- Volume 130, Issue 12, 1984
Volume 130, Issue 12, 1984
- Biochemistry
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Purification and Characterization of an Extracellular and a Cellular a-Glucosidase from Bacillus licheniformis
More Lessα-Glucosidase has been purified from culture fluid and from lysed cells of Bacillus licheniformis NCIB 6346. The enzymes from these two sources were virtually identical in molecular weight as judged by SDS-PAGE (63000) and catalytic properties. The enzymes were unstable at high temperature and lost all activity after incubation at 60°C for 10 min. Of the substrates examined, isomaltose gave maximal activity, followed by maltotriose, p-nitrophenyl α-d-glucopyranoside, sucrose and maltose. With isomaltose or maltotriose as substrate, transglucosylation activity was evident.
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Hyperproduction of Exocellular Levansucrase by Bacillus subtilis: Examination of the Phenotype of a sacUh Strain
More LessThe induction parameters of levansucrase synthesis were the same in Bacillus subtilis strain 168 Marburg and in a derivative, hyperproducing (sacU h) strain. However, only the hyperproducing strain showed an induction lag period. The kinetics of appearance of functional levansucrase mRNA was established. Strain 168 did not release levansucrase, but washing the cells with high ionic strength buffer released different proteins of which levansucrase represented 2%. In contrast, the great majority of levansucrase synthesized by the hyperproducer was released in a homogeneous form into the culture medium. In this case high ionic strength treatment caused the cells to release the remaining levansucrase but not other proteins. A Triton X-100 sensitive form of levansucrase was isolated by phenol treatment of the sacU h strain; this form was absent in strain 168. We suggest that the sacU gene product possibly controls the synthesis of cellular components with which levansucrase is associated and thus its release is normally prevented.
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Purification and Properties of Glycerol Dehydrogenase from Candida valida
More LessGlycerol : NAD+ 2-oxidoreductase (EC 1.1.1.6) was purified to homogeneity from the non-methylotrophic yeast Candida valida H 122. Results of electrophoresis in polyacrylamide gels, gel filtration and ultracentrifugation were compatible with the enzyme’s consisting of two subunits with a molecular weight 38000. No heterogeneity was observed by isoelectric focusing. The pH optima were10·0 for glycerol oxidation and 7·5 for dihydroxyacetone reduction. The K m values for glycerol, dihydroxyacetone, NAD+ and NADH were 5·8 10−2 m, 7·7 10−4 m, 1·4 10−4 m and 4·8 10−5 m, respectively. 1,2-Propanediol also served as substrate in the forward and dl-glyceraldehyde in the reverse reaction. The oxidation product of glycerol was identified as dihydroxyacetone. An ordered bi-bi mechanism was deduced from product-inhibition studies. Of several anions and cations tested, pyrophosphate and ammonium ions stimulated the dehydrogenating activity the most. 2-Mercaptoethanol, ethylene glycol and Tris inhibited activity; an inhibition was also observed by phosphorylated coenzymes and substrates. The purified enzyme, which is labile at low concentrations, was stabilized by the addition of neutralized filtrate from the culture liquid.
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Phosphofructokinase and the Regulation of the Flux of Carbon from Glucose to Lipid in the Oleaginous Yeast Rhodosporidium toruloides
More LessChanges in cell composition of Rhodosporidium toruloides CBS 14 were monitored during growth of batch cultures with NH4Cl and glutamate as nitrogen sources. Carbohydrate was synthesized at the expense of lipid in NH4 +-grown cells, whereas in glutamate-grown cells lipid accumulation was predominant. Total biomass and protein concentration were similar in both cultures. Uptake of [U-14C], [1-14C] and [6-14C]glucose, and evolution of 14CO2 from these sources, by washed suspensions of cells grown on glutamate revealed the flux of carbon during glucose dissimilation was principally via the Embden-Meyerhof pathway (72%), with 28% being metabolized by the pentose phosphate pathway. Both urea and glutamate, when added to the cell suspensions, significantly stimulated glucose catabolism, with the flux of carbon via the former pathway increasing to about 89% of the total. Phosphofructokinase (PFK) was implicated as the likely controlling enzyme to explain these events.
