- Volume 131, Issue 8, 1985
Volume 131, Issue 8, 1985
- Sgm Special Lecture
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Little Fleas and Lesser Fleas
More LessSummary: Ruder heads stand amazed at these prodigeous pieces of nature, whales, elephants, dromedaries, and camels; these, I confess, are the colossus and majestick pieces of her hand; but in these narrow engines there is more curious mathematicks; and the civility of these little citizens more easily sets forth the wisdom of their Maker.
Sir Thomas Browne, 1642
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- Biochemistry
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An Inducible Xylanase of the Mushroom Termitomyces clypeatus Differing from the Xylanase/Amylase Produced in Dextrin Medium
More LessSummary: The mushroom Termitomyces clypeatus produces a single endoxylanase (1,4-β-D-xylan xylano-hydrolase, EC 3.2.1.8) in the presence of either dextrin or xylan as sole source of carbon. The enzymes produced in the two conditions are different. The enzyme induced by xylan has been purified 67-fold from the culture filtrate of T. clypeatus. The enzyme preparation gave a single protein band on SDS-PAGE, corresponding to a molecular weight of about 24000. The enzyme has an isoelectric point at pH 4·0 and acts on arabinoxylan and arabinogalactan, but not amylopectin or galactomannan. It shows maximum activity on xylan (1,4-β-linked D-xylopyranose units) at pH 3·5 and 55°C and is fairly stable up to 60°C. The K m for xylan is 4 mg ml−1. Hg2+, Fe2+ and Ag+ are the most potent inhibitors of the enzyme. The pH optimum and molecular weight of this inducible xylanase differ from those of the enzyme produced by the same organism grown in dextrin medium.
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One-dimensional Peptide Mapping of the Subunits of Pertussis Toxin
More LessSummary: Pertussis toxin (pertussigen) purified from the cytoplasmic fraction of Bordetella pertussis strain 18334, phase 1, consisted of five subunits which included an additional subunit (S1a) not previously reported. Subunits S1, S1a and S2 showed extensive structural homology when analysed by one-dimensional peptide mapping, indicating that the latter two were probably derived from proteolytic cleavage of the largest subunit, S1. Subunits S3 and S4,5 generated only a limited number of peptides following chemical and enzymic degradation, but these subunits differed structurally from each other and from those showing structural homology.
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Glutamine Synthetase of Streptomyces cattleya: Purification and Regulation of Synthesis
More LessSummary: Glutamine synthetase (GS; EC 6.3.1.2) from Streptomyces cattleya was purified using a single affinity-gel chromatography step, and some of its properties were determined. Levels of GS in S. cattleya cells varied by a factor of 8 depending upon the source of nitrogen in the growth medium. Of 24 nitrogen sources examined only glutamine or NH4Cl utilization resulted in very low GS activity. Addition of NH4Cl to a culture with high GS levels appeared to stop further synthesis and resulted in a progressive decrease in the specific activity of the enzyme. The GS inhibitor methionine sulphoximine (MSX) inhibited GS activity but had no effect on exponentially growing cells. The presence of MSX either lengthened or shortened the period between spore inoculation and initiation of exponential growth, depending on the source of nitrogen. In glutamine minimal medium MSX produced earlier and more efficient spore germination while in glutamate or nitrate minimal medium germination was delayed by its presence.
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Purification and Molecular and Catalytic Properties of Bromoperoxidase from Streptomyces phaeochromogenes
More LessSummary: A bromoperoxidase has been isolated and purified from the chloramphenicol-producing actinomycete Streptomyces phaeochromogenes. The purified enzyme was homogeneous as determined by polyacrylamide gel electrophoresis. The prosthetic group of the bromoperoxidase was ferriprotoporphyrin IX. Based on gel filtration results the molecular weight of the enzyme was 147000 ± 3000. Sodium dodecyl sulphate-polyacrylamide gel electrophoresis showed a single band having the mobility of a 72500 molecular weight species. Therefore, in solution at neutral pH, the bromoperoxidase behaved as a dimer. The isoelectric point was 4·0. The spectral properties of the native and reduced enzyme are reported. The homogeneous enzyme also had peroxidase and catalase activity.
