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Volume 135,
Issue 6,
1989
Volume 135, Issue 6, 1989
- Sgm Special Lecture
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Molecular Characterization of Bacterial Virulence Factors and the Consequences for Vaccine Design The 1988 Fleming Lecture
More LessThe pathogenesis of bacterial infections involves a complex series of interactions between the pathogenic micro-organisms and the host. The outcome of an infection will depend on a combination of factors including the virulence of the bacterial pathogen, the immune status of the host and the innate resistance of the host. In recent years we have learned to apply molecular techniques to the study of bacterial virulence. Molecular approaches allow us to identify and characterize at a structural, biochemical and immunological level particular bacterial virulence factors. The information we gain from such studies can be used together with in vivo and in vitro models to investigate the immune response to individual bacterial antigens. In the future these studies may lead to the development of novel immunoprophylactic, chemotherapeutic and diagnostic reagents. In this lecture I will present a personalized review of recent molecular studies on bacterial pathogens with particular reference to the potential for vaccine development.
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- Biochemistry
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Chitinase Activity from Candida albicans and its Inhibition by Allosamidin
More LessCandida albicans chitinase isolated using the Dyno-Mill disruption technique was characterized using an improved radiometric assay procedure. The enzyme had apparent temperature and pH optima of 45 °C and 65, respectively. The preparation yielded an apparent K m of 3·9 mg chitin ml−1 [17·6 mm-N-acetylglucosamine (GlcNAc) equivalents] and V of 23 nmol GlcNAc formed min−1 (mg protein)−1. The potential of the streptomycete antibiotic allosamidin as an antifungal agent is discussed in view of its dose-dependent inhibition of C. albicans chitinase activity (IC50 = 0·3 μm). Allosamidin was a potent competitive inhibitor of enzyme activity (K i = 02·3 μm).
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Ammonium Assimilation by Candida albicans and Other Yeasts: Evidence for Activity of Glutamate Synthase
More LessActivities and properties of the ammonium assimilation enzymes NADP+-dependent glutamate dehydrogenase (GDH), glutamate synthase (GOGAT) and glutamine synthetase (GS) were determined in batch and continuous cultures of Candida albicans. NADP+-dependent GDH activity showed allosteric kinetics, with an S0·5 for 2-oxoglutarate of 7·5 mm and an apparent K m for ammonium of 5·0 mm. GOGAT activity was affected by the buffer used for extraction and assay, but in phosphate buffer, kinetics were hyperbolic, yielding K m values for glutamine of 750 μm and for 2-oxoglutarate of 65 μm. The enzymes GOGAT and NADP+-dependent GDH were also assayed in batch cultures of Saccharomyces cerevisiae and three other pathogenic Candida spp.: Candida tropicalis, Candida pseudotropicalis and Candida parapsilosis. Evidence is presented that GS/GOGAT is a major pathway for ammonium assimilation in Candida albicans and that this pathway is also significant in other Candida species.
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13C-NMR Studies of Acetate and Methanol Metabolism by Methylotrophic Pseudomonas Strains
More LessThe metabolism of [2-13C]acetate by Pseudomonas M27(Icl−) and Pseudomonas MA(Icl+) was studied in vivo using 13C-NMR spectroscopy. The flux of 13C-label into bicarbonate, glutamate and citrate was observed in both organisms. In addition 13C-labelled α, α-trehalose was synthesized as a major metabolite by Pseudomonas M27 but not by Pseudomonas MA. The presence of this disaccharide in cell extracts of Pseudomonas AM1(Icl−) grown with [13C]methanol was also observed. The data from analysis of the trehalose multiplet signal observed in the spectra of Pseudomonas M27 cell extracts were consistent with the absence of the glyoxylate cycle in this methylotroph.
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Cepabactin from Pseudomonas cepacia, a New Type of Siderophore
More LessIn iron-deficient conditions of growth Pseudomonas cepacia ATCC 25416 excreted both pyochelin and a low-molecular-mass compound which strongly chelated iron(III), and facilitated iron translocation as demonstrated by growth and uptake experiments. The name cepabactin is proposed for this new siderophore. Comparisons of UV-visible spectra and chromatographic behaviour, together with 1H-NMR spectra, led to the conclusion that cepabactin is 1-hydroxy-5-methoxy-6-methyl-2(1H)-pyridinone, a compound which can be considered as a cyclic hydroxamate, but also as a heterocyclic analogue of catechol. This pyridinone has already been described by other workers as an antibiotic produced by Pseudomonas alcaligenes, and by a soil isolate closely related to Pseudomonas cepacia. Thus, cepabactin appears to act as a siderophore for more than one species of non-fluorescent pseudomonad.
