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Volume 133,
Issue 1,
1987
Volume 133, Issue 1, 1987
- Biochemistry
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The Isolation of Plasma Membrane from the Diatom Phaeodactylum tricornutum Using an Aqueous Two-Polymer Phase System
More LessPlasma membrane of the marine diatom Phaeodactylum tricornutum was purified by the application of the microsomal fraction to an aqueous two-polymer phase system containing 5·7% (w/w) each of polyethylene glycol (m r 3340) and dextran T500, plus 50 mm-NaCl in a Tris/maleate buffer (pH 7·3) with 500 mm-sorbitol. 5′-Nucleotidase, used as a marker for the plasma membrane, partitioned differentially into the upper phase, and chlorophyll into the lower phase. In sectioned intact cells only the plasma membrane and tonoplast stained with periodic acid-chromic acid-phosphotungstic acid as viewed by electron microscopy. All of the membranous material which partitioned into the upper phase of the two phase system stained with this procedure, whilst much of the material in the lower phase did not. This result indicated the presence of plasma membrane and/or tonoplast only in the upper phase; the increased specific activity of 5′-nucleotidase indicated a 25-fold purification of plasma membrane in this fraction compared to broken cells.
In order to differentiate between plasma membrane and tonoplast, vanadate-sensitive, nitrate-insensitive, azide-insensitive, molybdate-insensitive K+, Mg2+-ATPase was used as an additional marker for plasma membrane, and acid phosphatase and nitrate-sensitive ATPase were used as markers for tonoplast. Use of these markers indicated the presence of some tonoplast membrane in the upper phase. It is concluded that the procedure used separates plasma membrane almost completely from organelle membranes and partially from tonoplast.
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Changes in Membrane Lipid Composition of Clostridium acetobutylicum during Acetone-Butanol Fermentation: Effects of Solvents, Growth Temperature and pH
More LessChanges in membrane lipid composition of Clostridium acetobutylicum were studied during acetone-butanol fermentation. Large changes were found in phospholipid composition and in fatty acid composition, the latter characterized mainly by a decrease in the unsaturated/saturated fatty acid (U/S) ratio. The effects of the addition of alcohols (ethanol, butanol, hexanol and octanol) and of acetone were also studied. In all cases, large changes were observed in the U/S ratio but with differences which were related to the chain lengths of the alcohols. The effect of solvents appears to account for a large part of the changes in lipid composition observed during the fermentation. The pH was also important, a decrease in pH resulting in a decrease in the U/S ratio and in an increase in cyclopropane fatty acids. The effect of increasing temperature was mainly to increase fatty acid chain lengths.
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Isolation and Characterization of an Extracellular Lipase from the Conidia of Neurospora cvassa
More LessA triacylglycerol lipase (EC 3.1.1.3) from the conidia of Neurospora crassa was purified and characterized. The enzyme was purified by Sephadex G-100 column chromatography. Homogeneity was checked by PAGE, and isoelectric focusing gave a single band corresponding to a pI of 6·4. The enzyme had an apparent M r 54000 ± 1000 as determined by gel filtration. SDS-PAGE gave a single band of M r 27000, suggesting the presence of two identical subunits. This lipase preferred triglycerides with C16- and C18-fatty acyl chains. It cleaved only the primary groups of triglycerides. The lipase also exhibited a marked preference for substrates containing endogenously occurring fatty acids and so may prove useful in detailed studies on the physiological relevance of fatty acyl specificity of lipases. The enzyme was not affected by detergents, or thiol-binding agents. Modification of free amino groups caused 90 % inhibition, suggesting a role of these groups in the maintenance of lipase activity.
