- Volume 83, Issue 1, 1974
Volume 83, Issue 1, 1974
- Biochemistry
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Partial Characterization of the Intra- and Extracellular Acid Phosphatase of an Alga, Ochromonas danica
More LessSUMMARY: Intra- and extracellular acid phosphatases were purified 88- and 65-fold respectively from photoheterotrophic Ochromonas danica Pringsheim. The purified enzymes differed in heat inactivation, substrate specificity, and inhibition by several divalent cations and NaF. Intracellular enzyme lost only 30 % of its activity by heating at 60 °C for 200 min whereas the extracellular enzyme lost 80 %. Both enzymes were active over a broad pH range from 2·2 to 5·2 and had an optimum pH of 4·8. Both had broad substrate specificity and differed in their relative ability to hydrolyse β -glycerophosphate, phenolphthalein diphosphate, glucose-1 -phosphate, fructose-1,6-diphosphate, ADP and ATP. Both were inhibited by Co2+, Zn2+, Hg2+, Fe3+, arsenate, tartrate and fluoroacetate but differed in their inhibition by Cu2+, Hg2+ and NaF. Intracellular acid phosphatase was more susceptible to inhibition by Hg2+ and NaF, while extracellular acid phosphatase was more susceptible to inhibition by Cu2+. pChloromercuribenzoate and urea had no effect on either enzyme’s activity. EDTA stimulated the activity of both enzymes. The Km for the intra- and the extracellular enzymes was 0·5 and 0·33 mm respectively with p-nitrophenyl phosphate as the substrate.
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Purification and Mode of Action of Two Bacteriocins Produced by Serratia marcesens HY
More LessSUMMARY: Serratia marcescens strain hy was found to produce marcescin B in addition to the production of marcescin A which was already known. The synthesis of both marcescins was inducible by mitomycin C, but A was induced much less than B. Both marcescins were isolated from the culture fluid of a mutant deficient in exocellular protease, and purified by ammonium sulphate precipitation, gel- filtration, and ion-exchange chromatography on hydroxylapatite and DEAE- cellulose. Marcescin A has a molecular weight of 2 × 106, is resistant to trypsin, attacks some S. marcescens as well as Escherichia coli strains, and its mode of action resembles, in many respects, that of colicin E2, i.e. it inhibits the synthesis of DNA, RNA and protein in sensitive cells and, furthermore, causes degradation of DNA; at high concentrations it also degrades RNA. Marcescin B, on the other hand, has a low molecular weight (43000), is sensitive to trypsin, does not act on S. marcescens strains, and its mode of action seems to be similar to that of colicin E1, i.e. it inhibits the synthesis of DNA, RNA and protein in sensitive cells without DNA-degradation. Thus, several properties of the two marcescins produced by strain hy agree well with the (few) data already published on marcescins derived from other strains. The synthesis of marcescin B resembles that of marcescin A as regards the pleiotropic effect of nucsu mutations.
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Antisera Produced to Purified Extracellular Pectolytic Enzymes from Sclerotinia fructigena
More LessSUMMARY: Purified isoenzymes of α-l-arabinofuranosidase (AF I, pI value 3·0) and polygalacturonase (PG III, pI 5·5; PGIV, pI 9·7) separated from culture filtrates of Sclerotinia fructigena by isoelectric focusing and gel chromatography were used to produce antisera in sheep. The serum produced to AF I was specific as judged by immunodiffusion, immunoadsorbent column chromatography and binding of fluorescein-conjugated serum to the antigen separated by polyacrylamide gel electrophoresis. Fluorescein-labelled anti-AF I serum was bound to mycelium producing this isoenzyme in liquid culture, and binding was decreased when AF production was repressed with glucose; no labelling occurred on mycelium infecting apple fruit tissue.
Data for the anti-PG sera were less conclusive. The antigens gave precipitin lines only with homologous sera, but an anti-PG III immunoadsorbent column bound PG III as well as a smaller proportion of PG IV, while an anti-PG IV column bound neither antigen. Fluorescein isothiocyanate-labelled anti-PG III serum was bound to mycelium of S. fructigena grown in liquid culture and to mycelium-infected apple fruit tissue, but labelled anti-PG IV serum was not bound.
