A New Simple Method for Isolating Multistress-Tolerant Semidominant Mutants of Saccharomyces cerevisiae by One-Step Selection under Lethal Hydrogen Peroxide Stress Condition

A tetrathionate-decomposing enzyme that catalyzes the decomposition of tetrathionate into thiosulfate and sulfate was purified to homogeneity from tetrathionate-grown Thiobacillus thiooxidans. The enzyme had an apparent molecular weight of 104,000, and was composed of two identical subunits (MW = 58,000) as estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The enzyme had an isoelectric point at 9.6 and was most active at pH 3.0–3.5 and 40°C. Enzyme activity was increased approximately 100-fold in the presence of 400 mm sulfate ion. The Michaelis constant of this enzyme for tetrathionate in the presence of 20, 50, and 200 mm of sulfate ion was 2.4 mm. Mercuric and ferric ions completely inhibited the enzyme activity at 1 mm. Though cupric ion up to 0.01 mm markedly stimulated the activity in the presence of 20 mm sulfate ion, a higher concentration (1 mm) rather strongly inhibited the activity. Ethylenediaminetetraacetic acid (EDTA) strongly inhibited the activity, but this inhibiton was completely restored by cupric ion.     

Sublethal effects of cupric ion activity on the phototaxis of three calanoid copepods

Using wavelengths near maximal photosensitivity, phototactic responses of two estuarine calanoid copepods (Acartia tonsa, Acartia hudsonica) and one nearshore, neritic copepod (Temora longicornis) were measured after 24 h exposures to sublethal concentrations of free cupric ions. A nitrilotriacetate-trace metal ion buffer system was used to control the free cupric ion activity (pCu = negative log of the free cupric ion activity), which determines organismic response. All three species exhibited positive phototaxis at pCu 13.0 reported for unpolluted surface sea waters and estuarine waters. As cupric ion activity increased, percent positive phototactic response decreased, indicating a strong sublethal effect of free cupric ions on photobehavior. Changes in photobehavior occurred at cupric ion activities that have been reported for many estuaries and coastal waters near urban and industrialized areas. Temora longicornis was much less phototactically sensitive than the two estuarine species. It also exhibited phototactic sign switching as pCu changed.     

In vitro culture of Vitis: the effects of light spectrum,manganese sulfate and potassium iodide on morphogenesis

The effects of blue and red light, manganese sulfate concentration (100 and 5 M), and potassium iodide (5 and 0 M) on shoot and root production from subcultured shoots of the Vitis hybrid Remaily Seedless were studied.Shoot production was greater in blue light. It was increased by lowering the manganese sulfate concentration. The response to manganese in blue light was greatest when there was no potassium iodide addition.Root production was decreased by red light and lower manganese concentration.The effects of manganese, iodide and light spectrum on morphogenesis are discussed in relation to their known effects on IAA metabolism.     

Uptake of sulfate,sulfite and thiosulfate by proton-anion symport in Desulfovibrio desulfuricans

pH changes and sulfide production upon addition of sulfate, sulfite or thiosulfate to non-buffered H₂-saturated cell suspensions of Desulfovibrio desulfuricans were studied by means of electrodes. The addition of these electron acceptors resulted in a rapid alkalinization of the suspension which was accompanied by sulfide production. At-2° C, alkalinization without immediate sulfide production could be obtained. After addition of ³⁵S-labelled sulfate at-2° C, the label was found to be concentrated 7,500-fold in the cells, while 2 protons per sulfate molecule had disappeared from the outer bulk phase. Alkalinization and sulfide production from micromolar electron acceptor additions depended on the transmembraneous proton gradient ( pH), and were reversibly inhibited in alkaline solution (pH>8.0) or by the protonophore carbonylcyanide m-chlorophenylhydrazone (CCCP). Protonophore-inhibited sulfide production from sulfite or thiosulfate could be restored if the cell membranes were permeabilized by the detergent cetyltrimethylammonium bromide (CTAB), or if downhill transport was made possible by the addition of electron acceptors at millimolar concentrations. Sulfate was not reduced under these conditions, presumably because the cells did not contain ATP for its activation. K⁺-and Na⁺-ionophores such as nigericin, valinomycin or monensin appeared to be of limited efficiency in D. desulfuricans. In most experiments, sulfate reduction was inhibited by the K⁺–H⁺ antiporter nigericin in the presence of K⁺, but not by the thiocyanate anion or the K⁺-transporter valinomycin. The results indicate that sulfate, sulfite and thiosulfate are taken up by proton-anion symport, presumably as undissociated acids with an electroneutral mechanism, driven by the transmembraneous pH gradient ( pH) or by a solute gradient. Kinetics of alkalinization and sulfide production in cells grown with different electron acceptors revealed that D. desulfuricans has different specific uptake systems for sulfate and thiosulfate, and obviously also for sulfite. It is proposed that the electron acceptor transport finally will not consume net energy during growth in buffered medium: The protons taken up during active electron acceptor transport leave the cell with the reduced end-product by simple passive diffusion of H₂S.Abbreviations CCCP carbonyl cyanide m-chlorophenylhydrazone - FCCP carbonyl cyanide p-trifluoromethoxy phenylhydrazone - CTAB cethyltrimethylammonium bromide     

