- Volume 129, Issue 7, 1983
Volume 129, Issue 7, 1983
- Pathogenicity And Medical Microbiology
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Amino Acid Requirements of Strains of Chlamydia trachomatis and C. psittaci Growing in McCoy Cells: Relationship with Clinical Syndrome and Host Origin
I. Allan and J. H. PearceThe effects of omission of individual amino acids from growth medium on the multiplication of a range of Chlamydia trachomatis and C. psittaci strains in cycloheximide-treated McCoy cells have been assessed. Differences in requirements were revealed which for C. trachomatis strains correlated with clinical syndrome and for C. psittaci with host origin. All 11 strains of C. trachomatis examined showed a requirement for addition of histidine to the medium; this was not shown by any of four C. psittaci strains. Among the strains of C. trachomatis, three from cases of trachoma, representing serotypes A, B and C, showed a distinctive requirement for the addition of tryptophan to the medium, whilst six strains of oculogenital origin, representing serotypes D-I, exhibited no requirement for tryptophan or methionine; a lymphogranuloma venereum and a ‘fast variant’ strain both showed a requirement for methionine. Of the four C. psittaci strains from different hosts, three showed distinct patterns of amino acid requirements. All chlamydiae squired the addition of valine to medium and the majority required leucine, phenylalanine and also glutamine.
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Effect of Nutrient Depletion on Sensitivity of Pseudomonas cepacia to Phagocytosis and Serum Bactericidal Activity at Different Temperatures
More LessPseudomonas cepacia grown under different specific nutrient depletions in batch culture showed varying degrees of sensitivity to engulfment and killing by human polymorphonuclear leukocytes (PMN) and to killing by human serum. Resistance to killing by the combined action of PMN and serum in whole blood was found to increase in the following order of depletions: glucose < iron < sulphate < phosphate or ammonium < magnesium. There was also an increase in resistance to killing by whole blood with decrease in temperature, except that carbon-depleted cells remained very sensitive irrespective of temperature. Cells in the exponential phase of growth also showed a consistent increase in resistance as the temperature was decreased. Similar, but smaller effects were observed with oxygen-depleted cells. The increase in killing by whole blood as the phagocytic temperature was raised correlated with an increase in the number of bacteria ingested per PMN. A pattern of serum sensitivity was observed with cells grown under different nutrient depletions similar to that for whole blood. But in all cases whole blood was 6 to 10 times more effective than serum alone in killing the cells at 37 °C.
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Fimbrial Haemagglutinins in Enterobacter Species
More LessFifty-two strains from seven species of Enterobacter, grown under a variety of conditions, were examined in rocked-tile tests for production of haemagglutinins and with the electron microscope for fimbriae. Thirteen non-haemagglutinating strains were non-fimbriate. Most (33) of the 39 haemagglutinating strains produced only one kind of haemagglutinin, either the mannose-sensitive haemagglutinin associated with type-1 fimbriae or, the mannose-resistant, klebsiella-like haemagglutinin associated with type-3 fimbriae. Multiply haemagglutinating strains were most common in E. aerogenes, in which species a third kind of haemagglutinin, also mannose-resistant, was found. The findings are discussed briefly in the light of the current taxonomy of Enterobacter.
