- Volume 128, Issue 11, 1982
Volume 128, Issue 11, 1982
- Biochemistry
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6βHydroxylation of Steroids by Extracts of Aspergillus niger
More LessGrowing cultures of Aspergillus niger hydroxylate progesterone at the 6α and 11αpositions. The properties of the 6β-hydroxylase activity were studied in extracts of this organism using 11α hydroxyprogesterone as substrate. Maximum 6β-hydroxylase activity was obtained at pH 6·5. EDTA and thiophenol inhibited hydroxylation, while NADPH, 2-oxoglutarate and pyruvate stimulated it. Cobalt and cadmium ions inhibited the 6β-hydroxylase activity. The feasibility of introducing a hydroxyl group into the 6β-position of various steroid compounds was tested: the 3-Δ4 configuration appeared to exert a directional effect for 6β-hydroxylation, while an 11β-hydroxyl group exerted some kind of steric hindrance against 6β-hydroxylation.
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The Effect of Potassium Ionophores and Potassium on Cellular Calcium in the Yeast Saccharomyces cerevisiae
More LessThe efflux of Ca2+ from the yeast Saccharomyces cerevisiae is induced by the influx of mono- or divalent cations. The mechanism of the tight coupling between K+ influx and Ca2+ efflux was investigated. Two possibilities were examined: (1) Ca2+ efflux is induced only by the carriermediated and energy-dependent K+ uptake or (2) Ca2+ efflux can be induced by electric coupling to K+ influx which is driven by its inward electrochemical gradient in the presence of K+ ionophores. It was found that K+ influx in the presence of valinomycin or nigericin and in the absence of glucose did not induce Ca2+ efflux. It is suggested, therefore, that the coupling is mediated by antiport carriers which exchange Ca2+ and K+.
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Pathways of Glucose Assimilation in Puccinia graminis
More Less[U-14C]-D-Glucose (1 and 20 mm) was added to glucose-grown suspension cultures of Puccinia graminis, and the sequence of movement of the label into metabolic intermediates was determined under steady-state conditions. Radioactive label moved most rapidly into cellular pools of free glucose, amino acids and phosphate esters of trehalose and glucose, and less rapidly into free fructose. No lag was detected in the movement of label into any of these compounds. Thus fructose was the first free carbohydrate synthesized from glucose. Radioactive label moved more slowly, and with an initial lag phase, into trehalose, glucitol and mannitol; the lag was more pronounced for mannitol than glucitol. Kinetic data and specific activity measurements to identify precursors suggest that trehalose was formed via trehalose phosphate, mannitol via fructose, and glucitol via both glucose and fructose. Steady-state specific activities of all free intracellular carbohydrates were much lower (15-50%) than that of the exogenous glucose, indicating that unlabelled carbon was entering these pools from storage reserves or other components from the culture medium.
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Yeast 3-Hydroxy-3-methylglutaryl-CoA Reductase: an Enzyme Committed to Catabolite Derepression Before Exhaustion of Fermentable Substrate
More LessSaccharomyces cerevisiae grown on glucose (1 %, w/v) medium exhibited diauxic growth because of the transition from catabolite-repressed fermentative metabolism to derepressed oxidative metabolism. 3-Hydroxy-3-methylglutaryl-CoA reductase (HMG-CoA reductase; EC 1.1.1.34) was object to catabolite repression and required cytoplasmic protein synthesis for derepression. The addition of glucose (2 %, w/v) at up to 4 h prior to glucose exhaustion had no effect on subsequent derepression of HMG-CoA reductase, suggesting that the enzyme was irreversibly committed to derepression at this stage. However, other derepressible enzyme activities such as NAD-dependent glutamate dehydrogenase (EC 1.4.1.2) and malate dehydrogenase (EC 1.1.1.37) did not show a commitment to derepression at this time. In contrast, NADP-dependent glutamate dehydrogenase (EC 1.4.1.4) was induced by the addition of glucose.
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An Exopectate Lyase of Butyrivibrio fibrisolvens from the Bovine Rumen
More LessAn extracellular pectinolytic enzyme produced by Butyrivibrio fibrisolvens isolated from the bovine rumen was studied. The enzyme had a pH optimum of 8·0 to 8·5 and was stimulated by Ca2+ and inhibited by EDTA. The products of pectinolysis had an absorption peak at 235 nm and reacted with thiobarbituric acid, indicating a lyase type of action. The enzyme cleaved the substrates terminally from the reducing end; action on poly- and oligogalacturonates resulted in the formation of an unsaturated trigalacturonate. The enzyme was classified as an exopectate lyase (EC 4.2.2.9). A pectinesterase was also produced by B. fibrisolvens but polygalacturonase was not detected.
