Preparation and spectroscopic characterization of lyophilized Mo nitrogenase

Mo nitrogenase is the primary source of biologically fixed nitrogen, making this system highly interesting for developing new, energy efficient ways of ammonia production. Although heavily investigated, studies of the active site of this enzyme have generally been limited to spectroscopic methods that are compatible with the presence of water and relatively low protein concentrations. One method of overcoming this limitation is through lyophilization, which allows for measurements to be performed on solvent free, high concentration samples. This method also has the potential for allowing efficient protein storage and solvent exchange. To investigate the viability of this preparatory method with Mo nitrogenase, we employ a combination of electron paramagnetic resonance, Mo and Fe K-edge X-ray absorption spectroscopy, and acetylene reduction assays. Our results show that while some small distortions in the metallocofactors occur, oxidation and spin states are maintained through the lyophilization process and that reconstitution of either lyophilized protein component into buffer restores acetylene reducing activity. Graphic abstract

(349) Fertilizer-N Effectson Cycas–Nostoc Symbiosis

Cycasrevoluta Thunb., cultivated as an ornamental plant for indoor and outdoor use, is characterized by an extremely slow rate of growth. In spite of the occurrence in its coralloid roots of the nitrogen-fixing cyanobiont Nostoc, N fertilization is commonly used to accelerate Cycas growth. A greenhouse experiment was conducted to examine the effects of two forms of combined N on growth of Cycas plants and cyanobacterial nitrogenase activity, measured on intact plants. Cycas plants grown in pots were fertilized from June to September with nutrient solution containing macronutrients as P, K, Mg, and Hoagland's micronutrients. N (700 mg/plant) was supplied as KNO3, or applied as NH4NO3; control plants received nutrient solution without nitrogen. Treatments were applied monthly and repeated for three times. Each treatment consisted of 15 plants. The length of the new leaves was recorded during the growth cycle of the plants. The nitrogenase activity, based on acetylene reducing activity (ARA), was measured on the plants in situ in July and in October. N fertilization stimulated both the nitrogenase activity and the growth of the plants. In comparison with the control, average increases in ARA of more than 20% were observed in the treated plants. Nitrogenase activity was slightly better in the presence of NH4NO3 in July, whereas the values measured in October were about the same for two treatments. The two forms of nitrogen were the same also regarding the stimulus on growth: in N-treated plants the total length of the new leaves was more than double with respect to the control at the end of the growing season. Control plants grown without fertilizer N had a slow start to their growth cycles and were unable to recover and compensate later for the lack of transient N.

The Basis of Ammonium Release in nifLMutants of Azotobacter vinelandii

ABSTRACT In Azotobacter vinelandii, nitrogen fixation is regulated at the transcriptional level by an unusual two-component system encoded by nifLA. Certain mutations innifL result in the bacterium releasing large quantities of ammonium into the medium, and earlier work suggested that this occurs by a mechanism that does not involve NifA, the activator ofnif gene transcription. We have investigated a number of possible alternative mechanisms and find no evidence for their involvement in ammonium release. Enhancement of NifA-mediated transcription, on the other hand, by either elimination ofnifL or overexpression of nifA, resulted in ammonium release, correlating with enhanced levels of nifHmRNA, raised levels of nitrogenase and acetylene-reducing activity, and increased concentrations of intracellular ammonium. Up to 35 mM ammonium can accumulate in the medium. Where measured, intracellular levels exceeded extracellular levels, indicating that rather than being actively transported, ammonium is lost from the cell passively, possibly by reversal of an NH4 + uptake system. The data also indicate that in the wild type the bulk of NifA is inactivated by NifL during steady-state growth on dinitrogen.

NITROGEN FIXATION AND CARBON METABOLISM IN VICIA FABA ROOT NODULES OVER ONTOGENY

The evolution of growth, nodulation, nitrogen fixation, and activities of root-nodule enzymes related to sucrose breakdown (sucrose synthase, alkaline invertase), pentose phosphate pathway (glucose-6-phosphate dehydrogenase, 6-phosphogluconate dehydrogenase), malate dehydrogenase, phosphoenolpyruvate carboxylase, and NADP-dependent isocitrate dehydrogenase, were followed over the complete life-cycle of faba bean in a growth chamber. The aim was to study the ontogenic changes of these parameters to obtain information on the carbon metabolism in nodules ofVicia faba, an amide-exporting legume. The maximum values of the relative growth rate and the specific acetylene-reducing activity were registered during the vegetative period. At pod-filling, the specific and total acetylene-reducing activity per plant declined in parallel with the enzyme activities of carbon metabolism. Contrary to reports for other legume species, inV. fabaroot nodule activity of sucrose synthase exceeded alkaline invertase values by 2-fold or more during the vegetative period. The activity of the two enzymes was similar at flowering and pod formation. The enzymes of carbon metabolism registered two maxima, one before and one after a trough (day 32), which marked the change from the vegetative to the reproductive period.

