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Volume 129,
Issue 4,
1983
Volume 129, Issue 4, 1983
- Biochemistry
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The Methanol: Cytochrome c Oxidoreductase Activity of Methylotrophs
More LessBoth the soluble cytochromes c of the obligate methylotroph Methylophilus methylotrophus were rapidly autoreducible at high pH. The intramolecular autoreduction mechanism was also involved in the reduction of the cytochrome c L by methanol dehydrogenase which occurred in the absence of methanol. Pure soluble methanol dehydrogenase was shown to be able to catalyse the methanol-dependent reduction of pure cytochrome c from M. methylotrophus and from the facultative methylotroph Pseudomonas AM1 by coupling oxidation of the bacterial cytochrome to the reduction of a large excess of mammalian cytochrome c. Only one of the two cytochromes c (cytochrome c L of each organism) could react with methanol dehydrogenase to give methanol:cytochrome c oxidoreductase activity. This activity, using proteins from M. methylotrophus, was independent of pH between pH 7·0 and 9·0 and ammonia was not required. By contrast, the pH optimum for the system from Pseudomonas AM1 was 9.0 and activity was stimulated about fourfold by NH4Cl. The product of methanol oxidation was formaldehyde, which was also a substrate for the oxidoreductase system. During formaldehyde oxidation two molecules of cytochrome c were reduced for every molecule of formaldehyde oxidized. In a survey of methanol dehydrogenases and cytochromes c from Pseudomonas AM1, M. methylotrophus and the facultative autotroph Paracoccus denitrificans, it was shown that, of the two soluble cytochromes c found in each methylotroph only one was able to react with methanol dehydrogenase. The cytochrome c L from M. methylotrophus and the cytochrome c(2) of Pa. denitrificans were specific, only reacting with methanol dehydrogenase from the same organism, whereas the cytochrome c L of Pseudomonas AM1 reacted with all three methanol dehydrogenases tested.
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Dihydroxyacetone Synthase: a Special Transketolase for Formaldehyde Fixation from the Methylotrophic Yeast Candida boidinii CBS 5777
More LessThe transketolase synthesized by Candida boidinii CBS 5777 during growth on non-C1 substrates has been purified and shown to have a molecular weight of approximately 135000 and to consist of two subunits of approximate molecular weight 68000. The enzyme is active with xylulose 5-phosphate as glycolyl donor and ribose 5-phosphate as acceptor; under the conditions of assay formaldehyde was inactive as acceptor. Candida boidinii CBS 5777 also synthesizes a second transketolase during growth on methanol. This enzyme, which is unstable, has been purified to homogeneity. It has a wide substrate specificity towards glycolyl donors and acceptors; formaldehyde is a good acceptor and in terms of its physiological role during growth on methanol the enzyme can be described as a dihydroxyacetone synthase. This enzyme has a molecular weight of 105000-110000, with two subunits of molecular weight 62000-67000. Its properties have been compared with analogous enzymes purified elsewhere from methanol-grown Candida boidinii KD1 and Candida boidinii (Kloeckera sp.) 2201.
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Bacterial Metabolism of Aliphatic Diols. Function of Alcohol Oxidases and Catalase in Flavobacterium sp. NCIB 11171
More LessThe inducible diol-metabolizing oxidases from ethylene glycol-grown and propane 1,2-diol-grown Flavobacterium sp. NCIB 11171 have been partially purified and shown to be the same broad substrate specificity enzyme. The inducible catalase from diol-grown strain NCIB 11171 has been partially purified and shown to act on the hydrogen peroxide product of the alcohol oxidase with the peroxide acting as the electron plus proton donor. The partially purified catalase was unable to oxidize various alcohols in the presence of hydrogen peroxide.
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Distribution and Transport of Iron in Conidia of Colletotrichum musae in Relation to the Mode of Action of Germination Stimulants
More LessComparative studies on germination of conidia of Colletotrichum musae with low and normal iron contents indicated that in addition to lacking the normal requirement for a germination stimulant low-iron conidia germinated substantially faster. Parallel investigations on the iron flux in conidia during germination demonstrated that the presence of germination stimulants did not significantly influence iron uptake or release by conidia. In addition no major intracellular movement of iron between the organelle, wall and soluble fractions of conidia was detected. These findings imply that the action of chelating agents in promoting germination in conidia of C. musae involves the complexing of inhibitory iron at a site within the conidia, so releasing the germination mechanism. Selective accumulation of a germination stimulant (EDTA) within the organelle fraction during induction of germination in iron-replete conidia suggests that the site involved is located in this fraction, possibly on ribosomes. However, the marked differences observed between the relative dimensions of low-iron and iron-replete conidia could implicate the conidial wall as the site of iron complexed by chelating agents involved in stimulation of germination.
