- Volume 14, Issue 2, 1956
Volume 14, Issue 2, 1956
- Article
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Isolation of High Acid-yielding Mutants of Aspergillus niger by a Paper Culture Selection Technique
More LessSUMMARY: A method for isolating and identifying high acid-yielding mutants of Aspergillus niger is described. This involves cultivation of the organisms on absorbent paper soaked in an indicator medium. The advantages of the technique and the criteria used for selecting biochemically interesting mutants are briefly described. The greater acid production of mutants selected by paper culture has been confirmed by comparing yields of citric acid in surface and submerged fermentations with those given by the wild type strain.
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Production of Citric Acid by Mutants of Aspergillus niger
More LessSummary: Mutants of Aspergillus niger, Wisconsin strain 72–4, were produced by multiple X-ray and ultraviolet irradiation. The mutants differed from the parent strain culturally and in citric acid production. Comparison between the yields of citric acid from the parent strain and mutants showed significant increases with the latter. In crude sugar media, such as ferrocyanide-treated brown sugar, yields of citric acid equivalent to 80% of the sugar fermented were obtained in aerated culture, whereas only 21% was obtained with the parent strain.
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Nitrite Production by Heterotrophic Bacteria
More LessSUMMARY: Heterotrophic bacteria were studied which produced nitrite in the presence of ammonia and in the absence of nitrate. A soil extract medium was prepared which allowed good growth as well as nitrite production in the absence of nitrate. Quantitative data are recorded showing that ammonia decreases as nitrite accumulates when four different cultures are grown in the soil medium. Resting cell studies add further evidence that some heterotrophic bacteria can convert ammonia to nitrite.
A defined medium was prepared containing glucose or sodium acetate as the carbon source and NH4Cl as the nitrogen source. This medium supported growth and nitrite production; however, optimum conditions for growth were not established. Neither growth nor nitrite accumulation was as great in defined media as in soil-extract media. Results from defined media and from resting-cell studies rule out the possibility of any nitrate contamination.
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The Amino Acid Metabolism of Aspergillus flavus
More LessSUMMARY: A complete quantitative amino acid analysis of the free amino acid fraction and the mycelial residue fraction of the mould Aspergillus flavus during different stages of growth has been made by the circular paper chromatographic technique. A qualitative analysis of the amino acid composition of the culture fluid has also been accomplished. The changes of the individual amino acids present in the various fractions on different days of incubation were studied and their implications discussed.
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Conversion of Cholesterol into Coprosterol by Bacteria in vitro
A. Snog-Kjaer, I. Prange and H. DamSummary: Certain anaerobic bacteria from human faeces were found to be able to hydrogenate cholesteroli n vitro; some also decreased to a marked degree the total amount of sterols in the incubation mixture. Other organisms did not hydrogenate or decrease the amount of sterols under the conditions chosen; among these were; Clostridium welchii, C. sporogenes, Bacterium bifidum, various streptococci and micrococci, Escherichia coli, Aerobacter aerogenes. A germ-free filtrate from human faeces was inactive. In a series of incubated samples, where the Tschugaeff reaction was applied to the non-saponifiable matter, an orange colour developed, suggestive of the presence of lathosterol.
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The Virulence of Biochemical Mutants of Erwinia aroideae for Varieties of Radish and Turnip
More LessSummary: Biochemical mutants of Erwinia aroideae displayed a pattern of virulence and avirulence for varieties of radish and turnip similar to the pattern previously reported for different host species. Three types of host response were noted when slices of the fleshy storage organs were inoculated: uniformly resistant, uniformly susceptible, and a variable response in which individuals of a given variety may be resistant or susceptible to a specific mutant. Prototrophic reversions from an avirulent mutant requiring arginine and from an uncharacterized mutant with diminished virulence were as virulent as the parental strain for all varieties of radish and turnip.
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The Streptococci of Lancefield's Group E; Biochemical and Serological Identification of the Haemolytic Strains
More LessSummary: The non-pathogenic beta-haemolytic streptococci from milk, forming Lancefield’s group E, were physiologically defined as Streptococcus infrequens and S. subacidus. They were found to fall into four serological types. Fractionation with ethanol showed that both group- and type-specific fractions of these organisms are of polysaccharide nature which probably accounts for the success in using formamide extracts for the type-precipitin reactions. S. subacidus possesses two type antigens, only one of which is also found in S. infrequens.