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The Purification and Properties of Cellobiose Dehydrogenase from Sclerotium rolfsii and its Role in Cellulolysis
More LessSummary: An extracellular cellobiose dehydrogenase has been purified from the culture filtrates of Sclerotium rolfsii. The purified enzyme is homogeneous as determined by disc gel electrophoresis, with and without SDS, and by analytical isoelectric focusing in polyacrylamide gel. The enzyme is a single-subunit glycoprotein containing 8·9% total carbohydrate; its M r is 63000-64500, and its isoelectric point 5·18. The enzyme oxidized cellobiose, other cellodextrins and lactose whereas other disaccharides tested were not utilized as substrates. The rate of cellodextrin oxidation decreased and the K m increased with increasing degree of polymerization of the substrate. Cytochrome c was reduced though at a considerably lower rate than 2,6-dichlorophenolindophenol. The natural electron acceptor for the enzyme has not been identified.
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Purification and Properties of LIQ 4, an Antibacterial Substance Produced by Streptococcus faecalis var. liquefaciens K 4
More LessSummary: An antibacterial substance (LIQ 4) produced by Streptococcus faecalis var. liquefaciens K 4 was isolated in extracellular and cell associated form. It markedly inhibited the growth of Gram-positive bacteria, whereas only a few Gram-negative bacteria were susceptible. LIQ 4 was purified by hydrophobic chromatography (Servachrome XAD-2; octyl-Sepharose CL-4B; Sephadex LH 60) and Sephacryl S-200. TLC yielded a fluorescent spot as the active component and several inactive ninhydrin-positive substances. These contaminating peptides strongly adsorbed to LIQ 4 and could only be removed by repeated reversed phase HPLC. Furthermore, HPLC separated LIQ 4 into seven closely related substances. All showed strong fluorescence under UV light, stained yellow with ninhydrin, and contained aspartate and lysine after acid hydrolysis. The molecular weight was estimated by Amicon ultrafiltration to be less than 2000. LIQ 4 was stable at 80°C (30 min) and pH 2, but considerably inactivated above pH 8. It was apparently affected by proteolytic enzymes, but the activity could be fully restored upon heating.
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One or Two Low Affinity Penicillin-binding Proteins May Be Responsible for the Range of Susceptibility of Enterococcus faecium to Benzylpenicillin
More LessSummary: Three benzylpenicillin-resistant, clinical isolates of Enterococcus faecium (MIC values 16-64 μ;g ml−1) contained six penicillin-binding proteins (PBPs), of which PBP5 was the most abundant and had the lowest affinity for the antibiotic. Four benzylpenicillin-susceptible strains (MIC values 0·031-0·5 μ;g ml−1) were obtained as spontaneous derivatives from these above organisms. There were significant decreases in the amounts of PBP5 in each of the derivatives, with the concomitant appearance of a new, higher affinity PBP (5*) in three strains. Increased amounts of PBP5, with no changes in PBP5*, were found in several mutants with intermediate-level benzylpenicillin-resistance (MIC values 1-8 μ;g ml−1) selected from two of the susceptible strains. Examination of 18 other clinical isolates, with a wide range of susceptibilities to benzylpenicillin (MIC values 0·062-128 μ;g ml−1), showed that PBP5* was present in 13 strains, and PBP5 in all of them, but in differing amounts. The results concerning the relative amounts and relative affinities of PBPs 5* and 5 allowed the categorization of the various strains into six groups, within which organisms had somewhat similar susceptibilities to benzylpenicillin.
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Composition of Lipopolysaccharide from Strains of Pseudomonas syringae pv. morsprunorum of Differing Host Specificity and Virulence
More LessSummary: Lipopolysaccharide (LPS) from strains of Pseudomonas syringae pv. morsprunorum isolated from plum and cherry contained a uniform core region, which, in all cases, was composed of 2-keto-3-deoxyoctonic acid, heptose, glucosamine, galactosamine, rhamnose, glucose, alanine, ethanol-amine and phosphate. Lipid A contained glucosamine, phosphate and the fatty acids 3-OH 10:0, 12:0, 2-OH 12:0, and 3-OH 12:0. Sidechains of LPS from two virulent plum isolates were composed of glucose and rhamnose, and were susceptible to hydrolysis by rhamnanase from the typing phage A7, which uses LPS as its binding site. Phage A7-resistant mutants of the virulent cherry isolate C28 contained rough LPS, did not adsorb A7, and displayed reduced virulence towards cherry. An avirulent, phage A7-resistant mutant of a plum isolate contained smooth LPS with sidechains of a high glucose content that were resistant to phage hydrolysis. Loss or alteration of sidechains therefore correlated with loss of virulence.
