Incorporation of hygromycin resistance in Brassica nigra and its transfer to B. napus through asymmetric protoplast fusion

Summary With the idea to develop a selection system for asymmetric somatic hybrids between oilseed rape (Brassica napus) and black mustard (B. nigra), the marker gene hygromycin resistance was introduced in this last species by protoplast transformation with the disarmed Agrobacterium tumefaciens strain C58 pGV 3850 HPT. The B. nigra lines used for transformation had been previously selected for resistance to two important rape pathogens (Phoma lingam, Plasmodiophora brassicae). Asymmetric somatic hybrids were obtained through fusion of X-ray irradiated (mitotically inactivated) B. nigra protoplasts from transformed lines as donor with intact protoplasts of B. napus, using the hygromycin resistance as selection marker for fusion products. The somatic hybrids hitherto obtained expressed both hygromycin phosphotransferase and nopaline synthase genes. Previous experience with other plant species had demonstrated that besides the T-DNA, other genes of the donor genome can be co-transferred. In this way, the produced hybrids constitute a valuable material for studying the possibility to transfer agronomically relevant characters — in our case, diseases resistances — through asymmetric protoplast fusion.     

Growth and metabolic flexibility in groundwater bacteria

Groundwater bacteria isolated from an oligotrophic-saturated soil showed a mixed strategy of economized metabolism and migration when grown in a continuous-flow column system simulating poor or nutrient-amended growth conditions. The cells were generally <0.5m in diameter in pure groundwater, but doubled in size when the concentration of dissolved organic carbon and phosphate in groundwater was increased 10-fold. The biomass, estimated from analysis of muramic acid (MuAc) in cell wall peptidoglucans, increased at the same time by a factor of 5 when the solid support in the columns was gravel and by a factor of 10 when it was glass beads. Bacteria in pure groundwater stored 10 times more of the energy-rich polysaccharide, poly--hydroxybutyric acid (PHB), than bacteria in enriched groundwater, and those cells that were attached to the gravel stored 10 times as much as cells in the interstitial pore water. Once phosphate was added to groundwater, stored PHB was metabolized. The proportion of free-living to attached bacteria was 2 to 10 times higher in enriched compared with pure groundwater indicating a mass transport of cells as the carrying capacity of their habitat rose.     

Selective illumination of single photoreceptors in the house fly retina: local membrane turnover and uptake of extracellular horseradish peroxidase (HRP) and Lucifer Yellow

Summary Single photoreceptor cells in the compound eye of the housefly Musca domestica were selectively illuminated and subsequently compared electron-microscopically with the unilluminated photoreceptors in the immediate surroundings. The rhabdomeres of the illuminated cells remain largely unaffected, but the cells show an increase in the number of coated pits, various types of vesicles, and degradative organelles; some of the latter organelles are described for the first time in fly photoreceptors. Coated pits are found not only at the bases of the microvilli, but also in other parts of the plasma membrane. Degradative organelles, endoplasmic reticulum (ER) and mitochondria aggregate in the perinuclear region. The rough ER and smooth ER are more elaborate, the number of Golgi stacks, free ribosomes and polysomes is increased, and the shape and distribution of heterochromatin within the nuclei are altered. Illuminated photoreceptors also interdigitate extensively with their neighbouring secondary pigment cells. These structural changes in illuminated fly photoreceptor cells indicate an increase in membrane turnover and cellular metabolism. When applied to the eye, Lucifer Yellow spreads into the extracellular space and is taken up only by the illuminated photoreceptor cells. These cells show the same structural modifications as above. Horseradish peroxidase applied in the same way is observed in pinocytotic vesicles and degradative organelles of the illuminated cells. Hence, the light-induced uptake of extracellular compounds takes place in vivo at least partially as a result of an increase in pinocytosis.     

Glucocorticoid receptor binds cooperatively to adjacent recognition sites.