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The Role of the O and K Antigens in Determining the Resistance of Klebsiella aerogenes to Serum Killing and Phagocytosis
More LessThe presence of both K and O antigens of Klebsiella aerogenes was found necessary to protect the organism from either complement-mediated serum killing or phagocytosis in the absence of specific antisera. Optimal phagocytic ingestion of K. aerogenes NCTC 5055 could be achieved in the presence of either anti-K or anti-O sera or to a much smaller extent in antisera raised against a rough unencapsulated mutant (M10B) derived from NCTC 5055. Anti-O sera failed to opsonize a clinical klebsiella isolate (DL1) possessing immunologically identical lipopolysac-charide, but did so when the amount of capsule was physically reduced. The serum sensitivity of the encapsulated strains was unaffected by the addition of specific antisera. Fresh serum was bacteriostatic for an unencapsulated smooth mutant (M10) derived from NCTC 5055. This bacteriostatic effect was reduced by heat-inactivation of the serum or by the addition of anti-O serum. M10 was rendered sensitive to the bactericidal action of serum in the presence of antisera raised against M10B or after chelation with MgEGTA to isolate alternative complement pathway activity. The rough unencapsulated mutant (M10B) was rapidly killed by fresh serum, an effect which could be delayed by chelation with MgEGTA. The serum sensitivity of M10B was unaffected by the presence of anti-M10B sera. Thus, the O antigen, unlike the K antigen, of these klebsiella strains is not antiphagocytic but it does confer some protection against the rapid bactericidal activity of serum complement.
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Multiflagellate Variants of Vibrio anguillarum
More LessAn ultrastructural examination of six strains of Vibrio anguillarum of varying virulence for eels revealed an apparent correlation between pathogenicity and the possession of more than one flagellum. The relationship between V. anguillarum surface appendages and virulence is discussed.
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The Serological Properties of the Cell Surface Proteins of Vibrio cholerae
More LessThe serological properties of cell surface proteins of Vibrio cholerae belonging to both the biotypes (classical and El Tor) and the serotypes (Ogawa and Inaba) were investigated. Proteins were isolated by extracting V. cholerae with EDTA in the presence of sodium chloride. The surface localization of these proteins was confirmed with (a) radioiodinated protein A as an immunoprobe and (b) antiserum absorption studies with whole bacteria. There were similarities among the polypeptides of cell surface proteins isolated from various V. cholerae types. Antisera to these proteins agglutinated several V. cholerae strains, irrespective of biotype, serotype and antibiotic sensitivity. The antisera did not agglutinate pathogenic enteric bacteria such as enterotoxigenic Escherichia coli, Salmonella, typhi, Shigella spp., Aeromonas hydrophila and Yersinia enterocolitica. The cell surface proteins of V. cholerae were immunogenic in rabbits as high titres of anti-protein specific antibodies were detected by the ELISA technique in the immune sera. These results suggest that the cell surface proteins are common antigens of V. cholerae and can be developed as a potential vaccine candidate against cholera.
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- Physiology And Growth
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Regulation of Expression of Novel Mandelate Dehydrogenases in Mutants of Acinetobacter calcoaceticus
More LessWild-type strains of Acinetobacter calcoaceticus able to grow on only L(+)- or d(−)-mandelate gave rise to mutants that could grow on the other isomer of mandelate. Each mutant contained an additional mandelate dehydrogenase which was not expressed in the parent strain. The novel enzymes were shown to be controlled co-ordinately with the pre-existing enzymes for the conversion of mandelate into benzoate when induced with phenylglyoxylate, gratuitously induced with thiophenoxyacetate, subjected to anti-induction by 2-phenylpropionate or catabolite-repressed by succinate. Mutants which had been selected on the basis of possession of a constitutive phenylglyoxylate decarboxylase also constitutively expressed both the original and the novel mandelate dehydrogenases.
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Disruptive Effects of Tris and Sodium Lauroyl Sarcosinate on the Outer Membrane of Pseudomonas cepacia Shown by Fluorescent Probes
More LessThe disruptive effects of Tris buffer and sodium lauroyl sarcosinate (Sarkosyl) on the outer membrane (OM) of Pseudomonas cepacia were investigated with several fluorescent probes. Tris increased the permeability of the OM to 6-anilino-1-naphthalenesulphonic acid and 2-p-toluidinylnaphthalene-6-sulphonate. The degree of damage to the OM was enhanced when the pH was decreased. 3-(N-morpholino)propanesulphonic acid buffer had a small but significant effect at acid pH, while citrate/phosphate buffer showed insignificant effects. Sarkosyl released 3,3′-dipentyloxacarbocyanine iodide (CC5) from CC5-labelled OM or whole cells and altered OM fluidity as studied by fluorescence polarization.