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Effects of Nitrate, Nitrite and Diphenylamine on the Photosynthetic Apparatus of Rhodopseudomonas sphaeroides f. sp. denitrificans
More LessSummary: We have compared the effect of diphenylamine (DPA) on the pigment composition of Rhodopseudomonas sphaeroides f. sp. denitrificans grown in photoheterotrophic culture with the previously reported effect of nitrate. It was demonstrated that the effect of nitrate is due to the nitrite which is produced during denitrification. Both nitrite and DPA caused a decrease in the synthesis of spheroidene, and the accumulation of more-reduced precursors not normally seen. Nitrate (effectively nitrite) caused a decrease in the amount of bacteriochlorophyll, and the reaction centre from denitrifying cells did not contain the 28 kDal polypeptide (RC-H) subunit. These effects did not occur over a range of DPA additions (4 to 8 μ;g ml−1) to cells growing in the absence of nitrate. Denitrifying cells also had 40-50% lower activity of δ-aminolaevulinic acid synthase than those grown with or without DPA. Both nitrite and DPA treatments resulted in the loss of the B870 light-harvesting complex because of a failure to synthesize its 12 kDal polypeptide subunit.
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Studies of the 2':3'-Cyclic Nucleotide Phosphodiesterase of Haemophilus influenzae
More LessSummary: The 2':3'-cyclic nucleotide phosphodiesterase:3'-nucleotidase of Haemophilus influenzae was purified from a periplasmic preparation by affinity chromatographic techniques. The enzyme-catalysed hydrolysis of 2': 3'-cyclic AMP to adenosine without accumulation of the intermediate substrate 3'-AMP was demonstrated by high performance liquid chromatography. Competitive inhibition of the enzyme by a variety of nucleosides and mononucleotides indicated the presence of either purine or pyrimidine bases to be essential for selective interactions with the enzyme, and confirmed the need for a 3'-position phosphate for the functioning of mononucleotides as substrates for the enzyme. The enzyme had a molecular weight of 79000, was stable at low temperatures and was thermally denatured at temperatures above 50°C.
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Isolation and Characterization of the Pigment Esters of Xanthomonas juglandis (campestris)
More LessSummary: Separation of the pigment esters of Xanthomonas juglandis (campestris) on silica gel columns yielded four bands. Two of the bands, esters 1 and 2, made up over 95% of the mixture. Both esters were phospholipids, with ester 1 containing the alcohol moieties glycerol and sorbitol, and ester 2 only glycerol. Both appeared to be mixtures containing different xanthomonadin pigment molecules but the results suggested that ester 1 consisted of one pigment molecule esterified to glycerophosphoryl sorbitol, whereas ester 2 consisted of 2 pigment molecules esterified to glycerophosphate.
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An Exo-D-galacturonanase of Butyrivibrio fibrisolvens from the Bovine Rumen
More LessSummary: An intracellular pectinolytic enzyme was isolated from a cell extract of Butyrivibrio fibrisolvens and purified. The optimum pH for enzyme activity was 5·6. The enzyme preferentially degraded de-esterified substrates by hydrolysis of monosaccharide units from the non-reducing end; the only product of degradation was D-galacturonic acid. Values of K m and V max for oligo- and polygalacturonates indicated that the best substrate was digalacturonic acid; oligogalact-uronates containing either a saturated or a Δ4,5-unsaturated non-reducing end were both degraded. The enzyme was classified as an exo-D-galacturonanase [poly(1,4-α-D-galacturonide) galacturonohydrolase (EC 3.2.1.67)].
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- Development And Structure
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Purification and Characterization of Flagella from Roseburia cecicola, an Obligately Anaerobic Bacterium
More LessSummary: Flagella from Roseburia cecicola, an obligately anaerobic bacterium originally isolated from murine caecal mucosa, were purified by mechanical shearing followed by differential centrifugation. Purity of the flagellar preparation was determined by polyacrylamide gel electrophoresis, electron microscopy and chemical analysis. The flagella were composed of a single protein subunit (flagellin) with an estimated molecular weight of 42000. The amino acid composition of the flagellin was similar to that of some facultatively anaerobic and aerobic bacteria.
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- Genetics And Molecular Biology
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Identification of a New Locus in the Escherichia coli Cotransduction Gap that Represents a New Genetic Component of the L-Asparagine Utilization System
More LessSummary: A temperature-sensitive Escherichia coli mutant defective for the ability to utilize L-asparagine as a sole nitrogen source was isolated after N-methyl-N'-nitro-N-nitrosoguanidine mutagenesis. The mutation (asu) produces two distinct phenotypic effects. (1) Mutant strains grow poorly at high temperature on minimal plates containing asparagine as the sole nitrogen source; this effect is greatly exacerbated by the presence of methionine. (2) Mutant strains utilize L-asparagine as a nitrogen source three to four times more efficiently at permissive temperatures than the wild-type strains. The mutation maps at 32·4 min on the E. coli chromosome, within the E. coli cotransduction gap. Mutant strains produce normal amounts of thermo-stable L-asparaginase I activity. The mutation therefore affects a component of the asparagine utilization system other than the catabolism of asparagine within the cell; it probably affects asparagine uptake.