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Substrates for Myxococcus virescens with Special Reference to Eubacterial Fractions
More LessSummary: It was found that a mutant strain of Myxococcus virescens, able to grow dispersed in liquid media, oxidized the cytoplasmic lipids and proteins of Escherichia coli. Cell walls and nucleic acids of E. coli were not oxidized, and the acid soluble fraction was only slightly oxidized. It was also observed that the cytoplasmic contents of intact cells of E. coli were not available for oxidation. Glucose and starch were oxidized by the strain of Myxococcus virescens used.
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Biophysical Studies of the Virus System of Vesicular Stomatitis
More LessSummary: Biophysical studies of the virus system of vesicular stomatitis passaged in eggs showed that the major part of the infectivity was associated with a component of sedimentation coefficient 625S. A component of sedimentation coefficient 330 S was observed also, and is probably a non-infective product of the disintegration of the 625S component. These components contribute about 35% of the total complement-fixing activity of the virus system. All infective materials were handled in subdued light (Skinner & Bradish, 1954). Electron micrographs of concentrates of the infective fraction revealed rods, of length 175 mμ. and diameter 69 mμ., and almost spherical granules of diameter 65 m. These particles are identified with the 625S and 330S sedimentation components. The remaining 65% of the total complement-fixing activity was associated with two discrete components of sedimentation coefficients about 20S and 6S. The first of these components may contribute up to 0·1% of the total infectivity of the virus system. The structure of the virus system is discussed in relation to the data obtained.
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The Production of Hydrogen Sulphide from Thiosulphate by Escherichia coli
More LessSummary: Suspensions of non-proliferating Escherichia coli produced H2S from thiosulphate in the presence of pyruvate or acetaldehyde. Production of H2S was slight in the presence of α-ketoglutarate and α-ketobutyrate. Organic acids such as malate, fumarate, succinate, lactate, formate and acetate, aldehydes other than acetaldehyde and monohydric alcohols either had no effect or inhibited H2S production from thiosulphate. H2S was not produced from sulphite, bisulphite or sulphate, either in the presence or in the absence of the above-named compounds. Crude cell-free extracts of Escherichia coli produced H2S from thiosulphate in the presence of pyruvate. Experiments with dialysed extracts showed that inorganic phosphate, Mg ions and cocarboxylase were essential for H2S production. Treatment of the extracts with anion exchange resin revealed that in addition, coenzyme A was indispensable for H2S production from thiosulphate. The addition of DPN to extracts dialysed or treated with anion exchange resin did not influence H2S production to a marked degree.
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Inhibition of Coli Bacteriophage T2 by Apple Pectin
More LessSummary: Apple pectin in a complex organic medium partly protected Escherichia coli strain B from lysis by T2 phage. It was not bactericidal or virucidal. The rate of adsorption of the phage was unaltered, but part of the initially adsorbed phage could be eluted with distilled water at 0°, as the second irreversible step of adsorption was inhibited by pectin. It was shown in one-step growth and single cell burst experiments that phage multiplication was reduced. The release of any formed phage from the host was not affected. The protective effect of the pectin resulted from the failure of some of the phage particles to penetrate into the host cell and from its action in decreasing phage synthesis in those cells where penetration did take place. It is suggested that this non-specific polysaccharide may exert its protective action because of its polymeric electrolyte nature.
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Identification of Azotobacter Species by Fluorescence and Cell Analysis
More LessSummary: Eight strains of Azotobacter agile and A. vinelandii were studied for their ability to elaborate water soluble compounds with a fluorescence that would characterize each when observed under ultraviolet light of 3600 A. It was found that the material produced by A. agile fluoresced white, whereas that produced by A. vinelandii fluoresced green. Additional studies with iron and molybdenum showed that molybdenum enhanced synthesis of fluorescent material in both species and iron appeared to quench the fluorescence. A pH/fluorescence curve for the fluorescent material of each species showed that, although similarities were evident, sufficient difference existed to permit recognition of each. Analyses of dried cell material revealed a much higher protein content in A. agile than in A. vinelandii, but the amount of one of their amino acids, lysine, was essentially the same, on the basis of protein, for each species.
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Studies with a Pseudomonad able to Grow with Creatine as Main Source of Carbon and Nitrogen
More LessSummary: An organism able to grow in a simple medium with creatine as the main C and N source was isolated from garden soil and subsequently identified as Pseudomonas ovalis Chester. Organisms harvested from a creatine-containing medium destroyed creatine with the uptake of O2 and the formation of CO2, NH2 and urea. With the exception of urea the quantities of reactants fell short of those required by theory, and only part of the deficiency could be attributed to oxidative assimilation. Optimal conditions for the growth of active organisms and for the destruction of creatine were determined. In these conditions the organisms destroyed, in addition to creatine, only arginine and agmatine from a variety of compounds tested; compounds not attacked included creatinine and glycocyamine. Ability to oxidize creatine was partially lost during repeated washing and storage of the organisms, and was inhibited by p-chloromercuribenzoic acid and fatty acids.