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Microbial Transformation of Lucanthone by Growing Cultures, Washed Mycelia and Non-germinating Spores of Fungi from the Aspergillus Group
More LessWhole broth cultures, washed mycelia and non-germinating spores of 13 aspergilli scored from among 91 moulds isolated from soil and air transformed lucanthone (I) into three to five products with increased polarity. Biotransformations brought about by actively growing cultures were also performed by washed mycelia and non-germinating spores of the same strains. Lucanthone (I) was oxidized by growing cultures, washed mycelia and spore suspensions of an Aspergillus species (no. 2) into: hycanthone (II) as the main product, its aldehyde analogue (III) and its carboxylic acid derivative (IV). The pathway of lucanthone (I) oxidation by this strain involved hydroxylation of the 4-methyl group (to give hycanthone, II) followed by dehydrogenation of the resulting primary alcohol (to give the aldehyde, III). The aldehyde III was finally slowly oxidized to the corresponding carboxylic acid analogue IV. Evidence is presented to show that mycelial and spore enzymes effecting these oxidative reactions are intracellular and non-inducible in nature. Spore-mediated transformations were found not to require a source of energy and could be conducted in distilled water over a wide range of incubation temperature (from 4 to 37 °C). Use of the spores in successive transformations did not affect lucanthone (I) hydroxylation into hycanthone (II) or the dehydrogenation of the latter into the aldehyde analogue (III) but the ability of the spores to oxidize the aldehyde (III) to the carboxylic acid (IV) was lost.
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Alteration of Membrane Permeability in Bacillus subtilis by Clofoctol
More LessWhen Bacillus subtilis was treated with a bacteriostatic concentration of clofoctol [2-(2,4-dichlorobenzyl)-4-(tetramethyl-1,1,3,3-butyl)phenol], UV-absorbing material was released. Electron micrographs showed evidence of physical alteration of the bacterial envelope. The uptake of [14C]glutamate was strongly inhibited by clofoctol, and preloaded glutamate was found to leak from the bacteria. Clofoctol caused an immediate and dramatic decrease in the amount of intracellular ATP. This was neither the consequence of the stimulation of an ATPase, nor of the inhibition of bacterial respiration. Both the proton gradient and the potential gradient across the cytoplasmic membrane collapsed and this inhibition of energy metabolism was sufficient to account for the inhibition of growth by clofoctol. At the same bacteriostatic concentration complete permeabilization of the bacteria occurred.
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Characterization of Extracellular Alkaline Proteases and Collagenase Induction in Vibrio alginolyticus
More LessThe number and approximate molecular weights of extracellular alkaline proteases produced by Vibrio alginolyticus were determined by gelatin-PAGE. Three major bands of protease activity with apparent molecular weights of approximately 28000, 22500 and 19500 (proteases 1, 2 and 3, respectively) and two minor bands of protease activity with apparent molecular weights of approximately 15500 and 14500 (proteases 4 and 5, respectively) were obtained after gelatin-PAGE. The activities of the five proteases were inhibited by serine protease inhibitors but their activities were not affected by inhibitors of trypsin-like enzymes. Histidine, which inhibited V. alginolyticus collagenase, did not inhibit the activities of the alkaline serine proteases. The production of protease 1, however, was enhanced by histidine. Protease 1 production was also affected by temperature and production was depressed at 37 °C. Gelatin-PAGE of a commercial V. alginolyticus collagenase preparation revealed four bands of activity which were identified as collagenases with apparent molecular weights of approximately 45000, 38500, 33500 and 31000. The collagenase preparation was contaminated with two serine proteases. The release of [3H]proline from collagen matrices produced by smooth muscle cells was shown to be a sensitive assay for bacterial collagenases and was used to show that V. alginolyticus produced a basal constitutive level of extracellular collagenase. The constitutive levels of collagenase were affected by aeration.