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Transduction Complicates the Detection of Conjugative Ability in Lysogenic Salmonella Strains
More LessSummary: When lysogenic salmonella strains were examined for conjugative ability in tri-parental crosses, false positive results were sometimes obtained because phage carried by the lysogenic strains multiplied on the intermediate salmonella recipient strain and then transduced its streptomycin/sulphonamide resistance plasmid to the final salmonella recipient strain. Back transfer of the plasmid to the lysogenic strains was also detected.
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Resistance Plasmids of Aeromonads
More LessSummary: The resistance plasmids of isolates of Aeromonas hydrophila and A. salmonicida from Japan, France, the UK and Ireland have been characterized. Three classes of plasmids transmissible to Escherichia coli were found: plasmids of incompatibility group C, plasmids of incompatibility group U, and plasmids incapable of stable inheritance in E. coli, not belonging to any defined incompatibility group.
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Identification of Restriction Fragments from Two Cryptic Clostridium butyricum Plasmids That Promote the Establishment of a Replication-defective Plasmid in Bacillus subtilis
More LessSummary: Clostridium butyricum NCIB 7423 carries two cryptic plasmids, pCB101 (6·05 kbp) and pCB102 (7·8 kbp). Sites for the restriction enzymes EcoRI, EcoRV, HindIII, ClaI and PstI have been found in one or both of these plasmids and their relative positions determined. Restriction fragments from both plasmids have been inserted into a vector plasmid (pJAB1) that is able to replicate in Escherichia coli but not in Bacillus subtilis and the recombinant plasmids have been established in E. coli. A 3·3 kbp Sau3A fragment of pCB101 conferred upon the vector the ability to transform both Rec+and Rec−strains of B. subtilis. Plasmid pRB1, a representative chimaera carrying only the 3·3 kbp Sau3A fragment of pCB101, was successfully transferred from B. subtilis back to E. coli. Plasmid pRB1 was readily lost from B. subtilis in the absence of selection. This evidence, together with the results of hybridization experiments, suggests that pRB1 is present as a weakly replicating autonomous element in B. subtilis. A recombinant plasmid carrying a 2·0 kbp Sau3A fragment of pCB102 underwent integration into the B. subtilis chromosome.
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Genetic Analysis of Candida albicans Morphological Mutants
More LessSummary: In contrast to some other strains, Candida albicans 1001 gave rise, upon UV irradiation, to mutants displaying a ‘rough colony’ morphology associated with a permanent alteration in morphogenesis which determined growth of the cells mostly as pseudohyphae. One of these mutants, C. albicans 1001FR, could form sectored (rough/smooth) colonies spontaneously, and with increasing frequency treatment with mild UV doses (32-64 μ;J mm−2). Rough sectors corresponded to stable ‘rough-filamentous’ strains which never segregated smooth strains. On the other hand, smooth sectors consisted mainly of yeast cells which could occasionally revert to a rough-filamentous phenotype. We suggest that C. albicans 1001 is heterozygous for some gene involved in the control of morphogenesis, and that the described mutants should be of help in the characterization of the genetic control of dimorphism in C. albicans.
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- Immunology
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Immunomodulation by Myxospores of Myxococcus xanthus
More LessSummary: Glycerol-induced myxospores of Myxococcus xanthus caused non-specific modulation of humoral and cellular immune responses in laboratory animals. The number of cells which formed specific haemolysins in spleens of mice immunized with sheep erythrocytes was increased when 0.5 × 108 myxospores were inoculated 2 d after the erythrocytes, and decreased when myxospores were injected 2 d before or at the same time as the erythrocytes. Both the IgG primary response and the secondary response to erythrocytes were decreased in rabbits after pretreatment with 2 × los myxospores per rabbit. Delayed-type hypersensitivity to sheep erythrocytes was also suppressed in mice after intraperitoneal (i.p.) injection of 0.3 × 108 myxospores. One day after i.p. injection of myxospores, neither an inflammatory response nor bone marrow cell depletion was observed in mice. These results support the idea that M. xanthus myxospores possess diverse immunomodulation properties apparently due to factors different from the classical LPS of Gram-negative bacteria.
