001) greater than those at high light (mean = 106). The results indicate that M. laminosus nitrogenase activity is low light adapted and suggest that the temporal pattern for nitrogen fixation might be significantly different from that of thermophilic Calothrix.     

Ultrastructural analysis of the rehydration of desiccatedNostoc commune HUN (cyanobacteria) with particular reference to the immunolabelling of NifH

Summary Vegetative cells and heterocysts of the filamentous desiccation-tolerant cyanobacteriumNostoc commune HUN retain their ultrastructural organisation and the integrity of their intra- and extracellular membranes after two years of desiccation and subsequent rehydration. Immunogold-labelling of thin sections demonstrated the presence of NifH (Fe protein of nitrogenase) in vegetative cells and heterocysts within five minutes of the rehydration of dried colonies. Immunogold label accumulated in discrete areas vegetative cells within 5 minutes of the rewetting of cells, and after 30 minutes a conspicuous association of NifH protein with heterocyst ribosomes was detected. After longer periods of rehydration, the deposition of gold particles became more random within both cell types but occurred with a greater frequency in heterocysts. Up to 24 hours after the rewetting of cells, two morphologically-distinct forms of heterocyst could be discerned. NifH protein was detected through Western blotting (subunit M_r=33,800) in protein extracts from samples ofNostoc commune, collected in different parts of the world and including some which had been desiccated for periods of up to 10 years. The results are discussed in relation to the sequential recovery of metabolic functions, particularly nitrogen fixation, which occurs upon the rehydration of cells after their prolonged storage in the air-dry state.     

Interactive effects of elevated CO2, phosphorus deficiency, and soil drought on nodulation and nitrogenase activity in Alnus hirsuta and Alnus maximowiczii

We examined the interactive effects of elevated CO₂, soil phosphorus (P) availability, and soil drought on nodulation, nitrogenase activity, and biomass allocation in Alnus hirsuta and Alnus maximowiczii. Potted seedlings were grown in either ambient or elevated CO₂ (36 Pa and 72 Pa CO₂), with different levels of P (7.7 and 0.77 mgP pot^?1 week^?1 for high-P and low-P, respectively) and water supply in a natural daylight phytotron. Measurements of nitrogenase activity by an acetylene reduction assay failed to reveal significant effects of the treatments in any species. In high-P, nodule biomass increased under elevated CO₂ and decreased under drought. In low-P, nodule biomass decreased substantially compared to high-P, but the effect of elevated CO₂ on nodule biomass was unclear. Soil drought increased the partitioning of biomass into nodules, especially in A. hirsuta. These results suggest that with high P availability, elevated CO₂ could promote N₂ fixation by increasing nodule biomass even under drought. On the other hand, if soil P is limiting, elevated CO₂ may not enhance N₂ fixation because of the suppression of growth.     

Enhancement of specific nitrogenase activity inAzospirillum brasilense andKlebsiella pneumoniae,inhibition inRhizobium japonicum under air by phenol

Specific nitrogenase activity inAzospirillum brasilense ATCC 29145 in surface cultures under air is enhanced from about 50 nmol C₂H₄·mg protein^-1·h^-1 to 400 nmol C₂H₄ by the addition of 1 mM phenol. 0.5 and 2 mM phenol added increase the rate 5-fold and 4-fold. This enhancement effect is observed only between 2 and 3 days after inoculation, with only a small reduction of the growth of the cells by the phenol added. In surface cultures under 1% O₂, nitrogenase activity is slightly reduced by the addition of 1–0.01 mM phenol. Utilization of succinate is enhanced during the period of maximum enhancement of nitrogenase activity by 60% by addition of 1 mM phenol. The cells did not produce¹⁴CO₂ from [U-¹⁴C] phenol, neither in surface cultures nor in liquid cultures and less than 0.1% of the phenol was incorporated into the cells. A smaller but significant enhancement of nitrogenase activity by about 100% in surface cultures under air was found withKlebsiella pneumoniae K 11 after addition of 1 mM phenol. However, inRhizobium japonicum 61-A-101 all phenol concentrations above 0.01 mM reduced nitrogenase activity. With 1 mM phenol added activity was reduced to less than 10% with no effect on the growth in the same cultivation system. With thisRhizobium japonicum strain significant quantities of phenol (25 mol in 24 h by 2·10¹² cells) were metabolized to¹⁴CO₂, with phenol as sole carbon source. WithAzospirillum brasilense in liquid culture under 1% and 2% O₂ in the gas phase, no enhancement of nitrogenase activity by phenol was noticed.     

Nitrogenase activity of two genotypes of Acacia mangium as affected by phosphorus nutrition

The specific nodulation, nitrogenase activity (acetylene reduction) and budgets of carbon allocation to respiration by nodulated roots were examined in two provenances of Acacia mangium Willd. grown in a glasshouse for 17 weeks to investigate the effects of soil phosphorus and genotypes of the host plant on symbiotic nitrogen fixation. Application of phosphorus (0–80 mg P kg^-1 soil) increased specific nodulation (g nodule dry weight g^-1 plant dry weight) of provenance Ma11 by two-fold and the percentage of nodulated root respiration allocated to nitrogenase by 50%, but had no effect on specific activity of nitrogenase or specific respiration coupled with nitrogenase activity. Improved phosphorus nutrition increased the specific nitrogenase activity of provenance Ma9 by 2-fold, the percentage of nodulated root respiration allocated to nitrogenase, and specific nitrogenase-linked respiration by 50%, respectively, but had no effect on the specific nodulation. The percentage of respiration coupled with nitrogenase activity in nodulated root respiration by provenance Ma9 was 60–70% higher than that in provenance Ma11, regardless of phosphorus levels applied. At the optimal level of phosphorus addition (10 mg P kg^-1 soil), provenance Ma9 had a lower dry mass than provenance Ma11. This was accompanied by a lower nodulated root respiration and a higher percentage of nodulated root respiration allocated to nitrogenase activity in provenance Ma9.     