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Mycelial Growth and Branching of Streptomyces coelicolor A3(2) on Solid Medium
More LessEarly growth of the filamentous actinomycete Streptomyces coelicolor A3(2) on solid medium was investigated. Growth kinetics were similar to those of filamentous fungi in that total mycelial length and the number of branches increased exponentially at the same specific rate, with attainment of a constant hyphal growth unit. However, both germ tube and branch hyphae showed initial linear, rather than exponential extension. Logarithmic relationships were found between branch order and both branch length and branch number, although the constancy of these relationships varied during growth. Length ratio, obtained from branch pattern analysis, reached a constant value as did the hyphal growth unit, but branch ratio did not become constant during the period of study. There was some evidence of mycelial development analogous to that found in filamentous fungi.
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Ligninolytic Activity of Coriolus versicolor
More LessLignin degradation by Coriolus versicolor was investigated by culturing the fungus on spruce sapwood chips and extracting the extracellular proteins produced by an active lignin-degrading culture. The conditions for optimum ligninolytic activity were determined using a cultivation technique which simulated natural conditions, involving the use of cellulose in a growth medium placed beneath a lignin-impregnated glass fibre disc that served as a solid-phase support for hyphal growth. The extracellular proteins from the wood chip culture were shown to cause some modification of isolated milled wood lignin isolated from milled wood, namely an increase in hydroxyl groups in the molecule and a slight reduction in the mean molecular weight of the lignin polymers.
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Glycolysis and Respiration in Yeasts: the Effect of Ammonium Ions Studied by Mass Spectrometry
More LessAddition of NH+ 4 in the presence of glucose to washed suspensions of Saccharomyces uvarum, Schizosaccharomyces pombe or Candida utilis greatly increased glycolytic CO2 production and slightly stimulated respiration. In all three organisms the ammonium ion effect was distinguishable from the effect of an uncoupler of aerobic energy conservation (carbonyl cyanide m-chlorophenylhydrazone; CCCP). The Pasteur effect (aerobic inhibition of glycolysis) in the fermentative yeasts also proceeded independently of the ammonium ion effect. Possible control mechanisms are discussed.
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The Cell Cycle of the Budding Yeast Sterigmatomyces halophilus: Culture Fractionation by Zonal Centrifugation and the Accumulation of DNA, RNA and Protein
More LessSterigmatomyces halophilus is an unusual budding yeast in which daughter cells are formed, remote from the mother cell, on fine projections called sterigmata. Some fundamental properties of the cell cycle have been explored by separating cells from an exponentially growing culture into size, and thus age, classes by density-gradient centrifugation. Rate separations on high capacity, high resolution, equivolumetric gradients of sucrose, or, alternatively, isopycnic separations on gradients of Urografin revealed consistent and reproducible patterns of accumulation of DNA, RNA and protein through the cell cycle. Total DNA accumulation was stepwise, synthesis occurring late in the cycle, whilst protein accumulated continuously with no evidence for the discontinuities reported in some other lower eukaryotes. Total RNA accumulation, measured either colorimetrically or by long-term incorporation of radioactively-labelled uracil was transiently elevated early in the cycle and then accumulated continuously. A mathematical analysis of the volume distributions of the cells in fractions, from the gradients showed that there is a hyperbolic relationship between cell age and size but that, to a first approximation, measurements of cell size (and density) are direct measures of age. The results are discussed with reference to (1) the unusually high buoyant density of this yeast, (2) the resolution of zonal cell separation methods and (3) macromolecular accumulation in the cell cycles of other eukaryotic micro-organisms.