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Biosynthesis of Alkali-insoluble Cell-wall Glucan in Schizophyllum commune Protoplasts
More LessBiosynthesis of cell-wall glucans was studied during regeneration of protoplasts from Schizophylhum commune. Double-labelling experiments using [14C]glucose and [3H]glucose indicated a larger pool size of precursors for an alkali-insoluble (1 → 3)-β-glucan than for the other cell-wall components, (1 → 3)-β-glucan and chitin. Pulse-chase experiments established the existence of a water-soluble, partly alkali-soluble (1 → 3)-β-glucan as a precursor for the alkali-insoluble wall glucan, containing only (1 → 3)-β-linkages. Polyoxin D, an inhibitor of chitin synthase, completely arrested accumulation of alkali-insoluble (1 → 3)-β-glucan. This antibiotic did not inhibit the synthesis of the water-soluble glucan, but prevented the incorporation of this material into the alkali-insoluble glucan/chitin complex. Cycloheximide added at the start of regeneration prevented the synthesis of the alkali-insoluble (1 → 3)-β-glucan and of the water-soluble glucan precursor, whereas no effect on the formation of the alkali-insoluble glucan was observed when cycloheximide was added 3 h after the onset of the regeneration.
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Production of the Secondary Metabolite Cyanide by Extracts of Chromobacterium violaceum
More LessThe ability of cell-free extracts of Chromobacterium violaceum to form cyanide has been investigated. For maximal activity, extracts had to be prepared from bacteria harvested at the end of growth and stored under anaerobic conditions in the presence of dithiothreitol. Glycine was the substrate for cyanide formation by extracts and an electron acceptor such as phenazine methosulphate was required. Radiolabelling studies confirmed that cyanide was produced by decarboxylation of glycine. Formaldoxime, glyoxylic acid oxime, oxamic acid and N-hydroxyglycine, potential intermediates in the conversion of glycine to cyanide, were tested as substrates for cyanogenesis, but were inactive. However, N-hydroxyglycine was a noncompetitive inhibitor of cyanogenesis from glycine. Cyanogenic activity was found principally in the particulate fraction, but a significant amount was also present in the supernatant fraction from high-speed centrifugation. The activity in the particulate fraction was solubilized with detergents.
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Altered Lipid Composition in a Non-differentiating Derivative of Streptomyces hygroscopicus
More LessThe turimycin-producing Streptomyces hygroscopicus strain JA6599/NG60-93 (Tur+Amy+) and strain CCl, which is unable to produce antibiotic and aerial mycelium (Tur−Amy−), were compared with respect to their mycelial enzyme activities and cellular lipid composition. Changed activities of six enzymes of intermediary metabolism during submerged growth of strain CCl on chemically defined medium attest to alterations of the life cycle. In addition, strain CCl contained decreased amounts of 12-methyltetradecanoic acid in relation to 14-methylpentadecanoic acid (isopalmitic acid) and displayed a quantitatively altered phospholipid composition.
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Purification and Characterization of Extracellular and Intracellular Killer Toxin of Saccharomyces cerevisiae Strain 28
P. Pfeiffer and F. RadlerThe extracellular killer toxin of Saccharomyces cerevisiae strain 28 was concentrated by ultrafiltration of culture supernatants and purified by ion-exchange chromatography. Polyacrylamide gradient gel electrophoresis in SDS indicated that the toxin is a glycoprotein with a molecular weight of about 16000. Amino acid analysis revealed that the killer toxin contains 111 amino acid residues, equivalent to a molecular weight of 14045; the ratio of protein to carbohydrate in the molecule is therefore about 9 to 1. The isoelectric point of the killer toxin was pH 4·4 to 4·5. The toxin was unaffected by heating at 40 °C for 1 h and its maximum activity against sensitive yeast cells was observed at pH 5v0. Cell-free extracts prepared from well-washed cells of S. cerevisiae strain 28 were toxic for sensitive yeasts. The toxin present in these extracts (intracellular toxin) was partially purified by ultrafiltration and ion-exchange chromatography. The isoelectric points of the extracellular and intracellular killer toxin were similar.
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Inhibition by Adenosine of Aggregation Centre Initiation and Cyclic AMP Binding in Dictyostelium
More LessThe inhibitory action of adenosine on the initiation of aggregation centre formation in starving Dictyostelium discoideum was examined and used to test assumptions of theoretical models of cyclic AMP signalling. Adenosine at 5 mm strongly inhibited aggregation centre initiation with-out preventing signal relay. Inosine or AMP at 10 mm had no effect and did not prevent the effect of adenosine. Adenosine had no effect on cellular phosphodiesterase activity, contact site A formation, or chemotaxis to small drops of cyclic AMP. The chemotactically-related responses of rapid cyclic GMP formation and changes in light scattering after pulses of cyclic AMP did not show any inhibition or decrease in sensitivity to the signal in the presence of adenosine. However, binding of 3H-labelled cyclic AMP to cell surface receptors was strongly inhibited by adenosine. The maximum inhibition was approximately 90% and was non-competitive with respect to cyclic AMP. The role of cell surface cyclic AMP receptors in the mechanism of initiation and the implications of the effects of adenosine for the role of cyclic GMP as a secondary messenger are discussed.
