- Volume 135, Issue 7, 1989
Volume 135, Issue 7, 1989
- Genetics And Molecular Biology
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Escherichia coli tolQ Mutants Are Resistant to Filamentous Bacteriophages That Adsorb to the Tips, not the Shafts, of Conjugative Pili
More LessThe tolQ (previously fii) mutation in Escherichia coli K12 inhibits infection by filamentous bacteriophages f1 and IKe but not by RNA-containing phage f2. This work extends these observations to other plasmid-specific bacteriophages including various filamentous, RNA-containing, and lipid-containing isolates. Only tip-adsorbing filamentous phages were affected by tolQ and not shaft-adsorbing ones. Electron microscopy showed that RP4-specific filamentous phage Pf3 was one of the latter kind. Several tip-adsorbing filamentous phages inhibited conjugation between tolQ strains carrying their specific plasmids, implicating the phage receptors (conjugative pili) as mating organelles. tolQ mutant strains were as proficient as their parents in conjugation mediated by a wide range of plasmids.
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Mutations Affecting the Cytochrome d-Containing Oxidase Complex of Escherichia coli K12: Identification and Mapping of a Fourth Locus, cydD
More LessA mutant of Escherichia coli K12 has been isolated affected in a gene, designated cydD, distinct from the three previously described loci involved in the synthesis of assembly of the cytochrome bd oxidase complex. The mutant, obtained by nitrosoguanidine mutagenesis, lacks the spectroscopically detectable components of this oxidase, namely cytochromes b 558, b 595 and d. Cytochrome oxidase o is the sole CO-binding cytochrome in membranes of the mutant, but the soluble haemoprotein b-590 and catalase activity appear unaffected. Discrimination between Cyd+ and Cyd- strains is facilitated by the development of a defined low-phosphate medium that allows the inclusion of Zn2+ as well as azide, inhibitors of respiratory electron transfer particularly via cytochrome o. Mapping with F-prime factors and by P1 cotransductional frequencies shows the mutation to map near 19·3 min on the E. coli chromosome, distinct from cydC, which maps at 189 min. The gene order in this region was tested in a three-factor cross and demonstrates the order zbj::Tn10(YYC199)cydDaroA, consistent with cotransduction frequencies.
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The Genetics of Bile Acid Degradation in Pseudomonas spp.: Location and Cloning of Catabolic Genes
More LessFour Pseudomonas spp. capable of utilizing bile acids as sole carbon source were examined for the presence of plasmids. One plasmid was found in Pseudomonas sp. RAL8, but no plasmids could be detected in the other three strains. Mitomycin C curing of RAL8 did not affect the ability of the strain to grow on bile acids. This suggested that the genetic information for bile acid catabolism in all four strains was chromosomally located. To isolate bile acid catabolic genes, DNA from RAL8 was partially digested with Sau3A, then the DNA fragments cloned into the broad-host-range cosmid vector pMMB33. The resulting gene bank was screened by plate-mating with two stable RAL8 mutants. Four of the gene bank clones were found to give a positive complementation with one or both mutants. Examination of the plasmids in the four clones revealed that they were unstable, but detailed mapping enabled a 52 kb restriction map to be derived. Further complementation work showed that two of the bile acid catabolic genes are located close together on the map, and may be contiguous.
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- Immunology
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Relationship between Structure and Antigenicity of O1 Vibrio cholerae Lipopolysaccharides
More LessThe relationship between the release of fructose from 01 Vibrio cholerae lipopolysaccharides (LPS) by dilute acetic acid hydrolysis and the decrease in their antigenicity was examined. Decrease in the intigenicity of LPS was not parallel with the release of fructose, and occurred very much later than the latter. Periodate oxidation of LPS resulted in the total elimination of the fructose and glucose, and two-thirds of the heptose constituents, but no difference in the antigenicity of LPS was observed before and after oxidation. These findings indicate that the fructose present in O1 V. cholerae LPS is not substantially involved in their specific antigenicity. In the O1 V. cholerae LPS, the fructose is in the branch structure, most probably in the core region.
