The mechanism of arsenate inhibition of the glucose active transport system in Neurospora crassa.

The mechanism of arsenate inhibition of the glucose active transport system in wild-type cells of Neurospora crassa has been examined. Arsenate treatment results in approximately 65% inhibition of the glucose active transport system with only a small depression of cellular ATP levels. The transport system is not inhibited in cells treated with sodium arsenate in the presence of sodium azide. The transport inhibition is suppressed when orthophosphate is present during arsenate treatment, but is not reversed by orthophosphate when added after the arsenate treatment. The transport inhibition is completely reversed by treatment of the cells with mercaptoethanol. Gel chromatography of sonicates of intact cells which had been treated with [⁷⁴As]arsenate reveals three radioactive peaks, one with the elution volume of arsenate, one with the elution volume of arsenite, and a high molecular-weight radioactive fraction. Treatment of the high molecular-weight radioactive fraction with mercaptoethanol results in the production of radioactive arsenite. In view of these findings, it is proposed that arsenate inhibition of the glucose active transport system in Neurospora involves transport of arsenate into the cells, probably via the orthophosphate transport system, reduction of the transported arsenate to arsenite, and interaction of arsenite with some component of the glucose active transport system, presumably via covalent binding with vicinal thiol groups.     

Regulation of sulfate metabolism in Neurospora crassa: Transport and accumulation of glucose 6-sulfate

Neurospora crassa can utilize glucose 6-sulfate as its sole sulfur source, although this compound cannot serve as a carbon source for this organism. Neurospora possesses a transport system capable of glucose 6-sulfate uptake; the system is energy dependent, is inhibited by extracellular sulfate, and is clearly distinct from the permeases responsible for the uptake of glucose and those for sulfate transport. The metabolism of glucose 6-sulfate apparently involves its transport as an intact molecule, followed by a slow intracellular hydrolysis. Methionine, which represses the synthesis of a number of enzymes of sulfur anabolism, also represses the synthesis of the transport system responsible for glucose 6-sulfate uptake. A regulatory gene, cys-3, which controls the synthesis of aryl sulfatase, choline sulfatase, choline-O-sulfate permease, and two distinct permease species, also regulates the permease for glucose 6-sulfate.     

Endocytic uptake of Escherichia coli spheroplasts by Neurospora crassa slime cells

Summary Interaction of Escherichia coli spheroplasts with Neurospora crassa slime cells was examined by transmission electron microscopy after treatment with polyvinyl alcohol followed by dilution with the high pH-high Ca buffer. Bacterial spheroplasts were found either adhering to the flat surface, associating with the invaginating surface, or residing within the intracellular vesicle of fungal protoplasts. In addition, bacterial spheroplasts free of the surrounding vesicles and those in the course of breakdown were observed in the fungal cytoplasm. It was concluded that Escherichia coli spheroplasts are taken up by Neurospora crassa protoplasts almost exclusively via endocytosis. This is the first cytological evidence for the endocytic activity of fungal cells.     

Reconstitution of binding protein-dependent ribose transport in spheroplasts of Escherichia coli K-12.

Purified Escherichia coli K-12 ribose binding protein was used to reconstitute the high affinity ribose transport system in spheroplasts derived from ribose-induced cells. It was not possible to reconstitute ribose transport in spheroplasts derived from uninduced cells or from transport-negative mutant strains, suggesting that one or more additional inducible components are required for binding protein-dependent ribose transport. It was possible to reconstitute transport in a ribokinase-deficient mutant which constitutively transports but does not utilize ribose.     

Genetic and metabolic control of sulfate metabolism in Neurospora crassa: A specific permease for choline-O-sulfate

Neurospora crassa can use choline-O-sulfate as its sole sulfur source; the utilization of this compound involves its entry followed by intracellular hydrolysis. Neurospora possesses a transport system for the uptake of choline-O-sulfate which is specific for the sulfate ester and does not transport, nor is it inhibited by, either choline or inorganic sulfate. Mutant strains of Neurospora that are unable to transport or grow on inorganic sulfate can, nevertheless, utilize choline-O-sulfate for growth and transport the intact organic sulfate at a normal rate. Methionine, which represses a number of enzymes of sulfur anabolism, also represses the synthesis of the specific permease for choline-O-sulfate. A regulatory gene, cys-3, which controls the synthesis of choline sulfatase, aryl sulfatase, and several other related enzymes, also regulates the synthesis of the choline sulfate permease. Evidence is presented that the activity of choline sulfate permease is also regulated by a turnover process, the transport system having a functional half-life of approximately 3 hr.This investigation was supported by Public Health Service Grant 1 RO1 GM-18642 from the National Institute of General Medical Services.     