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Some Properties of Tyrosine Aminotransferase from Trichoderma viride
More LessTyrosine aminotransferase, induced in Trichoderma viride by l-tyrosine, was isolated from the culture medium, partially purified and characterized. The enzyme was inducible by both l- and dl-tyrosine, although l-tyrosine was the better inducer. The enzyme required l-tyrosine and a-ketoglutarate as amino donor and amino acceptor, respectively. Its pH optimum in 50 mm-potassium phosphate buffer was 7·6, the apparent Michaelis constants were 0·5 mm for l-tyrosine and 0·25 mm for a-ketoglutarate; it had a molecular weight of 110000.
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- Biotechnology
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Genetic and Biochemical Analysis of the Ability of Saccharomyces cerevisiae to Decarboxylate Cinnamic Acids
More LessDecarboxylation of ferulate to 4-vinylguaiacol is associated with the production of a phenolic off-flavour in beer. The ability (Pof+) of non-brewing strains of Saccharomyces cerevisiae to carry out this reaction has been assigned, by tetrad analysis, to a single nuclear gene. This gene (POF1) is dominant in heterozygous diploids and segregates independently from MATa/MATα, lys2 and DEX1. Therefore, elimination of the Pof+ phenotype from strains intended for brewing is feasible by either mutation or genetic segregation. Since ferulate was decarboxylated by cell-free supernatants derived from Pof+ strains, but not by similar fractions from Pof- strains, POF1 encodes production of a cellular decarboxylase. Strains carrying POF1 also decarboxylated coumarate and cinnamate in vivo, but with caffeate, 4-hydroxybenzoate, 4-hydroxyphenylacetate, styrylacetate, mandelate or phenylpropionate as substrates, the corresponding decarboxylation products were not detected. POF1 may confer resistance to inhibitory effects of naturally occurring cinnamates on yeast growth.
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- Development And Structure
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Isolation of Yeast Ascospores Free of Vegetative Cell Contamination
More LessA method has been devised for isolating yeast ascospores and asci free from vegetative cell contamination. By careful manipulation of the size and surface properties of the spheroplasted vegetative cells and asci, a pure preparation of ascospores could be recovered by sucrose gradient centrifugation.
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- Genetics And Molecular Biology
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Accumulation of 2′-O-Methylguanosine Deficient tRNATrp in Tryptophan Limited Saccharomyces cerevisiae
More LessSaccharomyces cerevisiae synthesizes one major tryptophan transfer ribonucleic acid (tRNATrp) species (isoacceptor A) carrying characteristic base modifications. Under tryptophan limited growth conditions wild-type strain X2180-1A exhibited a second important tRNATrp species (isoacceptor B). The total amount of tRNATrp, approximately 2.5 pmol (mg dry wt)-1, stayed essentially constant during amino acid shift-down experiments. The amount of isoacceptor B relative to total tRNATrp was 10 to 15% during amino acid sufficient exponential growth conditions, but increased during tryptophan limitation three- to four-fold. Analysis of the base compositions showed that isoacceptor B differed from isoacceptor A in one respect only: 2′-O-methylguanosine, a modified guanosine base occurring at position 17 of the major isoacceptor A tRNATrp, was not detectable in hydrolysates of purified isoacceptor B. The biological significance of isoacceptor B is discussed.
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Properties of Non-differentiating Derivatives of Streptomyces hygroscopicus
More LessThe properties of non-differentiating derivatives of Streptomyces hygroscopicus, which appeared spontaneously in continuous cultures of their parental strain, were investigated. These derivatives differ from the original strain by production of only trace amounts of the antibiotic turimycin, inability to form aerial mycelium and spores, and altered morphology of colonies and submerged mycelium. It is proposed that as a consequence of a genetic alteration the derivatives are impaired in a central regulatory function influencing differentiation processes by pleiotropic effects.
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‘Diad Analysis’ for Linkage Study in Yeast with Poor Spore Viability
More LessEquations were derived expressing the expected frequencies of the parental ditype (PD), non-parental ditype (NPD) and tetratype (T) as a function of the frequencies of 10 diad types with two spores from double heterozygous diploids. Using these equations, a novel procedure, ‘diad analysis’ was proposed that permits the estimation of centromere linkage from the frequencies of 10 diad types with two spores. To test the applicability of the diad analysis, the expected numbers of PD, NPD and T asci were calculated using these equations with simulated data for the 10 diad types, taken from tetrad data, and the results were compared, by x 2 statistics, with the observed data from the same tetrads. A good fit was found between the two. A comparison of diad analysis with tetrad analysis and random spore analysis is also discussed.
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The Diversity of Cyanobacterial Genomes with Respect to Ribosomal RNA Cistrons
More LessThe ribosomal RNAs were isolated from cyanobacteria (Anacystis nidulans, Anabaena cylindrica, Anabaena CA and Nostoc MAC) and labelled in vitro with [γ-32P]ATP and polynucleotide kinase. DNA from the corresponding species was isolated, digested with EcoRI or HindIII, and resolved on agarose gels. In situ hybridization was used to measure the number and location of the 16S and 23S ribosomal RNA genes. The comparative results are discussed in relation to the known genome size of these species and to their RNA content per cell.