Hormonal regulation of berberine production in cell suspension cultures of Thalictrum minus

Effects of auxin and cytokinin on cell growth and alkaloid production in cell suspension cultures of Thalictrum minus were examined in an attempt to increase the productivity of a medicinal compound, berberine. In Linsmaier and Skoog medium containing auxin such as 2,4-D (1 M), the cultured cells grew rapidly, producing little berberine. On the other hand, the berberine-producing activity was remarkably enhanced by simultaneous administration of auxin and cytokinin, although cell growth was inferior. In particular, for the combination of NAA (60 M) and 6-benzylaminopurine (10 M), the yield of berberine was as high as 20 mg/30 ml medium after 2 weeks of culture. Furthermore, most of the berberine produced by the cells was released into the liquid medium, in which an excess of berberine crystallized. The results of the present experiments are suggestive of an advantage in adopting a two-stage culture method for the production of berberine in fermentor systems.Abbreviations LS Linsmaier and Skoog - 2,4-D 2,4-dichlorophenoxyacetic acid - NAA 1-naphthaleneacetic acid - IAA indole-3-acetic acid - IBA indole-3-butyric acid - BAP 6-benzylaminopurine     

Effective extracellular trehalose production by<Emphasis Type="Italic"> Cellulosimicrobium cellulans</Emphasis>

A bacterium isolated from a petal of Casa Blanca Lily (ST26 strain) produced a marked amount of extracellular trehalose (-d-glucopyranosyl-[1,1]--d-glucopyranose) in culture medium containing glucose. 16S rDNA-based phylogeny showed that ST26 belongs to, or is related to, Cellulosimicrobium cellulans, a close relative of Cellulomonas spp. Various Cellulomonas strains obtained from culture collections also showed extracellular trehalose productivity, suggesting that trehalose production is a common property of this bacterial genus. ST26 accumulated trehalose in medium supplied with glucose but not with sucrose, glycerol or maltose. Effective extracellular trehalose production by ST26 was achieved by supplying 0.5–1% ammonium sulfate and 0.5–1% CaCO₃. The addition of CaCO₃ adjusted the pH of the culture to around 5.0. The optimized culture conditions yielded trehalose from glucose at a conversion rate of 61%. The addition of ammonium sulfate greatly reduced the dry cell weight of ST26 and intracellular content of trehalose, which suggests that the addition of ammonium sulfate makes ST26 cells leak trehalose into the medium. ST26 effectively propagated in minimal medium containing trehalose as a sole carbon source, which suggests that trehalose serves as a carbohydrate reserve of this organism.The nucleotide sequence of 16S rDNA of ST26 has been submitted to the DDBJ databank under accession number AB109293     

Dependence of the kinetics of secondary active transports in yeast on H+-ATPase acidification