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- Pathogenicity And Medical Microbiology
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Purification, Characterization and Immunological Properties of the Capsular Polysaccharide of Pasteurella haemolytica Serotype T15: Its Identity with the K62 (K2ab) Capsular Polysaccharide of Escherichia coli and the Capsular Polysaccharide of Neisseria meningitidis Serogroup H
More LessSummary: Capsular polysaccharide from two strains of Pasteurella haemolytica serotype T15 was purified and characterized by chemical analysis and NMR spectroscopy. The polymer, a teichoic acid, proved to be very similar in structure to the capsular polysaccharide of P. haemolytica serotype T4 and identical to the previously described K62 (K2ab) capsular polysaccharide of Escherichia coli. and the capsular polysaccharide of Neisseria meningitidis serotype H, i.e. (2-glycerol-3) (phosphate) (4-x-D-galactopyranose-1) with partial O-acetylation on the galactose residues. Electron microscopy with Protein A-gold labelled antisera showed that the polysaccharide was peripherally located on the surface of all three organisms. Chemical removal of O-acetyl groups from the polysaccharide yielded a structure identical to that previously described for E. coli K2 (K2a). Both O-acetylated and de-O-acetylated P. haemolytica T15 polymers, when absorbed on to sheep erythroyctes in passive haemagglutination assays, yielded identical antibody titres with sera raised against P. haemolytica T15, E. coli K 2 or N. meningitidis H whole cells. De-O-acetylation of the Pasteurella polysaccharide influenced its precipitability with immune sera, but this could not be related to the absence of O-acetyl groups because the non-acetylated E. coli K2 polymer readily precipitated with a line of ‘identity’ with the acetylated P. haemolytica T15 polymer.
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Motility as an Intestinal Colonization Factor for Campylobacter jejuni
More LessSummary: The colonization of the intestinal tract of suckling mice by Campylobacter jejuni was examined by orally challenging the mice with a wild-type strain and several nonmotile mutant strains which were isolated after treating the wild-type strain with mutagens. The wild-type strain had colonized the lower portion of the small intestine, the caecum and the colon 2 d after inoculation. Two nonmotile strains, one of which (M8) had lost all the flagellar structure including the filament, the hook and the basal structure, and the other (M1) which had lost only the filament region, were both cleared from the intestinal tract 2 d after challenge. Another nonmotile strain (M14), which had a complete flagellar structure like that of the wild-type strain, did not colonize and was cleared from the intestinal tract like the other nonmotile and nonflagellated strains. One atypically motile strain (M5), which had a shorter flagellar filament than that of the wild-type strain, colonized the intestinal tract only when mice were challenged with a large inoculum. None of the mice challenged with either the wild-type or any of the mutant strains showed signs of illness. We concluded that motility is an important factor in the colonization of the intestinal tract of suckling mice by C. jejuni.
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Chemical and Immunological Characterization of a Novel Amphipathic Antigen from Biotype B Streptococcus sanguis
More LessSummary: A new type of amphipathic antigen was extracted from whole cells of Streptococcus sanguis ATCC 10557 (biotype B, serotype II) by the phenol/water method. The extract was treated with nuclease P1, and was applied to a column of Sepharose 6B. Each fraction was checked by passive haemagglutination (PHA) and immunodiffusion tests against anti-10557 serum which was obtained by immunizing rabbits with whole cells of strain ATCC 10557. Strong PHA activity was demonstrated in the first hexose-containing peak (peak 1) eluted near the void volume, while the second hexose-containing peak (peak 2) produced a heavy band against anti-10557 serum in an immunodiffusion test. The third peak (peak 3) which partially overlapped with peak 2 reacted with concanavalin A, but not with the antiserum, in agar gel. Peaks 2 and 3 had no PHA activity. Peak 1 contained only 1% phosphorus, indicating that cells of strain ATCC 10557 possess an amphipathic antigen which differs from the lipoteichoic acids that are common in many Gram-positive bacteria. Peak 1 was a fatty acid-substituted heteropolysac-charide composed of glucose, galactose, mannose, glycerol and fatty acids in a molar ratio of approximately 1·0 :1·3 : 2·7 : 0·3 : 1·0. PHA activity was inhibited in the presence of polymerized mannose. Peak 2 was composed of glucose, galactose, rhamnose and N-acetylgalactosamine in a molar ratio of approximately 1·0:1·4:0·8:0·8, which was essentially identical to the serotype II carbohydrate antigen reported previously.