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An Oxidoreductive Pathway for d-Xylose Assimilation by Rhodosporidium toruloides
More LessExtracts of Rhodosporidium toruloides grown aerobically on xylose contained xylitol dehydrogenase and d-xylose reductase activities. Extracts of cells grown on glucose contained one-tenth as much xylose reductase and no detectable xylitol dehydrogenase. The xylitol dehydrogenase was purified to near homogeneity, and is a tetramer of 45 kDa subunits. This labile enzyme, could be stabilized by glycerol (25%) and was rapidly inactivated by 10 mm-EDTA. It catalyses the reversible, NAD+-dependent oxidation of xylitol to xylulose. Apparent K m values are 19 mm-xylitol and 0·3 mm-NAD+ at 30 °C, pH 8·5. Partially purified preparations of xylose reductase catalysed the NADPH-dependent reduction of d-xylose to xylitol, and were 16 times as active with 33 mm-dl-glyceraldehyde as with 33 mm-d-xylose. Apparently R. toruloides grown on xylose has the necessary enzymes to convert xylose to xylulose by the oxidoreductive pathway.
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- Development And Structure
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Ultrastructural Details of the Apparatus of Gliding Motility of Myxococcus fulvus (Myxobacterales)
More LessCell was preparations of the myxobacterium Myxococcus fulvus were fractionated by ion-exchange chromatography. Highly ordered chain-like structures were found in certain fractions when negatively stained specimens were inspected in the transmission electron microscope. The chain-like structures, which we call ‘strands’, were built up of two structural components: (1) a ring with an outer diameter of 12 to 15 nm which was regularly stacked with a periodicity of 14 nm. and (2) an elongated component, measuring 11·0 × 2·8 nm. Each ring appeared to be linked to the next one by two elongated components. Their orientation towards the elongated components was somewhat variable, which suggests a certain flexibility and a potential for conformational changes of the ‘strands’. Three or more of the ‘strands’ appeared to form a superstructure, which is defined as ‘ribbon’, which might undergo conformational changes leading to the observed strinkage in the transverse direction of about 40%. We suggest that the structures described crease contraction waves in the surface of the myxobacterial cell, and that those waves drag the cell along. The organization of the apparatus of gliding motility in the cell surface could only very rarely be seen. An exceptionally well-preserved whole cell in a negatively stained specimen showed a surface pattern consisting of parallel ridges, indicated by dark stain lines and 40 to 50 nm wade running helically around the cell pole. At a higher magnification these parallel ridges showed a distinct, periodic substructure normal to their long axis and with a spacing of 14 nm. which corresponds exactly to the periodicity of the stacked rings within the ‘strand’.
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Hyphal and Mycelial Responses Associated with Genetic Exchange Within and Between Species of the Basidiomycete Genus Stereum
More LessLiving hyphae of Stereum hirstutum behavaved similarly prior to fusion on a cellophane membrane whether they were genetically the same or different. Fusions were generally initiated within 5–10 h of mycelial interdigitation. A period (10–35 min) of interfacial expansion usually preceded opening of the fusion pore, but in certain combinations involving an Australian homokaryon, MP16–15, refractile sheaths developed around hyphal penetration pegs. Between somatically incompatible heterokaryons, the cytoplasm in fusion compartments and some adjacent compartments progressively lysed and was replaced by refractile globules and wall deposits. Self fusions other than clamp connections results little or unidirectional nuclear movement via the fusion pore followed by cycles of nuclear aggregation, division and septation. This sequence also followed fusion between mating-compatible pairs and ‘bow-tie’-forming pairs of sibling homokaryons for 2–3 h and 18–28 h respectively. before the onset of central or eccentric septal erosion and nuclear migration Following magration, numerous intercalary septa were formed and there was no repair of eroded septa. An extended non-septate phase occurred within the Australian homokaryon, MP 16–15, in which rapid (≥ 10 µm s−1), pulsed, unidirectional, longrange protoplasmic streaming occurred.
Certain combinations of homokaryons of different taxonomic species of Stereum, or of different breeding strategy or geographical origin, gave rise to unilaterally extensive degenerative reactions in plate culture accompanied by production of free crystals of (+)-torreyol. Sequential septal nuclear migration and protoplasmic lysis were all observed in the partner which become degenerate. Constitutive septal erosion, nuclear migration and intercalary septal synthess were observed in sectors of appressed mycelium in certain homokaryons after long-term.
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- Genetics And Molecular Biology
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cAMP- and RAS-independent Nutritional Regulation of Plasma-membrane H+-ATPase Activity in Saccharomyces cerevisiae
More LessThe plasma-membrane ATPase of Saccharomyces cerevisiae is a proton pump whose activity, essential for proliferation, is subject to regulation by nutritional signals. The previous finding that the CDC25 gene product is required for the glucose-induced H+-ATPase activation suggested that H+-ATPase activity is regulated by cAMP. Analysis of starvation-induced inactivation and glucose-induced activation of the H+-ATPase in mutants affected in activity of the RAS proteins, adenylyl cyclase or cAMP-dependent protein kinase showed that nutritional regulation of H+-ATPase activity does not depend directly on any of these factors. We conclude that adenylyl cyclase does not mediate all nutritional responses. This also indicates that the specific CDC25 requirement for the glucose-induced activation of the H+-ATPase identifies a new function for the CDC25 gene product, a function that appears to be independent of CDC25-mediated modulation of the RAS/adenylyl cyclase/cAMP pathway.