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Electron Transport Carriers Involved in Nitrogen Fixation by the Coliform, Klebsiella pneumoniae
More LessSummary: In cell-free extracts, pyruvate, formate, malate and NADPH provided reducing equivalents to the nitrogenase of the nitrogen-fixing colform, Klebsiella pnemoniae. Two electron carriers were isolated from extracts, both capable of mediating the transfer of reducing power between illuminated spinach chloroplasts and the nitrogenases of Klebsiella or Azotobacter vinelandii. One electron carrier was a flavoprotein, named Klebsiella flavodoxin (molecular weight about 21000); the second was not characterized. A similarity is suggested between Klebsiella and Escherichia coli in the generation and transport of low-potential reducing power from pyruvate.
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The Regulation of Naphthalene Oxygenase in Pseudomonads
More LessSUMMARY: Naphthalene oxygenase was induced in several pseudomonads when these were grown on salicylate as a carbon and energy source, or when salicylate was added to cultures growing on succinate. The enzyme was not induced in Pseudomonas strain ncib9816 when this was grown in the presence of catechol, although after the addition of this compound to cultures growing on succinate the levels of catechol 1,2-oxygenase and catechol 2,3-oxygenase were similar to those observed after the addition of salicylate. Furthermore, two structural analogues of salicylate, 2-aminobenzoic acid and 2-hydroxybenzyl alcohol, induced naphthalene oxygenase gratuitously. Therefore salicylate is probably the inducer of naphthalene oxygenase.
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- Development And Structure
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The Biology and Ultrastructure of Phagotrophy in Ochromonas danica (Chrysophyceae: Chrysomdnadida)
More LessSUMMARY Ochromonas danica can obtain energy and required nutrients by eating several different bacteria or brewer’s yeast. The phagocytic process includes aggregation of bacteria into clumps which contain and are surrounded by Ochromonas.
Bacteria were phagocytized too fast to be seen while yeast took at least 5 min to be engulfed. 2,4-Dinitrophenol or sodium azide, but not sodium fluoride or iodo-acetic acid, inhibited the phagocytic process; aerobic energy metabolism was necessary for phagocytosis. Food organisms were engulfed and the food vacuole migrated to the posterior end of the organism behind the leucosin vacuole. The bacteria were digested mainly in the food vacuole although secondary lysosomes were formed. Digestion of bacteria resulted in the proliferation of vesicles and membranes from the food vacuole membrane and the proliferation of membrane from the bacteria being digested.
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Biogenesis of Mitochondria in Trichoderma viride: Structural Changes in Mitochondria and Other Spore Constituents during Conidium Maturation and Germination
More LessSummary: Structural changes in mitochondria and other spore constituents during maturation and germination of Trichoderma viride conidia were investigated. During maturation there was an increase in spore volume and number of mitochondria, while endoplasmic reticulum and electron-dense bodies disappeared. The stellate, precursor-lipid-granules underwent structural and chemical changes, the smooth plasmalemma invaginated and the conidial wall became thicker and relatively impermeable. The resting conidium contained few internal structures - only a single nucleus, several ovoid mitochondria and a few lipid granules.
After inoculation in germination medium there was a 4 h lag period, during which the conidia did not increase significantly in size, take up cotton blue or undergo a change in mitochondrial number. However, metabolic activity was evident from the reappearance of the electron-dense bodies, a change in the shape of some of the mitochondria and an increased permeability to the various fixation and embedding reagents.
The period of conidium swelling was accompanied by a reappearance of the endoplasmic reticulum, often in association with and seemingly interconnecting the various organelles. The lipid granules gradually disappeared and the electron-dense bodies became partially evacuated. Mitochondria increased in number, many having cup-shaped configuration; an unbalanced growth of surface membranes with respect to matrix may have been the physical cause for this shape. The nucleus divided at least once before the emergence of the germ tube. Structural changes continued during germ-tube emergence. The mother conidium in Trichoderma retained its ultrastructural integrity, and did not vacuolate.
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The Activities of Glutamate Dehydrogenases during Mycelial Growth and Sporophore Development in Coprinus lagopus (sensu Lewis)
More LessSUMMARY: The activity of the NADP-linked glutamate dehydrogenase (GDHnadp) increased in the pileus during sporophore development but remained at a low level in the parent mycelium and in the sporophore stipe. In contrast, the NAD-linked enzyme (GDHnad) increased in mycelium, stipe and pileus. It is suggested that the increase in GDHnadp activity is a developmental phenomenon. In vegetative mycelia the two GDH enzymes appeared to be regulated reciprocally; GDHnadwas subject to catabolite repression and urea derepression, while GDHnad was catabolite derepressed and repressed by urea. It is suggested that GDHnad may be the enzyme normally involved in ammonia assimilation, GDHnadp being reserved for specific functions associated with developmental alterations in metabolism.