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A Dominance Modifier for Cycloheximide Resistance in Coprinus cinereus
More LessWild and laboratory strains of Coprinus cinereus show varying levels of sensitivity to cycloheximide (actidione). Mutants resistant to 1.0 g cycloheximide ml−1 were isolated from UV light-irradiated oidia of two strains unable to grow on more than 0·25 μg cycloheximide ml−1. Of 13 induced mutants, 12 fell into one complementation group, cy-2; mutants at this locus were usually recessive, but one of those isolated carried a dominance modifier gene, modcy +, which led to partial resistance in dikaryons heterozygous for cy-2 r. Modcy+ was dominant in dikaryons to its non-mutant allele modcy −, but it had no effect in diploids. However, it affected all cy-2 r alleles tested in dikaryons. Linkage data are presented for cy-2, modcy and another cycloheximide resistance gene, cy-1, which is found in wild strains.
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pHH502, a Plasmid with IncP and IncIα Characters, Loses the Latter by a Specific recA-independent Deletion Event
More LessPlasmid pHH502, of molecular weight 70 × 106, determined resistance to tetracycline, chloramphenicol, trimethoprim, sulphonamides and mercuric chloride and was incompatible with members of IncP and IncIα. It resembled other plasmids of IncIα in the following properties: it determined pili that were morphologically and serologically la pili, whose production was repressed in established plasmid-carrying (R+) cultures; its transfer was equally efficient in liquid or on solid medium; it exerted surface exclusion against other IncIα plasmids; it was non-transferable to Proteus. In a reproducible, recA-independent event, pHH502 gave rise to pHH502-1, a plasmid of molecular weight 40 × 106, lacking determinants for resistance to tetracycline and chloramphenicol and all detectable IncIα characteristics. pHH502-1 was incompatible only with IncP plasmids and resembled other IncP plasmids in determining constitutive production of rigid pili, in its surface exclusion, in transferring at greater frequency on solid than in liquid medium and in being transmissible to Proteus mirabilis. It differed from other IncP plasmids in the morphology and serological type of its pili and in failing to transfer to Pseudomonas aeruginosa or Acinetobacter calcoaceticus. Small numbers of pHH502-1 rigid pili were present on bacteria carrying pHH502. Possible mechanisms for the generation of pHH502 and pHH502-1 are discussed.
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Plasmid R394 is a Cointegrate
More LessPlasmids R394a and R394b which cointegrate to form R394 are described. They have molecular masses of 102 ± 4 and 11·0 ± 0·4 MDal, belong to the T and N incompatibility groups and confer resistance to kanamycin and ampicillin, respectively. R394a is self transmissible and mobilizes R394b, which is non-self transmissible. These findings clarify anomalies in the behaviour of R394 and support suggestions based on the properties of variant phages derived from R394.
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Phages Iα and I2-2: IncI Plasmid-dependent Bacteriophages
More LessPhage Iα was isolated from sewage from Windhoek, South West Africa. It formed relatively clear plaques about 2 mm in diameter, on sensitive strains of Escherichia coli K12 and Salmonella typhimurium LT2. The phage had an hexagonal outline with a diameter of about 24 nm, contained RNA and was resistant to chloroform. Phage Iα formed plaques or propagated only on organisms carrying I1 plasmids or the Iγ plasmid R621a. The efficiency of plating was higher on E. coli than on S. typhimurium hosts. The phage adsorbed along the length of shafts of I1 pili.
Phage I2-2 was isolated from Pretoria sewage. It was a filamentous virus and individual virions varied considerably in length. Phage I2-2 formed turbid plaques which varied from pin point to about 1 mm in diameter on all hosts. It was resistant to RNAase and sensitive to chloroform. Phage I2-2 had a spectrum of activity limited to strains harbouring I2 plasmids but the adsorption site could not be demonstrated. The phage was not related serologically to phages If1 or PR64FS.
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Isolation and Characterization of a Recombinant Plasmid Carrying a Functional Part of the Bacillus subtilis spoIIA Locus
More LessPlasmid pHM2 consists of a 3·3 kb insert of Bacillus subtilis DNA in the chimeric plasmid pHV33, and can replicate in Escherichia coli and B. subtilis. In B. subtilis, pHM2 complements the defects resulting from mutations spo-42, spo-50, spo-69 and sas-1 in the spoIIA locus. This complementation can occur in recE4 strains where recombination of the plasmid with the chromosome is prevented, and the chromosome retains the mutant allele. Thus the plasmid carries a functional part of the spoIIA locus; it does not contain the complete locus as it cannot complement several other spoIIA mutations. It is likely that the locus is complex, containing at least two genes.