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- Pathogenicity And Medical Microbiology
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Radiolabelling of Mycobacterium avium Oligosaccharide Determinant and Use in Macrophage Studies
More LessInternal radiolabelling procedures were used to radiolabel the oligosaccharide determinant of the glycopeptidolipids (GPL) from serovars 4 and 20 of the Mycobacterium avium complex. Mycobacteria were cultured in the presence of [6-3H]fucose, [2-3H]mannose or [methyl-3H]methionine, after which radiolabeled native lipid was extracted and distribution of radioactivity in native and deacetylated lipid was determined by thin-layer chromatographic methods. Incorporation of radiolabel was confirmed by examining acid hydrolysates of purified GPL for 3H-labelled sugars on cellulose thin-layer plates. Least incorporation of radiolabel into GPL was observed with [6-3H]fucose, whereas better incorporation was obtained with [2-3H]mannose and [methyl-3H]methiomne. Use of [methyl-3H]methiomne resulted in the radiolabelling of the methylated sugars in both the oligosaccharide determinant and the 3,4-di-O-methylrhamnose located at the terminus of the peptide core. Use of [2-3H]mannose resulted in the incorporation of radioactivity into the oligosaccharide determinant as 2-O-methylfucose, found in the GPL of both serovars 4 and 20. GPL radiolabeled with [2-3H]mannose were subsequently examined in macrophage cultures and found to be relatively inert to degradation by those phagocytic cells. These results substantiate earlier findings with the GPL of serovar 20 and indicate that these mycobacterial components may play a role in pathogenesis.
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Interaction of Candida albicans with Neutrophils: Effect of Phenotypic Changes in Yeast Cell-surface Composition
More LessThe susceptibility of four strains of Candida albicans to phagocytosis and intracellular killing by rabbit peritoneal neutrophils was investigated. Two of the strains, isolated from active infections, were known to synthesize a surface layer of mannoprotein fibrils in response to growth on 500 mm-galactose; the other strains, from asymptomatic carriers, lacked this capability. The presence of serum opsonins greatly enhanced phagocytosis of all four strains and, following opsonization, phagocytosis of an infective strain was equally rapid after growth on either 500 mm-galactose or 50 mm-glucose. In the absence of opsonins, galactose-grown infective strains were phagocytosed faster than either glucose-grown infective strains or galactose-grown carrier strains. These differences in phagocytic uptake were paralleled by differences in neutrophil chemiluminescence response. Intracellular killing of galactose-grown infective strains was only half that of glucose-grown infective strains or galactose-grown carrier strains after incubation for 60 min. Pretreatment of neutrophils with extracellular polymeric material, which contains the surface fibrils, completely inhibited intracellular killing. These results indicate that production of the fibrillar layer promotes yeast virulence by increasing resistance to intracellular killing, although it may enhance phagocytosis in locations where opsonic activity is poor.
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Location of a Blocking Epitope on Outer-membrane Protein III of Neisseria gonorrhoeae by Synthetic Peptide Analysis
More LessA series of overlapping peptides spanning the deduced amino acid sequence of outer-membrane protein PHI of Neisseria gonorrhoeae have been synthesized on solid-phase supports. The peptides were used in an attempt to locate the epitopes recognized by anti-PIII monoclonal antibodies with defined biological properties. Four bactericidal and two nonbactericidal antibodies were reacted with the synthetic peptides. None of the bactericidal antibodies reacted with the linear peptides. However, the two nonbactericidal antibodies were found to react within the disulphide loop thought to be exposed on the bacterial surface. Monoclonal antibody SM51 recognized a decapeptide corresponding to amino acid residues 24–33, while monoclonal antibody SM50 recognized an octapeptide contained within the decapeptide. The difference in the ability of the two antibodies to block the bactericidal effect of antibodies directed against other surface antigens therefore appears to be related to a difference in their ability to activate complement rather than to the location of the epitope recognized.
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Differentiation of Staphylococcal Species and Strains by Ribosomal RNA Gene Restriction Patterns
More LessStaphylococcal DNA was digested with endonucleases and probed with labelled ribosomal RNA (rRNA) from Escherichia coli. Reproducible restriction patterns containing between seven and 22 bands were obtained for seven different species of staphylococci. These profiles were species-specific with different strains of a particular species sharing an identical or similar restriction pattern. The results reported here indicate that rRNA gene restriction pattern analyses have an application in the taxonomy of staphylococci.
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Initial Phase of Infection with Tyzzer's Organism in Cultured Mouse Hepatocytes
More LessThe initial phase of infection with Tyzzer’s organisms in cultured mouse hepatocytes was observed using indirect immunofluorescence (IF) and a plaque assay. The organism, adhered poorly to aldehyde- or acetone-fixed cells, but once adhered to host cells, whether methanol-fixed or unfixed, they were not removed by methanol or acetone. By the IF as well as the plaqlue assay, both of which discriminated intra- and extracellularly located organisms the number of cell-associated organisms increased linearly up to 3 h post-inoculation. The number of intra-cellular organisms increased rapidly in the first 1 h, followed by linear increase at a much lower rate. Similar results were obtained by the plaque assay.