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Subcellular Fractionation of Candida stellatoidea after Growth with Glucose or n-Hexadecane
More LessSpheroplasts of glucose grown and n-hexadecane-grown Candida stellatoidea were prepared using snail-enzyme or Zymolyase-5000 and the resultant cell extracts fractionated on sucrose or metrizamide gradients. Organelles from n-hexadecane-grown cells were more fragile than those from glucose-grown cells and organelle integrity was maintained only after spheroplast formation using Zymolyase-5000. Isopycnic density gradient centrifugation through metrizamide gradients yielded more complex distributions and markedly higher percentage sedimentabilities of marker enzymes than with sucrose gradients. The zone containing cytochrome c oxidase and all tricarboxylic acid cycle enzymes assayed was readily identified. The density of microbodies appears to be similar to that of mitochondria on either gradient material; on metrizamide a second catalase peak at ρ = 1·07 g ml−1 was also observed. This zone was shown by electron microscopy to contain organelles up to 1 μm diameter, and activities of carnitine acetyltransferase and long chain alcohol and aldehyde dehydrogenases. The first enzyme was located mainly in zones containing mitochondria and microbodies; the last two enzymes were multinational and of differing distributions, but were found mainly in mitochondrial and microsomal fractions. The possibility that cells grown on n-hexadecane contain two populations of microbodies is discussed. Most lysosomes were disrupted on sucrose gradients but sedimented to a density of 1·12 g ml−1 on metrizamide gradients.
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- Development And Structure
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Chemical Composition of Cell Walls of Alkalophilic Strains of Bacillus
More LessCell walls of 10 alkalophilic Bacillus strains were prepared by inactivation of autolytic enzymes with SDS, disruption with a sonic oscillator, trypsin digestion and washing with SDS. The walls were composed of peptidoglycan and acidic compounds. The peptidoglycan was similar in composition to that of B. subtilis. After hydrolysis, the acidic compounds detected were glucuronic acid, glutamic acid and aspartic acid. The strains tested could be divided into three groups as follows. Group 1, the glucuronic acid and hexosamine contents were high; no growth was observed at pH 7·0; Na+ was essential for their growth. Group 2, large amounts of glutamic acid, aspartic acid and glucuronic acid were detected. Growth at higher pH values increased the content of acidic compounds; growth was observed at pH 7·0; Na+ was essential for their growth. Group 3, no remarkable difference was detected in the chemical components in comparison with B. subtilis; growth was observed in the presence of Na+ or K+ at pH 7·0 and 10·2.
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Synthesis of a Spore-specific Surface Antigen During Sporulation of Saccharomyces cerevisiae
More LessAntisera raised against purified yeast ascospores caused agglutination of both ascospores and vegetative cells. A spore-specific activity was obtained by absorbing out anti-vegetative activity with vegetative cells. The anti-vegetative cell activity was directed against mannan, and was probably due to exposure of some spore coat mannan at the spore surface since concanavalin A and lentil lectin also caused agglutination of ascospores. The spore-specific activity was probably determined by a protein or proteins, since extraction of spores with a mixture of sodium dodecyl sulphate and dithiothreitol markedly affected their agglutination by the spore-specific serum.
The spore-specific antigen was synthesized in a soluble form during sporulation several hours before the appearance of the spore surface and the pool of soluble antigen declined as the spore was assembled. Synthesis of the soluble antigen was inhibited by adding cycloheximide at all times up to its first appearance in the sporulating cell.
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Immunochemical Identification of the Major Cell Surface Agglutinogen of Acinetobacter calcoaceticus RAG-92
More LessThe immunochemical and immunocytochemical characteristics of three Acinetobacter calcoaceticus RAG strains were compared in order to clarify the relationship between antibody-induced agglutination and the production of polyanionic extracellular emulsifier (termed emulsan). In addition to the parent, RAG-92, two mutant strains were examined: (1) a nonagglutinating emulsan-producer (AB15), and (2) an agglutinating mutant (16TLU) defective in the production of emulsan. A combined genetic-immunochemical approach was employed. This included the comparison of crossed Immunoelectrophoresis patterns of parent and mutant supernates and the effect of absorption of anti-whole cell antiserum with mutant cells. In addition, agglutinability and competition studies were performed as well as electron microscopic cytochemistry. The results demonstrated that three major antigenic components were associated with the cell surface and the supernate. Mutant cells were altered both in their cell surface properties and in their extracellular products. One antigenic component, termed component C3, was the major cell surface agglutinogen; this component was absent in nonagglutinating mutants. Component C3 may be identical with or attached to the 300 nm projections on the parent cell surface, but it is not directly related to the presence of emulsan. It appears that emulsan plays little or no role in the phenomenon of antibody-induced agglutination of this organism.