Acidification of the external medium of the yeast Saccharomyces cerevisiae, mainly caused by proton extrusion by plasma membrane H⁺-ATPase, was inhibited to different degrees by D₂O, diethylstilbestrol, suloctidil, vanadate, erythrosin B, cupric sulfate and dicyclohexylcarbodiimide. The same pattern of inhibition was found with the uptake of amino acids, adenine, uracil, and phosphate and sulfate anions. An increase of the acidification rate by dioctanoylglycerol also increased the rates of uptake of adenine and of glutamic acid. In contrast, a decrease of the membrane potential at pH 4.5 from a mean of -40 to -20 mV caused by 20 mm KC1 had no effect on the transport rates. The ATPase-deficient mutant S. cerevisiae pmal-105 showed a markedly lower uptake of all the above solutes as compared with the wild type, while its membrane potential and pH were unchanged.Other types of acidification (spontaneous upon suspension; K⁺ stimulated) did not affect the secondary uptake systems.     

Induction of berberine biosynthesis by cytokinins in Thalictrum minus cell suspension cultures

Production of berberine could be induced by adding 6-benzylaminopurine (BAP) to Thalictrum minus cells, cultured in suspension in a medium containing 2,4-dichlorophenoxyacetic acid (2,4-D), early in the growth cycle. In the presence of BAP, the precursor, L-tyrosine, was rapidly converted into berberine which was then released into the medium, whereas substantial amounts of the intermediates, tyramine and dopamine, accumulated in non-berberine-producing cells grown in the same 2,4-D-containing medium without BAP. These results suggest that BAP activated enzymatic reactions subsequent to the formation of the amines in the biosynthesis of berberine.Abbreviations 2,4-D 2,4-dichlorophenoxyacetic acid - BAP 6-benzylaminopurine - NAA 1-naphthaleneacetic acid - IAP 6-isopentenylaminopurine - LS medium Linsmaier-Skoog medium - Growth medium LS medium containing 10^-6 M 2,4-D     

Methane production and sulfate reduction in two Appalachian peatlands

Anaerobic carbon mineralization was evaluated over a 1-year period in two Sphagnum-dominated peatlands, Big Run Bog, West Virginia, and Buckle's Bog, Maryland. In the top 35 cm of peat, mean rates of methane production, anaerobic carbon dioxide production, and sulfate reduction at Big Run Bog were 63,406 and 146 mol L^-1 d^-1, respectively, and at Buckle's Bog were 18, 486 and 104 mol L^-1 d^-1. Annual anaerobic carbon mineralization to methane and carbon dioxide at Big Run Bog and Buckle's Bog was 52.8 and 57.2 mol m^-2, respectively. Rates of methane production were similar to rates reported for other freshwater peatlands, but methane production accounted for only 11.7 and 2.8%, respectively, of the total anaerobic carbon mineralization at these two sites. Carbon dioxide production, resulting substantially from sulfate reduction, dominated anaerobic carbon mineralization. Considerable sulfate reduction despite low instantaneous dissolved sulfate concentrations (typically < 300 mol L^-1 of substrate) was apparently fueled by oxidation and rapid turnover of the reduced inorganic sulfur pool.The coincidence of high sulfate inputs to the Big Run Bog and Buckle's Bog watersheds through acid precipitation with the unexpected importance of sulfate reduction leads us to suggest a new hypothesis: peatlands not receiving high sulfate loading should exhibit low rates of anaerobic decomposition, and a predominance of methane production over sulfate reduction; however, if such peatlands become subjected to high rates of sulfur deposition, sulfate reduction may be enhanced as an anaerobic mineralization pathway with attendant effects on carbon balance and peat accumulation.     

l-Isoleucine Production by Analog-resistant Mutants Derived from Threonine-producing Strain of Corynebacterium glutamicum

A thiaisoleucine-resistant mutant, ASAT–372, derived from a threonine producer of Corynebacterium glutamicum, KY 10501, produced 5 mg/ml each of l-isoleucine and l-threonine. l-Isoleucine productivity of ASAT–372 was improved stepwise, with concurrent decrease in threonine production, by successively endowing it with resistivity to such substances as ethionine, 4-azaleucine and α-aminobutyric acid. The mutant strain finally selected, RAM–83, produced 9.7 mg/ml of l-isoleucine with a medium containing 10% (as sugar) molasses.

l-Isoleucine production was significantly affected by the concentration of ammonium sulfate in the fermentation medium. At 4% ammonium sulfate l-isoleucine production was enhanced whereas l-threonine production was suppressed. At 2% ammonium sulfate l-threonine production was stimulated while l-isoleucine production decreased.     