In order to define the mechanism of synergistic induction mediated by multiple glucocorticoid response elements (GRE), the affinity of the glucocorticoid receptor to a single or duplicated GRE was analyzed by gel retardation, nitrocellulose filter binding and by footprinting experiments. Direct measurement of the relative affinity and indirect determination by competition showed greater than 10-fold higher affinity of the glucocorticoid receptor to a duplicated GRE when compared to a single element. Maximal stability of the GRE-receptor complex was obtained using two closely spaced GREs positioned on the same side of the DNA helix. Increasing the distance or changing the helical position of the GREs considerably increased the off rate of the receptor. DNase I footprinting shows in addition to the protection of the GRE region, an altered pattern in the nonprotected intervening DNA indicating structural alteration of the DNA helix by the receptor bound to adjacent GREs.     

Effects of nucleotides and divalent cations on phospholipase activity in Saccharomyces cerevisiae

Divalent cations activate the lysophospholipase and transacylase reactions catalyzed by the same enzymes in the yeast Saccharomyces cerevisiae. The activation was observed at neutral pH, but not at the pH optimum of lysophospholipase/transacylase, near 3.5. Adenine nucleotides, especially AMP and ADP, are strong inhibitors of the same group of enzymes. Half maximal inhibition by AMP was found at a concentration of about 20 M. The inhibition by nucleotides in low concentrations is enhanced by divalent cations.     

Function of methanofuran,tetrahydromethanopterin, and coenzyme F420 in Archaeoglobus fulgidus

Archaeoglobus fulgidus is an extremely thermophilic archaebacterium that can grow at the expense of lactate oxidation with sulfate to CO₂ and H₂S. The organism contains coenzyme F₄₂₀, tetrahydromethanopterin, and methanofuran which are coenzymes previously thought to be unique for methanogenic bacteria. We report here that the bacterium contains methylenetetrahydromethanopterin: F₄₂₀ oxidoreductase (20 U/mg), methenyltetrahydromethanopterin cyclohydrolase (0.9 U/mg), formyltetrahydromethanopterin: methanofuran formyltransferase (4.4 U/mg), and formylmethanofuran: benzyl viologen oxidoreductase (35 mU/mg). Besides these enzymes carbon monoxide: methyl viologen oxidoreductase (5 U/mg), pyruvate: methyl viologen oxidoreductase (0.7 U/mg), and membranebound lactate: dimethylnaphthoquinone oxidoreductase (0.1 U/mg) were found. 2-Oxoglutarate dehydrogenase, which is a key enzyme of the citric acid cycle, was not detectable. From the enzyme outfit it is concluded that in A. fulgidus lactate is oxidized to CO₂ via a modified acetyl-CoA/carbon monoxide dehydrogenase pathway involving C₁-intermediates otherwise only used by methanogenic bacteria.Non-standard abbreviations APS adenosine 5-phosphosulfate - BV benzyl viologen - DCPIP 2,6-dichlorophenolindophenol - DMN 2,3-dimethyl-1,4-naphthoquinone - DTT DL-1,4-dithiothreitol - H₄F tetrahydrofolate - H₄MPT tetrahydromethanopterin - CH₂ H₄MPT, methylene-H₄MPT - CH H₄MPT, methenyl-H₄MPT - Mes morpholinoethane sulfonic acid - MFR methanofuran - Mops morpholinopropane sulfonic acid - MV methyl viologen - Tricine N-tris(hydroxymethyl)-methylglycine - U mol product formed per min     

Pelodictyon phaeoclathratiforme sp. nov., a new brown-colored member of the Chlorobiaceae forming net-like colonies

A new strain of the green sulfur bacteria was isolated from the monimolimnion of Buchensee (near Radolfzell, Lake Constance region, FRG). Single cells were rod-shaped, nonmotile and contained gas vacuoles. Typical net-like colonies were formed by ternary fission of the cells. As photosynthetic pigments bacteriochlorophylls a, e, isorenieratene and -isorenieratene were present. Sulfide, sulfur and thiosulfate were used as electron donors during anaerobic phototrophic growth. Besides carbon dioxide, acetate and propionate could serve as carbon sources under mixotrophic conditions in the light. Like all other members of the green sulfur bacteria, the new bacterium is strictly anaerobic and obligately phototrophic. The possession of gas vacuoles and the formation of net-like colonies and the guanine plus cytosine content of the DNA (47.9 mol% G+C) are typical characteristics of the genus Pelodictyon. Because of its photosynthetic pigments which differ from those of Pelodictyon clathratiforme, strain BU 1 represents a new species, P. Phaeoclathratiforme sp. nov.     