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Exochelin-mediated Iron Acquisition by the Leprosy Bacillus, Mycobacterium leprae
More LessSUMMARY: Exochelins, water-soluble siderophores of mycobacteria, were isolated and partially purified from culture filtrates of iron-deficiently grown cultures of Mycobacterium neoaurum NCTC 10439 and an armadillo-derived Mycobacterium (ADM 8563). Two biologically active fractions mediating iron uptake were isolated from each bacterium which not only were able to transport iron into the producing organism but also into suspensions of Mycobacterium leprae isolated from armadillo liver. The rate of exochelin-mediated iron uptake into M. leprae was about 1.5% of the rate observed into the producing organisms. The process of iron uptake appears to be by facilitated diffusion as it was not inhibited by HgCl2, NaN3, KCN, dinitrophenol or carbonyl cyanide m-chlorophenylhydrazone. Since no uptake of iron occurred into iron-sufficient ADM cells, this may indicate that M. leprae, as recovered from an animal tissue, had been growing iron-deficiently in order for iron uptake to have been demonstrated in vitro.
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Detection of Trehalose Monomycolate in Mycobacterium leprae Grown in Armadillo Tissues
More LessSUMMARY: Trehalose-6-monomycolate (TMM) was isolated from the lipids of armadillo-derived Mycobacterium leprae. Only meagre amounts of this glycolipid were recovered, but its structure was unequivocally established. Only α-mycolates were detected in the TMM by 252Cf plasma desorption mass spectrometry. Electron impact mass spectrometry showed the alpha branch to be principally C20. Trehalose dimycolate (cord factor) was not detectable. Since we have also found TMM in M. lepraemurium and in every Mycobacterium species so far examined, we suggest that this glycolipid is truly ubiquitous amongst mycobacteria.
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- Biotechnology
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Expression and Stability of a Recombinant Plasmid in Zymomonas mobilis and Escherichia coli
More LessA recombinant plasmid was constructed by ligating EcoRI digests of the plasmid cloning vector pBR325 and pZMO2, one of the natural plasmids of Zymomonas mobilis ATCC 10988. This vector, named pDS212 (total size 7·9 kb), which was able to transform Escherichia coli efficiently, was also transferred to Z. mobilis hosts by mobilization during conjugation using the helper plasmid pRK2013. pDS212 was inherited stably in both E. coli and Z. mobilis hosts and could be recovered intact from them. Markers of pBR325 and pRK2013 were also transferred in Z. mobilis but at very low frequencies. Neither pBR325 nor pRK2013 could be recovered intact from the Z. mobilis hosts. It is proposed that expression and stability of pDS212 in Z. mobilis is due to the origin of replication of pZMO2 that it carries, and that it may be used for developing a gene transfer system in Z. mobilis.
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- Development And Structure
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Regulation of Gene Expression during Aerobic Germination of Mucor racemosus Sporangiospores
More LessThe pool of mRNA stored in dormant sporangiospores of Mucor racemosus and expressed during early germination in air has been investigated. Total RNA was extracted from dormant and germinating spores and translated in a cell-free rabbit reticulocyte system containing l-[35S]methionine. Isotopically labelled in vitro translation products were analysed by PAGE and autoradiography and were compared with labelled proteins synthesized in vivo at the same stages of development. This comparison revealed several significant findings about the fates of individual mRNA populations as templates in translation: (i) a pool of mRNA, presumably represented entirely or in part by a recoverable polyadenylated RNA fraction, can be extracted from dormant spores in a translatable form; (ii) most of the differential gene expression displayed at the level of protein synthesis during germination results from concomitant changes in functional mRNA levels; (iii) some of the stored mRNA species may be activated and others inactivated by post-transcriptional processing mechanisms; and (iv) a small population of gene products may be regulated at the level of selective translation of pre-existing messages.
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- Genetics And Molecular Biology
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Uptake of Galactose and Lactose by Kluyveromyces lactis: Biochemical Characteristics and Attempted Genetical Analysis
HÉLÈne Boze, G. Moulin and P. GalzySUMMARY: Study of the lactose and galactose transport systems in Kluyveromyces lactis has shown that lactose uptake is by active transport. The transport system is under monogenic control and is inducible. Galactose uptake is also by active transport but the system is controlled by two genes which, in the four strains we studied, are present only in K. lactis CBS 2359. Galactose uptake in the other K. lactis strains is by a simple diffusion process.