SURFACTANT PROMOTION OF THE INHIBITORY EFFECTS OF CUPRIC GLUTAMATE ON THE DINOFLAGELLATE ALEXANDRIUM1

We studied cupric glutamate as a novel algicide for marine harmful algae and hexadecyltrimethyleamine bromide (HDTMAB) as an accelerant. Cupric glutamate had an excellent ability to inhibit the growth of Alexandrium sp. LC3, but the inhibition efficiency did not increase with higher cupric glutamate concentration. The studies on the inhibition ofAlexandrium sp. LC3 by cupric sulfate or cupric glutamate showed that cupric glutamate had a higher inhibition rate than cupric sulfate (P < 0.05). HDTMAB could significantly enhance the inhibition by cupric glutamate (P < 0.05). Ultrastructural changes of Alexandrium sp. LC3 under cupric sulfate, cupric glutamate, and cupric glutamate–HDTMAB combined treatment were studied with TEM. Under these stresses, the integrity of the cell plasma membranes (cell plasma membrane, chloroplast and mitochondria membranes) was destroyed. The degree of damage under cupric glutamate–HDTMAB combined treatment was more severe than under the other stresses. These results indicated that mechanistically cupric glutamate inhibits algal growth by destroying the cell membranes, and that HDTMAB promotes this process, which induced mass extravasation of intracellular components and more copper ion entry into the plasma.     

Improved plant regeneration from barley callus cultures by increased copper levels

Incorporation of cupric sulfate into callus induction, maintenance, and regeneration media significantly enhanced plant regeneration from callus cultures of barley (Hordeum vulgare L.) immature embryos. Embryos from the cultivars Hector and Excel were cultured on MS medium containing 0, 0.1 (MS level), 0.5, 1.0, 5.0, 10.0, 50.0, or 100.0 M cupric sulfate. Plants were regenerated beginning at 8 weeks and continuing through 36 weeks. For Hector, medium containing 50 M copper regenerated significantly more plants than any other medium, with an average of 17 plants per embryo. In comparison, medium with MS copper levels (0.1 M) regenerated only 5 plants per embryo. For Excel, medium containing 5.0 M copper was the best, regenerating 1.4 plants per embryo. No Excel regenerants were obtained on medium with MS copper levels. Increased copper levels also increased the percentage of embryos that regenerated at least one plant, in both cultivars. The results indicate that MS copper levels are not optimized for barley callus cultures, and that improved plant regeneration can be obtained at higher copper concentrations.Abbreviations MS Murashige & Skoog (1962) - 2,4-d 2,4-dichlorophenoxyacetic acid The US Government's right to retain a non-exclusive royalty-free license on and to any copyright is acknowledged     

Sulfate assimilation in Rhodopseudomonas globiformis

Rhodopseudomonas globiformis is able to grow on sulfate as sole source of sulfur, but only at concentrations below 1 mM. Good growth was observed with thiosulfate, cysteine or methionine as sulfur sources. Tetrathionate supported slow growth. Sulfide and sulfite were growth inhibitory. Growth inhibition by higher sulfate concentrations was overcome by the addition of O-acetylserine, which is known as derepressor of sulfate-assimilating enzymes, and by reduced glutathione. All enzymes of the sulfate assimilation pathway. ATP-sulfurylase, adenylylphosphate-sulfotransferase, thiosulfonate reductase and O-acetylserine sulfhydrylase are present in R. globiformis. Sulfate was taken up by the cells and the sulfur incorporated into the amino acids cysteine, methionine and homocysteine. It is concluded, that the failure of R. globiformis to grow on higher concentrations of sulfate is caused by disregulation of the sulfate assimilation pathway. Some preliminary evidence for this view is given in comparing the activities of some of the involved enzymes after growth on different sulfur sources and by examining the effect of O-acetylserine on these activities.Abbreviations DTE dl-dithioerythritol - APS adenosine 5-phosphosulfate, adenylyl sulfate - PAPS 3-phosphoadenosine 5-phosphosulfate, 3-phosphoadenylylsulfate     