PFK was only detected in extracts prepared from the yeast grown in a carbon-limited (nitrogen-excess) medium; no activity was detected in extracts of cells grown in nitrogen-limited medium. The presence of a protease in these latter extracts was revealed. PFK was purified 92-fold to a final specific activity (in the presence of 10 mm-NH4 +) of 4·2 units (mg protein)−1 and exhibited one broad band on polyacrylamide gel electrophoresis. The apparent mol. wt (mt ) of the enzyme was approx. 700000. The major properties of the enzyme were examined to determine its regulatory role during lipid biosynthesis. Unlike the enzyme from Saccharomyces cerevisiae, no inhibition was found with 10 mm-ATP. ADP was not inhibitory either. NH4 + ions increased activity 11-fold by increasing the affinity of the enzyme for both fructose 6-phosphate and ATP. K+ ions also stimulated activity but to a lesser extent. Activity was severely inhibited by citrate, isocitrate and cis-aconitate but this inhibition was dramatically alleviated by NH4 +. Inhibition by citrate was competitive with fructose 6-phosphate in the absence of NH4 + ions. The K i values for citrate were 1·0 mm (with no NH4 +) and 7·2 mm (with 10 mm-NH4 +). Long-chain fatty acyl-CoA esters had no significant inhibitory effect. It is concluded that the interplay between the prevailing intracellular concentrations of NH4 + and citrate is the major determinant of the activity of PFK in vivo and thus governs the extent to which glucose is converted either to lipid or carbohydrate.
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Production of Extracellular Glutathione by Candida tropicalis Pk 233
More LessCandida tropicalis Pk 233 produced extracellular glutathione during growth in filamentous form caused by adding ethanol (⩾ 2·5%, v/v). The intracellular concentration of glutathione also was greater in cultures with added ethanol. myo-Inositol added at a physiological concentration (5 µg ml−1) prevented the ethanol-induced production of extracellular glutathione as well as the morphological change. In batch culture, production of extracellular glutathione was optimal at an ethanol concentration of 5%, and reached a maximum concentration of 42 mg ml−1 after 96 h cultivation, before decreasing gradually.
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ATP and GTP Stimulate Membrane-bound but not Digitonin-solubilized β-Glucan Synthases from Saprolegnia monoica
More LessGlucan synthases of Saprolegnia assayed in the presence of MgCl2 [(1→4)-β-glucan production] were stimulated by ATP or GTP, while the non-phosphorylating nucleotide analogues adenylyl (β,γ-methylene)diphosphonate and guanylyl (·γ-methylene)diphosphonate did not stimulate glucan synthesis. The analogues adenosine 5′-O-(3-thiotriphosphate) and guanosine 5′-O-(3-thiotriphosphate) stimulated the enzyme activity but to a lesser extent than the nucleotides. The activators stimulated membrane-bound enzymes, particularly those equilibrating at the endoplasmic reticulum density in sucrose gradients. Glucan synthases solubilized by digitonin were not stimulated by nucleotides. Nucleotides, whose action seems to be related to the integrity of the phosphate groups, may produce their stimulative effect by increasing the translocation of the substrate through membranes to the enzymes.
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Analysis of Wall Glucans from Yeast, Hyphal and Germ-tube Forming Cells of Candida albicans
More LessAcid-soluble and alkali-insoluble glucan fractions were prepared from yeast, hyphal and germ-tube forming cells of Candida albicans. Alkali-insoluble glucan was also extracted from purified yeast cell walls. Paper chromatography of partial acid hydrolysates confirmed that the glucan preparations contained β(1→3)-and β(1→6)-chains but no mixed intra-chain β(1→3)/β(1→6) linkages. Methylation and 13C-NMR analyses showed that the acid-soluble glucan consisted of a highly branched polymer composed mainly (67·0% to 76·6%) of β(1→6)-linked glucose residues. The alkali-insoluble glucan from yeast and hyphal cells contained from 29·6% to 38·9% β(1→3) and 43·3% to 53·2% (1→6) linkages. Alkali-insoluble glucan from germ-tube forming cells consisted of 67·0% β(1→3) and 14% β(1→6) linkages. Branch points accounted for 6·7%, 12·3% and 17·4% of the residues in the alkali-insoluble glucan of yeast, germ-tube forming and hyphal cells, respectively.
