- Volume 133, Issue 5, 1987
Volume 133, Issue 5, 1987
- Pathogenicity And Medical Microbiology
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Production of Cuticle-degrading Enzymes by the Entomopathogen Metarhizium Anisopliae during Infection of Cuticles from Calliphora Vomitoria and Manduca Sexta
More LessSUMMARY: A biochemical and histochemical investigation with specific substrates and inhibitors was used to visualize protease, esterase and aminopeptidase activities produced in situ during penetration of Calliphora vomitoria and Manduca sexta cuticles by hyphae of the entomopathogenic fungus Metarhizium anisopliae. Two endoproteases, and aminopeptidase and esterase activities, were mainly localized in simple and complex appressoria and germinating conidia. The effect of inhibitors on two characterized proteases (Pr1 and Pr2) and aminopeptidase activity in appressorial plates was quantified by microdensitometric measurement of reaction products. Pr1 and Pr2 activities were differentially inhibited by various protease inhibitors. Pr1, Pr2, esterase, aminopeptidase and N-acetylglucosaminidase (exochitinase) activities were present during penetration as detected directly following desorption from fungal and cuticle components. The proteases produced in situ were fractionated, and were shown by immunological and enzymological criteria to be the same as those produced in culture media. The sequence of enzyme appearance in situ showed that production of proteolytic enzymes precedes exochitinase production. No production of endochitinase was found before or during hyphal penetration of the cuticle.
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- Physiology And Growth
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Sulphur Sources for Growth of Chlorella Fusca and Their Influence on Key Enzymes of Sulphur Metabolism
More LessThe green alga Chlorella fusca strain 211–8b (algal collection of Göttingen) is able to grow on more than 100 different sulphur sources such as mercaptides, disulphides, thioethers, sulphinic and sulphonic acids including ‘Good buffers’, sulphoxides, sulphones and sulphate esters. This suggests that green algae might be of importance for degradation of xenobiotics and natural compounds in the overall sulphur cycle. The data presented describe the growth of C. fusca on such sulphur compounds and the influence of these compounds on enzymes of the assimilatory sulphate reduction sequence.
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Thermotolerance of the Respiratory Chain of Bacillus Coagulans
More LessThe thermostability of the respiratory chain of Bacillus coagulans grown at different temperatures was similar to the thermotolerance of the primary dehydrogenases. NADH was the major electron donor to the respiratory chain in cells grown at both 37 °C and 55 °C. The respiratory chain from cells grown at 55 °C exhibited slightly greater thermostability than that from cells grown at 37 °C. NADH-supported respiration, as well as NADH dehydrogenase activity, was much more thermotolerant than that with succinate in cells grown at both temperatures. Membrane-bound succinate dehydrogenase could be stabilized by the addition of 10% (w/v) NaCl while NADH dehydrogenase exhibited intrinsic thermostability.
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Effect of Osmotic Shock and Shearing Forces on Ferric Enterochelin Transport in Escherichia Coli K12
More LessThe fes mutation in Escherichia coli K12, which inactivates enterochelin esterase, allows the cell to accumulate ferric enterochelin. The ferric complex of enterochelin was released in significant quantities from a fes mutant after osmotic shock. Analysis of the effects of the individual stages of the shock procedure in wild-type cells showed that prior exposure of cells to sucrose and EDTA was not required, careful dilution of cells into a hypo-osmolar medium being sufficient to induce efflux of Fe3+. Prior treatment with EDTA or exposure to shearing forces served either to enhance efflux or to induce efflux in isotonic media. Neither vitamin B12 nor 5′-nucleotidase was released from the periplasm by these procedures. The release observed under mild conditions was stimulated specifically by Co2+, did not occur at 0 °C, and was inhibited by 2,4-dinitrophenol at 37 °C. From these observations, it was concluded that the efflux of Fe3+ represents a physiological response of the cell to exposure to a hypo-osmolar medium. Such changes may enhance survival following physicochemical stressing of the bacterial outer membrane.
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The Involvement of Glutamine Synthetase/Glutamate Synthase in Ammonia Assimilation by Aspergillus Nidulans
More LessWild-type Aspergillus nidulans grew equally well on NH4Cl, KNO3 or glutamine as the only nitrogen source. NADP+-dependent glutamate dehydrogenase (EC 1.4.1.4) and glutamine synthetase (GS; EC 6.3.1.2) activities varied with the type and concentration of nitrogen source supplied. Glutamate synthase (GOGAT) activity (EC 1.4.7.1) was detected but it was almost unaffected by the type and concentration of nitrogen source supplied. Ion exchange chromatography showed that the GOGAT activity was due to a distinct enzyme. Azaserine, an inhibitor of the GOGAT reaction, reduced the glutamate pool by 60%, indicating that GOGAT is involved in ammonia assimilation by metabolizing the glutamine formed by GS.