A sulfated galactan from the mucilaginous sheath of the red filamentous alga Chroodactylon ornatum (Stylonematophyceae,Rhodophyta)

The pseudofilamentous red alga Chroodactylon ornatum was grown in f/2 culture medium with the addition of 10, 34, and 100 μM (nominal concentration) cupric sulfate. The bioassays were terminated at two selected end points (days 4 and 12). Growth inhibition, changes in pigment composition, and oxidative stress indicators such as phenolic compounds and lipid peroxidation (dosed as thiobarbituric reactive substances) were observed in cultures with 34 and 100-μM cupric sulfate. Quinacrine (Atebrin) and chlorotetracycline fluorochromes showed abundant vacuoles of acidic content, related with mucilage secretion. Structural analyses by methylation, desulfation-methylation, alkaline treatment, and NMR spectroscopy revealed that the mucilaginous sheath of C. ornatum contains a sulfated galactan with a backbone of alternating 3-linked β-d-galactopyranose and 4-linked α-l-galactopyranose moieties, i.e., an agaran. The absence of 3,6-anhydrogalactose and of its precursor unit (α-galactose 6-sulfate) were confirmed. The highly sulfated polysaccharide contained these ester groups on the C-2 and C-4 hydroxyl groups of the 3-linked unit and on C-3 of the 4-linked units. The large proportion of sulfate esterification in this polysaccharide can be related to the extracellular biosorption of copper divalent cation (2.5?±?0.4 mg per gram of dry weight) and to copper tolerance in bioassays.     

Biodegradation of the organochlorine insecticide, endosulfan, and the toxic metabolite, endosulfan sulfate, by Klebsiella oxytoca KE-8

Biodegradation of endosulfan, a chlorinated cyclodiene insecticide, is generally accompanied by production of the more toxic and more persistent metabolite, endosulfan sulfate. Since our reported endosulfan degrader, Klebsiella pneumoniae KE-1, failed to degrade endosulfan sulfate, we tried to isolate an endosulfan sulfate degrader from endosulfan-polluted soils. Through repetitive enrichment and successive subculture using mineral salt medium containing endosulfan or endosulfan sulfate as the sole source of carbon and energy, we isolated a bacterium capable of degrading endosulfan sulfate as well as endosulfan. The bacterium KE-8 was identified as Klebsiella oxytoca from the results of 16S rDNA sequence analysis. In biodegradation assays with KE-8 using mineral salt medium containing endosulfan (150 mg l^–1) or endosulfan sulfate (173 mg l^–1), the biomass was rapidly increased to an optical density at 550 nm of 1.9 in 4 days and the degradation constants for - and -endosulfan, and endosulfan sulfate were 0.3084, 0.2983 and 0.2465 day^–1, respectively. Analysis of the metabolites further suggested that K. oxytoca KE-8 has high potential as a biocatalyst for bioremediation of endosulfan and/or endosulfan sulfate.     

Effects of several metals on spore,biomass, and geosmin production byStreptomyces tendae andPenicillium expansum

Cultures ofStreptomyces tendae andPenicillium expansum grown on Actinomyces and Czapek's media, respectively, were exposed to 5 mg L^–1 of manganese, magnesium, iron, cobalt, nickel, copper and zinc, supplied as sulfate salts. Only copper markedly increased geosmin (1, 10-dimethyl-9-decalol), biomass, and spore production. inductively coupled plasma-atomic emission spectrometric analysis ofS. tendae andP. expansum cells did not indicate an accumulation of copper. Both 1 and 5 mg L^–1 copper, as copper sulfate, increased total geosmin production in cultures ofS. tendae on several media, but decreased production on others, suggesting that substrate composition affects responses to copper.     