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- Pathogenicity And Medical Microbiology
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Carbohydrate Mediation of the Biological Activities of the Glycolipoprotein of Pseudomonas aeruginosa
More LessThe glycolipoprotein (GLP) extracted from the surface slime of Pseudomonas aeruginosa produces effects in mice similar to those of the viable cell. The lethal activity has been located in the lipid moiety; however, degradation of the carbohydrate moiety with sodium metaperiodate reduced the antigenicity and abolished the lethality of the GLP. Similar degradation with a phage-induced polysaccharide depolymerase reduced the antigenicity only slightly but reduced the lethality over 60%. The neutral sugar composition of the isolated polysaccharide moiety was shown to be that of the parental GLP. Of the component neutral sugars, mannose and its derivatives were capable of inhibiting the agglutination of erythrocytes coated with GLP. Inhibition also occurred with a soluble mannose polymer from the cell walls of yeast. Antiserum to GLP and to its isolated polysaccharide moiety agglutinated yeast cells, whereas antiserum to a glycolipid fragment of the GLP lacking mannose did not. The lethality of the GLP was reduced by degradation with α-mannosidase or by blocking the mannose residues with concanavalin A, and the glycolipid fragment showed less lethality than the native GLP. We conclude that mannose, in addition to being an immunodominant sugar, is an effector sugar in the expression of GLP lethality.
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- Physiology And Growth
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On the Growth and Form of Escherichia coli
More LessThe shape of many micro-organisms can be understood in terms of the general surface stress hypothesis that hydrostatic pressure forces newly formed wall to expand in a particular direction. What distinguishes one type of organism from another is the regions of the cell where new wall growth occurs. For several classes of organisms, the pattern of growth deduced from the shape agrees with biochemical, morphological and physiological studies. Gram-negative rods, as typified by Escherichia coli, have a morphology that may be explained in several ways by this general hypothesis.
In the present paper, the morphological, autoradiographic and biochemical data concerning E. coli are reviewed. Thirteen models are considered; there is reason to reject most of them but one model that includes two others appears more satisfactory. All the models conform to the biophysical principles that it is impossible to turn over stress-bearing peptidoglycan without shape change and that growth of the sacculus requires the introduction of new oligosaccharides and their covalent linkage before cleavage of stress-bearing bonds. The model that best accounts for the experimental data assumes that, while addition of peptidoglycan all over the cell is possible and does occur, the effective surface tension is higher (and therefore the rate of wall growth is very small) in old poles than on the sides, and very much lower at the developing division sites (where the rate of wall growth is high). It is shown that, when the hydrostatic pressure is constant throughout the cell and during a large part of the cell cycle, changes in the biochemical mechanism of wall growth which correspond to a decrease in surface tension would lead to an invagination of the stress-bearing wall. An additional mechanism may, however, be needed for final closure and for the splitting of the last few covalent bonds holding the two nascent cells together.
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Spatial Resolution of Autoradiograms of Rod-shaped Organisms
More LessIsotope-containing rod-shaped bacteria approximate a line-segment source for purposes of autoradiography. This is because the width is small and bacteria are usually cylindrically symmetric. For this reason published work has only classified the silver grains longitudinally, independent of the transverse position. While the theoretical integral distribution from a variety of geometric shapes has been calculated, the line-segment has been overlooked because long narrow objects well separated from other sources occur infrequently in eukaryotic biology. This oversight is corrected here. In addition, a simple program for line-segments and ways to combine distributions from separate line-segment sources is presented. Some typical distributions are depicted. The program is used elsewhere in the analysis of wall growth patterns of bacteria.
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Effect of Plasma-membrane Phospholipid Unsaturation on Solute Transport into Saccharomyces cerevisiae NCYC 366
More LessA comparison was made of kinetics of solute accumulation by Saccharomyces cerevisiae NCYC 366 grown anaerobically under conditions that lead to enrichment of the plasma membrane with ergosterol and either oleyl or linoleyl residues. Values for K T and V max were identical for accumulation of l-asparagine, l-glutamine, H2PO4 2-, Ca2+ and SO4 2-, while for accumulation of d-glucose, the value differed slightly but not significantly. Values for K T for accumulation of l-lysine, by both the low-and high-affinity systems, decreased when oleyl residues were replaced by linoleyl residues. Under these conditions, V max values for the high-affinity system decreased while that for the low-affinity system increased. An Arrhenius plot for accumulation of lysine by the high-affinity system revealed a discontinuity when membranes were enriched in linoleyl residues. However, no discontinuity was evident on plots of lysine accumulation when membranes were enriched in oleyl residues. Similar plots for accumulation of l-asparagine, which was used as a control, showed that substitution of linoleyl for oleyl residues significantly raised the transition temperature, but had little effect on the activation energy at temperatures below the discontinuity. When palmitoleyl residues were incorporated into the yeast plasma membrane, the K T value for l-lysine accumulation by the high-affinity system was hardly altered, although the V max value was lowered, as compared with organisms with membranes enriched in oleyl residues. Replacement of oleyl by palmitoleyl residues lowered both the K T and V max values for accumulation of l-asparagine. A modified statistical method is described for calculating confidence limits for transition points on Arrhenius plots.