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The Action of 2-Deoxy-d-glucose on the Incorporation of Glucose into (1→3)-β-Glucan in Stationary Phase Cultures of Candida albicans
More Less2-Deoxy-d-glucose added to cultures of Candida albicans in the stationary phase of growth inhibited the incorporation of glucose into the (1→3)-β-glucan fraction of the organisms. In the presence of ATP and cell extracts it was converted to 2-deoxy-d-glucose phosphate and when UTP was also present, material with the electrophoretic properties of UDP-2-deoxy-d-glucose was formed. In similar conditions glucose formed glucose phosphates, UDP-glucose and other products. Evidence was obtained that the analogue, after conversion to a phosphate derivative, was an inhibitor of phosphoglucomutase.
When C. albicans was grown in the presence of 2-deoxy-d-glucose for 24 h, analogue residues became incorporated into the (1→3)-β-glucan fraction and the subsequent rate of incorporation of glucose into that fraction was enhanced. The rate of turnover of glucose in this β-glucan fraction was greater than in controls. Pretreatment of cultures with β-glucanase, or incubation under conditions known to stimulate endogenous β-glucanases, increased the subsequent rate of glucose incorporation and this increase was enhanced by growth in the presence of 2-deoxy-d-glucose. The analogue thus had the effect of altering the stability and glucose-acceptor function of (1→3)-β-glucan chains. This could affect the properties of the polymer network leading to the known effect of the analogue in delaying the onset of phenotypic resistance to amphotericin methyl ester in stationary phase cultures of C. albicans.
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The Roles of Cytochrome c in Membranes of Methylophilus methylotrophus
More LessThe soluble and membrane-bound cytochromes c of methylotrophic bacteria were investigated in order to determine their relationships with each other, and their roles in electron transport. The proportions of soluble cytochromes cH and cL in various methylotrophs were not markedly dependent on the type of methylotroph nor on conditions of growth. Between 30 and 50% of the cytochrome c of Methylophilus methqdotrophus was bound tightly to membranes; 8% of this was cytochrome cH, 37% was cytochrome cLand 55% was the cytochrome c component of the oxidase, cytochrome co. These cytochromes were purified and characterized. It was concluded that cytochrome cH has no role in the membrane. By contrast, the membrane cytochrome cL has a separate role from that of soluble cytochrome cL; the membrane-bound cytochrome cL may play a role analogous to that of mitochondria1 cytochrome c1 in mediating between cytochrome b and the terminal oxidase during the oxidation of NADH.
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- Development And Structure
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Morphogenetic Effects of Mutations at the A and B Incompatibility Factors in Coprinus cinereus
More LessMutated A and B incompatibility factors of Coprinus cinereus (Amut and Bmut) were recovered from fruit bodies produced on common-A and common-B heterokaryons, respectively, following mutagenesis. The Amut hyphal cells were either uninucleate or binucleate and had pseudo-clamps irregularly scattered along the hyphae. The Bmut hyphal cells were predominantly uninucleate and had no clamp structures. Amut Bmut double mutants constructed from these Amut and Bmut strains were predominantly binucleate, had true clamps, and gave rise to fertile fruit bodies indistinguishable from those of wild-type dikaryons. Although these Amut Bmut strains resembled in most respects a normal dikaryon, they produced abundant oidiophores and oidia like the monokaryons. The oidia were uninucleate, and possessed the potential to grow into fertile homokaryons with the above characteristics.
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- Ecology
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A Comparison of the Growth Characteristics and Spatial Distribution of Hypolimnetic Ciliates in a Small Lake and an Artificial Lake Ecosystem
More LessWithin the anaerobic hypolimnion of a small eutrophic lake, a well defined and stable community of large ciliates was present throughout the period of summer stratification. Peak ciliate densities occurred at the oxic/anoxic interface; ciliate numbers then declined in an approximately exponential manner with increasing depth. Two thermally stratified 2 m high artificial lake ecosystems were constructed in which an anaerobic hypolimnion developed. Within these model lakes, populations of hypolimnetic ciliates were maintained for many months. Community structure, growth characteristics and spatial distribution of the ciliates were similar to those in the natural environment.