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Conditions for Mutagenesis of the Nitrogen-fixing Cyanobacterium Anabaena variabilis
More LessChemically induced mutation in the cyanobacterium Anabaena variabilis was studied using resistance to the pyrimidine analogue 5′-fluorocytosine as a genetic marker which can be selected positively. Cytosine is metabolized through uracil and the UMP pyrophosphorylase ‘salvage’ pathway in this photoautotroph, as it is in enteric bacteria. Treatment with various concentrations of N-methyl-N′-nitro-N-nitrosoguanidine (MNNG) gave the highest frequencies of 5FC-resistant mutants when lethality approximated 99%, irrespective of the exposure time or mutagen concentration. The pH of the incubation medium strongly influenced mutation; exposure to MNNG at pH 6·0 yielded 13-fold higher frequencies of mutants than at pH 7·5. The greatest frequency of resistant cells was found after cultures had undergone six or more doublings following mutagenesis. The mutation frequencies obtained by treatment with MNNG were approximately 4- and 25-fold higher than those after exposure under empirically defined conditions to diethyl sulphate or nitrous acid, respectively, and 1·4 × 103-fold higher than the frequency of spontaneous mutation. Neither chloramphenicol-inhibited nor caffeine-sensitive systems capable of repairing MNNG-induced DNA damage were observed.
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Molecular Cloning and Isolation of a Cyanobacterial Gene Which Increases the UV and Methyl Methanesulphonate Survival of recA Strains of Escherichia coli K12
More LessThe unicellular cyanobacterium Gloeocapsa alpicola contains both photoreactivation and excision repair mechanisms for correcting UV-induced damage to its cellular DNA. An 11·5 kb EcoRI fragment was isolated from a cosmid bank of G. alpicola and was shown to complement a recA deletion in Escherichia coli S. 17 and JC10289. These recA strains showed increased survival to UV and methyl methanesulphonate (MMS) when transformed with the cyanobacterial DNA fragment, and also showed filamentation in response to UV irradiation. Preliminary analysis of the protein encoded by the cyanobacterial DNA fragment indicated a major protein of 39000 Da; this is very similar in size to the recA protein of E. coli.
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The cdc30 Mutation in Saccharomyces cerevisiae Results in a Temperature-sensitive Isoenzyme of Phosphoglucose Isomerase
More LessThe cdc30 mutation in the yeast Saccharomyces cerevisiae causes cell cycle arrest late in nuclear division when cells are shifted from the permissive temperature of 25 °C to the restrictive temperature of 36·5 °C. Cell cycle arrest at 36·5 °C is dependent upon the carbon source used: a shift-up in glucose containing media results in cell cycle blockade, whereas a shift-up in ethanol, fructose, glycerol, glycerol plus ethanol, or mannose does not. Metabolite analyses showed accumulation of glucose 6-phosphate in a cdc30-bearing strain after a temperature shift-up in glucose-containing medium. Thermal denaturation studies and kinetic measurements indicate the existence of two isoenzymes of phosphoglucose isomerase (EC 5.3.1.9); one of which is apparently altered in the temperature-sensitive cell cycle mutant. We propose that the gene products of both the CDC30 and PGI1 genes are required for cell cycle progression in glucose media and that the PGI1 gene product has a regulatory function over the CDC30 gene product.
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Determination of Genome Size and DNA Homology between an Unclassified Mycobacterium Species Isolated from Patients with Crohn's Disease and Other Mycobacteria
More LessSUMMARY: The genome size of an unclassified Mycobacterium species, isolated from a patient with Crohn’s disease, was determined by measurement of DNA renaturation kinetics as 3·1 × 109 Da. The percentage of DNA homology of this organism to DNA of related mycobacteria was evaluated by measurement of DNA renaturation with heterologous DNA. These studies supported the classification of this organism as Mycobacterium paratuberculosis but failed to distinguish between M. paratuberculosis and organisms of the Mycobacterium avium-intracellulare groups.
