- Volume 129, Issue 7, 1983
Volume 129, Issue 7, 1983
- Biochemistry
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Terminal Oxidase of Crithidia fasciculata. Reactions with Carbon Monoxide and Oxygen at Subzero Temperatures and Photochemical Action Spectra
More LessRoom temperature CO-difference spectra of whole cells ofCrithidia fasciculata show two CO-reactinghaemoproteins. The reaction of cytochrome a/a 3 with CO is complete within 1 min of bubbling with CO; that of cytochrome b takes longer than 40 min. A non-photodissociable O2-containing compound of cytochrome a/a 3 was formed in whole cell suspensions at −112 °C after photolysis of CO in the presence of 200 μm-O2. No O2-cytochrome b compound was observed under these conditions. Photochemical action spectra for the relief of CO-inhibited respiration, obtained at different O2 tensions, indicate cytochrome a/a 3 to be the major haemoprotein terminal oxidase; no evidence for a b-type cytochrome oxidase has been found.
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Unusual Ergot Alkaloid Biosynthesis in Sclerotia of a Claviceps purpurea Mutant
More LessA strain of Claviceps purpurea, which has consistently failed to elaborate ergot alkaloids when growing as a parasite, has been shown to perform only the first step of the ergoline biosynthetic pathway catalysed by dimethylallyltryptophan (DMAT) synthetase. The next step, involving N-methylation of DMAT, did not operate. [14C]Agroclavine and lysergic acid, normally intermediates in alkaloid biosynthesis, were accepted by parasitic sclerotial tissue as substrates and were metabolized to lysergic acid amide (LAA). This amide therefore constituted a previously unreported end product for C. purpurea. It is concluded that the mutant has a metabolic block in the pathway following DMAT and, while enzymes for several subsequent steps are present, the fungus seems unable to form the usual cyclic tripeptide ergot alkaloids. The steps involved in metabolizing agroclavine to LAA were insensitive to 1 m-phosphate, while this concentration of phosphate completely inhibited DMAT synthetase. This double mutant therefore has unique potential for exploring control mechanisms in ergot alkaloid biosynthesis.
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The Membrane-bound Cytochromes of an Aerobically Grown, Extremely Thermophilic Bacterium, PS3: Characterization by Spectral Deconvolution Coupled with Potentiometric Analysis
More LessCytochromes of the a, b and c types in membranes from the thermophilic bacterium PS3 have been characterized with respect to their spectral, potentiometric and ligand-binding properties. The integrated approach used (a) conventional potentiometric analysis with non-linear least-square analysis, (b) incorporation of the potentiometric procedures into a spectral decomposition protocol at 298 and 77 K, (c) fourth-order finite difference and fourth derivative analysis, and (d) photodissociation studies of the CO-liganded components. Either one c-type cytochrome with a split α-band (547, 552 nm at 77 K) or two cytochromes c with similar redox potentials (approx. +230 mV) were found, together with at least three b-type cytochromes (554, 557 and 561 nm at 77 K). On the basis of its high redox potential (+104mV), cytochrome b 557 was tentatively equated with the o-type oxidase of this organism. A c-type cytochrome that formed a photodissociable complex with CO was also detected. The α-band of the previously purified cytochrome c oxidase was resolved into two components, having spectral and potentiometric properties remarkably similar to those of the analogous mitochondrial oxidase.
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Enzymes of Malate Oxidation in Mycobacterium leprae Grown in Armadillo Livers
More LessA NAD-dependent malate dehydrogenase is the principal enzyme for malate oxidation by Mycobacterium leprae, FAD-dependent malate-vitamin K reductase was detected at about 1% the level of the NAD-dependent activity. Both enzyme activities were detected in extracts from M. leprae treated with NaOH to abolish host-derived activities which might be adsorbed to the bacteria and the NAD-dependent enzyme was shown to be electrophoretically distinct from the host-tissue enzyme, thus establishing that these were both authentic bacterial enzymes. Mycobacterium leprae does not possess malic enzyme.
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- Development And Structure
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Electron Microscopic Observations of Cell Division in Mycobacterium vaccae V1
More LessCell division of Mycobacterium vaccae was initiated by deposition of new wall material in the cross wall. The surface layers of the old wall remained continuous until septum formation was complete. Subsequently, rupture of the outer cell wall layers occurred circumferentially, leaving rings on the cell wall. The two daughter cells remained connected with each other at the new pole and bent to form V-shaped structures at the connecting point.