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Transhyphal Electrical Currents in Fungi
More LessRepresentative mycelial fungi from the phycomycete, ascomycete and basidiomycete groups (Achlya bisexualis, Neurospora crassa, Aspergillus nidulans, Schizophyllum commune and Coprinus cinereus) all generated steady electrical currents around their hyphal tips; the generation of a transhyphal ion current may therefore be a universal characteristic of hyphal growth. As with all other tip growing organisms, positive current always entered apically and left distally; non-growing hyphae did not drive transcellular currents. The current density, measured approximately 30 m from the membrane surface at the hyphal tips, varied between 0·05 and 0·60 μA cm−2 in different fungi and tended to be larger in wider, rapidly extending hyphae than in thinner, slow growing hyphae. The possibility that these currents serve to localize growth at the apex is discussed.
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- Genetics And Molecular Biology
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Nitrogen Regulation of Synthesis of the High Affinity Methylammonium Transport System of Escherichia coli
More LessUptake of 14CH3 NH+ 3 (methylammonium) was measured as a probe of NH+ 4 transport in intact Escherichia coli cells and derivatives impaired in the Ntr regulatory system. The results suggest that expression of the high affinity 14CH3 NH+ 3 transport system (a) requires de novo polypeptide synthesis, (b) is activated the glnG and glnF regulatory products under nitrogen limitation, and (c) is repressed under nitrogen excess by the glnL product. Cells deficient in glutamate synthase activity by virtue of their harbouring the gltB31 mutation were unable to activate synthesis of 14CH3NH+ 3 transport. This could explain the inability of cells carrying gltB mutations to grow on low concentrations of NH+ 4.
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Complementation Analysis of the Aliphatic Amidase Genes of Pseudomonas aeruginosa
More LessA plasmid, pCL34, capable of autonomous replication in Escherichia coli and Pseudomonas aeruginosa has been constructed which carries the promoter and structural gene (amiE) for P. aeruginosa amidase, but not the regulator gene (amiR). Plasmid pCL34 has been mobilized from E. coli to P. aeruginosa using the broad host range plasmid RP4. Complementation studies were performed in P. aeruginosa strains carrying various amidase mutations. Measurements of amidase activity in the recipients under inducing, non-inducing and repressing conditions showed trans-complementation by the chromosomally located regulator gene product. These results confirmed the positive control model for amidase gene expression. Levels of amidase expression seen during these studies were approximately threefold higher than in the parental, amidase-positive strains.
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Two-dimensional Gel Electrophoretic Separation of the Proteins Present in Chromatin of Escherichia coli
More LessThe polypeptides present in 35S-labelled chromatin prepared from Escherichia coli cells, and polypeptides present in the DNA and RNA complexes obtained by micrococcal nuclease digestion of the chromatin, were analysed by two-dimensional non-equilibrium polyacrylamide gel electrophoresis. Three hundred and thirty-five 35S-labelled polypeptides were detected in the chromatin whereas the DNA- and RNA-containing fractions of the micrococcal nuclease digest contained 126 and 183 polypeptides respectively. The major basic low-molecular-weight polypeptides were found in the DNA-containing fractions.
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Analysis and Detection of Chlamydial DNA
More LessElementary bodies of lymphogranuloma venereum (LGV) strains of Chlamydia trachomatis contain, in addition to the genomic DNA, a 6·7 kb plasmid. The plasmid from serovar L2 (434-B) was cloned at the BamHI site of pBR327 into Escherichia coli and a restriction cleavage map of this pLGV125 recombinant plasmid determined. All 15 C. trachomatis serovars contained DNA sequences that hybridized with pLGV125. When total DNA from L2 elementary bodies was used as a probe in Southern blotting and spot hybridization, serovars L1, L2 and L3 exhibited significant homology. The detection level of homologous DNA was 100 pg and LGV DNA was detectable in infected cells when total L2 probe was used in the nucleic acid hybridization test. These DNA probes may be useful as investigative and diagnostic reagents for C. trachomatis.
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On the Role of Pili in Transformation of Neisseria gonorrhoeae
More LessTransformation of competent transformable Neisseria gonorrhoeae F62 to streptomycin resistance was unaffected by antibodies directed against the pilus protein (pilin) of this organism. The pilin component of either crude or purified pilus preparations, separated by SDS gel electrophoresis and transferred to nitrocellulose, failed to bind detectable amounts of DNA; DNA binding to other gonococcal polypeptides was observed under these conditions. These results suggest that gonococcal pilin does not play a direct role in gonococcal transformation.