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The Pathway of Creatine Catabolism by Pseudomonas ovalis
More LessSUMMARY: Creatine is oxidized by suspensions of a strain of Pseudomonas ovalis Chester harvested from media containing creatine as main source of carbon and nitrogen. A possible pathway of degradation of at least part of the creatine is: (a) hydrolysis of creatine to sarcosine and urea, (b) oxidation of sarcosine to glycine and formaldehyde, (c) oxidation of the latter products to CO2 and NH3 with considerable concurrent oxidative assimilation.
Soluble enzyme preparations catalysing stage (a) were obtained by aqueous extraction of either acetone-dried or toluene-treated organisms. The enzyme for stage (b) was present in the insoluble residue from the latter organisms.
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Influence of Aliphatic Organic Acids and Metal Ions on Numbers of Local Lesions Produced by a Tobacco Necrosis Virus
More LessSUMMARY: Many aliphatic organic acids, when sprayed on the leaves of bean plants, decreased the numbers of local lesions produced following inoculation with a tobacco necrosis virus. Citric and succinic acids were effective only when applied before or during the period of virus establishment. The inhibitory effect of these acids could be annulled by certain metal nitrates.
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The Wavelengths of Helical Bacterial Flagella
More LessSUMMARY: During normal movement most motile bacteria carry a straight tail, which, when the movement slows, stiffens into helical structures commonly called flagella. The helices of many kinds of bacteria were photographed with a sunlight dark-ground microscope, and their wavelengths measured. Mean values and standard deviations were calculated for each strain and then for the species. ‘Biplicity’ (two wavelengths per bacterium, one twice the other) was observed frequently. Each strain appears to have its own constant wavelengths. The wavelength differs in different kinds of bacteria from 0·60 to 5·058μ., the distribution over the various species not revealing a distinct pattern nor any obvious correlation with other characteristics. The wavelength is affected by temperature, pH value, and colloid content of medium. These features, and the effects of drying, make stained preparations useless for measuring.
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Inhibition of the Growth of Fungi by Streptomyces spp. in Relation to Nutrient Conditions
More LessSUMMARY: Several species of soil actinomycetes arrested the growth of fungi by antibiotic secretions on agar media containing 10 g. glucose/1. On media of lower glucose concentration, the fungi continued to grow in the presence of the actinomycetes, but there was evidence that traces of antibiotic substances were still being formed. In sand moistened with liquid medium containing glucose, S. albidoflavus limited early growth of Fusarium culmorum by antibiotic action and also attacked preformed fungus mycelium directly. The effectiveness of these antagonistic mechanisms was decreased when the glucose concentration was lowered. The fungus and the actinomycete grew together on a variety of natural organic materials, but only when dried grass was used did the actinomycete arrest growth of the fungus at a distance.
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The Effect of Adding Clays to Mixed Cultures of Streptomyces albidoflavus and Fusarium culmorum
More LessSUMMARY: An antibiotic present in culture filtrates of Streptomyces albidoflavus was inactivated by clays and by suspensions and extracts of soils. When this actino-mycete was grown with Fusarium culmorum in a sand + bentonite mixture moistened with nutrient solution, it did not antagonize the fungus by antibiotic secretions. However, suppression of fungus growth was observed even in the presence of bentonite particularly when glucose was present in abundance; this effect was attributed to competition between the organisms for limiting nutrients. The actinomycete also lysed the contents of the fungus mycelium in sand culture but not when bentonite was added. The lytic agent appeared to differ from the antibiotic. Neither antibiotic action nor direct (lytic) attack on the fungus was demonstrated in sterilized soil.
