Hemoglobin in Frankia, a Nitrogen-Fixing Actinomycete

Frankia strain CcI3 grown in culture produced a hemoglobin which had optical absorption bands typical of a hemoglobin and a molecular mass of 14.1 kDa. Its equilibrium oxygen binding constant was 274 nM, the oxygen dissociation rate constant was 56 s⁻¹, and the oxygen association rate constant was 206 μM⁻¹ s⁻¹.     

Formation and Regeneration of Methanococcus voltae Protoplasts

Methanococcus voltae cells were converted into protoplasts by suspension in anaerobic 0.1 M Tris-HCl buffer containing 0.4 M sucrose and 0.05 M NaCl as osmoprotectants. Protoplast formation was monitored microscopically by observing the conversion of the typical irregularly shaped (uneven peripheries) coccoid whole cells to rounded forms with smooth peripheries. Although the procedure resulted in about 50% lysis of the initial number of cells, the remainder were converted to the rounded form. Analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and electron microscopy of negatively stained cell preparations indicated that the treatment removed the wall layer from whole cells to yield protoplasts. Protoplast regeneration was evaluated by using optimized plating conditions and an anaerobic microplating technique. Between 50 and 63% of the initial number of protoplasts regenerated as colonies on agar medium (35°C, 7 days). The colony and cell morphologies of the regenerated protoplasts were indistinguishable from those of whole cells plated under identical conditions.     

Isolation of Frankia Strains from Alder Actinorhizal Root Nodules

A simple procedure, based on the rapid filtration and washing of Frankia vesicle clusters, was devised for the isolation of Frankia strains from alder actinorhizal root nodules. Of 46 Alnus incana subsp. rugosa nodules prepared, 42 yielded isolates. A simple medium containing mineral salts, Casamino Acids, and sodium pyruvate proved to be the most effective for isolation. In general, colonies appeared 6 to 20 days after inoculation. On the basis of hyphal morphology, two distinct types of Frankia strains were characterized. Randomly selected isolates were tested for infectivity, and all formed root nodules on A. glutinosa. Because of its simplicity and efficiency, the procedure is an improved method for the study of Frankia diversity in alder root nodules.     

Effectiveness of Different Frankia Cell Types as Inocula for the Actinorhizal Plant Casuarina

The soil bacterium Frankia of the Actinomycetales, capable of forming N₂-fixing symbiotic root nodules on a diverse array of actinorhizal plants, has several morphological forms when grown in pure culture. Fresh hydrated preparations of whole cells, hyphae, and spores were all infective on seedlings of Casuarina at different dilutions. Desiccated hyphae showed no infection capacity, while desiccated spores remained infective, although at a reduced level. On the basis of most-probable-number statistics, spore suspensions were 3 orders of magnitude more infective than hyphae.     

Natural Diversity of Frankia Strains in Actinorhizal Root Nodules from Promiscuous Hosts in the Family Myricaceae

Actinorhizal plants invade nitrogen-poor soils because of their ability to form root nodule symbioses with N₂-fixing actinomycetes known as Frankia. Frankia strains are difficult to isolate, so the diversity of strains inhabiting nodules in nature is not known. To address this problem, we have used the variability in bacterial 16S rRNA gene sequences amplified from root nodules as a means to estimate molecular diversity. Nodules were collected from 96 sites primarily in northeastern North America; each site contained one of three species of the family Myricaceae. Plants in this family are considered to be promiscuous hosts because several species are effectively nodulated by most isolated strains of Frankia in the greenhouse. We found that strain evenness varies greatly between the plant species so that estimating total strain richness of Frankia within myricaceous nodules with the sample size used was problematical. Nevertheless, Myrica pensylvanica, the common bayberry, was found to have sufficient diversity to serve as a reservoir host for Frankia strains that infect plants from other actinorhizal families. Myrica gale, sweet gale, yielded a few dominant sequences, indicating either symbiont specialization or niche selection of particular ecotypes. Strains in Comptonia peregrina nodules had an intermediate level of diversity and were all from a single major group of Frankia.     