Morphology of slime variants of Neurospora crassa growing on a glass surface in liquid medium

Two Neurospora crassa strains designated slime, FGSC no. 1118 and 326, were observed during their growth on glass surfaces in liquid medium by phase contrast microscopy, and following fixation, dehydration and critical point drying, by scanning electron microscopy. Both strains were found to grow by several processes:

1. Production of microhyphae with terminal bulbs which often contained nuclei and could change into spheroplasts once the subapical microhyphal stalk was severed.
2. Amoeboid movement of large flattened spheroplasts extended on the glass surface like a pancake, followed by pinching off of beaded spheroplasts from extended flattened spheroplasts. A number of different drugs caused rapid bead formation from flattened spheroplasts. These included inhibitors known to cause disaggreation of cytoplasmic microfilaments such as cytochalasins A and B, inhibitors of respiration and oxidative phosphorylation such as azide, cyanide, dinitrophenol, phenazinemethosulfate, antimycin A, and arsenate, and ion carriers and ions such as carbonyl cyanide m-chlorophenylhydrazone, potassium chloride (0.15 M), ethylenediaminetetraacetate, and sodium pyrophosphate. Similar effects were observed after application of reagents affecting cyclic nucleotides such as dibutyryl cyclic adenosine-3:5-monophosphate and caffeine, and sulfhydryl reagents such as N-ethylmaleimide, iodoacetate, p-chloromercuribenzoate.Cyanide treatment resulted in a biphasic response: initially bead formation, followed by retraction of protoplasm of peripheral beads into a central large spheroplast. The second phase may require energy from glycolysis since it was inhibited by fluoride. The pretreatment of spheroplasts by concanavalin A prevented this change in every case, probably because of a cross-linking of membrane components. The rapid contraction and movement of these cells is consistent with the presence of contractile proteins similar to actin and myosin in Slime and suggests a search for such proteins in Neurospora. During growth of slime 1118, wall-like material accumulated in the medium. Such material was isolated and found to contain protein (51.5%); polysaccharides appearedto be the major form-giving component. This dried powdered wall-like material also contained 11–15 μg of lipid phosphorus per mg. A 10 min 3/4 inch television cassette offering time lapse photographic sequences of the processes described is available from the authors.

     

A rapid and efficient method for the purification of tyrosinase from Neurospora.

A short and efficient procedure consisting of two chromatographic steps is described for the isolation of tyrosinase from Neurospora. The first step, Celite-column chromatography, resulted in the isolation of four copper-containing proteins from the crude mycelial extract. Anion-exchange chromatography on DEAE-Sephadex of these proteins resulted in the isolation of electrophoretically and serologically pure tyrosinase. More than 70% of the initial tyrosinase activity was recovered in the final enzyme preparation, which had a specific activity of 450 units/mg.     

Specificity and regulation of peptide transport on Neurospora crassa.

The tripeptide, glycyl-d,l-leucyl-l-tyrosine was chemically synthesized in radioactive form and used to directly study the specificity, regulation, and properties of an oligopeptide transport system in Neurospora. Transport activity is sensitive to azide but does not result in the accumulation of the intact peptide; rather, the radioactive label is accumulated as free tyrosine. Inhibition studies suggest that the transport system probably has a relatively wide range of specificity and is responsible for uptake of short oligopeptides of quite distinct sequences. However, free amino acids and dipeptides are not transported significantly, if at all, by the oligopeptide transport system. A free amino group appears to be a requirement for peptide transport. A mutant strain that is unable to use various peptides for growth is further described and shown to be reduced greater than 90% in transport of the tripeptide.     

Efflux of potassium ions from cells and spheroplasts of <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis> yeast treated with silver and copper ions

Silver ions induce the efflux of potassium from cells of the yeast Saccharomyces cerevisiae but have no such effect on spheroplasts. Copper ions and the natural fungicide 2-O-3-hydroxyhexanoyl-β-D-glucopyranosyl-(1→4)-(6-O-acetyl-β-D-glucopyranosyl-(1→16)-2,15,16-trihydroxyhexadecanoic acid) induce the efflux of potassium ions from both cells and spheroplasts of S. cerevisiae. Silver and copper ions inhibit the activity of the plasma membrane H⁺-ATPase during the treatment of both cells and spheroplasts. It is supposed that the inability of silver ions to stimulate potassium efflux from spheroplasts results from damage to some components of K⁺ transport systems during preparation of spheroplasts.     