Nontoxic and toxic oligopeptides with D-amino acids and unusual residues in Microcystis aeruginosa PCC 7806

Toxic and nontoxic peptides were isolated from the cyanobacterium Microcystis aeruginosa PCC 7806 by a procedure including extraction of cells with water-saturated 1-butanol, chromatography of the extract on silica gel plates and high performance liquid chromatography (HPLC) on Partisil-5. The toxin was shown to be only a minor constituent, being negatively charged and thus separable by electrophoresis, within the HPLC-purified fraction. It contained erythro-β-methyl-D-Asp, D-Glu, D-Ala, L-Leu, and L-Arg known to be part of the Microcystis peptide-toxin with M_r 994. The major part of the HPLC-purified fraction was assigned, however, to a nontoxic peptide with a M_r of 956. Partial hydrolysis studies of the nontoxic peptide(s) revealed amino acid sequences composed of D-Glu, N-methyl-Phe, and 3,4-dehydro-Pro, aside from the common L-amino acids. Cyclic linkage in the nontoxic peptide(s) appears likely.     

Continuous microbial desulfurization of coal--application of a multistage slurry reactor and analysis of the interactions of microbial and chemical kinetics

Microbial desulfurization of coal by pyrite oxidizing bacterial enrichment cultures has been studied in air-agitated slurry reactors of 4- and 20-L volumes. Batch experiments showed that inoculation with an active bacterial culture is essential to minimize the lag phase, although a considerable number of pyrite oxidizing bacteria was found on the coal prior to desulfurization. For detailed investigations of kinetics, energy requirements, and technical applicability, a bioreactor equipment consisting of a cascade of eight stages was developed and operated continuously. Microbial desulfurization of coal-monitored by measuring the axial profile of dissolved iron concentration, real and maximum oxygen consumption rates, and cell concentration-at pulp densities to 30% was performed over a period of 200 days without any disturbances concerning the aeration system, fluidization, transport of solids and microbial growth. At a pulp density of 20%, a pyrite conversion of 68% was achieved after the third reactor stage at a total residence time of five days in the first three stages. The kinetics of pyrite degradation were found to be well described by a rate equation of first order in pyrite surface area concentration if the pyrite is directly accessible for microbial attack. Rate constants were determined to 0.48 mg pyrite/(cm(2) day) in the first and to 0.24 mg pyrite/(cm(2) day) in the following reactor stages. Kinetic models taking into account adsorption/desorption as well as growth kinetics failed to describe the observed reaction rates. However, a model treating pyrite degradation and microbial growth kinetics formalistically seems to be applicable when backmixing between the reactor stages can be avoided. The advantage of a multistage reactor in comparison to single-stage equipment was shown by calculation. To obtain a pyrite conversion of 68%, a three-stage reactor would require only 58% of the volume of single-stage equipment.Measurement of oxygen consumption rates proved to provide quickly and easily measurable parameters to observe microbial coal desulfurization in technical scale: the real oxygen consumption rate is correlated to the pyrite oxidation rate and the maximum oxygen consumption rate is correlated to the concentration of viable cells. The Y(o/s) coefficient for the amount of oxygen consumed per mass unit of pyrite oxygen was determined to approximately 0.33 in comparison to 1.0 which can be calculated from stoichiornetry. This could yet not be explained. Chemical leaching experiments as well as sulfur analyses of desulfurized coal samples showed that the microorganisms play the main role in degradation of pyrite from coal and that pyrite oxidation by ferric iron can be neglected.