Identification by Suppression Subtractive Hybridization of Frankia Genes Induced under Nitrogen-Fixing Conditions

Frankia is an actinobacterium that fixes nitrogen under both symbiotic and free-living conditions. We identified genes upregulated in free-living nitrogen-fixing cells by using suppression subtractive hybridization. They included genes with predicted functions related to nitrogen fixation, as well as with unknown function. Their upregulation was a novel finding in Frankia.Frankia is a Gram-positive actinobacterium that establishes symbiosis with several angiosperms termed actinorhizal plants and forms nitrogen-fixing nodules on their roots (20). Frankia also fixes nitrogen in free-living culture under nitrogen-free conditions (19). Induction of the nitrogen-fixing ability is accompanied by differentiation of vesicles (19). Vesicles are spherical cells specialized to nitrogen fixation and are surrounded by multilayered lipid envelopes by which nitrogenase is protected from oxygen (3). Frankia plays an important role in the global nitrogen cycle, yet little is known about the genes involved in the induction of nitrogen-fixing activity. Recently, three Frankia genome sequences were determined (15), which facilitates the genetic dissection of Frankia biology. In this study, we identified Frankia genes induced in nitrogen-fixing cells under free-living conditions by using suppression subtractive hybridization (SSH) (4).     

Callus Formation from Protoplasts and Plant Regeneration from Tissue Culture of Silybum marianum Gaertn

Leaf calli of Silybum marianum Gaertn. subcultured for one year were used for protoplast isolation and culture. First division was observed three days after culture on medium M12, and the highest division frequency was 35.4%. One to three months later, small ralli were seen with naked eyes, and grew up gradually. Upon transferring them onto D6 differentiation medium, the green bud apices were observed two months later. However, no shoot differentiation was obtained. Hypocotyl calli were induced on MS+NAA 0.8mg/1, 6-BA 0.5mg/1. Two months after transferring calli onto D6 medium, shoots were regenerated from the surface of the calli. The freqency of shoot differentiation was 75%. On a MS rooting medium containing NAA 0.5 mg/1, IBA 0.1 mg/1, whole plants with healthy roots were obtained.     

链霉菌11371原生质体的制备与再生

为选育链霉菌11371的高产Tetramycin菌株,摸索该菌株的原生质体的制备与再生。结果表明,链霉菌11371原生质体制备和再生的最佳条件为菌丝生长培养液中甘氨酸浓度为0．7％,培养温度为28℃,培养时间为42h,溶菌酶浓度为3mg／mL,酶解温度为37℃,酶解时间为90min。最佳再生培养基为R2YE培养基。     

Production and Regeneration of Lactobacillus casei Protoplasts

Methods for the production and regeneration of Lactobacillus casei protoplasts are described. Protoplasts of L. casei strains were obtained by treatment with mutanolysin or with mutanolysin and lysozyme together in a protoplast formation buffer containing 0.02 M HEPES (N-2-hydroxyethylpiperazine-N′-2-ethanesulfonic acid) (pH 7.0), 1 mM MgCl₂, 0.5% gelatin, and 0.3 M raffinose. Cells were regenerated on a complex medium supplemented with bovine serum albumin, MgCl₂, CaCl₂, gelatin, and raffinose. Lengthy digestion with lytic enzymes inhibited the capacity of protoplasts to regenerate. The optimum conditions of protoplast formation varied from strain to strain. Using predetermined optimal conditions it was possible to prepare protoplasts of several L. casei strains and regenerate them with 10 to 40% efficiency. The methods were applicable to other species of lactobacilli as well.     

Preparation and Regeneration of Chalara paradoxa Protoplasts

A lignan mixture from sesame salad oil containing episesamin and sesamin as major components was fed to rats. Lignans at the dietary level of approximately 0.2% tended to decrease plasma and liver cholesterol levels with an accompanying increase in the fecal excretion of neutral steroids, particularly when the dietary fat source was evening primrose oil containing γ-linolenic acid. There was a decreasing trend in the specific activity of Δ5-desaturase in liver microsomes whereas that of Δ6-desaturase tended to increase, in particular in rats fed with safflower oil. The proportion of dihomo-γ-linolenate increased in response to the reduction of Δ5-desaturation activity, and that of docosapentaenoate (n-6) decreased in liver phosphatidylcholine in both groups of rats, suggesting that lignans interfered with various steps of linoleate metabolism. However, the production by the aorta of prostacyclin and by platelets of thromboxane A₂ was not influenced by lignans. Thus, episesamin and/or sesamin functioned as a regulator of cholesterol and linoleate metabolism in rats.     