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Differential Protein Synthesis in Candida albicans during Blastospore Formation at 24·5 °C and during Germ Tube Formation at 37 °C
More LessTo identify proteins synthesized during blastospore to germ tube transformation in Candida albicans, membrane and cytoplasmic protein fractions labelled with 14C were analysed by SDS-PAGE and autoradiography. Four cytoplasmic proteins were detected in pulse-labelled lysates prepared from cells forming blastospores and germ tubes at 37 °C, but not in pulse-labelled lysates prepared from cells forming blastospores at 24·5 °C. Three proteins detectable in 24·5 °C lysates were diminished in 37 °C lysates.
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- Ecology
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Variability among Isolates of Pseudomonas syringae pv. savastanoi from the Phylloplane of the Olive
More LessLeaves of three or four different ages were taken from olive plants quarterly in 1974–1980. One thousand and fifty isolates of Pseudomonas syringae pv. savastanoi from the phylloplane were tested for virulence to the olive and subjected to numerical phenetic analysis using 60 unit characters. The data were analysed using unweighted average linkage (UPGMA) and single linkage clustering on the simple matching (S SM) and pattern (S P) coefficients. The isolates obtained from leaves of a given age at a given, time of the year shared higher percentage similarity (S) values between themselves than with the others. Cluster composition was only marginally affected by different coefficients and methods of clustering. UPGMA analysis on the S SM coefficient recovered 92% of the isolates in 10 major clusters at 75% S. Of the isolates from leaves of the same age collected at the same time of the year, 81–99% fell in the same cluster. Conversely, 91–97% of the isolates in five of the major clusters were from leaves of the same type. Of the isolates in the other major clusters, 95–98% were from two different sources but most of the isolates from leaves of one type segregated into discrete subclusters at 85–90% S. Hypothetical median organisms (HMOs) were constructed to represent all the isolates obtained from the leaves of each type each year. The resulting relationships between the HMOs confirmed those described above for the individual isolates.
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The Endoparasitic Nematophagous Fungus Meria coniospora Infects Nematodes Specifically at the Chemosensory Organs
More LessConidia of the endoparasitic fungus Meria coniospora infected the bacterial-feeding nematode Panagrellus redivivus at specific sites, namely the mouth region and in male nematodes also in the tail. Plant-parasitic nematodes were also infected in other parts of the body. The specific infection sites in P. redivivus were the sensory organs, which are the sites of chemoreception. Blocking of chemoreceptors by adhered conidia of M. coniospora caused a complete loss of the ability of nematodes to be attracted to different sources of attractants. Inhibition of conidial adhesion by means of sugar haptens suggested a lectin-carbohydrate interaction. Out of 21 sugars only N-acetylneuraminic acid inhibited the adhesion, indicating the importance of sialic acids in the infection process. Reduction of conidial adhesion by treatment of nematodes with neuraminidase further supported this view.
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Clostridium thermosulfurogenes sp. nov., a New Thermophile that Produces Elemental Sulphur from Thiosulphate
More LessA new Clostridium species is described that was isolated from a thermal, volcanic, algal-bacterial community via selective enrichment procedures with pectin as energy source. Clostridium thermosulfurogenes sp. nov. deposits elemental sulphur on the cell surface and in the culture medium from thiosulphate transformation. This species stained Gram-negative, but electron micrographs revealed a double-layered wall without the presence of an outer membranous layer. Thin sections displayed numerous internal membranes and sulphur granules were not discernible. The organism was motile and formed distinctly swollen sporangia with terminal, white-refractile, spherical spores. The temperature range for growth was >35 °C and <75 °C, the pH range was between 4·0 and 7·5. The DNA base composition was 32·6 ± 0·04 mol% guanosine plus cytosine. Fermentable carbohydrates included pectin, starch, xylose, glucose, mannose, cellobiose, maltose, arabinose and sucrose. The doubling time on glucose or pectin was about 2 h. The production of ethanol, H2/CO2, acetate and lactate accounted for a balanced fermentation of glucose, whereas methanol and isopropanol were also produced during pectin fermentation. The taxonomic relationships of C. thermosulfurogenes to other thermophilic Clostridia and its biological role in a thermal microbial community are discussed.