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- Pathogenicity And Medical Microbiology
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Cleavage of the Protein III and Major Iron-Regulated Protein of Neisseria gonorrhoeae by Lysosomal Cathepsin G
More LessIncubation of either 125I-labelled or unlabelled Neisseria gonorrhoeae with enzymically active preparations of human polymorphonuclear leucocyte lysosomal cathepsin G revealed that surface-exposed outer-membrane proteins were susceptible to proteolytic modification. Electroimmunoblotting experiments confirmed that outer-membrane protein III (PIII) and the major iron-regulated protein (MIRP), two conserved gonococcal proteins, were cleaved by cathepsin G. A direct relationship was observed between susceptibility to the antibacterial properties of cathepsin G and cleavage of PIII among isogenic strains differing in their level of resistance to the bactericidal activity of cathepsin G. Although the antibacterial property of cathepsin G is known to be independent of serine-esterase activity, the data suggest that gonococcal outer-membrane proteins are involved in the binding of cathepsin G, and that variation in the level of resistance reflects the degree to which target outer-membrane proteins such as PIII are exposed.
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Identification of the Outer Membrane Proteins of Campylobacter pyloridis and Antigenic Cross-reactivity between C. pyloridis and C. jejuni
More LessThe outer membrane and surface exposed proteins of four strains of the gastric Campylobacter-like organism Campylobacter pyloridis were identified by SDS-PAGE of Sarkosyl-insoluble membranous material and 125I-surface-labelled whole bacteria. Although constant outer membrane proteins (molecular mass 61, 54 and 31 kDa) were observed in these strains, several variable 125I-labelled surface proteins were detected. C. pyloridis does not appear to express a single surface-exposed major outer membrane protein like that of C. jejuni and C. coli. Putative flagella proteins were identified from isolated flagella and acid-extractable surface material and by immunoblotting with anti-flagella antibodies. Several major protein antigens were observed by immunoblotting with anti-C. pyloridis antisera. At least two of these antigens cross-reacted with anti-C. jejuni antiserum. This cross-reaction appears to be caused primarily by flagellar antigens. However, one major protein antigen (61 kDa) was not cross-reactive with C. jejuni and may, therefore, be useful in serological tests for the specific diagnosis of C. pyloridis infections.
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Immunochemical Characterization of a Polysaccharide Antigen of Bacteroides fragilis with an IgM Monoclonal Antibody
More LessAn IgM mouse monoclonal antibody (McAb) Bf4 was produced to a surface polysaccharide of Bacteroides fragilis NCTC 9343. Immunoblotting showed that McAb Bf4 reacted strongly with a high molecular mass structure which was sensitive to oxidation with periodate but resisted protease treatment. An inhibition enzyme-linked immunosorbent assay (ELISA) indicated that McAb Bf4 did not cross react with the sixteen Bacteroides species and strains tested. Cells of B. fragilis NCTC 9343 recovered from the various interfaces of a Percoll discontinuous density gradient were tested in the inhibition ELISA. Bacteria from the 0–20 %, 20–40 % and 40–60 % interfaces inhibited the ELISA; however, cells from the 60–80 % interface did not. Electron microscopy with immunogold labelling showed that McAb Bf4 did not react with the extracellular fibrous network on bacteria recovered from the 0–20 % interface, or the extracellular electron dense layer on cells from the 60–80 % interface; however, it was associated with a surface structure on cells from the 20–40 % interface. Growth in vivo did not enrich for bacteria with this structure.