Nitrogenase activity in Rhodopseudomonas sulfidophila

The marine purple nonsulfur bacterium, Rhodopseudomonas sulfidophila, strain W4, was capable of photosynthetic growth on dinitrogen and malate. Higher growth rates were observed when either glutamate or ammonia replaced dinitrogen as nitrogen source and when bicarbonate was omitted from the culture medium. Although ammonia was released from cells growing on malate and N₂, no nitrogenase activity could be detected unless -ketoglutarate was added to the culture medium. No nitrogenase activity was found in cultures grown in the presence of NH ₄ ⁺ . In cultures grown on glutamate as nitrogen source, nitrogenase and hydrogenase activities were found to be 5.4 nmol C₂H₂ reduced · min^-1 · mg^-1 dry weight and 50 nmol methylene blue reduced · min^-1 · mg^-1 dry weight respectively. Such activities are significantly lower than those observed for other members of the Rhodospirillaceae e.g. Rhodopseudomonas capsulata. However, the hydrogenase activity would be sufficient to recycle all H₂ produced by nitrogenase. It was indeed observed that growing cells did not evolve molecular hydrogen during photoheterotrophic growth and that H₂ stimulated nitrogenase activity in resting cells of R. sulfidophila. The nitrogenase from this bacterium proved to be extremely sensitive to low concentrations of oxygen, half-inhibition occurring at between 1–1.5% O₂ in the gas phase, depending on the bacterial concentration. Light was essential for nitrogenase activity. No activity was found during growth in the dark under extremely low oxygen concentrations (1–2% O₂), which are still sufficient to support good growth. Resting cell suspensions prepared from such cultures were unable to reduce acetylene upon illumination. Optimum nitrogenase activities were broadly defined over the temperature range, 30–38°C, and between pH 6.9 and 8.0. The results are discussed in comparison with the non-marine purple nonsulfur bacterium, R. capsulata, which somewhat resembles R. sulfidophila.     

Growth kinetics and nitrogenase induction inFrankia sp. HFPArI 3 grown in batch culture

Summary Kinetics of growth and nitrogenase induction inFrankia sp. Ar13 were studied in batch culture. Growth on defined medium with NH ₄ ⁺ as the N source displayed typical batch culture kinetics; however, a short stationary phase was followed by autolysis. Removal of NH ₄ ⁺ arrested growth and initiated vesicle differentiation. Vesicle numbers increased linearly and were paralleled by a rise in nitrogenase (acetylene reduction) activity. Nitrogenase activity (10 nM C₂H₄·mg protein^–1·min^–1) was sufficient to support growth on N₂ and protein levels rose in parallel with nitrogenase induction. Optimal conditions for vesicle and nitrogenase induction were investigated. Maximum rates of acetylene reduction were obtained with 5 to 10 mM K₂ HPO₄/KH₂PO₄, 0.1 mM CaCl₂ and MgSO₄. The optimum pH for acetylene reduction and respiration was around 6.7. The amount (5 to 10 g protein/ml) and stage (exponential) of growth of the ammonium-grown inoculum strongly influenced the subsequent development of nitrogenase activity. Propionate was the most effective carbon source tested for nitrogenase induction. Respiration in propionate-grown cells was stimulated by CO₂ and biotin, suggesting that propionate is metabolized via the propionyl CoA pathway.     

Coexisting Bacterial Populations Responsible for Multiphasic Mineralization Kinetics in Soil

Experiments were conducted to study populations of indigenous microorganisms capable of mineralizing 2,4-dinitrophenol (DNP) in two soils. Previous kinetic analyses indicated the presence of two coexisting populations of DNP-mineralizing microorganisms in a forest soil (soil 1). Studies in which eucaryotic and procaryotic inhibitors were added to this soil indicated that both populations were bacterial. Most-probable-number counts with media containing different concentrations of DNP indicated that more bacteria could mineralize low concentrations of DNP than could metabolize high concentrations of it. Enrichments with varying concentrations of DNP and various combinations of inhibitors consistently resulted in the isolation of the same two species of bacteria from soil 1. This soil contained a large number and variety of fungi, but no fungi capable of mineralizing DNP were isolated. The two bacterial isolates were identified as a Janthinobacterium sp. and a Rhodococcus sp. The Janthinobacterium sp. had a low μ_max and a low K_m for DNP mineralization, whereas the Rhodococcus sp. had much higher values for both parameters. These differences between the two species of bacteria were similar to differences seen when soil was incubated with different concentrations of DNP. Values for μ_max from soil incubations were similar to μ_max values obtained in pure culture studies. In contrast, K_s and K_m values showed greater variation between soil and pure culture studies. The results of this study help to confirm predictions that two physiologically distinct bacterial populations are responsible for the multiphasic mineralization kinetics observed in the soil studied.