The purification of hydrogenase II (uptake hydrogenase) from the anaerobic N2-fixing bacterium Clostridium pasteurianum

The H₂ uptake activity (units/mg protein) of Clostridium pasteurianum cells with methylene blue as the electron acceptor increases with cell density independent of the growth conditions. The H₂ evolution activity (units/mg protein) of the same cells with reduced methyl viologen as the electron donor remains fairly constant under all growth conditions tested. Cells grown under N₂-fixing conditions have the highest H₂ uptake activity and were used for the purification of hydrogenase II (uptake hydrogenase). Attempts to separate hydrogenase II from hydrogenase I (bidirectional hydrogenase) by a previously published method were unreliable. We report here a new large-scale purification procedure which employs a rapid membrane filtration system to fractionate cell-free extracts. Hydrogenases I and II were easily filtered into the low-molecular-weight fraction (M_r less than 100 000), and from this, hydrogenase II was further purified to a homogeneous state. Hydrogenase II is a monomeric iron-sulfur protein of molecular weight 53 000 containing eight iron atoms and eight acid-labile sulfur atoms per molecule. Hydrogenase II catalyzes both H₂ oxidation and H₂ evolution at rates of 3000 and 5.9 μmol H₂ consumed or evolved/min per mg protein, respectively. The purification procedure for hydrogenase II using the filtration system described greatly facilitates the large-scale purification of hydrogenase I and other enzymes from cell-free extracts of C. pasteurianum.     

Turnover and transport of quinolizidine alkaloids. Diurnal fluctuations of lupanine in the phloem sap,leaves and fruits of Lupinus albus L.

Quinolizidine alkaloids formed in the leaves of Lupinus albus L. are translocated via the phloem to the other plant organs, especially the maturing fruits. Compared with amino-acid transport in the phloem, the alkaloids contribute about 8% to the overall nitrogen being exported from the leaf. Since it is likely that the alkaloids are subsequently degraded in the target tissues a minor role of quinolizidine alkaloids might be nitrogen transport. A marked diurnal fluctuation of alkaloids was observed in the leaves, the phloem sap, the roots and the fruits with an increase during the day and an amplitude of several hundred percent thus providing evidence for a rapid turnover of endogenous alkaloids.Abbreviations QA quinolizidine alkaloids - GLC gas-liquid chromatography     

Nitrogen fixation efficiency,natural15N abundance,and morphology of mesquite (Prosopis glandulosa) root nodules

Summary The relative nitrogen fixation efficiencies (RE 1-[H₂ evolved÷C₂H₂ reduced]·100) of four mesquite (Prosopis glandulosa var.torreyana) rhizobia (Strains WR 1001, WR 1002, L5, L9) and a cowpea rhizobia (Strain 176A32) on mesquite were evaluated in a glasshouse experiment. Plant yield, shoot N accumulation, and the natural¹⁵N abundance (¹⁵N) of nodule tissue were determined. Strain WR 1002 failed to nodulate mesquite and strain L5 produced ineffective nodules. Among the three effective strains (WR 1001, L9, 176A32) the cowpea strain (176A32) and strain L9 had significantly higher RE than strain WR 1001. Differences in RE, however, were not accompanied by significantly higher plant yield and shoot N accumulation. The difference in¹⁵N abundance between foliar tissue and nodules (nodules minus leaves) was 0.47 ¹⁵N for the ineffective L5 nodules, while for the effective WR 1001, L9, and 176A32 nodules, respectively, this difference was 8.35, 7.81, and 8.35 ¹⁵N. This indicates a similar relationship between N₂-fixing effectiveness and natural¹⁵N enrichment of nodules that was previously observed in soybeans (Glycine max, L. Merr.). Strains WR 1001 and L9 produced elongate, indeterminate nodules typical for mesquite. The ineffective L5 nodules had few infected cells and an abundance of cortical amyloplasts. Mesquite nodules produced by the cowpea strain were spherical and were somewhat more similar in internal morphology to determinate nodules typical of cowpea than indeterminate nodules normally associated with mesquite.     

Rice response to inoculation with N2-fixing and P-solubilizing microorganisms

Summary Inoculation effect ofA. chroococcum, P. striata andA. awamorii on yield and nutrients uptake in rice was studied under green house conditions. The organisms appreciably increased the yield and uptake of nutrients with or without chemical fertilizers. Phosphorussolubilizing microorganisms and a mixture of the three showed better response than the rest of the treatments among single and mixed culture inoculations respectively. Chemical fertilizers further improved the yield and nutrients uptake. The yield response remained unaffected by replacing superphosphate with rock phosphate and microbial inoculations.     

Growth,nitrogen fixation and relative efficiency of nitrogenase inAlnus incana grown in different cultivation systems

Summary Three cultivation systems were compared. In one system the alders were grown hydroponically. In the two other systems the alders were planted in gravel and either given water and nutrients at intervals or the nutrient solution was continuously supplied. Alders continuously supplied with nutrients and water showed a significantly more rapid growth, higher biomass production and higher nitrogen content than did alders given nutrients and water at intervals or alders hydroponically grown. Alders continuously supplied with water and nutrients had a constant RE (relative efficiency of nitrogenase) of about 0.80 throughout the experimental period while alders supplied with water and nutrients at intervals showed a slight decrease in RE at the end of the experimental period. No strict relationship was found between RE and nitrogen content or between RE and plant productivity.     