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Canavanine Resistance and the Mechanism of Arginine Uptake in the Fission Yeast Schizosaccharomyces pombe
More LessThe mechanism of resistance to the arginine analogue l-canavanine, and of arginine uptake, were examined in the fission yeast Schizosaccharomyces pombe. Two mutants with increased resistance to canavanine were analysed genetically: both were double mutants, and in each case one mutation conferred resistance to canavanine, while the other enhanced this resistance. Evidence is presented that can1.1 strains are defective in one system for arginine uptake, which presumably prevents entry of canavanine into the cell. This system operates in the wild-type whether the nitrogen source supplied is ammonium or glutamate. Double mutants carrying can1.1 and an arginine requirement are unable to grow on ammonium medium even when supplied with exogenous argine, while growth can occur on glutamate plus arginine. This suggested the existence of a second uptake system for arginine which is absent during growth on ammonium, and direct measurements of the rates of arginine uptake under various conditions confirmed this. Our observations closely parallel those made on the budding yeast Saccharomyces cerevisiae. The ability to select for or against function of the can1 gene should facilitate certain types of genetical analysis in S. pombe.
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- Physiology And Growth
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Nitrogenase Synthesis in Klebsiella pneumoniae: Enhanced nif Expression without Accumulation of Guanosine 5′-Diphosphate 3′-Diphosphate
More LessDerepression of nitrogen fixation (nif) genes in Klebsiella pneumoniae following transfer from NH+ 4-sufficiency to N-free medium was preceded rapid expansion of the guanosine 5′-di-phosphate 3′-diphosphate (ppGpp) pool. When derepressed in N-free medium supplemented with glutamine (600 μg ml−1), expression from the nifH and nifL promoters, determined as β-galactosidase activity in nif: :lac merodiploid strains, was stimulated 7-fold and nitrogenase activity 26-fold; ppGpp did not accumulate, remaining at the levels found in NH+ 4-repressed populations. The relaxed mutant K. pneumoniae relA40, which accumulates only very low levels of ppGpp, showed partial derepression of nitrogenase activity in the presence of glutamine, thus ppGpp is unlikely to be an effector of nif expression. ATP and GTP levels were elevated under conditions where nif expression was enhanced, consistent with previous data suggesting that maintenance of ATP levels is a prerequisite for the expression of nif genes in K. pneumoniae.
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The Response of Mg2+-limited Anacystis nidulans to Alterations in Photon Fluence Rate When Grown in Chemostats: a Suitable System for Studies of Mg2+-controlled Cell Division
More LessThe effect of photon fluence rate on Anacystis nidulans grown in Mg2+- and non-Mg2+-limited continuous cultures was studied. A decline in photon fluence rate from 445 to 30 μE m−2 s−1 resulted in a decrease of the mean cell volume from 2 to 0·8 μm3 in the Mg2+-limited culture. Compared with non-Mg2+-limited cultures, this was the only obvious difference found when decreasing the photon fluence rate. Altering the photon fluence rate for a Mg2+-limited chemostat therefore seems to be a suitable system for studies of the Mg2+-controlled events in cell division of A. nidulans. At constant dilution rate, the DNA content per unit cell volume was independent of photon fluence rate, while the RNA content decreased with this factor. The RNA/protein ratio decreased by a factor of 3 in both Mg2+- and non-Mg2+-limited cultures, when decreasing the photon fluence rate from 445 to 100 μE m−2 s−1. The RNA efficiency therefore varied with photon fluence rate and the organism seemed to have surplus RNA at high light intensities.
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Mechanism of Mycobacteriocin and Tween 80 Mediated Antimycobacterial Activity
More LessRapidly growing mycobacteria (14 species, 67 strains) showed a highly significant correlation between their susceptibility to smegmatocin (a Tween-hydrolysing esterase of Mycobacterium smegmatis exhibiting antimicrobial activity in the presence of Tween 80) and sensitivity to oleic acid (a hydrolysis product of Tween 80). Polyoxyethylenesorbitan ether, the other product of Tween 80 hydrolysis, also showed some toxic effect on smegmatocin-sensitive mycobacteria. From Mycobacterium diernhoferi ATCC 19340, a representative smegmatocin-sensitive strain, mutant strains with smegmatocin-resistant phenotypes were isolated. These mutants were more resistant to oleic acid than the parent strain, although their oleic acid resistance was somewhat lower than their smegmatocin resistance.