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Properties of Serratia marcescens Isolated from Diseased Honeybee (Apis mellifera) Larvae
More LessSUMMARY: Twenty-three strains of Serratia marcescens were isolated in pure culture from diseased honeybee (Apis mellifera) larvae in the Sudan. All the strains belonged to biotype A4(b). DNAase and lipase production, pectolytic activity, and citrate utilization were useful criteria for distinguishing S. marcescens from closely related bacteria. A challenge experiment using pure cultures of S. marcescens biotype A4(b) proved this organism to be pathogenic for honeybee larvae.
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- Physiology And Growth
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Physiological Role of Glutaminase Activity in Saccharomyces cerevisiae
More LessSUMMARY: The participation of glutaminase activity in glutamine degradation was studied in a wild-type strain (S288C) of Saccharomyces cerevisiae. Evidence is presented that this strain has two glutaminase activities, a readily extractable form (glutaminase B) and a membrane-bound enzyme (glutaminase A). Glutaminase A and B activities could also be distinguished by their thermostability, pyruvate sensitivity and pH optimum. Glutaminase B activity was negatively modulated by some 2-oxo acids, and in vivo pyruvate accumulation inhibited this activity. A mutant strain (CN10) with an altered glutaminase B activity was isolated and partially characterized. Its glutaminase B activity was more sensitive to inhibition by pyruvate and 2-oxoglutarate than the wild type, thus resulting in inactivation of this enzyme in vivo. The physiological role of glutaminase activity is discussed with regard to the phenotype shown by the mutant strain.
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Glutamine Degradation Through the ω-Amidase Pathway in Saccharomyces cerevisiae
More LessSUMMARY: A glutamine transaminase activity has been identified in Saccharomyces cerevisiae, and the existence of the ω-amidase activity previously described in this yeast has been confirmed. The glutamine transaminase utilizes different 2-oxo acids as substrates, including pyruvate and glyoxylate, and is regulated by the available nitrogen source. The glutamine transaminase activity decreases when lysine or glycine is added to the medium; the inhibition by lysine diminishes under microaerophilic culture conditions.
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Requirement for Vitamin B1 for Growth of Euglena gracilis
More LessSUMMARY: Euglena gracilis Z showed an absolute requirement for vitamin B1 for growth. Increase of cell number in vitamin B1-deficient cultures occurred on the addition of vitamin B1 and depended on the amount added. The phosphate esters of vitamin B1 also supported growth. E. gracilis cells exhaustively took up vitamin B1 within about 2 h of addition. Of the total amount of vitamin B1 taken up, as much as 96% existed in the free form irrespective of the forms added. The addition of 4-amino-5-hydroxymethyl-2-methylpyrimidine, but not 5-(2-hydroxyethyl)-4-methylthiazole, to the vitamin B1-deficient cells enhanced cell number at the same rate as the addition of vitamin B1, indicating that E. gracilis Z is unable to synthesize the pyrimidine moiety of vitamin B1.
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Regulation of Carbohydrate Utilization in Clostridium pasteurianum
More LessSUMMARY: Clostridium pasteurianum is capable of fermentative growth on a number of carbohydrate compounds. Several, including glucose, fructose and sorbitol, are accumulated via a phosphoenolpyruvate-dependent phosphotransferase system (PTS), while the uptake of galactose and gluconate is protonmotive-force-dependent. We have examined the utilization of these substrates by cultures of C. pasteurianum growing on carbohydrate mixtures to determine whether the organism displays preferences for one carbon source over the others; such a preference may indicate the operation of specific mechanisms for regulation of carbohydrate metabolism. In most cases the carbohydrates were co-metabolized. Glucose was utilized together with fructose, gluconate and galactose, although galactose appeared to be favoured over glucose. This preference was not due to repression of synthesis of the glucose PTS. On the other hand, glucose prevented induction of the sorbitol PTS, and led to strong inhibition of sorbitol utilization in uninduced cells. Pre-adaptation of cells to growth on sorbitol counteracted the inhibition by glucose resulting in utilization of glucose and sorbitol at approximately equal rates.
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