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Accumulation of L-Asparagine by Saccharomyces cerevisiae X-2180
More LessAt concentrations up to 1 mm, l-asparagine was accumulated by Saccharomyces cerevisiae X-2180 at 30 °C against a concentration gradient. Values for K T and V max were, respectively, 3.·5 × 10−4 M and 33 nmol (mg dry wt)−1 min−1. At concentrations below 0·1 mm, a convex curve was obtained on a Woolf-Hofstee plot, possibly indicating the presence of two l-asparagine-binding sites. Autoradiograms of extracts of organisms that had accumulated labelled l-asparagine revealed only one spot with an R F value identical with that of l-asparagine. Four mutant strains lacking the ability to synthesize the general amino acid permease system grew and accumulated l-asparagine at similar rates to the parent. The rate of accumulation of l-asparagine from a 0·2 mm solution was greatest at pH 4·5, with the decrease in accumulation rate greater at values below than above 4·5. l-Glutamine, l-histidine, l-methionine, l-threonine and l-tryptophan caused appreciable inhibition of the rate of l-asparagine accumulation. With the exception of l-methionine, the inhibition caused by these amino acids was competitive. Several other amino acids, including d-asparagine and l-aspartic acid, caused little or no inhibition of l-asparagine accumulation.
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Accumulation of Intracellular Solutes by Two Filamentous Fungi in Response to Growth at Low Steady State Osmotic Potential
More LessGlycerol and the solutes used to control the steady state osmotic potential of the medium, either glucose, glucose and fructose, or KC1, accumulated in the hyphae of Penicillium chrysogenum and Chrysosporium fastidium as the external potential was lowered. When the osmoticum was KC1, nearly all the observed hyphal osmotic potential of P. chrysogenum could be accounted for from the measured solute concentrations, but the proportion decreased when a glucose osmoticum was used, to 56% in the case of P. chrysogenum and to 65% for C. fastidium, when grown at an external potential of − 10 MPa. Higher polyols were present in considerable quantity in both species but did not appear to be involved in osmotic adjustment.
Potassium was the predominant cation in both species when grown on glucose, but chloride was insufficient to maintain electroneutrality. The potassium to sodium ratios were considerably higher in C. fastidium than P. chrysogenum, and inversely related to the external potential in the former species only.
Glycerol and glucose were present in greater amounts at the margin than in older parts of large colonies of P. chrysogenum. High turgor potentials were apparently maintained throughout these large colonies which meant that the observed osmotic potential could not be accounted for by the solutes measured in older parts of the colony.
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Effect of Osmotic Shock on Some Intracellular Solutes in Two Filamentous Fungi
More LessHypoosmotic and hyperosmotic shock experiments confirmed the importance for osmoregulation in Penicillium chrysogenum and Chrysosporium fastidium of glycerol and the osmoticum used in the medium. Growth ceased following both types of shock treatment for a time, depending on the magnitude of the shock and the species. Regrowth following hypoosmotic shock took place some distance behind burst tips. Following hyperosmotic shock, growth was initiated by branching at the apex, but if the magnitude of the shock was greater than 10 MPa, it did not occur in either species although osmotic adjustment was observed. Shock experiments did not implicate the higher polyols in osmotic adjustment. Hypoosmotic shock to C. fastidium resulted in a rapid loss of all internal solutes, while decrease in glycerol in P. chrysogenum following this treatment was more gradual. Transfer of C. fastidium, a species which does not grow on media of high salt concentration, to isoosmotic KCl did not result in loss of turgor although growth was inhibited. Glucose was lost from the hyphae but K+ and Cl− were taken up.
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Growth and Accumulation of Solutes by Phytophthora cinnamomi and Other Lower Fungi in Response to Changes in External Osmotic Potential
More LessProline and the solute used to control the osmotic potential of the medium, either sucrose or KCl, accumulated in the hyphae of Phytophthora cinnamomi as the steady state external potential deceased. Proline also accumulated in three other lower fungi. Very little polyol was detected in any of the species analysed. Proline was lost following hypoosmotic shock and accumulated after hyperosmotic shock in P. cinnamomi. The time taken for growth to resume after shock treatment was variable, but less than 5 h. Potassium was the predominant cation in hyphae grown on sucrose but chloride was insufficient to maintain electroneutrality. The potassium to sodium ratio followed a similar pattern to the radial growth rate with respect to external potential, with maxima at a potential slightly below the highest potential tested. When the osmoticum was sucrose, the calculated internal potential was 20% greater than the observed osmotic potential, but when the osmoticum was KCl, the calculated potential was too high by a factor of more than two, probably indicating error in assumptions concerning the location of ions within the hyphae.
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Thiamin Accumulation and Growth Inhibition in Yeasts
More LessThiamin caused depression of growth, a marked decrease in cellular vitamin B6 content and cytochrome oxidase activity in Saccharomyces yeasts growing in a vitamin B6-free medium under aerobic conditions but had practically no effect in Kluyveromyces, Schizosaccharomyces and Candida spp. Pyridoxine added concomitantly with thiamin permitted the thiaminsensitive yeasts to grow normally with increased activity of cytochrome oxidase. δ-Aminolaevulinate also caused the increase in cytochrome oxidase activity but growth was only partially improved by the addition of this precursor of haem biosynthesis. These phenomena were similar to those found previously in Saccharomyces carlsbergensis strain 4228 (ATCC 9080) ( Nakamura et al., 1981 ). Thiamin-sensitive yeasts accumulated thiamin more than 24-fold when compared with the thiamin-insensitive cells. Thiamin transported into the thiamin-sensitive yeasts was recovered only in the non-esterified form. Thiamin added to the growth medium was also accumulated by growing cells of the thiamin-sensitive yeasts especially during the lag and early exponential phases of growth. Pyridoxine did not affect either thiamin accumulation or the intracellular form of the transported thiamin.