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Factors Affecting the Biosynthesis of l-Tryptophan by Genetically Modified Strains of Escherichia coli
More LessDerivatives of Escherichia coli strain W3110 with increased tryptophan synthase (TS) activity were constructed. The biosynthesis of serine was shown to limit tryptophan production in minimal medium with indole as precursor. In the presence of serine and indole we obtained a correlation between the specific activity of TS and the specific productivity (q p) of tryptophan. Supplementation of the growth medium with glycine enhanced q p two-fold. In a strain with high serine hydroxymethyltransferase (SHMT) activity no such increase of tryptophan productivity was observed, although crude extracts from these cells were shown to produce tryptophan with indole, one-carbon units and glycine as precursors. Growth of the strain with high SHMT activity was inhibited in a medium with high glycine concentration. This inhibition could not be released by addition of isoleucine and valine. In a buffer system with permeabilized cells high in specific TS and SHMT activities we did not obtain any tryptophan production in presence of indole, glycine, one-carbon units and cofactors. On the other hand, in a buffer system with indole and serine as precursors we obtained high q p of tryptophan [13·3 g tryptophan (g dry wt cells)−1 h−1], which was correlated to the TS specific activity.
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Germination of Bacillus cereus Spores: Critical Control by DL-Alanine Racemase
More LessEffects of dl-alanine racemase, D-alanine, heat-activation and ammonia on germination of Bacillus cereus T spores were studied. Michaelis-Menten parameters for dl-alanine racemase activity in intact spores were: K m (l-alanine)=3·6 mm; K m (d-alanine) = 1·2 mm; V max (l- to d-alanine) = 1·6 μmol min−1 (mg dry spores)−1; V max (d- to l-alanine) = 0·53 μmol min−1 (mg dry spores)−1. Racemase activity was irreversibly inhibited with pseudo first order kinetics by incubating intact spores with 10 mm-d-cycloserine. (Aminooxy)acetate (50 μm) completely inhibited racemase activity with 100 mm-l-alanine as substrate. d-Cysteine was a moderately effective racemase inhibitor. Comparisons of the effects of racemase inhibitors, d-alanine and spore concentration on germination rates in 1 mm-l-alanine indicated that racemization of l-alanine accounted for nearly total inhibition of germination at spore concentrations exceeding about 50 μg ml−1, in the range conventionally used for spectrophotometric germination assays. Heat-activation decreased the inhibitory effect of d-alanine, increasing the germination rate in 2 mm-racemic alanine by a factor of 26. Using spores with an inhibited alanine racemase, germination rates in 1 mm-l-alanine were stimulated by 40 mm-NH4Cl to an extent comparable to the stimulation obtained by heat-activating the spores. Germination of unactivated spores in 1 mm-l-alanine + 40 mm-NH4Cl was completely inhibited by 1 mm-d-alanine. These results clarified the roles of d-alanine and dl-alanine racemase in controlling the germination of unactivated spores and suggested that the germinative functions of ammonia and the stereoisomers of alanine may be related.
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The Ability of Sordaria fimicola to Take up and Metabolize Glucose and Sucrose
More LessGlucose, fructose and trehalose, but not sucrose, supported the growth of Sordaria fimicola. Mycelial suspensions rapidly metabolized [14C]glucose, which was shown to be taken up by a mechanism saturated at 1 mm. Comparable experiments with [14C]sucrose were complicated by contaminating [14C]hexose but provided no conclusive evidence for sucrose uptake. Mycelial extracts hydrolysed sucrose at 7% of the rate found with trehalose. It is suggested that the fungus cannot grow on sucrose because it lacks a mechanism that permits uptake of sucrose carbon.
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Mitochondrial Activities in the Methylotrophic Yeast Kloeckera sp. 2201 During Growth with Glucose and/or Methanol
Th. Egli and N.D. LindleyThe methylotrophic yeast Kloeckera sp. 2201 was grown in carbon-limited chemostat cultures with glucose and/or methanol as the carbon sources, to investigate the regulation of the synthesis of enzymes involved in the tricarboxylic acid (TCA) cycle and the electron transport chain.