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Scanning Electron Microscopic Studies on the Growth Features of Mycobacterium lepraemurium in Cell-free Medium
Z. Yoshii and M. NakamuraSUMMARY: The morphology of Mycobacterium lepraemurium, Hawaiian strain, cultivated on a glass slide in a cell-free liquid medium (NC5) at 30 °C for 12 weeks, was observed every 2 weeks with a scanning electron microscope. Flourishing growth and microcolonial growth occurred in the peripheral and central areas, respectively, of a smear. Elongation, septum formation, division, budding and branching in the bacteria were seen between the second and fourth weeks of incubation, which may correspond to the beginning of the exponential phase. After this period, the number of bacilli suddenly increased, and intertwined, elongated cells with large granules appeared. These features gradually increased until the stationary phase was reached after 12 weeks of incubation. These observations are the first scanning electron microscopical descriptions of the growth patterns of Mycobacterium lepraemurium in vitro.
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Sporulation in Protoplasts of the Yeast, Saccharomyces cerevisiae
More LessSUMMARY Protoplasts of Saccharomyces cerevisiae cultivated in sporulation medium with an osmotic stabilizer at pH 7·0 were studied by means of light and electron microscopy. After 15 h, mature ascospores with complete cell walls were formed in the protoplasts. During sporulation, protoplasts regenerated only the fibrillar wall component and were osmotically sensitive. By using snail enzymes to block fibril regeneration on the protoplast surface, it was proved that sporulation was quite independent of such regeneration. Most of the ascospores produced by protoplasts were viable, osmotically resistant and gave rise to new yeast cells. Meiotic division and sporulation in the yeasts are processes quite independent of the presence of the ascus wall.
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- Genetics And Molecular Biology
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Progress of Competence Development in Bacillus subtilis
More LessSUMMARY: B. subtilis can develop the capacity to take up transforming DNA and at the same time continue to grow for about 1½ to 3 h. Such growing competent cells are susceptible to penicillin. The competent cells, if allowed further growth, eventually become penicillin insensitive.
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A Search for Transmissible Pathogenic Characters in Invasive Strains of Escherichia coli: the Discovery of a Plasmid-controlled Toxin and a Plasmid-controlled Lethal Character Closely Associated, or Identical, with Colicine V
More LessSummary: Most invasive strains of Escherichia coli from man and domestic animals were lethal for chickens and mice. The lethal characteristic was not, in general, transferred when invasive strains were grown in mixed culture with non-pathogenic strains of E. coli, although two transmissible plasmids coding for pathogenic properties were discovered.
One plasmid, designated Vir, was found in an E. coli strain causing bacteraemia in a lamb. It transferred at high rate to several strains of E. coli, including a rec A− k12 strain, to Salmonella typhi, Salm. typhimurium and Shigella sonnei. Culture filtrates and, especially, bacterial ultrasonicates of Vir+ strains were toxic for chickens, mice and rabbits. The toxin was heat-sensitive, acid-sensitive and non-diffusible. Organisms producing it were agglutinated by specific Vir+ antisera; their toxic activity was not neutralized. The transfer factor of the Vir plasmid was fi+ and could transfer antibiotic-resistance determinants in addition to the Vir determinant.
The other plasmid was first discovered in an E. coli strain f120, isolated from an outbreak of bacteraemia in chickens. Organisms of E. coli k12 and of other E. coli strains acquiring this plasmid during mixed culture with f120 were increased in lethality for chickens and mice; this was associated not with toxic activity but with greater ability to survive in blood and peritoneal fluids. Strain f120 possessed transmissible ColV and Collb plasmids; increased lethality was closely associated with the ColV plasmid. When the ColV plasmids of another six wild strains of E. coli of varied origin were transferred to organisms of E. coli k12, the lethality increase was similar to that for ColV transfer from f120.No lethality change accompanied transfer of other Col plasmids. It was concluded that colicine V itself might be responsible for the increased lethality.
Strains of E. coli associated with bacteraemia in man and animals commonly produced colicine V.