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- Ecology
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Antimicrobial Properties of Calcium Peroxide in Relation to Its Potential Use as a Seed Dressing
More LessCalcium peroxide, in a formulation with calcium hydroxide, inhibited the growth of fungi and bacteria in liquid shaken culture. The formulation (‘CaO2’) was effective against spore germination, but less so against hyphal growth on solid or liquid media. With the exception of Mucor hiemalis, similar amounts of Ca(OH)2 alone were not effective; the antimicrobial activity of the formulation depends only partly on its alkalinity. Of the fungi tested, M. hiemalis had the greatest peroxidase activity and showed most resistance to ‘CaO2’ The production of H2O2 and free radicals by ‘CaO2’ may in part account for its action.
‘CaO2’ increased overall carbon release from axenic wheat seed, especially at 17 and 21% (v/v) O2 concentrations, but fructose and glucose exudations were inhibited. Total carbohydrate released was not affected. Sucrose exudation was detected from ‘CaO2’-treated seed, but not from untreated seed. The major form of carbon released from seeds was not carbohydrate and possibly originated from the seed coat. Although exudates supported substantial fungal and bacterial growth, the antimicrobial properties of the formulation suggest that it has potential as a plant protection chemical.
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- Genetics And Molecular Biology
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β-Lactam Antibiotic Production by Streptomyces clavuligerus Mutants Impaired in Regulation of Aspartokinase
More LessThis work describes isolation and characterization of Streptomyces clavuligerus mutants resistant to the lysine analogue S-(2-aminoethyl)-l-cysteine (AEC). The mutation to AEC resistance was shown to affect the feedback regulation of aspartokinase; 70% of the mutants isolated had aspartokinase activity insensitive to concerted feedback inhibition by lysine plus threonine. Among these mutants, 70% (about 50% of the total AEC-resistant strains isolated) showed significant overproduction of β-lactam antibiotics.
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The Formation of an R-prime Carrying the Fraction I Dehalogenase Gene from Pseudomonas putida PP3 using the IncP Plasmid R64.44
More LessPlasmid R68.44 was transferred to Pseudomonas putida PP3 at a frequency of approximately 10−6. In the new strain, P. putida PPW1, the plasmid, designated pUU1, lost a 2·0 kb fragment corresponding to IS21 which had previously been implicated in enhanced chromosome mobilization, but gained between 3·4 and 5·7 kb of DNA. Plasmid pUU1 was used to mobilize the fraction I dehalogenase gene at a low frequency of approximately 10−1 into another strain of P. putida. The R-prime plasmid, designated pUU2, contained an additional 9·2 to 11·0kb fragment which carried the fraction I dehalogenase.
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Discriminated Induction of SOS Functions in Escherichia coli by Alkylating Agents
More LessTreatment of Escherichia coli with the alkylating agents diethyl sulphate, ethyl methane-sulphonate and N-methyl-N′-nitro-N-nitrosoguanidine produces a different pattern of expression of SOS functions. There is a full induction of recA-dependent inhibition of cell respiration, a slight induction of lambda prophage, and no inhibition of cellular division. In a comparative study with bleomycin; an agent which is able to induce these three SOS functions, we have also shown that the differences in expression of SOS functions are not due to any variation in the pattern of DNA synthesis, or DNA degradation after treatment with alkylating agents. These results suggest that the kind of damage induced in the DNA may be important in determining which SOS function is expressed.
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Variety of Sporulation Phenotypes Resulting from Mutations in a Single Regulatory Locus, spoIIA, in Bacillus subtilis
More LessClosely linked mutations in either of the two putative genes of the sporulation locus spoIIA can affect, in quite diverse ways, spore incidence, the production of alkaline phosphatase and DNAase, and the stability of the cells in sporulation medium. It is concluded that the locus has a regulatory function affecting the activation or induction of at least two, and possibly more, sporulation-associated operons.
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Parasexual Analysis of Aspergillus parasiticus
More LessTen genetic markers were located on six linkage groups in Aspergillus parasiticus by means of the parasexual cycle. Diploids, selected by complementation of spore colour and auxotrophic markers, were subjected to induced haploidization by treatment with benlate. Haploid segregants, including recombinants resulting from mitotic crossing over, were distinguished from diploids by their stability and growth in the presence of benlate. Evidence is presented for the linkage of two genetic markers, norA and verA, which affect the biosynthesis of aflatoxin.