Morphology,physiology and infectivity of twoFrankia isolates An 1 and An 2 from root nodules ofAlnus nitida

Summary Two different strains, An 1 and An 2, were obtained from root nodules ofAlnus nitida Endl., collected from one locality in the area of its natural habitat near Bahrin, District Swat, Pakistan. The light and electron microscopy of the isolates revealed the occurrence of septate and branched hyphae bearing sporangia and vesicles. The strains differed in their growth requirements, nitrogen-fixing ability and production of extracellular pigments, thus indicating the existence of more than oneFrankia strain in the same locality. In the absence of combined nitrogen in the medium strain An 1 formed vesicles and fixed N₂ (up to 200 nmol C₂H₄. mg protein^–1.h^–1), while strain An 2 under the experimental conditions formed only few vesicles and fixed N₂ at a very low rate (ca 10 nmol C₂H₄. mg protein^–1 .h^–1). The nitrogenase activity of strain An 1 was strongly affected by the O₂ concentration.Frankia An 1 and An 2 were infective and effective onA. nitida andA. glutinosa but not onDatisca cannabina andElaeagnus umbellata. Both An 1 and An 2 strains were more infective and effective onA. glutinosa thanFrankia strains AvcIl and CpI1.     

The uptake of soil and fertilizer-nitrogen by barley growing under Scottish climatic conditions

Summary Field experiments were carried out using¹⁵N-labelled calcium nitrate, to investigate the relative uptake by barley of fertilizer-N and soil-N. On imperfectly drained till soils uptake of soil-N increased with increasing rate of fertilizer, but remained constant on a brown sand, possibly due to more efficient root exploration in the latter soil. In four out of five seasons, late uptake of soil-derived N was a major feature, and uptake from ploughed soil as compared with uptake from direct-drilled soil was correlated with seasonal rainfall patterns. Significantly higher quantities of both fertilizer- and soil-derived N were taken up by winter barley than by spring barley, reflecting the longer growth period and higher dry matter yield from the former crop.     

Nodulation studies on legumes exotic to Australia: Hedysarum coronarium

Abstract Symbiotic experiments in glasshouse, controlled environment cabinet, and field were conducted with four lines of sulla ( Hedysarum coronarium ) and 15 strains of Rhizobium spp. This plant is highly Rhizobium -specific and appropriate strains are most unlikely to occur naturally in Australia. Under several sets of experimental conditions, H. coronarium nodulated abundantly and effectively with homologous rhizobia introduced from Spain and Italy. The optimum temperature for nitrogen fixation was relatively low (approx. 21°C) but significant interactions between line of host, strain of rhizobia, and growth temperature were frequent. The rhizobia were persistent in soil.     

Änderungen in der Stickstoffversorgung auf Dauerflächen im Brachland

Summary Starting in 1968 plant succession and nitrogen status in the top soil of an old field was investigated. The soil is a calcareous loam which was sterilized by heating. In the absence of human influence distinct successional stages with characteristic floristical and physiognomical features were observed. During the first six years vegetational development was characterized by competition between therophytes and hemicryptophytes. Agricultural treatments such as annual ploughing (spring, summer) and cutting (autumn) were found to change the trend of the succession.Five years after starting the experiment total nitrogen content in the top soil of the control area had increased slightly, while the ploughed plots persisted in their low values. A comparison of the nitrogen mineralisation between 1970 and 1974 showed decreasing amounts of mineral nitrogen in the later stage (1970: 67 kg N_min/ha/30 weeks; 33 kg N_min/ha/30 weeks). On the other hand, mineral nitrogen supply on the ploughed plots was not significantly different after the five-years-period. It is suggested that in old-field vegetation the external nitrogen cycle of the early therophyte stage changed to an internal one when long-lived hemicryptophytes, rhizome- and root-budding geophytes became dominant.Tables 1 to 4 show the vegetation development under different treatments; the figures represent average cover values per vegetation season using Londo's (1975) scale. Table 5 contains the total nitrogen concentration values in the uppermost of 10 cm of mineral soil, and Table 6 lists the values for soil volume, mean soil temperatures, average water content, and mineral nitrogen supplies for 1970 and 1974.

Contribution to the Symposium of the Working Group for Succession Research on Permanent Plots, held at Yerseke, the Netherlands, October 1975.     

Effect of soil moisture and clipping stresses on the nutrient (N,P and K) concentration,uptake and use efficiency in one temperate and two tropical grasses

Summary The concentration, uptake and element use efficiency of N, P and K in one C₃ annual (Polypogon monspeliensis) and two C₄ (Echinochloa colonum, an annual, andDichathium annulatum, a perennial) grasses were determined during winter and summer seasons in monocultures raised in field plots at three moisture levels,viz. full, half and one-fourth of field capacity. At each moisture regime the plants were clipped thrice at moderate and severe levels corresponding to 40 and 80% of live green. The concentration of these elements was characteristic of the growth habit of these plants;e.g. the build up of concentration was maximum in leaf of the annuals while it was comparable in crown and leaf of Dichanthium. The N level was maximum in Polypogon. The nutrient use effiency was comparable in the two annuals and maximum K and N use were obtained in Polypogon and Dichanthium, respectively.