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- Physiology And Growth
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Events Surrounding the Early Development of Euglena Chloroplasts: Experiments with Streptomycin in Non-dividing Cells *
More LessSUMMARY: The concentration of streptomycin (Sm) which selectively inhibits light-induced chloroplast development in non-dividing Euglena is the same as that which induces the loss of green-colony forming ability in dividing organisms. This concentration of Sm has no effect on division or viability. Chlorophyll synthesis is insensitive to streptomycin for the first 12 h of development but is strongly inhibited after this time. Between 72 and 96 h after the beginning of chloroplast development, Sm-treated organisms contain 10 % of the chlorophyll and 24 % of the carotenoids of algae developing in the absence of the antibiotic. The chlorophyll-to-carotenoid ratio in treated organisms at 72 to 96 h is 0·9, the same as is found at 12 h for organisms developing in the absence of Sm. In the presence of streptomycin, Euglena never develops the ability to fix CO2 photosynthetically, although CO2 fixation after 12 h of development in the absence of the antibiotic can be readily detected. At 12 h of chloroplast development the following parameters are at comparable levels in Sm-treated and untreated organisms: the bound forms of chlorophyll, concentration of cytochrome 552, the activities of ribulose diphosphate carboxylase, NADP-triose phosphate dehydrogenase, the enzymes converting ribose-5-phos- phate to ribulose diphosphate, and photosystem II activity measured as dye reduction.
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Events Surrounding the Early Development of Euglena Chloroplasts: I Cellular Origins of Chloroplast Enzymes in Euglena*
More LessSUMMARY: During chloroplast development, the large increases in ribulose diphosphate carboxylase (RUDPCase) activity and cytochrome 552 concentration follow the pattern of chlorophyll synthesis, in that the formation of these two enzymes is inhibited by streptomycin (Sm) and by chloramphenicol (Cm) beyond 12 h of development. Neither enzyme can be detected in w 3 bul, a mutant of Euglena in which chloroplasts and chloroplast DNA are undetectable. In contrast, the NADP-linked triose phosphate dehydrogenase (NADP-TPDase), another plastid- localized enzyme, increases in activity without the 12 h lag normally observed for chlorophyll synthesis; this increase in activity is not inhibited by Sm and Cm, but is inhibited by cycloheximide, an antibiotic which acts on 87 S cytoplasmic ribosomes. NADP-TPDase activity is present at the same level in w 3 bul as in the dark-grown wild-type organisms. These data are interpreted to mean that NADP-TPDase is coded in the nuclear DNA, and is translated on 87 S cytoplasmic ribosomes. The sensitivity of the increase in cytochrome 552 and RUDPCase activities to Sm and Cm indicates that they are translated, at least in part, on the 68 S ribosomes of the chloroplast. Thus, chloroplast differentiation in Euglena is dependent upon information and synthetic machinery from both the plastid and the rest of the cell. Since total cellular protein does not change significantly during chloroplast development in resting cells, we conclude that protein turnover probably occurs.
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Nitrogen Fixation and Co-oxidation of Ethylene by a Methane-utilizing Bacterium
More LessSUMMARY: A methane-oxidizing bacterium, isolated from soil, was capable of fixing nitrogen. Nitrogenase activity could be assayed by acetylene reduction when the bacterium was growing on methanol but not when growing on methane, although 15N2 was fixed. Bacteria growing on methane co-oxidized ethylene but methanolgrowing cells did not. The organism was extremely sensitive to oxygen when dependent on N2 as nitrogen source, a consequence of the sensitivity of its nitrogenase towards oxygen.
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The Production of β-1,3 Glucanase by a Thermophilic Species of Streptomyces
G. Lilley and A. T. BullSUMMARY A β-1,3 glucanase-producing, thermophilic Streptomyces species was grown successfully in a chemically defined medium. Enzyme synthesis was semi-con stitu- tive and subject to catabolite repression by metabolizable carbon substrates; it was inducible by gentiobiose, a molecule structurally unlike its substrate. The enzyme was truly extracellular. Maximum batch production of the glucanase was obtained by adding the inducer late in the fermentation.
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- Short Communications
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Plasmid-determined Fusidic Acid Resistance in the Enterobacteriaceae
More LessSummary: A number of R factors have been shown to determine resistance to fusidic acid, an antibiotic effective against Gram-positive bacteria. Among several R factors examined for this property only one was fi−; the remainder were fi+ and of the FII compatibility group.
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Volumes and issues
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