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Semi-continuous and Continuous Production of Aspergillus niger Spores in Submerged Liquid Culture
More LessSporulation of Aspergillus niger was induced in continuous tower fermenters by restricting growth with nitrate limitation. Semi-continuous production of spores was achieved in a single-stage fermentation by alternating full-nutrient and nitrogen-deficient media to the culture. Continuous spore production was obtained in a two-stage fermentation system using the first stage for the growth of vegetative mycelium under optimum conditions and the second, larger, stage for sporulation induction in a constant nitrogen-deficient environment. Spores were produced from much simplified sporulation structures. Using a new sporulation index (Ω), which relates spore numbers produced to the quantity of substrate utilized, the average production efficiency of the two-stage continuous system was shown to be more than twice that of the semi-continuous production system. The tower fermenter was shown to be ideal for controlling organism morphology, thus providing conditions which allowed the development of continuous fungal sporulation.
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Changes in Intracellular pH Accompanying Chemoreception in the Plasmodia of Physarum polycephalum
More LessA new method for measuring intracellular pH, employing the intrinsic fluorescent pigments of the plasmodia of the myxomycete Physarum polycephalum, was used to study the role of pH in chemotactic transduction in the plasmodia. The cell became acidified following stimulation with the attractants alanine, glucose, galactose and maltose when their concentrations exceeded the respective thresholds of chemoreception and taxis. The degree of cell acidification paralleled the relaxing tendency in tension generation. A non-metabolizable attractant. 2-deoxyglucose, also acidified the cell. However, the repellent salts NaCl, KCl and CaCl2 did not change the intracellular pH. Our results suggest that the effects of attractants are mediated by intracellular pH, while the effects of repellents are transduced by ATP as reported previously.
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Inhibition of Nicotinic Acid and Nicotinamide Uptake into Bordetella pertussis by Structural Analogues
More Less3-Pyridine-carboxaldehyde and 3-pyridine-aldoxime were effective and specific inhibitors of the uptake of both nicotinic acid (NA) and nicotinamide (ND) by Bordetella pertussis, although neither compound inhibited the growth of the bacteria in liquid medium or the oxidation of glutamate by washed suspensions. In contrast, the following pyridine derivatives did not inhibit uptake of NA or ND: iso-NA, iso-ND, isoniazid, 6-amino-NA and 6-amino-ND, 3-acetyl-pyridine, 3-pyridyl-acetic acid, N,N-diethyl-ND and 3-pyridine-sulphonic acid. 3- Pyridyl-carbinol was inhibitory, but less so than the first listed compounds.
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Differentiation of Saccharomyces cerevisiae at Slow Growth Rates in Glucose-limited Chemostat Culture
More LessSaccharomyces cerevisiae was grown in a glucose-limited chemostat at dilution rates equivalent to mass doubling times of 2 to 16 h. Phase contrast microscopy revealed that cultures with mass doubling times greater than 4·5 h contained two sub-populations of cells, phase-dark and phase-light. The latter were shown to be predominantly unbudded daughter cells, which were not proliferating, and had properties similar to those of stationary phase cells in batch culture. Phase-dark cells were equivalent to cells growing exponentially in batch culture. The results suggest that the formation of phase-light cells represents cell cycle specific differentiation into stationary phase. The consequences of this phenomenon for the interpretation of results obtained with S. cerevisiae in the chemostat are discussed.
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Localization of Carpophore Initiation in Coprinus congregatus
More LessThe differentiation of mushroom primordia in young mycelia of Coprinus congregatus is a localized event: the mycelium is not uniformly competent to differentiate, or even to be induced, in all areas. A narrow zone corresponding to the 24 h growth of the youngest hyphae is the only area capable of receiving the obligatory light induction and of eventually differentiating into primordia. The inducible zone migrates outwards with the peripheral hyphae as the mycelium expands and previously competent zones become inactive and non-inducible. Light induction of a zone fixes the zone of differentiation in space and prevents subsequent zones from becoming induced. The light receptor system appears to involve two independent processes. One is activated by very low levels of light, fixes the zone in space and prevents subsequent zones from becoming induced, but does not permit differentiation. A second, requiring higher light levels, triggers development of the primordia. The induction of a responsive zone can occur at 20 °C, but actual development requires 25 °C.
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The Role of Laccase in Carpophore Initiation in Coprinus congregatus
More LessThe mycelium of the basidiomycete Coprinus congregatus is not uniformly competent to differentiate. Differentiation occurs only in localized areas of young mycelia. Studies on laccase activities from whole mycelium extracts suggest a correlation between overall laccase levels and the development of mushroom primordia. However, studies of higher resolution on the localized areas of developmental competence, under a variety of light, nutrient and temperature regimes, indicate that laccase is probably not involved in the actual development of the primordia. If laccase is involved in C. congregatus differentiation, it is only at the initial, light-requiring, step.