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Genetic Loci Associated with Altered Resistance to Microtubule Inhibitors and with Spore Shape in Dictyostelium discoideum
More LessMutations at 18 loci which map to five linkage groups in Dictyostelium discoideum are shown to affect resistance to antimicrotubule agents (including coumarin). The resistance or sensitivity mutations are classified according to whether their effects are limited to antimicrotubule agents, or whether cross-resistance or sensitivity to other compounds (notably acriflavin and cycloheximide) is observed. The latter class is likely to include mutations affecting permeability and is probably not of interest in the search for mutations directly affecting the cytoskeleton. All four acriflavin-resistance loci (acrA to acrD), many coumarin-sensitivity loci (couB to couE, couI), and both arsenate-resistance loci (arsA, arsB) fall into this category. Many mutants isolated on the basis of resistance to antimicrotubule agents (e.g. benlate, thiabendazole) in fact map at acrA or acrC. Some mutations affecting resistance to microtubule inhibitors also affect spore shape. Analysis of mutations such as couG370 and couH361, which affect coumarin sensitivity, spore shape, temperature sensitivity and, in the case of couH361, thiabendazole sensitivity provides the possibility of an ultrastructural approach to the study of the cytoskeleton in D. discoideum.
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The Tail Fibre of Bacteriophage T4 is Sensitive to Proteases at Elevated Temperatures
More LessBacteriophage T4 wild-type is not sensitive to heating at 60 °C. Trypsin at this temperature quickly inactivates the bacteriophage, with first-order kinetics for about the first 60 min. The half-inactivation period is around 15 min. The characteristics of this inactivation reaction have been studied. Inactivation of T4 particles is paralleled by a loss in ability to adsorb to bacteria. SDS-PAGE reveals a ‘clipping’ of gp 37, the protein of the distal part of the long tail fibres, during the inactivation reaction, gp 37 is the only protein to be modified under these conditions. Mutants have been isolated which resist this modification; they map in gene 37.
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Cloning of the Bacillus subtilis lys and spoIIIB Genes in Phage ø105
More LessThe lys gene of Bacillus subtilis was inserted into prophage 𝜙105. The recombinant phage (𝜙105dlys) contained DNA which was about 2 MDal smaller than the wild-type phage DNA, and the phage particles had no tails. The phage did not plaque but, when provided with tails in vitro, it transduced both lys-1 and lys-3 strains of B. subtilis to Lys +. The lys + gene was located on a 2·5 MDal EcoRI restriction fragment. Subsequently this phage was used to clone, using a similar technique, the spoIIIB gene(s). The second recombinant phage, 𝜙105dspoIIIB, was also defective, i.e. without tails. The DNA was 1.5 MDal smaller than the wild-type phage DNA and the spoIIIB2 + gene was located on a 3 MDal EcoRI fragment. When provided with tails in vitro, phage 𝜙105dspoIIIB transduced cells of a spoIIIB2 recipient to Spo+. In these transductants the spoIIIB2 mutation was complemented, and the cells sporulated normally.
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Isolation and Partial Characterization of the Cell Wall of Pseudomonas syringae pv. syringae HS191: Comparison of Outer Membrane Proteins of HS191 with Those of Two Plasmidless Derivatives
More LessThe outer membrane (OM) proteins of a Pseudomonas syringae pv. syringae strain (HS191), capable of causing holcus leaf spot of corn and other grasses, and two plasmidless derivatives of HS191, one avirulent (AO111) and one with reduced virulence (PSG100), were isolated by selective solubilization of the cytoplasmic membrane (CM) with 0·4 % (w/v) SDS or by centrifugation on sucrose density gradients. OM preparations were enriched in hexosamine and 2-keto-3-deoxyoctonate and contained little of the CM enzymes NADH oxidase and malate dehydrogenase. Characterization of OM preparations by SDS-PAGE and isoelectric focusing indicated a minimum of 20 OM proteins; protein bands 8 and 9 quantitatively predominated. In addition, some of the proteins were modified by heat and others by 2-mercaptoethanol. Although protein 5 was absent in both plasmidless strains, AO111 differed from PSG100 in lacking protein 5a and having two proteins (8b′ and 8c′) with different pi values. It is suggested that plasmid pCG131 coded for protein 5 (mol. wt 68000) and repressed the synthesis of proteins 4a (78000) and 5a (63000). The relationship of specific changes in OM protein composition to virulence is discussed.