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Relation between pH, Hydrogenase and Nitrogenase Activity, NH+ 4 Concentration and Hydrogen Production in Cultures of Rhodobacter Sulfidophilus
Y. Peng, P. Stevens, P. De Vos and J. De LeyRhodobacter sulfidophilus, a sulphide-tolerant, salt-tolerant member of the Rhodospirillaceae, was shown to produce molecular hydrogen on a mineral medium with lactate as electron donor and glutamate as nitrogen source. A maximum production of about 1·1 mmol H2 (mmol added lactate)−1 was found at pH 6·75. The in vivo activity of hydrogenase and nitrogenase showed that hydrogenase activity (and thus the H2 recycling system) was greatest at pH 6·5 and 6·75 and thus, at least partially, was responsible for the lack of H2 production below 6·75, but that nitrogenase activity was greatest at between pH 6·5 and 7·0, and decreased to zero at pH 8·0, resulting in reduced H2 production at pH 7·5 and none at pH 8·0. When sufficient NH+ 4 had accumulated in the culture, nitrogenase activity remained below the maximum value and no H2 production occurred.
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Chemolithotrophic and Autotrophic Growth of Thermothrix thiopara and Some Thiobacilli on Thiosulphate and Polythionates, and a Reassessment of the Growth Yields of Thx. thiopara in Chemostat Culture
More LessSeveral thiobacilli and Thermothrix thiopara were grown in chemostat culture with inorganic sulphur compounds as growth-limiting energy substrates for autotrophic growth. Thiobacillus neapolitanus, Thiobacillus thiooxidans and Thiobacillus acidophilus were all able to use thiosulphate, trithionate or tetrathionate as sole energy substrates, as was Thx. thiopara grown at 72 °C. ‘True growth yields’ (Y max) were estimated for the organisms and showed yields of T. neapolitanus to be the same on trithionate and thiosulphate, thereby suggesting that only oxidative, and not substrate-level, phosphorylation is involved in energy conservation in this organism. The yield data suggested that substrate-level phosphorylation could, however, be significant in T. acidophilus. Thiobacillus versutus only grew with thiosulphate, with which it gave yield values similar to those of T. neapolitanus and T. thiooxidans. Thx. thiopara exhibited maximum specific growth rates on thiosulphate, trithionate and tetrathionate of 0·5, 0·23–0·25 and <0·06 h−1 respectively. Its yields on all three were much greater than those of the thiobacilli: the Y max on thiosulphate was in the range 18·0–22·8 g mol−1, but the organisms contained only about 29% carbon and 48% protein. These values do, however, make Thx. thiopara the highest-yielding inorganic-sulphur-oxidizing chemolithoautotroph yet described.
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Oxygen Profiles in, and in the Agar Beneath, Colonies of Bacillus Cereus, Staphylococcus Albus and Escherichia Coli
More LessThis paper reports the use of microelectrodes to measure O2 penetration in different aged colonies of Bacillus cereus, Escherichia coli and Staphylococcus albus. In young (18 h) colonies of B. cereus and E. coli O2 disappeared at depths of 25–30 μm and 35–40 μm respectively. In young S. albus colonies, O2 reached a minimum but was never completely absent. As colonies aged (24–168 h) the depth to which O2 penetrated increased.
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Bacteriolytic Activity of Seminalplasmin
More LessSeminalplasmin, an antimicrobial protein from bovine seminal plasma, lysed both Gram-positive and Gram-negative bacteria but not Candida albicans. The lytic activity was not lysozyme-like and was not affected by inhibitors of RNA or protein synthesis or by azide; it was strongly inhibited by divalent cations like Ca2+, Mn2+ and Mg2+ at millimolar concentrations. Maximum lysis of Escherichia coli was obtained at 37°C; heat treatment of E. coli drastically reduced its susceptibility to lysis by seminalplasmin. E. coli cells in the stationary phase of growth were lysed much less than those in the exponential phase, and those grown in an enriched medium were lysed much more than those grown in a minimal medium. It appears that the lytic activity of seminalplasmin is due to the activation of an autolysin.
