Three hydroxylations incorporating molecular oxygen in the aerobic biosynthesis of ubiquinone in Escherichia coli.

The biosynthetic origin of the oxygen atoms of ubiquinone 8 from aerobically grown Escherichia coli was studied by 18O labeling. An apparatus was developed which allowed the growth of cells under a defined atmosphere. Mass spectral analysis of ubiquinone 8 from cells grown under highly enriched 18O2 showed that three oxygen atoms of the quinone are derived from molecular oxygen. It was established that the molecular oxygen is incorporated into the two methoxyl groups (at C-5 and C-6) and one of the carbonyl positions of the ubiquinone molecule by demonstrating that only one of the incorporated oxygens will exchange with water under acidic conditions that specifically catalyze the exchange of carbonyl, but not methoxyl, oxygens. That the C-4 carbonyl oxygen is derived from molecular oxygen was shown by the incorporation of three atoms of 18O2 into ubiquinone 8 biosynthesized from added 4-hydroxybenzoic acid. Comparison of ubiquinone 8 and menaquinone 8 from E. coli grown under 18O2 confirmed that the labeled carbonyl oxygen of the [18O2]ubiquinone 8 is incorporated biosynthetically and not by chemical exchange in the cell. It is concluded that the three hydroxylation reactions involved in the pathway for the aerobic biosynthesis of ubiquinone are all catalyzed by monooxygenases. The implications of this study for the anaerobic biosynthesis of ubiquinone 8 in E coli are discussed.     

The partial replacement of the serum growth factor requirement of SV3T3 cells by lactalbumin hydrolyzate.

Lactalbumin hydrolyzate (LH), a commercially prepared, enzymatic digest of a milk protein fraction, can partially replace the serum requirement of SV3T3 cells. In a low, but obligatory, background of calf serum (0.15% v/v), LH carses a large increase in the final cell density (5-10 x over the control) while modestly steimulating the actual growth rate. Lactalbumin hydrolyzate does not contain survival activity for SV3T3 cells and does not affect the growth of 3T3 cells. Since even prolonged exposure to pH 4 or 2 results in complete abolishment of the growth-rate stimulatory capability without affecting the capacity to increase the final cell density, it is possible that the LH growth activity may consist of two dissociable components. All of LH growth activity is water soluble, autoclavable, and resists proteolytic treatment. On Sephadex G15 growth activity appears as a single peak at the void volume but on G25 it is retained beyond the void volume as a broad, skewed peak. The relevant molecular weight range lies between 1,500 and 4,000. The putative low pH resistant material is strongly adsorbed to Dowex 1 x 2 and can be displaced from the column by a reduction in the pH. A comparison of the properties of the LH factors with those of known growth promoting agents isolated from serum and various enzymatic digests indicates that these new factors do not correspond to any of these agents.     

Degradation of feather keratin by culture filtrates of Streptomyces fradiae.

Treatment of milled defatted capon feathers with filtrates of Steptomyces fradiae ATCC 10745 that had been concentrated 20 times showed that the most keratin degradation in 0.5h occurred at pH 8.0 and 60degrees C. If assay time was extended, however, adecrease in reaction rate occurred and appeared to result from keratinase instability at elevated temperatures. Exposure of commercial feather meal to unconcentrated culturefiltrates for 15 h produced greatest degradation at 50 degrees C.     

Seasonal and diurnal changes in the water temperature of a temperate pond (England) and a tropical pond (Kenya)

Seasonal changes in water temperatures, embracing periods of low, rising, high and declining temperatures, were recorded in both a temperate and a tropical pond. In the course of a year, the range between the lowest and highest average weekly temperatures was greater in the temperate pond. The spans between the average minimum and the average maximum weekly water temperatures in the warmest months of the year in the tropical pond were greater than those found at any time of the year in the temperate pond. The average weekly air and water temperatures showed the same pattern of seasonal fluctuations; in the tropical pond the average weekly air temperatures were always less than the average minimum weekly water temperatures, whereas they were below, within or above the spans between the average minimum and average maximum weekly aquatic temperatures, according to the time of year, in the temperate pond.In both ponds, diurnal fluctuations were absent during the cooler months; the amplitudes of the fluctuations in the warmer months varied according to the time of year, and were greater, during the warmest months, in the tropical pond. In both ponds lowest temperatures were recorded sometime between 0200 and 1000 hours and highest between 1200 and 2000 hours.The influence of temperature on the life-cycles of invertebrates in both ponds is discussed briefly.

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Zusammenfassung Jahreszeitlich bedingte Schwankungen in der Wassertemperatur, welche Perioden mit niedriger, ansteigender, hoher und abfallender Temperatur einschlossen, wurden in einem gemäßigten und in einem tropischen Teich festgestellt. Im Laufe eines Jahres war der Unterschied zwischen den tiefsten und höchsten wöchentlichen Durchschnittstemperaturen im gemäßigten Teich größer. Hingegen war der Abstand zwischen Mindest- and Höchsttemperatur des Wassers im Wochendurchschnitt während der warmsten Monate des Jahres im tropischen Teich größer als zu irgendeiner Jahreszeit im gemäßigten Teich. Der Wochendurchschnitt der Luft- und Wassertemperaturen wies das gleiche jahreszeitlich bedingte Schwankungsmuster auf. In dem tropischen Teich war die wöchentliche Durchschnittstemperatur der Luft stets niedriger als die Mindestdurchschnittstemperatur des Wassers in einer Woche. Jedoch lag sie — je nach Jahreszeit — unterhalb, auf gleicher Höhe oder oberhalb der Spannweite der niedrigsten and der höchsten wöchentlichen Dutchschnittstemperaturen des Wassers im gemäßigten Teich.In beiden Teichen traten die taglichen Schwankungen während der kÜhleren Monate nicht auf. Der Umfang der Schwankungen in den wärmeren Monaten variierte je nach Jahreszeit and war in den wärmsten Monaten in dem tropischen Teich größer. In beiden Teichen wurden die tiefsten Temperaturen zwischen 2.00 and 10.00 Uhr and die höchsten zwischen 12.00 and 20.00 Uhr gemessen.