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The Cell Cycle of the Budding Yeast Sterigmatomyces halophilus: Oscillations in the Amounts and Activities of the Terminal Components of the Respiratory Chain
More LessCells harvested from exponentially growing cultures of the budding yeast Sterigmatomyces halophilus were fractionated according to their age in the cell cycle by isopycnic-zonal centrifugation in Urografin gradients. Activities of five enzymes were assayed in cell-free extracts, prepared using a French pressure cell. The activities of catalase and three enzymes of the mitochondrial inner membrane (NADH dehydrogenase, succinate dehydrogenase and cytochrome c oxidase) doubled during the cell cycle but showed complex oscillatory patterns. Acid phosphatase activity increased continuously during the cell cycle. Fourth-order finite difference analysis of low-temperature difference spectra showed that the levels of three b-type cytochromes increased continuously during the cycle. In contrast, amounts of cytochromes c 1, c and aa 3 oscillated over one cycle, with cytochromes c and aa 3 rising to two maxima per cycle in phase with cytochrome c oxidase activity. Liganding of redox-active metals, and discontinuous synthesis and proteolysis of apocytochromes are discussed as possible mechanisms for cell cycle-dependent fluctuations in the composition and function of the respiratory chain.
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The Cell Cycle of the Budding Yeast Sterigmatomyces halophilus: Levels of Mitochondrial Components in Mother and Bud Cells
More LessSterigmatomyces halophilus is an unusual budding yeast in which daughter cells are formed remote from the mother cell on fragile projections called sterigmata. These sterigmata are readily disrupted by ultrasonication, allowing easy detachment of immature buds from their mother cells. Fractions containing cells at different stages of the cell cycle, obtained by isopycnic fractionation of exponentially growing cultures, were treated ultrasonically to produce a mixture of immature buds, mothers and mother-bud doublets. Rate-sedimentation of these suspensions using sucrose gradients produced three discrete bands corresponding to each of these populations. The activities of succinate dehydrogenase and cytochrome c oxidase were measured in extracts prepared from (1) the cells recovered from the slowest sedimenting band (i.e. immature buds) and (2) the original population (i.e. mother-bud doublets). The activity of each enzyme, expressed on a per cell basis, varied in phase with the observed activity in the mother-bud doublets during the cell cycle, and when expressed as specific activity was the same in both daughter and mother-daughter pairs. This indicates that these two enzymes (and by implication inner mitochondrial membrane) are evenly distributed between mother and developing daughter cells during the cell cycle.
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Preliminary Identification of a Possible Cell-division Protein in the Cyanobacterium Anacystis nidulans
More LessCultures of the cyanobacterium Anacystis nidulans were grown under conditions of Mg2+-limitation and Mg2+-excess. Cell-free extracts, obtained after sonication and centrifugation (45000 g , 60 min), were analysed on polyacrylamide gels. Mg2+-limited cells, in which cell division was inhibited, accumulated a protein of molecular weight 50 103. Only small amounts of this protein were detected in non-Mg2+-lirnited cultures. A protein of molecular weight 36 103, found in non-Mg2+-limited cells, was not detected in Mg2+-limited cells. When a Mg2+ shift-up from 5 m to 1 mM was carried out in a chemostat, synthesis of the 36 103 protein was initiated and the amount of the 50 103 protein decreased whilst the main protein pattern remained unaltered. The possibility that the two proteins are involved in cell division is discussed.