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A Nitrate Non-utilizing Mutant of Gibberella Zeae
More LessA nitrate non-utilizing mutant of Gibberella zeae (Fusarium graminearum) was isolated following UV mutagenesis and filtration enrichment. In preliminary reports, this mutant was designated gdh, but it has now been renamed nnu. This mutant has a complex pleiotropic phenotype which affects most aspects of nitrogen utilization. The wild-type parent could use nitrate, nitrite, ammonium, urea, polyamines, and most amino acids and purines as sole source of nitrogen, but the nnu mutant could use only putrescine, anthranilic acid, and the amino acids asparagine, aspartic acid, glutamic acid, glutamine, ornithine, proline and tyrosine. Most of these compounds could also serve as the sole source of both carbon and nitrogen. Addition of 1 mM-glutamine or 1 mm-proline to the medium resulted in a synergistic growth response if spermidine, arginine, citrulline, histidine, phenylalanine or tryptophan was present as the primary nitrogen source. Addition of 1 mM-ammonium to the medium repressed growth when putrescine, aspartic acid, asparagine, glutamic acid or tyrosine was the primary nitrogen source. The addition of ammonium did not repress growth when anthranilic acid, glutamine, ornithine or proline was the primary nitrogen source. The nnu mutant grew more rapidly than its wild-type parent in race tubes on some complex media. The complexity and diversity of the alterations in nitrogen catabolism caused by the nnu mutation suggest that this locus is important in the regulation of nitrogen catabolism by G. zeae.
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The Role of Exopolysaccharides in Adhesion of Freshwater Bacteria
More LessCultures of two strains of freshwater bacterial isolates adhered readily to inert glass surfaces exposed in the growth medium. The process of microbial film formation could be followed by a new staining technique based on congo red, a dye specific for carbohydrate material. In conjunction with a chemical assay for total carbohydrate, the association of extracellular polysaccharides with attached cells was demonstrated. Under optimal growth conditions, the involvement of exopolysaccharide in the adhesion process appeared to follow the initial attachment of bacterial cells, leading to the formation of microcolonies enmeshed in polysaccharides. A non-polysaccharide-producing mutant attached to glass slides in numbers similar to the wild-type bacteria, but did not form microcolonies. Growth conditions such as glucose or Ca2+ limitation which affected polysaccharide synthesis in the wild-type prevented microcolony formation, but not cell attachment. It is proposed that exopolysaccharide production is involved in the development of the surface films, but possibly not in the initial adhesion-process. In those strains which do produce polysaccharide, the cells which attach develop into microcolonies.
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Protein Synthesis and Degradation during the Differentiation Cycle of Rhodomicrobium Vannielii Swarmer Cells
More LessSUMMARY: Rhodomicrobium vannielii swarmer cells, when synchronized by selective filtration, differentiate synchronously with the loss of motility and synthesis of a prostheca, and subsequently form a daughter cell. Synchronized swarmer cells synthesized an 11·5 kDa protein under dark anaerobic conditions which disappeared from the soluble fraction when the culture was subsequently incubated in the light and the differentiation cycle thus initiated. The protein did not accumulate under dark aerobic conditions and it may play a role in the inhibition of differentiation in the light limited and thus energy limited swarmer cell.
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- Systematics
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SIMCA Pattern Recognition in the Analysis of Streptomycete Fatty Acids
More LessBiomass from representative strains of Streptomyces cyaneus and related taxa was degraded using an alkaline methanolysis procedure. The resultant fatty acid methyl esters were analysed by gas chromatography and the quantitative data examined using the SIMCA statistical package. The S. cyaneus strains were recovered in two major clusters. The larger of these contained sixteen strains which had been previously classified as S. cyaneus in a numerical phenetic survey. The remaining S. cyaneus strains formed a heterogeneous aggregate cluster together with representatives of other streptomycete species. A third cluster encompassed a number of blue-spored streptomycetes from soil. Disjoint principal components analysis in conjunction with cross-validation analyses demonstrated that the S. cyaneus group and the blue-spored soil isolates could be represented by statistically stable principal component models. The fact that the initial, numerically circumscribed, S. cyaneus taxon was only partially recovered by the numerical analysis of fatty acid data supports the view that this taxon contains strains which are not entirely typical of the cluster, despite their high degree of similarity with other members. These preliminary findings suggest that models based on fatty acid data could be used for identifying unknown streptomycetes to established taxa and for the definition of novel streptomycetes.
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