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- Short Communications
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Facile in vivo Transfer of Mutations Between the Bacillus subtilis Chromosome and a Plasmid Harbouring Homologous DNA
More LessTransformation of Bacillus subtilis strains carrying the point mutations spoIIA42 or spoIIA69 by a plasmid, pHM2, carrying the spoIIA + allele gave occasional recombinants in which the plasmid had acquired the chromosomal mutation. Transformation of a spo + strain by these derivative plasmids gave occasional recombinants in which the chromosome of the recipient had acquired the plasmid-borne spoIIA mutation. Both types of behaviour were recE + dependent. Mutations spoIIA42 and spoIIA69 fell into a single complementation group.
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Rapid Tissue Culture Method for Detection of Mycoplasma hyorhinis
More LessAn easy, rapid tissue culture method for detection of Mycoplasma hyorhinis is described. The organism induces morphological changes in mink S + L - cells. This effect was not observed in eight other animal cell lines infected by M. hyorhinis and it did not occur in the mink cells infected with six other strains of mycoplasma. This cell system should be useful in research laboratories which do not have other standard techniques available for monitoring the presence of M. hyorhinis.
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- Taxonomy
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Fatty Acid Composition of Some Mycolic Acid-containing Coryneform Bacteria
More LessThe fatty acid profiles of 74 strains of mycolic acid-containing coryneform bacteria were examined by gas-liquid chromatography. All of the strains contained major amounts of straightchain and monounsaturated fatty acids although some also possessed substantial amounts of 10-methyloctadecanoic acid. Iso- and anteiso-branched acids were not present. Five distinct fatty acid patterns were evident: (i) Corynebacterium diphtheriae, C. pseudotuberculosis and C. ulcerans strains contained major amounts of hexadecanoic and hexadecenoic acids; (ii) C. glutamicum, C. xerosis and related saprophytic and animal-associated strains, predominantly hexadecanoic and octadecenoic acids; (iii) C. bovis, major amounts of octadecenoic and 10-methyloctadecanoic acids; (iv) C. mycetoides, significant amounts of heptadecanoic acid as well as hexadecanoic and octadecenoic acids; and (v) strains related to Rhodococcus possessed significant quantities of 10-methyloctadecanoic acid in addition to straight-chain and monounsaturated acids.
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Deoxyribonucleic Acid Sequence Relatedness Between Thermophilic Members of the Genus Campylobacter
More LessThe genomic relatedness among 28 catalase-positive Campylobacter strains was assessed by determination of DNA base composition, by DNA: DNA hybridization followed by S1 endonuclease digestion of single-stranded DNA, and by determining the thermal stability of homologous and heterologous double-stranded DNA. The catalase-positive Campylobacter strains were shown to comprise 3 species, C. coli, C fetus and C. jejuni; each included the type strain (respectively, CIP 7080, CIP 5396 and CIP 702).
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Classification of the F38 Group of Caprine Mycoplasma Strains by DNA Hybridization
More LessRepresentatives of groups of mycoplasmas which have antigens in common with Mycoplasma mycoides (as demonstrated by different serological tests) were compared by nucleic acid hybridization. Determinations of DNA homology were performed by filter hybridization as well as hybridization in solution; no differences were revealed between the two methods. Genetic relatedness was not demonstrated between M. primatum and strain F38. The antigenic similarities between strain F38 and the type strain of M. primatum (HRC292) may be due to common epitopes. DNA from strain F38 hybridized with DNA from M. capricolum (California kid) to 80%, but only to about 40% with the two M. mycoides subspecies, a result which can justify the classification of the F38 group as a variant of M. capricolum. The representative strain of bovine serogroup 7 of Leach was equally distant from F38, M. capricolum and the two subspecies of M. mycoides (approximately 60% hybridization).
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Characterization of Propionispira arboris gen. nov. sp. nov., a Nitrogen-fixing Anaerobe Common to Wetwoods of Living Trees
More LessA new species in the family Bacteroidaceae is described, isolated from alkaline wetwoods of poplar trees. It was a non-sporing, curved rod that formed long spiral filaments, especially when grown with N2 as the sole nitrogen source. This obligate anaerobe had peritrichous flagellation and an outer-wall membranous layer. The DNA G + C content was 36·7 ± 1·0 mol %. Cell extracts displayed absorption peaks for type b cytochromes in oxidized versus reduced difference spectra. Nitrogenase was detected by acetylene reduction in cells grown on glucose in the absence of combined nitrogen. This species fermented lactate and a variety of saccharides. Propionate, acetate and CO2 were major products, with succinate and ethanol formed in trace quantities. The optimal pH and temperature for growth were 6·0–6·5 and 3033 C, respectively. The name Propionispira arboris gen. nov. sp. nov. is proposed for the type strain 12B4, which has been deposited as ATCC 33732.
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- Corrigendum
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