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The Action of Isonicotinic Acid Hydrazide on the Metabolism of Mycobacterium smegmatis
More LessSUMMARY: The effect of various substrates on the oxygen uptake of Mycobacterium smegmatis grown in the presence of glucose and glycerol was investigated; isoniazid had no effect on these oxidations. Three different effects of isoniazid on the metabolism of this organism were demonstrated, (i) Growth of the organism in a minimal medium in the Warburg apparatus was prevented by the addition of isoniazid, the amount required being related to the number of organisms present, their age and their sensitivity to the drug as measured by serial dilution in the test tube, (ii) In confirmation of the report of Zeller, Barsky, Berman & Fouts (1952) it was found that isoniazid inhibited the oxidation of putrescine by M. smegmatis. (iii) Following the work of Gray (1953) the effect of isoniazid on the oxidation of various substrates by M. smegmatis grown in the absence of glucose and glycerol was studied. Isoniazid caused a 30 % inhibition of the oxidation of acetate. The amount of isoniazid required to inhibit the oxidation of putrescine and acetate was the same whether the strain used was sensitive or resistant to isoniazid as measured by growth in the test tube.
In an attempt to discover the mechanism by which isonicotinic acid hydrazide (isoniazid) inhibits the growth of mycobacteria, the effect of isoniazid on their respiration was investigated. Preliminary work involved a study of the oxidative metabolism of the saprophytic organism Mycobacterium smegmatis.
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The Effect of Isonicotinic Acid Hydrazide on the Oxidative Metabolism of Mycobacterium tuberculosis var. bovis B.C.G
More LessSUMMARY: Isoniazid inhibited the oxidation of acetate in the Warburg apparatus by Mycobacterium tuberculosis B.C.G. and other mycobacteria but not by any of the other organisms tested. This effect was investigated in more detail with B.C.G. Inhibition of acetate oxidation was obtained by isoniazid whatever medium had been used for growth. The amount of isoniazid required for inhibition was related to the number of organisms present and their sensitivity to isoniazid as measured by the usual test-tube method. None of the substances reported to antagonize isoniazid inhibition was effective in annulling inhibition by isoniazid in this acetate oxidation system. The effect of various mixtures of drugs on acetate oxidation was tested. Concentrations of isoniazid and streptomycin which when used singly were insufficient to inhibit acetate oxidation, were inhibitory when used together. Similarly, mixtures of isoniazid + p-aminosalicylic acid (P.A.S.) or terramycin, or of strepto-mycin + P.A.S., at concentrations which were subinhibitory when used singly, were also effective in inhibiting acetate oxidation. It is suggested that the action of drugs or drug mixtures could usefully be investigated by this or similar techniques which have the advantage of largely eliminating the selection of resistant strains.
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Some Factors Affecting Lactase Formation and Activity in Saccharomyces fragilis
More LessSUMMARY: A method for determining the lactase activity of Saccharomyces fragilis is described. The yeast is treated with cetyltrimethylammonium bromide, and the products of lactose hydrolysis are fermented with S. mandshuricus. The formation of lactase in S. fragilis was studied by using the continuous culture technique. Lactase formation was inhibited by the presence of sugars in the medium at concentrations greater than 0·001% (w/v), but the magnitude of the inhibition and the range of activity over which it occurred depended on the nature of the sugar in the medium. With media containing glucose or sucrose, activities up to 10 units lactase/mg. dry weight were found when the sugar concentration was less than 0·001% (w/v), while at high concentrations lactase activity was almost completely absent. With media containing galactose or lactose at concentrations less than 0·001% (w/v) activities of approximately 100 units lactase/mg. dry weight were observed, while at concentrations greater than 0·01% (w/v) the activity was less. The mean generation time of the organisms and the concentrations of growth factors, ammonium and hydrogen ions had, over the range tested, no significant effect on lactase formation. The lactase activity found in intact organisms was always lower than the activity found in disrupted organisms, irrespective of the conditions under which the yeast had been grown. Possible interpretations of this phenomenon are discussed.
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Antibiotic Activity of Actinomycetes in Soil and their Controlling Effects on Root-rot of Wheat
More LessSUMMARY: A study has been made of conditions affecting the production of antibiotics in three soils by a number of unidentified Streptomyces spp. capable of inhibiting a variety of test organisms in vitro. In actinomycete-inoculated soils, antibiotic production was demonstrated only in sterile soils supplemented with a suitable organic source. The greatest accumulations of antibiotics were found in a neutral soil with added glucose (2·5%) while under similar conditions, no antibioties, or only traces were recovered from acid and alkaline soils. Antibiotics, however, could be recovered from inoculated acid soil, following neutralization and the addition of glucose. Fresh grass (3%), clover (3%) and soybean meal (2%) were also suitable supplements for antibiotic production by the majority of the actinomycetes, though the amounts of antibiotics were considerably less than in glucose-treated soils.