Nitrate transport system in Neurospora crassa

Nitrate uptake in Neurospora crassa has been investigated under various conditions of nitrogen nutrition by measuring the rate of disappearance of nitrate from the medium and by determining mycelial nitrate accumulation. The nitrate transport system is induced by either nitrate or nitrite, but is not present in mycelia grown on ammonia or Casamino Acids. The appearance of nitrate uptake activity is prevented by cycloheximide, puromycin, or 6-methyl purine. The induced nitrate transport system displays a K_m for nitrate of 0.25 mM. Nitrate uptake is inhibited by metabolic poisons such as 2,4-dinitrophenol, cyanide, and antimycin A. Furthermore, mycelia can concentrate nitrate 50-fold. Ammonia and nitrite are non-competitive inhibitors with respect to nitrate, with K_i values of 0.13 and 0.17 mM, respectively. Ammonia does not repress the formation of the nitrate transport system. In contrast, the nitrate uptake system is repressed by Casamino Acids. All amino acids individually prevent nitrate accumulation, with the exception of methionine, glutamine, and alanine. The influence of nitrate reduction and the nitrate reductase protein on nitrate transport was investigated in wild-type Neurospora lacking a functional nitrate reductase and in nitrate non-utilizing mutants, nit-1, nit-2, and nit-3. These mycelia contain an inducible nitrate transport system which displays the same characteristics as those found in the wild-type mycelia having the functional nitrate reductase. These findings suggest that nitrate transport is not dependent upon nitrate reduction and that these two processes are separate events in the assimilation of nitrate.     

Glucose transport in membrane vesicles from Azotobacter vinelandii,: Properties of the glucose carrierin situ

The active transport of d-glucose by membrane vesicles prepared from Azotobactervinelandii strain O is coupled to the oxidation of l-malate. The glucose carrier, but not the energy coupling system of the vesicles, is induced by growth of the cells on d-glucose medium. Vesicles isolated from A. vinelandii grown in the presence of sucrose or acetate accumulate glucose at less than 7% of the rate observed for vesicles from glucose-grown cells. Nevertheless, vesicles from sucrose- or acetate-grown cells transport sucrose or calcium, respectively, in the presence of malate.The transport system expressed in vesicles from glucose-cultured cells is highly specific for d-glucose. Studies of glucose analog uptake and of the competitive effect of analogs reveal that: (i) The glucose carrier is stereospecific. (ii) The affinity of hexoses for the transport system is inversely related to the bulk of substituents on the pyranose ring, especially at the C-1 and C-2 positions, (iii) The most effective competitors, 6-deoxyglucose and 2-deoxyglucose, exhibit affinities only 10–20% that of d-glucose for the transport system, (iv) Phloretin, but not phlorizin, is a competitive inhibitor of glucose transport, having an apparent K_i of 9 μm at pH 7.0. These latter findings suggest a similarity of the glucose transport system of fxA. vinelandii and those of eukaryotes with regard to the glucose carrier.     

Role of periplasmic trehalase in uptake of trehalose by the thermophilic bacterium Rhodothermus marinus

Trehalose uptake at 65°C in Rhodothermus marinus was characterized. The profile of trehalose uptake as a function of concentration showed two distinct types of saturation kinetics, and the analysis of the data was complicated by the activity of a periplasmic trehalase. The kinetic parameters of this enzyme determined in whole cells were as follows: K_m = 156 ± 11 μM and V_max = 21.2 ± 0.4 nmol/min/mg of total protein. Therefore, trehalose could be acted upon by this periplasmic activity, yielding glucose that subsequently entered the cell via the glucose uptake system, which was also characterized. To distinguish the several contributions in this intricate system, a mathematical model was developed that took into account the experimental kinetic parameters for trehalase, trehalose transport, glucose transport, competition data with trehalose, glucose, and palatinose, and measurements of glucose diffusion out of the periplasm. It was concluded that R. marinus has distinct transport systems for trehalose and glucose; moreover, the experimental data fit perfectly with a model considering a high-affinity, low-capacity transport system for trehalose (K_m = 0.11 ± 0.03 μM and V_max = 0.39 ± 0.02 nmol/min/mg of protein) and a glucose transporter with moderate affinity and capacity (K_m = 46 ± 3 μM and V_max = 48 ± 1 nmol/min/mg of protein). The contribution of the trehalose transporter is important only in trehalose-poor environments (trehalose concentrations up to 6 μM); at higher concentrations trehalose is assimilated primarily via trehalase and the glucose transport system. Trehalose uptake was constitutive, but the activity decreased 60% in response to osmotic stress. The nature of the trehalose transporter and the physiological relevance of these findings are discussed.     