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Plasmid Replication in a Temperature-sensitive Chromosome Replication Mutant of Staphylococcus aureus
More LessReplication of the antibiotic resistance plasmids pI258, pT10501 and pC221 has been investigated in a mutant of Staphylococcus aureus NCTC 8325, which is temperature-sensitive for the initiation of chromosome replication. Replication of pI258 stopped rapidly at the non-permissive temperature, whilst replication of pT10501 and pC221 continued (although at a lower rate than in the wild-type). It is proposed that the product of the mutant gene may be required directly for pI258 replication, but not for replication of pT10501 or pC221.
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Plasmids, Recombination and Chromosome Mapping in Streptomyces lividans 66
More LessStreptomyces lividans 66 was shown to harbour two self-transmissible plasmids: SLP2, which acts as a sex factor, and SLP3. Derivatives of this strain which had lost both plasmids were used as host strains to study a range of Streptomyces plasmids for their ability to promote their own transfer and to mobilize chromosomal markers. A linkage map of the S. lividans chromosome containing ten markers was derived from the results of matings using several different sex plasmids, and protoplast fusions. SLP2 was transferred interspecifically to S. parvulus ATCC 12434 and to S. coelicolor A3(2); in the latter it acted as a fertility factor. Interspecific crosses also led to the discovery of a further plasmid, SLP4, from S. coelicolor. SLP2, SLP3 and SLP4 could not be visualized on agarose gels using standard plasmid isolation procedures, but their presence was detected by transformation into S. lividans.
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Bacteriophage M: an Incompatibility Group M Plasmid-specific Phage
More LessPhage M was specific for bacterial strains, of various genera, harbouring plasmids of the M incompatibility group. It formed turbid plaques which varied from pin point to more than 2 mm in diameter on all hosts where plaques were detected. The phage had an hexagonal outline with a diameter of 27 nm. It contained RNA but differed from other plasmid-dependent RNA phages in being sensitive to chloroform. It adsorbed along the length of shafts of M pili.
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Incompatibility Properties of the IncFIII/FIV Haemolytic Plasmid pSU316 when Integrated in the Escherichia coli Chromosome
More LessThe haemolytic plasmid pSU316 is incompatible with members of the IncFIII and IncFIV incompatibility groups. Plasmid pSU307 (pSU316 hlyC :: Tn5) was inserted by integrative suppression into the chromosome of JW112, a temperature-sensitive dnaA mutant of Escherichia coli. The incompatibility properties of this strain (SU51) were studied and it was found that: (1) plasmid pSU306 (pSU316 hlyA :: Tn802) was rapidly lost from strain SU51 both at 30 °C and 42 °C; (2) the IncFIII plasmid pSU397 (ColB-K98 :: Tn802) was lost from strain SU51 at 42 °C but not at 30 °C; and (3) the IncFIV plasmid R124 was stably maintained in strain SU51 at both temperatures. Revertants of pSU307 to the autonomous state could be obtained from SU51. These revertants exerted incompatibility towards the prototype plasmids pSU306, pSU397 and R124 in the same way as pSU307 itself. Thus, strain SU51 provided a suitable method for distinguishing the three different incompatibility determinants of plasmid pSU316.
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- Pathogenicity And Medical Microbiology
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Differential Amino Acid Utilization by Chlamydia psittaci (Strain Guinea Pig Inclusion Conjunctivitis) and its Regulatory Effect on Chlamydial Growth
I. Allan and J. H. PearceThe effect of omission of individual amino acids from growth medium on the multiplication of Chlamydia psittaci (strain guinea pig inclusion conjunctivitis) in cycloheximide-treated McCoy cells has been examined. Marked differences were observed in the amounts of particular amino acids required for normal chlamydial multiplication: omission of either leucine, phenylalanine or valine completely inhibited multiplication, whereas absence of any one of another 10 amino acids had no effect on numbers of cells infected. Threshold concentrations of 80, 80 and approx. 8 nmol ml−1 for leucine, valine and phenylalanine, respectively, were needed for normal chlamydial multiplication. These requirements could not be related either to unusually high content in the whole organism, to degradation in the medium, or, from studies with leucine, to deficient association of leucine with host cells. Leucine deprivation at late stages of the developmental cycle also appeared to regulate multiplication. Possible mechanisms responsible for these effects are discussed.
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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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- Corrigenda
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