In greenhouse experiments the assessment of root damage to wheat seedlings in sterile soil demonstrated that all the actinomycetes tested significantly reduced the degree of root-rot caused by Helminthosporium sativum. In the neutral and alkaline soils a relationship was evident between disease incidence and degree of antagonism exhibited by actinomycetes in vitro, suggesting that antibiotics were responsible. No such relationship was observed between disease control and the antibiotic-producing abilities of the actinomycetes in soil as determined by standard assay procedures.
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Effects of Clupein and of its Degradation Products on a Rhizobium Bacteriophage, on its Host Bacterium and on the Interaction between the two
More LessSUMMARY: Clupein at 0·02–0·05% in the liquid nutrient medium used to cultivate nodule bacteria, rapidly killed the bacteria and slowly inactivated a bacteriophage that attacked them. When added to a bacterial culture in liquid medium before adding phage, clupein prevented phage and bacteria from combining; when added after the two had combined, clupein interrupted further stages of phage-host interaction. Clupein at 0·0016% acted bacteriostatically and slowed phage multiplication but did not stop it.
Trypsin and chymotrypsin hydrolyse clupein, trypsin breaking about twice as many peptide bonds as chymotrypsin. At a concentration corresponding to 0·02–0·05% clupein, the peptides produced by chymotrypsin acted bacteriostatically in the liquid nutrient medium; the peptides inactivated phage much more slowly than did intact clupein, and they inhibited phage multiplication by interfering with the combination between phage and host. When added after phage and bacteria had combined, the peptides did not interfere with further stages of phage-host interaction. The smaller peptides produced by trypsin had no effect on host bacteria, phage, or phage/host interaction.
Phage preparations partially inactivated by clupein had their activity partially restored by incubation with trypsin or chymotrypsin.
Clupein, but none of its hydrolytic products, made phage with much non-phage material sedimentable by slow-speed centrifugation.
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Observations on the Anomalous Proteins Occurring in Extracts from Plants Infected with Strains of Tobacco Mosaic Virus
More LessSUMMARY: When extracts from plants infected with various strains of tobacco mosaic virus were ultracentrifuged, the non-infective supernatant fluids still contained 0·5-5% of the protein serologically related to the viruses. The small, mostly spherical, particles aggregated to form short rods as the antigen was progressively purified by precipitation with acid or salts. It formed long rods when heated in pH 5·5 buffer or when incubated with trypsin. As the particles increased in length, their serological behaviour in precipitation tests changed from ‘somatic’ to ‘flagellar’ type.
Purified preparations of the unsedimented antigen from plants infected with either of two virus strains contained 0·1–0·2% phosphorus, seemingly in the form of a ribose nucleic acid. No evidence was obtained that the preparations were mixtures containing some particles with the 0·5% phosphorus characteristic of infective virus and some particles of protein free from nucleic acid.
One virus strain produced a higher ratio than the others of unsedimented to sedimented antigen. The amount of unsedimented antigen was correlated with the total content of anomalous protein when the protein was increasing rapidly, but later it fluctuated unpredictably. No conditions were found that consistently favoured its accumulation, but when plants systemically infected with the type strain were kept at 36 °, the total amount of antigen decreased, while the amount unsedimented sometimes increased.
The proportion of the total antigen now obtained as poorly infective nucleoprotein is much less than 10 years ago, when a third of it sedimented in the ultracentrifuge but failed to compact into a pellet. Now the uncompacted sediment, with all the host plants and virus strains used, contains only a trivial part of the total antigen. The virus released into sap when leaves are minced is, weight for weight, more infective than the virus that remains in the leaf residues until it is released by fine grinding.
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The Kinetics of Urease Activity in Corynebacterium renale
More LessSUMMARY: The urease activity of Corynebacterium renale has been studied in washed suspensions and in cell-free extracts of the micro-organism. The urease is constitutive, and whole organisms have a urease activity of 17,000-22,000μg. NH3/mg. dry wt. organisms/hr. in 0·5m-urea in phosphate buffer at pH 7·0. In a cell-free extract the enzyme is optimally active at pH 7·5, has a Michaelis constant of 0·030m, and a temperature velocity constant of 7800 cal.; the activity is inhibited by atmospheric oxygen and by thiourea, but not by sixteen other analogues of urea tested.
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Saccharomyces pretoriensis n.sp.—from South African Soil
More LessSUMMARY: A new Saccharomyces species has been isolated from soil. It is distinguished from other species of the genus by its ability to ferment glucose, galactose, sucrose, maltose and raffinose 1/3, as well as by its small cells and the formation of protuberances resembling conjugation tubes during sporulation.
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