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Plasmids Mediating Iron Uptake in Vibrio anguillarum Strains Isolated from Turbot in Spain
More LessSummary: Vibrio strains isolated from diseased turbot in an experimental fish farm on the Atlantic coast of northwest Spain were identified as Vibrio anguillarum. The isolates shared many biochemical characteristics with V. anguillarum strains obtained from other sources, and harboured a plasmid species that showed extensive homology with plasmid pJM1, carried by V. anguillarum strain 775 isolated from an epizootic in North America. Restriction endonuclease analysis showed that the two plasmids were very similar albeit not identical. The presence of the plasmid in the turbot isolates was associated with their ability to cause disease in fish. Plasmid-carrying bacteria could also grow under conditions of iron limitation. Two outer membrane proteins, of 86 and 79 kDal, were induced, and a similar siderophore activity to that produced by V. anguillarum 775 was also detected under these conditions. The 86 kDal outer membrane protein cross-reacted immunologically with antiserum raised against the outer membrane protein OM2 produced by strain 775. Nonvirulent plasmidless derivatives were unable to grow under iron-limiting conditions, and were also unable to produce either siderophore activity or the 86 kDal outer membrane protein, suggesting the plasmid-mediated nature of these components.
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- Physiology And Growth
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Kinetics of Utilization of Organic Substrates by Mycoplasma mycoides subsp. mycoides in a Salts Solution: a Flow-microcalorimetric Study
More LessSummary: The metabolism of various organic substrates by suspensions of Mycoplasma mycoides subsp. mycoides in a salts solution was followed by microcalorimetry. Enthalpy changes associated with metabolism were in good agreement with theoretical values. Substrate utilization showed Michaelis kinetics, allowing saturation constants (K m) and maximum specific rates of substrate utilization (V max) to be determined. In cells grown on a complex medium containing glucose, K m values were: glucose, fructose, N-acetylglucosamine, glycerol and pyruvate, <5 μ;M; lactate, 20 μ;m; glucosamine, 130 μ;m, and mannose, 1 mM. Values of V max for glycerol, pyruvate and lactate were similar and approximately twice those for glucose, mannose, glucosamine and N-acetylglucosamine; V max for fructose was one-quarter of that for glucose. In cells grown on complex medium in which glucose was replaced by mannose, glucosamine or N-acetylglucos-amine, V max and K m for the respective growth sugars and for glucose were not significantly affected. However, in cells grown in the presence of fructose, V max for fructose increased to the value observed for glucose. It is suggested that M. mycoides is adapted to, and is constitutive for, the utilization of a single sugar (glucose), and a single amino sugar (N-acetylglucosamine), but that in the presence of fructose a fructose-utilizing pathway is induced.
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Effect of Tunicamycin on Germ Tube and Yeast Bud Formation in Candida albicans
More LessSummary: Tunicamycin is an antimicrobial agent which inhibits the first reaction of the dolichol pathway leading to N-glycosylation of proteins. The effect of tunicamycin on the growth of the dimorphic fungus Candida albicans differed depending on the growth phase of the organism. Addition of tunicamycin to stationary phase yeast cells inhibited the resumption of growth of those cells in either morphology, as cultures failed to initiate either yeast bud or germ tube formation. When tunicamycin was added to growing cells, growth was inhibited but not immediately. When it was added to germ tube cultures, nuclear division and septum formation continued for some time before ceasing. Addition of the drug to exponential phase yeast cultures resulted in an approximately 45% increase in cell number before cell division ceased and yeast accumulated in both budded and unbudded stages of the cell cycle. Accumulation of trichloroacetic acid precipitable radiolabelled protein and nucleic acid continued unchanged for some time following addition of tunicamycin; however, after a while a reduced rate of accumulation was noted.
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Induction of Nectria galligena Mutants Resistant to Benzoic Acid and Study of Their Aggressiveness Towards Immature Apples
More LessSummary: The isolation of Nectria galligena mutants resistant to benzoic acid (BA), the phytoalexin of immature apples, is reported. Those in which pathogenicity on mature apples was similar to the wild-type were inoculated in immature fruits. One mutant resistant to BA was more aggressive than the wild-type in young apples at the end of their maturation cycle. Aggressiveness of this mutant and the wild-type could be increased by growing the fungus before inoculation into the fruit on a medium supplemented with BA. Amounts of BA in mutant-infected and wild-type-infected tissues were similar. We conclude that BA is involved in the resistance of immature apples infected by N. galligena.
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Nitrate Assimilation in the Basidiomycete Yeast Sporobolomyces roseus
More LessSummary: Ammonium-grown cultures of Sporobolomyces roseus developed the capacity to assimilate nitrate when they were incubated in nitrate or nitrogen-free medium. Cycloheximide, 6-methyl purine and antimycin A inhibited this process. Nitrate assimilation required aerobic energy metabolism and was inhibited completely by ammonium. The rapid inhibition of nitrate assimilation by ammonium was not the result of an inhibition of nitrate reductase (NR) activity. Nitrite also inhibited nitrate assimilation. NR in cell-free extracts of S. roseus was NADPH-specific and its activity was repressed in cultures containing ammonium and derepressed during nitrogen starvation. Nitrate stimulated the appearance of NR in these cultures. Nitrate assimilation was not limited by apparent (potential) NR activity.