Formation and regeneration of Frankia protoplasts

A technique for forming protoplasts from Frankia cells and regenerating them to the normal hyphal mode of growth is described. Electron microscopy proved that protoplasts were studied and not spores or small hyphae. Regenerated colonies were investigated for genetic markers. One ArI3 colony had been cured of its plasmids without being affected in its symbiotic properties.     

Plant Regeneration from Protoplasts of Coriandrum sativum

Calli produced from stem segments of seedling of Coriandrum satwum which were cultured on MS agar medium containing NAA 1.0mg/L. The embryogenic cell colony suspension was estabilished on MS liquid medium containing NAA 1.0mg/L%2,4-D 0.2mg/L+BA 0.5 mg/L. The cell suspension culture was used for protoplast preparation. Protoplasts were obtained in the enzyme mixture containing 2.0% Onozuka R-10, 1.0% pectinase, 0.5% snailase, 0.5% dextran sulfate potassium Salt, 0.6mol/L mannital CPW solution at pH 5.8 and 25℃. Cultured in a KM8P liquid medium containing NAA 1.0mg/L+2,4-D 0.2mg/L+6-BA 0.5 mg/L, glucose 0.4mol/L and CM 20mi/L; the protoplasts entered the stage of derision after three days, cell clusters formed in 10 days and calli formed after about 50 days. When the calli were transferred to MS agar medium containing many growth substances, they differentiated into embryoids, and then developed into plantlet with many green leaves and roots on the 1/2 MS agar medium.     

Plant Regeneration from Protoplasts of Talinum paniculatum

The protoplasts of Talinum paniculaturn (Jaeq.) Gaertn. were isolated from leaves and calli. The mesophyll protoplasts did not undergo normal division and lived one week at the longest in culture. However, the callus protoplasts, cultured in P4 medium (K8p+2, 4-D 0.2 mg/L, NAA 1.0 mg/L, ZT 0.5 mg/L, coconut milk 50 mL/L, glucose 0.5 mol/L), underwent first division after 3 d of culture. The division frequency was 36.7 % after 7 d of culture. The regeneration frequencies of callus were 0.31% in liquid culture and 0.34% in double-layer culture. Shoots differentiated on regeneration media and rooted on R3 and R7 media. Mature plants were obtained 2～3 months after transplanting the protoplast-derived plantlets into flower pot or successive subculturing in test tubes. The results also indicated that: (1) Too long a period of callus culture in liquid medium or in solid proliferation medium was unfavorable to differentiation. (2) Low concentration of 6-BA in medium was suitable for callus differentiation. (3) GA3 promoted development of young adventitious bud. (4) Multi-effect triazole significantly strengthened sprout and root development in test tube cultures.     

Regeneration of Mycelial Protoplasts from Lyophyllum shimeji

A new HPLC system for simultaneous analysis of NAD⁺, NADH, NADP⁺, and NADPH was developed and used to measure the transhydrogenase activity of spinach ferredoxin-N ADP ⁺ reductase (EC 1.18.1.2, FNR). The system is based on a reverse-phase HPLC with isocratic elution on an ODS column (4.6 × 50 mm). The four nucleotides were completely separated by developing the column with 0.15 m sodium phosphate/citrate buffer (pH 6.8) containing 1 mm EDTA at 40°C with a flow rate of 1 ml/min. The four nucleotides can be simultaneously assayed within 13 min by monitoring the effluents with a UV detector. It was also indicated that the transhydrogenase activity of spinach FNR can be advantageously assayed by measuring the nucleotides by this method.     

Plant Regeneration from Protoplasts of Medicago lupulina

Protoplasts isolated from suspension cell lumps of Medicago lupulina L. started to divide after 2 clays in K8p culture medium containing 0. 1～2.0 mg/L of 2, 4-D, with a maximum division frequency of 38. 35%. After S weeks of culture, the protoplast-derived cell lumps were transferred to liquid/solid double-layer media for microcallus regeneration, with a maximum frequency of 0.58%. The whole plants were regenerated from protoplastderived calli via somatic embryogenesis and organogenesis. In somatic embryogenesis, the embryoids were induced on MS and W14 media with rather wide range (1. 0420.0 mg/L) of 2, 4-D concentration. The highest induction frequency of embryoids was 71.0%. In organogenesis, the differentiation media containing lower concentration of 6-BA (0. 5～0. 7 mg/L) were suitable for adventitious bud formation. The highest frequency of adventitious bud formation from calli was 27. 8%. The mature protoplast-regenerated plants were obtained 3 months after transplanting the plantlets into soil.