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The Effect of Magnesium and Manganese Ion Concentrations and Medium Composition on the Production of Extracellular Enzymes by Lysobacter enzymogenes
More LessThe effect of medium composition on the production of four types of extracellular enzymes by Lysobacter enzymogenes was investigated. The nuclease, RNAase, alkaline phosphatase and proteases were produced in good yield after growth in tryptone broth. Much higher yields of the proteases, but low yields of the other three enzymes, were obtained using a skim milk/yeast extract or a chemically defined medium. The addition of NH+ 4, HPO2 4, ribonucleosides, Mg2+ or Mn2+ to tryptone broth reduced the production of some of the enzymes rather specifically. Of 12 monovalent and divalent metal ions tested, Mg2+ and Mn2+ had the greatest effect. Mg2+ at concentrations greater than about 0.01 mM inhibited the production of the nuclease, RNAase and the phosphatase but increased the proteases two- to threefold. Mn2+ at concentrations greater than about 0.01 mM inhibited production of the three enzymes more severely but did not stimulate protease synthesis. The extracellular enzymes produced with or without added Mg2+ or Mn2+ were analysed by PAGE and the activities associated with the cells and a shock fluid were determined. In addition, the effect of adding Mg2+ or Mn2+ to a growing, extracellular enzyme-producing culture was determined. The results suggest that the nuclease, RNAase and phosphatase are produced by a different mechanism than the proteases and that the metal ions interfere specifically with their production rather than with their release or by causing inhibition.
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Uptake of Vapour Phase [14C]Dodecane by Whole Mycelia of Cladosporium resinae
More LessThe kinetics of uptake of n-alkanes by a filamentous fungus, Cladosporium resinae, were shown to be similar to hydrocarbon uptake kinetics described for yeasts. Transport of n-alkanes across the cell membrane was accompanied by the partitioning of substantial amounts of hydrocarbon on to the cell surface. Metabolic inhibitors significantly reduced uptake rates. Most [14C]dodecane was taken up by dodecane-grown mycelia; slower uptake was observed with mycelia grown on other n-alkanes or glucose. Uptake of dodecane was not restricted to specific regions of the mycelium, although higher concentrations of 14C-labelled compounds were observed at the hyphal tips.
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- Short Communication
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Production of Contiguously Arranged Chlamydospores in Candida albicans
More LessContiguous arrangements of two to three chlamydospores occurred in a clinical isolate of Candida albicans. Time-lapse photography showed that the terminal cell of a cell chain was first transformed into a chlamydospore and such transformation proceeded centripetally to the next cell in the chain. Ultrathin sections revealed that the outermost layer of the three-layered chlamydospore wall was continuous throughout the interconnected spores, with the other layers surrounding each spore separately. Chlamydospore chains were common in this organism.
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- Taxonomy
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Chemotaxonomic Study of an Alkalophilic Bacterium, Exiguobacterium aurantiacum gen. nov., sp. nov
More LessChemical studies were performed on a Gram-positive alkalophilic bacterium of uncertain taxonomic position. On the basis of the present and earlier studies it is suggested that the alkalophilic bacterium be classified in a new genus Exiguobacterium, as Exiguobacterium aurantiacum gen. nov., sp. nov.
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Numerical Taxonomy of Bacterial Isolates Associated with a Freshwater Fishery
More LessPhenetic data on over 600 aerobic, heterotrophic, aquatic bacteria from a freshwater fish farm were collected and examined by numerical taxonomy procedures using 124 unit characters. Reference cultures representing 36 taxa were included in the analyses. The data were examined using the simple matching (SSM) and Jaccard (S J) coefficients, and clustering was achieved using unweighted average linkage. At similarity values of 70% or above as defined with the S J coefficient, 82% of the environmental isolates were recovered in 14 major and 56 minor phena. The major phena were equated with Acinetobacter calcoaceticus, Aeromonas hydrophila, Aeromonas salmonicida, Alcaligenes sp., coryneforms, Enterobacter aerogenes, Escherichia coli, Hafnia alvei, Pseudomonas fluorescens, Pseudomonas spp. (two groups), Serratia sp., Vibrio fluvialis and Yersinia sp. The minor phena comprised Agrobacterium, Arthrobacter, Bacillus, Bordetella, Cytophaga, Erwinia, Flavobacterium, Flexibacter, Klebsiella, Micrococcus, Moraxella, Staphylococcus and Vibrio. Aeromonas salmonicida was recovered only from moribund and dead rainbow trout (Salmo gairdneri) during an outbreak of furunculosis. In contrast, coryneforms (phenon 2) and Alcaligenes (phenon 14) were found exclusively in water samples.
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