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Copper Uptake by Penicillium ochro-chloron: Influence of pH on Toxicity and Demonstration of Energy-dependent Copper Influx Using Protoplasts
G. M. Gadd and C. WhiteSummary: The existence of energy-dependent copper influx was demonstrated in protoplasts of Penicillium ochro-chloron. Protoplasts and mycelium were tolerant of copper over the pH range 3·0 to 5·5 but sensitive at pH 6·0. Uptake of copper was approximately ten times greater at pH 6·0 than in the lower range. At pH 3·0, influx showed saturation kinetics with a half-maximal influx at an external Cu2+ concentration of 390 μ;M and a maximum influx rate of 22 nmol h−1 (107 cells)−1. Saturation kinetics were not observed at pH 6·0.
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Mechanism of Self-protection in a Puromycin-producing Micro-organism
More LessSummary: Puromycin is a potent inhibitor of bacterial protein synthesis, but puromycin-producing Streptomyces alboniger KCC S-0309 is tolerant to the antibiotic in vivo. Puromycin bound to both 30S and 50S ribosomal subunits from S. alboniger and inhibited polyuridylate-directed polyphenylalanine synthesis by the ribosomes. However, the organism possessed a novel puromycin-inactivating enzyme which acetylated the antibiotic at the 2″-NH2 group of the O-methyltyrosine moiety.
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Properties of Organic Acid Utilization Mutants of Rhizobium leguminosarum Strain 300
More LessSummary: Mutants of Rhizobium leguminosarum 300 which were unable to utilize one or more organic acids as growth substrates were obtained by Tn5 mutagenesis. Mutant strain MNF3080 was defective in dicarboxylate transport and was unable to grow on succinate. Strain MNF3085 was defective in phosphoenolpyruvate carboxykinase and hence could not carry out gluconeogenesis. This strain did not grow on pyruvate, succinate, glutamate or arabinose but grew on glucose and on glycerol. Strain MNF3075 was unable to utilize pyruvate; the biochemical lesion in this mutant was not identified. MNF3085 and MNF3075 were symbiotically effective. MNF3080 nodulated peas, but the nodules were ineffective in N2 fixation and displayed morphological abnormalities. These data support previous findings which suggest that utilization of exogenous dicarboxylates is essential for effective nodule development by R. leguminosarum.
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Gluconeogenesis in Rhizobium leguminosarum MNF3841
More LessSummary: Gluconeogenesis in Rhizobium leguminosarum MNF3841 takes place via the derepressible enzymes phosphoenolpyruvate carboxykinase (PEPCK) and fructose bisphosphate aldolase. A Tn5-induced PEPCK deficient mutant (MNF3085) fails to grow on a wide range of simple carbon sources. PEPCK and fructose bisphosphate aldolase are rapidly derepressed when cells are transferred from a sugar to an organic acid as the sole carbon source. The addition of 0·1 mM-sucrose causes an 80% inhibition of PEPCK and fructose bisphosphate aldolase synthesis, and 0·4 mM-sucrose causes a complete inhibition of PEPCK synthesis. Pea bacteroids of MNF3841 purified on a Percoll gradient contain low levels of PEPCK and fructose bisphosphate aldolase. This bacteroid-associated PEPCK activity is clearly of bacterial not plant origin because of its nucleotide requirement, and the fact that bacteroids of MNF3085 contain no PEPCK activity at all. Although the mutant lacks PEPCK it is still able to nodulate and fix N2 as effectively as the parent. The capacity to synthesize sugars via gluconeogenesis is not required for an effective symbiosis. Furthermore, these data suggest that pea bacteroids receive sufficient sugar to compensate for the gluconeogenic defect in strain MNF3085, but insufficient to completely repress the synthesis of PEPCK in the wild-type.
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- Systematics
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An Evaluation of Some Physiological and Biochemical Methods as an Aid to the Characterization of Species of Penicillium Subsection Fasciculata
More LessSummary: Physiological and biochemical test methods have been evaluated for use in the taxonomy of filamentous microfungi, with specific reference to isolates of Penicillium subsection Fasciculata. Physiological and enzymic tests such as growth in the presence of inhibitory substances, pH limits of growth and the hydrolysis of a variety of substrates proved to be relatively easy to perform and showed promise as differential characters. Carbon and nitrogen source assimilations were technically difficult to perform and few substrates gave differential results. In the few cases where assimilation tests were successful they often involved substrates that may otherwise be inhibitory, such as organic acids and sodium nitrite. Results from assimilation tests appeared to be differential at around species level and positive results were often associated with a significant rise in growth medium pH. Many of the tests described have not previously been applied to filamentous fungi and may have applications in the study of other genera.