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Ultrastructural Studies on the Intracellular Fate of Chlamydia psittaci (Strain Guinea Pig Inclusion Conjunctivitis) and Chlamydia trachomatis (Strain Lymphogranuloma Venereum 434): Modulation of Intracellular Events and Relationship with Endocytic Mechanism
More LessPrevious observations on the highly infectious LG V strain 434 of Chlamydia trachomatis and the guinea pig inclusion conjunctivitis (GPIC) strain of C. psittaci (which requires centrifugation of inocula with host cell monolayers for maximum infectivity) indicated that infectivity differences were expressed, not at entry, but at an intracellular stage affecting multiplication. Centrifugation increased the potential of internalized chlamydiae to undergo productive infection. Here, analysis of the intracellular fate of chlamydiae by ultrastructural methods indicates that strain GPIC exhibits two patterns of behaviour depending on the mode of inoculation. Strain GPIC showed limited entry, with 47 % of intracellular organisms becoming associated with thorotrast-labelled lysosomes, following static incubation with monolayers. In contrast, with centrifugation, entry was not limited and association with lysosomes was reduced to 12 %; strain 434 behaved similarly but independently of the mode of inoculation. The different results for strain GPIC correlated with distinct entry mechanisms. Entry during static incubation was unimpaired either by treatment with cytochalasin D or by temperature reduction to 20 °C, suggesting that it was pinocytic. Entry during centrifugation was markedly impaired by both treatments, suggesting that it was phagocytic. The data lead to two novel conclusions: first, that chlamydiae can apparently enter cells by both pinocytic and phagocytic mechanisms; second, that the entry mechanism influences intracellular fate. It is suggested that entry mechanism is linked to selection of the vesicle membrane forming around the internalizing chlamydiae. This, in turn, may influence both intracellular translocation and subsequent inhibition or promotion of multiplication of the internalized parasite.
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- Physiology And Growth
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A Role for G-proteins and Inositol Phosphate Signalling in Dictyostelium discoideum Slug Behaviour
More LessPhototaxis by Dictyostelium discoideum slugs was disoriented in the presence of pertussis toxin, suggesting a role for pertussis-toxin-sensitive G-proteins in slug behaviour. The decrease in orientation accuracy was dependent on the concentration of this bacterial toxin. Pertussis toxin did not significantly affect slug thermotaxis, while cholera toxin had no significant effects on either slug phototaxis or thermotaxis. The addition of micromolar concentrations of lithium salts (LiCl or LiBr) resulted in disorientation of both phototaxis and positive thermotaxis by slugs. The corresponding sodium salts had no effect on slug behaviour, confirming that the effects were specific to lithium ions. The effects of lithium ions suggest a role for inositol phosphates as possible second messengers in Dictyostelium slug behaviour. Except for slight impairment of thermotaxis by d-sphingosine, we found no effects on slug behaviour of either phorbol 12-myristate 13-acetateorsphingolipids. We thus found little pharmacological evidence for a role in slug signal transduction of the diacylglycerol branch of the inositol lipid pathway.
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Cationic Interactions Regulate the Initiation and Termination of Zoospore Activity in the Water Mould Achlya heterosexualis
More LessThe zoosporic phase of the water mould Achlya heterosexualis, initiated when zoospores emerge from cysts, is strongly influenced by cations. The concentrations of Ca2+ and K+ present in the medium can determine whether or not zoospores will be released from cysts, whether zoospore motion will be approximately linear or circular, rapid or slow, and when zoospore activity will be terminated by encystment. Ionic effects on zoospore motility are rapid and reversible over short time spans; within a 2 min period motility can be strongly suppressed by increasing the [K+] and then restored by increasing the [Ca2+]. Trifluoperazine, considered to be an anti-calmodulin drug, both inhibits zoospore release, and arrests zoospore motility.