Except for malate dehydrogenase, enzymes of the TCA cycle were repressed during growth with methanol to specific activities of 20–40% of those found in cells growing under identical growth conditions but with glucose as the carbon source. In methanol-grown cells, the specific activities of the enzymes of the respiratory chain were also repressed compared to cells grown with glucose [60–70% for NADH: cytochrome c (cyt c) oxidoreductase and cyt c oxidase; approximately 40% for succinate:cyt c oxidoreductase]. The measurement of cytochrome oxidized-minus-reduced spectra was impossible in methanol-grown cells because of interference of the chromophoric group of the peroxisomal enzyme alcohol oxidase under these growth conditions. The relative cytochrome c content of mitochondria from methanol-grown cells, separated from peroxisomes by density gradient centrifugation, was significantly lower (cyt aa 3:cyt b:cyt c = 1:1.24:0.86) than in mitochondria from glucose-grown cells (cyt aa 3: cyt b :cyt c = 1:1.27:2.20). The results are discussed with respect to the generation of energy for biosynthesis during growth with glucose and/or methanol.
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Protein Synthesis and Amino Acid Pool during Yeast-Mycelial Transition Induced by N-Acetyl-D-glucosamine in Candida albicans
More LessProtein synthesis at different stages of yeast-mycelial transition induced by N-acetyl-D-glucosamine in Candida albicans was evaluated by following incorporation of radioactive amino acids into the acid-insoluble cellular material. In passing from the early germ-tube formation (60–90 min) to the mature hyphal cell (240–270 min) there was a marked decrease in the capacity for protein synthesis. Apparently, this decrease was not due to a decreased amino acid uptake into the soluble cellular pool or to exhaustion of carbon/energy source in the inducing medium with consequent arrest of growth. Protein synthesis, however, did not decay when amino acids at high concentration were added to the medium fostering the yeast-mycelial transition and this effect was potentiated by glucose.
Analysis of the intracellular amino acid pool showed that both germ-tubes and hyphal cells were relatively depleted of several amino acids as compared to the yeast-form cells, whereas in the hyphae there was a higher concentration of glutamic acid/glutamine, the latter being the predominant component. These modulations in amino acid pool composition were not seen when yeasts were converted to hyphae in an amino acid-rich induction medium. This study emphasizes that yeast-form cells of C. albicans may efficiently convert to the mycelial form even under a progressively lowered rate of protein synthesis, and suggests that initiation of hyphal morphogenesis in the presence of N-acetyl-d-glucosamine is somehow separated from cellular growth.
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Substrate Specificities of the Soluble and Particulate Methane Mono-oxygenases of Methylosinus trichosporium OB3b
More LessThe intracellular location of methane mono-oxygenase (MMO) (soluble or particulate) in Methylosinus trichosporium OB3b is dependent on the availability of copper in the growth medium. Raising the Cu2+ concentration from 1 μm to 5 μm effected a transition from soluble to particulate MMO activity, and changes in major cell polypeptides were observed on SDS-polyacrylamide gels. Organisms containing soluble MMO oxidized a wide range of substrates including n-alkanes, n-alkenes, aromatic and alicyclic compounds. By contrast, organisms containing particulate MMO did not oxidize aromatic or alicyclic compounds. These observations provide further evidence that the two types of MMO are fundamentally different.
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- Systematics
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Discriminant Analysis of Volatile Fatty Acids Produced in Culture Medium: a Novel Approach to the Identification of Pseudomonas Species
More LessThe volatile fatty acids produced in culture medium by 357 Pseudomonas strains belonging to eight species were determined quantitatively by GLC. The resultant chromatograms were submitted to discriminant analysis. Stable discriminant functions were computed and included in a computerized identification system which also involved some distinctive volatile fatty acids regarded as two-state qualitative characters (presence or absence characters). Using a test group of 249 strains belonging to the studied species, more than 89% of the identifications made by this system agreed with those made by conventional biochemical methods despite the relatively poor differentiation between P. putida and P. fluorescens. When the individual species within the matrices were weighted with prior probabilities reflecting results given by two simple biochemical tests, 96% of the 249 strains were correctly identified.