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The Free Lipids of Mycobacterium leprae Harvested from Experimentally Infected Nine-banded Armadillos
More LessSummary: The free lipids of a sample of Mycobacterium leprae were extracted by a procedure designed to produce separate non-polar and polar fractions. The composition of these lipids was analysed semi-quantitatively by five special thin-layer chromatographic systems covering the total range of mycobacterial lipid polarities. In order of increasing polarity, the major lipids were dimycocerosates of phthiocerol A, phthiocerol B and phthiodiolone A, glycosyl phenolphthiocerol dimycocerosates and phospholipids, including monoacylphosphatidylinositol di- and pentamannosides. The diacylated forms of these latter lipids, found in most mycobacteria, were not present. The composition of the free lipids of the leprosy bacillus, surveyed over the total polarity range for the first time, showed that the patterns were particularly related to those of Mycobacterium bovis, Mycobacterium kansasii and Mycobacterium marinum.
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Quantitative Comparison of the Mycolic and Fatty Acid Compositions of Mycobacterium leprae and Mycobacterium gordonae
More LessSummary: The mycolic and fatty acids of three samples each of Mycobacterium leprae and Mycobacterium gordonae were compared. Acids released by whole-organism alkaline hydrolysis were converted to 4-nitrobenzyl esters and mycolic acids were further derivatized to t-butyldimethylsilyl ethers. Thin-layer chromatography of the derivatized long-chain extracts showed that all three M. leprae preparations contained so-called α-mycolates and ketomycolates but that the M. gordonae samples had a methoxymycolate in addition to the above types. Silica gel normal-phase high-performance liquid chromatography of the total mycolic acid derivatives confirmed the lack of detectable amounts of methoxymycolates in M. leprae and reverse-phase chromatography of the individual mycolate types demonstrated the homogeneity of the chain lengths of the mycolic acids in each species. Non-hydroxylated fatty acid 4-nitrobenzyl esters were transformed to methyl esters and examined by gas chromatography. Tuberculostearic (10-methyloctadecanoic) acid was a major component of the lipids of all three M. leprae preparations but it was absent in one M. gordonae strain and a very minor component in the other representatives of this latter species. On the basis of fatty and mycolic acid compositions, therefore, a previously suggested close relationship between M. leprae and M. gordonae was not supported.
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Numerical Analysis of Fatty Acid Profiles in the Identification of Staphylococci
More LessSummary: Representative strains of coagulase-positive and coagulase-negative staphylococci were degraded by acid methanolysis and the resultant fatty acid methyl esters analysed by gas chromatography. The quantitative data obtained were examined by cluster analysis. The coagulase-positive strains formed six major and one single-member cluster at the 90% S-level. The Staphylococcus intermedius aggregate cluster included the single-member cluster and major clusters 1 and 2. The four remaining clusters contained S. aureus strains and were homogeneous and distinct. The coagulase-negative strains were recovered in ten major and three single-member clusters at the 90% S-level. Five of the ten major clusters were reasonably homogeneous with respect to the existing classification. Thus, three S. capitis strains and five of the six S. epidermidis strains, two of the three S. hominis strains and five of the six S. simulans strains were recovered in separate clusters. Cluster 7 was divided into two subclusters; one contained five of the six S. hyicus strains and the other contained the two representatives of S. lentus. The remaining clusters were heterogeneous with regard to the named strains they contained.
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- Short Communications
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Construction of a Mutant of Escherichia coli That Has Deletions of Both the Penicillin-binding Protein 5 and 6 Genes
More LessSummary: A mutant of Escherichia coli has been constructed with deletions of the genes encoding penicillin-binding protein 5 (dacA) and penicillin-binding protein 6 (dacC). The construction of this mutant establishes that the complete loss of the two most abundant species of penicillin-binding proteins can be tolerated by E. coli. Moreover, the double deletion mutant had the same growth rate and morphology as an isogenic dacA+dacC+ strain.
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Diazotrophy within Desulfovibrio
More LessSummary: Use of a pyruvate-based medium in which the supply of combined N limited growth enabled detection of diazotrophy (as C2H2 reduction) in batch cultures of 12 out of 15 strains of Desulfovibrio representing 5 species and including 2 strains hitherto believed to be non-diazotrophic.
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