Isolation and characterization of mutants of Rhizobium meliloti unable to synthesize poly-β-hydroxybutyrate

The isolation and characterization of four mutants of Rhizobium meliloti unable to synthesize poly-β-hydroxybutyrate (PHB) are reported. The mutants were independently obtained via Tn5 transposon mutagenesis and exhibited physiological and cytomorphological characteristics similar to those of the parental strain, as well as overlapping DNA profiles. These were assessed at both the plasmid and total genome level, using for the latter the sensitive technique of pulsed-field gel electrophoresis in a contour-clamped homogeneous electric field. With respect to the parental PHB+ strain, the loss of PHB-synthesizing ability in the four mutants was demonstrated by gas chromatography, transmission electron microscopy, and enzymatic tests. Localization studies of Tn5 insertion showed that the PHB− phenotype had, in all mutants, a transposon insertion in the same region, although not in the same position. The symbiotic traits (nodule-inducing ability on Medicago sativa and acetylene-reducing activity of nodules) of the mutants did not differ significantly from those of the parental R. meliloti.Key words: Rhizobium meliloti, poly-β-hydroxybutyrate (PHB), PHB synthase.

Symbiotic Effectivity of Four Phaseolus vulgaris Genotypes after Inoculation with Different Strains of Rhizobium under Controlled Conditions

Four varieties of P. vulgaris L. were tested for their symbiotic nitrogen fixation effectivity in combination with nine different strains of Rhizobium leguminosarum bv. phaseoli and Rhizobium tropici. Plants were grown under controlled conditions and harvested 23 days after planting. Acetylene reducing activity, total N-content and dry weight of individual plant components were determined. Significant differences due to plant x bacterium interaction were assessed by ANOVA, especially for the total nodule mass per plant and the acetylene reducing activity per nodule dry weight. Data for acetylene reducing activity per plant correlated highly with the corresponding data for the total N-content. The comparison of the total N-content in symbiotically grown plants, lacking supply of mineral N, with plants luxuriously supplied with mineral N (relative N-accumulation rate) revealed high values (between 60% and 70% of maximal N-uptake) for some symbiotically active plant/bacterium combinations for this early developmental stage of the symbiosis N2 fixation potential for such symbioses.

Temperature sensitivity of nitrogen fixation in Azospirillum spp.

Azospirillum brasilense and Azospirillum lipoferum showed optimum acetylene-reducing activity at 25 and 30 °C, respectively, although both the bacteria grew optimally at 35 °C. Azospirillum halopraeferens displayed optimum growth and acetylene-reducing activity at 40–41 °C. Our experiments indicated that expression of nif genes was generally more sensitive to temperature than was nitrogenase activity. The NifA-dependent activation of a heterologous nifH–lacZ fusion was used to assess the impact of temperature on native NifA activity of A. brasilense and A. lipoferum. Maximum NifA activity was observed at 25 °C in A. brasilense and at 30 °C in A. lipoferum. Key words: temperature, nitrogen fixation, nifH–lacZ fusion, NifA activity, Azospirillum.

N2 fixation in a young Alnus incana stand, based on seasonal and diurnal variation in whole plant nitrogenase activity

N2 fixation by grey alder, Alnus incana (L.) Moench, was studied in the field during two growing seasons in northern Sweden. Alders were planted in a nitrogen-poor soil. Each alder had its root system enclosed in an open-ended cylinder that was closed with a gas-tight lid around the stem base to serve as cuvette during nitrogenase activity (acetylene reducing activity) measurements. To follow the seasonal variation, nitrogenase activity was measured at noon on 15 occasions for each alder in 1987 and on 15 occasions in 1988. Diurnal variation in nitrogenase activity was studied at six occasions, but no obvious pattern in the diurnal variation was found. Nitrogenase activity began shortly after leaf emergence at the very end of May, increased in June, stayed high although with some variation through July and August, declined during September, and was zero in early October. Cumulative nitrogenase activity over the season was converted to cumulative N2 fixation after determination of molar ratio nitrogenase activity to N2 fixation. This conversion was facilitated as the Frankia chosen as symbiont was lacking hydrogenase activity. Control experiments showed that the introduced symbiont was the only infective Frankia in the soil. N2 fixation was estimated to be 0.23 and 2.83 g N/(alder∙year) in the 1st and 2nd year, respectively. Despite its young age, A. incana was apparently capable of high N2 fixation rates at the high latitude studied. Key words: Alnus incana, hydrogenase, intact plants, N2 fixation, seasonal variation, spreading of Frankia.