     

Proton magnetic relaxation studies of the interaction of D-xylose and xylitol with D-xylose isomerase. Characterization of metal-enzyme-substrate interactions.

The interaction of D-xylose isomerase purified from two sources with Mn2+ and D-xylose or the competitive inhibitor xylitol has been examined by nuclear magnetic resonance. A greater paramagnetic effect of enzyme-bound Mn2+ on the alpha anomer of D-xylose than on the beta anomer was observed, providing independent evidence for the specificity of D-xylose isomerase for the alpha anomeric form of D-xylose. The exchange rate of alpha-D-xylose into the ternary complex, determined from the normalized paramagnetic contribution to the transverse relaxation rate (1/fT2p) of the carbon 1 proton of alpha-D-xylose, exceeds Vmax for the enzymatic reaction by 3 orders of magnitude. The amount of xylitol necessary to displace alpha-D-xylose from the substrate-enzyme-Mn2+ complex is consistent with the Km value for alpha-D-xylose and the inhibitor constant Ki for xylitol previously determined by the methods of enzyme kinetics. These results suggest that the NMR experiments observe complexes of D-xylose isomerase which are kinetically and thermodynamically competent to participate in catalysis. From the frequency dependence of the paramagnetic contribution to the longitudinal relaxation rate (1/T1p) of the carbon 1 proton of alpha-D-xylose, the correlation time (tauc) which modulates the dipolar interaction between enzyme-bound Mn2+ and alpha-D-xylose has been determined (5.1 x 1o(-10) s). From these observations a range of calculated distances between enzyme-bound Mn2+ and the carbon 1 proton of alpha-D-xylose (9.1 +/- 0.7 A) has been found. The enzyme-bound Mn2+ has comparable effects on the carbon 1, carbon 2, and carbon 5 protons of alpha-D-xylose, suggesting that these protons of the enzyme-bound substrate are equidistant from the bound Mn2+. A similar distance (9.4 +/- 0.7 A) between the enzyme-bound Mn2+ and the terminal methylene protons of xylitol, an analog of the open chain intermediate in the reaction, has been determined. The results of the present substrate relaxation and previous water relaxation studies suggest that two small ligands such as water molecules or a large portion of the protein intervene between the bound metal ion and the bound substrate in the active ternary complex.     

Biological soil crusts in ecological restoration: emerging research and perspectives

Drylands encompass over 40% of terrestrial ecosystems and face significant anthropogenic degradation causing a loss of ecosystem integrity, services, and deterioration of social‐ecological systems. To combat this degradation, some dryland restoration efforts have focused on the use of biological soil crusts (biocrusts): complex communities of cyanobacteria, algae, lichens, bryophytes, and other organisms living in association with the top millimeters of soil. Biocrusts are common in many ecosystems and especially drylands. They perform a suite of ecosystem functions: stabilizing soil surfaces to prevent erosion, contributing carbon through photosynthesis, fixing nitrogen, and mediating the hydrological cycle in drylands. Biocrusts have emerged as a potential tool in restoration; developing methods to implement effective biocrust restoration has the potential to return many ecosystem functions and services. Although culture‐based approaches have allowed researchers to learn about the biology, physiology, and cultivation of biocrusts, transferring this knowledge to field implementation has been more challenging. A large amount of research has amassed to improve our understanding of biocrust restoration, leaving us at an opportune time to learn from one another and to join approaches for maximum efficacy. The articles in this special issue improve the state of our current knowledge in biocrust restoration, highlighting efforts to effectively restore biocrusts through a variety of different ecosystems, across scales and utilizing a variety of lab and field methods. This collective work provides a useful resource for the scientific community as well as land managers.     

Solid cultures of thrips-pathogenic fungi Isaria javanica strains for enhanced conidial productivity and thermotolerance

Entomopathogenic fungi have great potential to control agricultural and horticultural insect pests, however optimizing conidial production systems to demonstrate high productivity and stability still needs additional efforts for successful field application and industrialization. Although many virulent entomopathogenic fungal isolates have been viewed as potential candidates in a laboratory environment, very few of the isolates are being used in practice for application in agricultural fields as commercial products. I. javanicus is an entomopathogenic fungus that is parasitic to various diverse coleopteran and lepidopteran insects and thought good candidate as biopesticdes. In this work, the basic characteristics of two entomopathogenic fungi, I. javanica FG340 and Pf04, were investigated in morphological examinations, genetic identification, and virulence against Thrips palmi, and then the feasibility of various grains substrates for conidial production was assessed, particularly focusing on conidial productivity and thermotolerance. Isaria javanica FG340 and Pf04 conidia were solid-cultured on 12 grains for 14?days in a Petri dish. Of the tested Italian millet, perilla seed, millet and barley-based cultures showed high conidial production. The four-grain media yielded >1?×?10⁹ conidia/g of I. javanica FG340 and Pf04. Pf04 strain had enhanced thermotolerance up to 45?°C when cultured on Italian millet. In application, it was easy to make a conidial suspension using the cultured grains, and several surfactants were tested to release the conidia. This work suggests several possible inexpensive grain substrates by which to promote conidial production combined with enhanced stability against exposure to high temperature.