Stability of the Plasma Membrane in Saccharomyces rouxii and Its Relationship to Glucose Tolerance

The stability of spheroplasts from the osmotrophic yeast Saccharomyces rouxii was studied in buffered solutions of mannitol and glucose. The plasma membranes from cells grown in high glucose concentrations were more stable to osmotic lysis than were membranes from cells grown in lower glucose concentrations. Mannitol was a better osmotic stabilizer than glucose, except when the cells were grown in a high glucose concentration. Spheroplasts from a glucose tolerant-deficient mutant were much less stable than the corresponding spheroplasts from the parent strain, especially when suspended in glucose solutions. These results suggest an involvement of the plasma membrane in the glucose-tolerant mechanism of S. rouxii.     

Organization of the glycolytic enzymes in Escherichia coli

Differential centrifugation of osmotically lysed lysozyme-EDTA spheroplasts from Escherichia coli sedimented 50–70% of the glycolytic activities examined in a low speed pellet; the remaining activity, occurring in a high speed supernatant, contained the soluble enzymes of the cell. The distribution pattern of the enzymes could be altered by extrusion of the spheroplasts through the French Press or by lysis at different pH values. Electron micrographs of the pellet fraction revealed lysed spheroplasts mostly devoid of cellular constituents but consisting of cytoplasmic membranes surrounded by partially degraded cell wall fragments. Washing of the pellet showed that the enzymes were not all bound to the same degree to the membrane fraction. Throughput activity of the glycolytic pathway was demonstrated for the membrane fraction, but none was observed for the soluble fraction of the cell (i.e. for enzymes present in the supernatants) unless these were first concentrated by ultrafiltration. The supernatant from the lysed spheroplasts, together with a further supernatant obtained by washing the membrane pellet, was concentrated by ultrafiltration and chromatographed on a Bio-Gel column. The eluate contained glycolytic activities both in fractions corresponding to relatively high and relatively low molecular weight material The high molecular weight species, containing a proportion of all the enzymes studied, had a molecular weight of at least 1.2 × 10⁶. A multienzyme aggregate containing one each of the glycolytic enzymes would have a molecular weight of ～ 1.3 × 10⁶. The specific rate of pyruvate formation from glucose by the high molecular weight species was similar to that obtained from a preparation in which the fractions containing all the low molecular weight material enzyme activities were pooled and concentrated by ultrafiltration. Using the high molecular weight material, studies were made of the ability of added unlabelled glycolytic intermediates to compete for catalytic sites with intermediates produced endogenously from [¹⁴C₆] glucose. The relatively weak competition observed indicated a high degree of protection afforded the labelled intermediates derived from [¹⁴C₆] glucose.     

Selection and characterization of chlorella mutants deficient in amino Acid transport : further evidence for three independent systems

Six amino acids are transported at high rates across the plasmalemma of Chlorella vulgaris only after the induction of two specific transport systems. Induction is achieved either by pretreatment with glucose, glucose analogs, or by nitrogen starvation. Mutants for these transport systems were obtained after incubation of Chlorella cells in the presence of acridine orange or ethidium bromide, followed by a selection procedure using the toxic amino acid analogs l-canavanine (for l-arginine), and l-azetidine-2-carboxylic acid (for l-proline). Mutants isolated by this method had lost their ability to induce the corresponding transport system. Double mutants deficient in transport of both these amino acids still possess the general amino acid transport system, a third system which was described previously. Evidence for additional amino acid transport systems in Chlorella is discussed.     