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The Role of Cytochromes and Blue Copper Proteins in Growth of an Obligate Methylotroph on Methanol and Methylamine
More LessThe aim of this work was to determine the extent to which the periplasmic cytochromes and blue copper proteins could replace each other during growth of an obligate methylotroph (organism 4025) on methanol and methylamine. This was done by varying the relative concentrations of these proteins as a result of varying the amount of copper added to the growth medium during growth in oxygen-limited conditions. It was shown that the amount of added copper required for maximum growth was greater during growth on methylamine than on methanol. The concentrations of membrane-bound cytochromes b, c and o were not markedly affected by the copper concentration added to the growth medium whereas the concentrations of soluble cytochromes and blue copper proteins varied considerably. The concentrations of cytochrome c L were highest at the copper concentrations giving maximum growth; this was more obvious during growth on methanol (when this cytochrome has a specific function) than on methylamine. The concentrations of blue copper proteins were highest at the copper concentrations which supported maximum growth (except for the absence of amicyanin during growth on methanol). In the absence of added copper, amicyanin could not be detected, and in the absence of added iron neither amicyanin nor ‘azurin’ was detectable. The most important conclusions are that high concentrations of amicyanin may not be essential for methylamine oxidation, that ‘azurin’ may replace cytochrome c for some electron-transport functions, and that iron is required for synthesis of blue copper proteins.
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Formation of Gas Vesicles in Phosphorus-limited Cultures of Microcystis aeruginosa
More LessThe formation of gas vesicles in samples taken from phosphorus-limited cultures of Microcystis aeruginosa was studied after a phosphate pulse. A light period of at least 10 h after the pulse was needed before gas vesicles were synthesized de novo, and formation did not occur in the dark. The length of the light period correlated with the increase in gas vacuolation. When samples were subjected to 5 h light periods at different times after the addition of phosphate, formation of gas vesicles was only observed when the light period started at least 5 h after the addition of phosphate. Gas vesicle formation was saturated at a photon flux density of approximately 50 μmol m-2 s-1. Synthesis of gas vesicles was not detected when the cultures were treated with chloramphenicol, rifampicin or 3-(3,4-dichlorophenyl)-N, N′-dimethylurea (DCMU); total gas vesicle volume did not decrease under these conditions, suggesting that turnover of gas vesicles occurs very slowly, if at all.
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Interaction of the Fluorescent Dye l-N-Phenylnaphthylamine with Escherichia coli Cells during Heat Stress and Recovery from Heat Stress
More LessThe fluorescent dye l-N-phenylnaphthylamine permeated Escherichia coli cells after exposure to a heat stress at 55 °C in Tris/Mg2+ buffer, pH 8·0. The rate of dye permeation increased with time during heat treatment and decreased gradually during subsequent incubation at 37 °C in a minimal medium. The initial level of rapid adsorption of the dye also increased with heating time, although it remained roughly constant during post-heating incubation. The results obtained suggest that the permeability barrier to the dye in the outer membrane was damaged by heat stress and was repaired after sublethal heating. RNA, protein and lipid syntheses, as well as an energy-yielding process, appeared to be necessary for the repair of impermeability to the dye.
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- Plant-Microbe Interactions
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A Pyrimidine Auxotroph of Sclerotinia sclerotiomm for Use in Biological Weed Control
More LessAn auxotroph of Sclerotinia sclerotiomm, A1-pyr, was isolated after mutagenesis of ascospores with ultraviolet light. Addition of cytosine and to a lesser extent uracil, but not thymidine, to minimal medium permitted growth of this auxotroph. Protoplasts of Al-pyr failed to regenerate on Czapek agar medium without cytosine supplementation (20 mg 1−1); on cytosine-supplemented medium 0·1 % of protoplasts successfully regenerated. The mutant was avirulent on four of seven susceptible hosts unless an exogenous cytosine source was applied at the inoculation site. The requirement for external cytosine makes A1-pyr a potential candidate for use in biological weed control. While isolates of this fungus obtained from nature attack numerous beneficial crop and native plants, A1-pyr would be limited to the area of cytosine application, thus reducing the threat to beneficial plants.
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- Systematics
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Inter- and Intrageneric Relationships of the Genus Propionibacterium as Determined by 16S rRNA Sequences
More LessThe intra- and intergeneric position of Propionibacterium was determined by comparing reverse transcriptase sequences of 16S ribosomal ribonucleic acid. Propionibacterium jensenii. P. thoenii and P. acidipropionici formed a phylogenetically tight cluster, while P. freudenreichii, P. acnes and P. propionicus were as remotely related among each other as each of them was to the first three species. The genus Propionibacterium represents a well defined taxon that stands isolated among other major groups of the actinomycetes. Its phylogenetic neighbours are the genera Nocardioides and Terrabacter.
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Classification of Pseudomonas aminovorans and Some Related Methylated Amine Utilizing Bacteria
P. N. Green and M. GillisEight strains of facultatively methylotrophic budding bacteria which utilized mono- and trimethylamine as sole carbon and energy source were shown to belong to the Agrobacterium-Rhizobium complex in rRNA superfamily IV. It is considered that these organisms probably represent a new taxon; a description is provided, and the taxonomy is discussed.
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