Characterization of Coptis japonica cells with different alkaloid productivities

Several cell lines of Coptis japonica with different alkaloid productivities were characterized to obtain information on how a high metabolite production is established. High and low metabolite producing cells, except those from one cell line, showed similar growth kinetics and a similar pattern of nutrient uptake. Amino acid contents, especially that of tyrosine, differed between cell lines, but no correlation was found between the amino acid or tyrosine levels and alkaloid production. Since the addition of tyrosine did not increase the production of berberine, this primary substrate is apparently not the limiting factor for high production in cultured Coptis cells. The addition of berberine to the medium revealed that low-producing cells also have the ability to store alkaloid, and that low productivity is not due to decomposition of alkaloids which have been produced. The direct measurement of the biosynthesis of berberine using ¹⁴C-tyrosine clearly showed that high-producing cells had a higher biosynthetic activity of berberine from tyrosine than low-producing cells. The measurement of enzyme activities in berberine biosynthesis indicated that the early steps of berberine biosynthesis are important in the increased production of berberine.     

Lysosomal sulfate efflux following glycosaminoglycan degradation: measurements in enzyme-supplemented Maroteaux-Lamy syndrome fibroblasts and isolated lysosomes

Studies using lysosomal membrane vesicles have suggested that efflux of the sulfate that results from lysosomal glycosaminoglycan degradation is carrier-mediated. In this study, glycosaminoglycan degradation and sulfate efflux were examined using cultured skin fibroblasts and lysosomes deficient in the lysosomal enzymeN-acetylgalactosamine-4-sulfatase. Such fibroblasts store dermatan sulfate lysosomally, which could be labelled biosynthetically with Na ₂ ³⁵ SO₄. The addition of recombinantN-acetylgalactosamine-4-sulfatase to the media of³⁵S labelled fibroblasts degraded up to 82% of the stored dermatan [³⁵S] sulfate over a subsequent 96 h chase and released inorganic [³⁵S] sulfate into the medium. In the presence of 4-acetamido-4-isothiocyanatostilbene-2,2-disulfonic acid (SITS), sulfate was reused to a minor extent in newly synthesized proteoglycan. Isolated granules from recombinant enzyme supplemented fibroblasts degraded stored dermatan [³⁵S]sulfate to sulfate which was rapidly released into the medium at a rate that was reduced by the extra-lysosomal presence of the lysosomal sulfate transport inhibitors SITS, Na₂SO₄ and Na₂MoO₄. SITS also inhibited dermatan sulfate turnover, although it had no effect on the action of purified recombinant enzymein vitro. These data imply that sulfate clearance occurred concomitantly with dermatan sulfate turnover in the lysosome even at high substrate loading, and that lysosome-derived sulfate, while available, is reutilized minimally in synthetic pathways.Abbreviations SITS 4-acetamido-4-isothiocyanatostilbene-2,-2-disulfonic acid - GAG glycosaminoglycan - 4S N-acetylgalactosamine-4-sulfatase - r4S recombinant humanN-acetylgalactosamine-4-sulfatase - PBS phosphate buffered saline - BME basal modified Eagle's medium - FBS fetal bovine serum - GalNAc4S-GlcA-GalitolNAc4S -(N-acetyl-d-galactosamine-4-sulfate)-(1–4)--d-glucuronic acid)-(1–3)-N-acetyl-d-[1-³H]galactosaminitol-4-sulfate - DS dermatan sulfate - MPS mucopolysaccharidosis     

Inhibition of Photosystem II in the Green Alga Ankistrodesmus falcatus by Copper

Copper strongly inhibited 2,6-dichloroindophenol (DCIP) photoreduction in the broken cells of the green alga Ankistrodesmus falcatus (C303), and the activity lost could not be restored by adding 1,5-diphenylearbazide (DPC). Inactivation of the DCIP Hill reaction reached 45% after incubation with 10 μM cupric sulfate for 20 min. In the same time, copper (13 μg/mg chlorophyll) was bound to the broken cells. Addition of 10 mM KCl reduced copper binding by about 53%. Fluorescence intensity at room temperature decreased upon addition of cupric sulfate and was partially restored by adding 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU), These results suggest that copper inactivates electron transport between the oxidizing side of the reaction center of photosystem II and the electron-donating site of DPC. Further, the effect of light intensity shows that copper mostly affected the reaction rate of the dark step and had less inhibitory effect on the quantum efficiency of the primary reaction of electron transport in photosystem II.