Glucose Transport in Escherichia coli Mutant Strains with Defects in Sugar Transport Systems

In Escherichia coli, several systems are known to transport glucose into the cytoplasm. The main glucose uptake system under batch conditions is the glucose phosphoenolpyruvate:carbohydrate phosphotransferase system (glucose PTS), but the mannose PTS and the galactose and maltose transporters also can translocate glucose. Mutant strains which lack the enzyme IIBC (EIIBC) protein of the glucose PTS have been investigated previously because their lower rate of acetate formation offers advantages in industrial applications. Nevertheless, a systematic study to analyze the impact of the different glucose uptake systems has not been undertaken. Specifically, how the bacteria cope with the deletion of the major glucose uptake system and which alternative transporters react to compensate for this deficit have not been studied in detail. Therefore, a series of mutant strains were analyzed in aerobic and anaerobic batch cultures, as well as glucose-limited continuous cultivations. Deletion of EIIBC disturbs glucose transport severely in batch cultures; cyclic AMP (cAMP)-cAMP receptor protein (CRP) levels rise, and induction of the mgl operon occurs. Nevertheless, Mgl activity is not essential for growth of these mutants, since deletion of this transporter did not affect the growth rate; the activities of the remaining transporters seem to be sufficient. Under conditions of glucose limitation, mgl is upregulated 23-fold compared to levels for growth under glucose excess. Despite the strong induction of mgl upon glucose limitation, deletion of this transport system did not lead to further changes. Although the galactose transporters are often regarded as important for glucose uptake at micromolar concentrations, the glucose as well as mannose PTS might be sufficient for growth at this relatively low dilution rate.     

Carbohydrate Catabolism in Azospirillum amazonense

The nitrogen fixer Azospirillum amazonense grew on the various disaccharides, hexoses, and pentoses tested in this study but not on polyols and on some tricarboxylic acid cycle intermediates. An active transport system was detected for sucrose and glucose but not for mannitol and 2-ketoglutarate. Six A. amazonense strains were examined for 16 carbon-metabolizing enzymes, and the results indicate that these strains employ the Entner-Doudoroff pathway to catabolize sucrose, fructose, and glucose. The hexose monophosphate and Embden-Meyerhof-Parnas pathways were not detectable.     

Role of quinones in electron transport to oxygen and nitrate in Escherichia coli. Studies with a ubiA− menA− double quinone mutant

A ubiA^? menA^? double quinone mutant of Escherichia coli K12 was constructed together with other isogenic strains lacking either ubiquinone or menaquinone. These strains were used to study the role of quinones in electron transport to oxygen and nitrate. Each of the four oxidases examined (NADH, d-lactate, α-glycerophosphate and succinate) required a quinone for activity. Ubiquinone was active in each oxidase system while menaquinone gave full activity in α-glycerophosphate oxidase, partial activity in d-lactate oxidase but was inactive in NADH and succinate oxidation. The aerobic growth rates, growth yields and products of glucose metabolism of the quinone-deficient strains were also examined. The growth rate and growth yield of the ubi⁺ menA^? strain was the same as the wild-type strain, whereas the ubiA^? men⁺ strain grew more slowly on glucose, had a lower growth yield (30% of wild type) and accumulated relatively large quantities of acetate and lactate. The growth of the ubiA^? menA^? strain was even more severely affected than that of the ubiA^? men⁺ strain.Electron transport from formate, d-lactate, α-glycerophosphate and NADH to nitrate was also highly dependent on the presence of a quinone. Either ubiquinone or menaquinone was active in electron transport from formate and the activity of the quinones in electron transport from the other substrates was the same as for the oxidase systems. In contrast, quinones were not obligatory carriers in the anaerobic formate hydrogenlyase system. It is concluded that the quinones serve to link the various dehydrogenases with the terminal electron transport systems to oxygen and nitrate and that the dehydrogenases possess a degree of selectivity with respect to the quinone acceptors.     

Ubiquitin Ligase Components Cullin4 and DDB1 Are Essential for DNA Methylation in Neurospora crassa

DNA methylation and H3K9 trimethylation are involved in gene silencing and heterochromatin assembly in mammals and fungi. In the filamentous fungus Neurospora crassa, it has been demonstrated that H3K9 trimethylation catalyzed by histone methyltransferase DIM-5 is essential for DNA methylation. Trimethylated H3K9 is recognized by HP1, which then recruits the DNA methyltransferase DIM-2 to methylate the DNA. Here, we show that in Neurospora, ubiquitin ligase components Cullin4 and DDB1 are essential for DNA methylation. These proteins regulate DNA methylation through their effects on the trimethylation of histone H3K9. In addition, we showed that the E3 ligase activity of the Cul4-based ubiquitin ligase is required for its function in histone H3K9 trimethylation in Neurospora. Furthermore, we demonstrated that Cul4 and DDB1 are associated with the histone methyltransferase DIM-5 protein in vivo. Together, these results suggest a mechanism for DNA methylation control that may be applicable in other eukaryotic organisms.