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Microbial Ecology of Volcanic Sulphidogenesis: Isolation and Characterization of Thermodesulfobacterium commune gen. nov. and sp. nov
More LessMicrobial sulphate reduction was examined in thermal waters, sediments and decomposing algal-bacterial mats associated with volcanic activity in Yellowstone National Park. In vivo radioactive tracer studies which demonstrated biological [35S]sulphkie production from [35S]sulphate at temperatures higher than 50 C but less than 85 C, correlated with the presence of a unique sulphate-reducing bacterium. This new species proliferated at temperatures above 45 C but below 85 C, and had an optimum growth temperature of 70 C. The organism was a small Gram-negative, straight rod which displayed an outer-wall membranous layer in thin sections. This obligate anaerobe utilized pyruvate, lactate or H2 as electron donors and sulphate or thiosulphate as electron acceptors for growth and sulphide formation. Pyruvate alone was fermented during growth to hydrogen, acetic acid and CO2. Cell extracts contained cytochrome c 3 but lacked a desulphoviridin-type bisulphite reductase. The DNA guanosine plus cytosine content was 34.4 1.0 mol%. Other unusual biochemical features of this extreme thermophile are discussed. Strain YSRA-1 is described as the type strain of the new genus and species Thermodesulfobacterium commune.
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- Genetics And Molecular Biology
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Characterization of a Pleiotropic Succinate Dehydrogenase-negative Mutant of Bacillus subtilis
More LessA succinate dehydrogenase-negative mutant of Bacillus subtilis is described which lacks all three subunits of the membrane-bound succinate dehydrogenase complex: flavoprotein, iron protein, and cytochrome b 558. The corresponding mutation is revertible and it maps at one extreme of the sdh region. The results presented suggest that the structural genes for the subunits of the succinate dehydrogenase complex are part of one operon.
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Deficiency in Homogentisic Acid Oxidase Activity Associated with the Brown Phenotype of Dictyostelium discoideum
More LessMutations at the bwnA locus of Dictyostelium discoideum were found to result in drastically reduced levels of activity of the enzyme homogentisic acid oxidase. It is proposed that the brown phenotype associated with these mutations results from the accumulation of an intermediate in the tyrosine degradative pathway, homogentisic acid, and its subsequent spontaneous oxidation. This knowledge is used to devise improved screening procedures potentially useful in the parasexual genetics system of this organism.
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Plasmid Content of Streptococcus faecalis Strain 39-5 and Identification of a Pheromone (cPD1)-induced Surface Antigen
More LessStreptococcus faecalis 39–5 is a haemolytic, bacteriocinogenic strain harbouring six plasmids. One of these plasmids, pPD1 (36·4 MDal) determines a bacteriocin and encodes a conjugative response to the sex pheromone cPD1 excreted by recipient (plasmid-free) strains. The pheromone response is characterized by the formation of mating aggregates of donors (responders) with recipients. Aggregation required the presence of phosphate and divalent cations and was inhibited by agents or conditions that destroy protein structure. Aggregation was postulated to be due to synthesis of a new proteinaceous molecule on the donor cell surface. Referred to as ‘aggregation substance’, such a material was identified and found to exhibit antigenic properties not associated with uninduced cells; it could be detected by immunoelec-tron microscopy. Aggregation substance could be extracted from induced cells but not uninduced cells as demonstrated by crossed immunoelectrophoresis. Antibody raised against the aggregation substance controlled by pPD1 cross-reacted with aggregation substance determined by other plasmid systems which respond to pheromones unrelated to cPD1.
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On the Origin of the Chloramphenicol Resistance Transposon Tn9
More LessThe widely studied chloramphenicol resistance (Cmr) transposon Tn9 came from phage P1Cm0. This phage, however, had acquired its Cmr marker from the R plasmid pSM14. The analysis of the physical structure of pSM14 has now revealed that this plasmid already carried Tn9 and also the tetracycline resistance transposon Tn10. Physical and functional studies indicated that Tn9 of pSM14, although capable of transposition, probably translocated to the P1 genome by reciprocal recombination processes.
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