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Mycoplasma cavipharyngis, a New Species Isolated from the Nasopharynx of Guinea-pigs
More LessTwo mycoplasma strains isolated from the nasopharynx of guinea-pigs in two separate colonies were biochemically and serologically identical, and distinct from 80 Mycoplasma and Acholeplasma spp. One of them, strain 117C (NCTC 11700), is designated the type strain of a new species Mycoplasma cavipharyngis.
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A Study by High-resolution Two-dimensional Polyacrylamide Gel Electrophoresis of Relationships between Neisseria gonorrhoeae and Other Bacteria
More LessHigh-resolution two-dimensional polyacrylamide gel electrophoresis was used to analyse the soluble proteins from seven strains of Neisseria gonorrhoeae, six strains of Neisseria meningitidis and one or two strains of twelve other species. Approximately 200 individual polypeptides could be visualized as Coomassie Blue stained spots on an electrophoretogram of N. gonorrhoeae and similar numbers were found for the other bacteria. Each species of bacterium had a distinctly different pattern of spots which could be recognized. Quantitative comparisons of 48 selected spots derived from one strain of N. gonorrhoeae with those of five other strains of gonococcus, three strains of N. meningitidis and one of Branhamella catarrhalis, showed relationships in agreement with their current taxonomic classification but with a higher level of discrimination than that of previously used methods. It was also possible to distinguish the individual gonococcal strains. It is suggested that the method could be useful for bacterial classification and identification.
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Some Biochemical and Morphological Characteristics of Serotypes of Verticillium albo-atrum from Hop
More LessSingle-spore strains of Verticillium albo-atrum from hop, which had previously been classified into three serotypes by immunoelectrophoretic and immunofluorescence methods, have been further characterized using a series of morphological and biochemical parameters. Serotype 1 strains were characterized by high sporulation and low polygalacturonase activity when cultured on hop root cell walls, the presence of certain protein bands on isoelectric focusing of mycelial extracts, and a characteristic dark-pigmented mycelial morphology on prune/lactose/yeast extract agar. It is suggested that the expression of serotype 1 characteristics is controlled by the previously reported hyl+ cytoplasmic factor. Serotypes 2 and 3 were both associated with low sporulation and high polygalacturonase activity and a fully hyaline fluffy mycelium, although many serotype 3 strains were only partially hyaline. Serotype 2 was associated with a characteristic pattern of mycelial proteins on isoelectric focusing gels: serotype 3 strains showed a heterogeneous series of patterns. None of the characters studied could be correlated with virulence for hop but the results do allow the selection for future studies of well-characterized strains which are extremely similar in many respects and yet still differ markedly in virulence.
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- Short Communication
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Use of a Synthetic Oligonucleotide to Identify a Chromosomal Gene for Chloramphenicol Acetyltransferase in a Plasmid-bearing Flavobacterium
More LessA micro-organism previously designated Flavobacterium sp. CB60 is resistant to chloramphenicol as a consequence of antibiotic acetylation by the enzyme chloramphenicol acetyltransferase and subsequent degradation of the acetylated product by co-metabolism. Although a 15·6 kb plasmid (pCB60) was demonstrated in this Flavobacterium strain, it did not appear to play a role in chloramphenicol acetylation. DNA hybridization was used to identify a fragment of DNA presumptively carrying the cat gene. The sequence of the synthetic probe was based upon known nucleotide sequences corresponding to the highly-conserved active site region of several chloramphenicol acetyltransferase variants. The structural gene for the enzyme in strain CB60 appeared to be chromosomal since the radioactive probe hybridized with a unique restriction fragment from chromosomal DNA but failed to do so with the DNA of plasmid pCB60.
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Genetic Analysis of Three Additional fla Genes in Salmonella typhimuvium
More LessIn Salmonella typhimurium, 27 fla responsible for formation of flagella have been identified and assigned to three regions on the genetic map, termed fla regions I to III. By genetic analysis of 1984 non-flagellate mutants obtained from a phase-1 stable strain of S. typhimurium, SJW 1103, three additional fla genes were identified; one, termed flaW, was assigned to fla region I and the other two, termed flaV and flaX, toflaA region III. By intergeneric complementation tests, the flaW, flaV and flaX genes were shown to be functionally homologous with flaS,flaC and flaP of Escherichia coli, respectively. Electron microscopy showed that flaW and flaV mutants carried hook-basal body structures.
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