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Cloning and Expression of Rhodococcus Genes Encoding Pigment Production in Escherichia coli
More LessPigment was produced by Escherichia coli cells carrying recombinant plasmids pNIL100, pNIL200 and pNIL400 containing DNA from Rhodococcus sp. E. coli cells containing pNIL100 or pNIL200 (with DNA inserts from Rhodococcus sp. JL10 and Rhodococcus sp. ATCC 21145 respectively) produced both blue and pink pigments, while cells containing pNIL400 (with a DNA insert from Rhodococcus sp. ATCC 21145) produced only pink pigment. Colonies of E. coli (pNIL100) and E. coli (pNIL200) were dark blue, whereas E. coli (pNIL400) colonies were pink. No pigment was detected in Streptomyces griseus transformants containing pNIL100, pNIL200 or pNIL400. Restriction endonuclease mapping indicated that the cloned DNA fragments were different. The pigment gene(s) in pNIL200 producing both the blue and pink pigments were contained within a 2·8 kb DNA fragment. The pigments produced by E. coli transformants containing pNIL200 were characterized by visible and UV spectroscopy. No similar pigments were detected in Rhodococcus sp. ATCC 21145.
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Analysis of Separate Isolates of Bordetella pertussis Repeated DNA Sequences
More LessTwo independent isolates of a Bordetella pertussis repeated DNA unit were sequenced and shown to be an insertion sequence element with five nucleotide differences between the two copies. The sequences were 1053 bp in length with near-perfect terminal inverted repeats of 28 bp, had three open reading frames, and were each flanked by short direct repeats. The two insertion sequences showed considerable homology to two other B. pertussis repeated DNA sequences reported recently: IS481 and a 530 bp repeated DNA unit. The B. pertussis insertion sequence would appear to comprise a group of closely related sequences differing mainly in flanking direct repeats and the terminal inverted repeats. The two isolates reported here, which were from the adenylate cyclase and agglutinogen 2 regions of the genome, were numbered IS481v1 and IS481v2 respectively.
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Nucleotide Sequence of the Neopullulanase Gene from Bacillus stearothermophilus
More LessThe gene (nplT) for a new type of pullulan-hydrolysing enzyme, neopullulanase, from Bacillus stearothermophilus TRS40 was sequenced. The DNA sequence revealed only one large open reading frame, composed of 1764 bases and 588 amino acid residues (M r 69 144). Although the thermostable neopullulanase contained eight cysteine residues, they did not provide conformational stability by disulphide bonds. A comparison was made of the amino acid sequences of α-amylase, neopullulanase, isoamylase, pullulanase and cyclodextrin glucanotransferase. All the enzymes examined contained four highly conserved regions which probably constitute the active centres of the enzymes. The amino acid residues required for the specificity of neopullulanase are compared with those of α-amylase and other amylolytic enzymes.
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Transmission Pattern of Mitochondrial DNA during Plasmodium Formation in Physarum polycephalum
More LessThe transmission pattern of mitochondrial DNA (mtDNA) was studied during plasmodium formation in Physarum polycephalum. Plasmodia were generated by matings between pairs of amoebal strains carrying mtDNA molecules that were distinguishable by restriction endonuclease digestion. The transmission of mtDNA was uniparental in every case; the plasmodia always carried mtDNA with the restriction pattern of only one of the two parental types. In each mating pair, one strain consistently acted as mtDNA donor, but this strain did not always act as mtDNA donor when combined in other mating pairs. The identity of the mtDNA donor in each pair was not determined by the different types of mtDNA molecules present or by different alleles of matB or matC, two mating-type loci which regulate amoebal fusion. The results suggested that alleles of a third mating-type locus, matA, which controls zygote development, might form a hierarchy such that the mtDNA donor in any cross would be the strain of higher status. The deduced hierarchy was matA2 > matA11 > matA12 > matA1.
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Growth, Development and Genetic Characteristics of Physarum polycephalum Amoebae Able to Grow in Liquid, Axenic Medium
More LessAmoebae from natural isolates of Physarum polycephalum, unlike the plasmodial phase, are unable to grow in axenic medium. A mutant strain of amoebae, CLd-AXE, can be cultured in the liquid, semi-defined medium used for plasmodial culture but lacks some of the properties required for studies of development and gene expression. From crosses of CLd-AXE with wild-type amoebae, new amoebal strains able to grow in axenic medium have been isolated; some of these can also undergo the reversible amoeba-flagellate transformation and apogamic plasmodium development in axenic conditions. Amoebae maintained in active growth in liquid culture for several months showed little change in their properties. Subcultures made with diluted inocula indicated that the same growth rate was achieved even when single amoebae were inoculated in liquid medium. All strains produced colonies with high efficiency when replated on bacterial lawns. Measurements of DNA content by flow cytometry indicated that the majority of amoebae in liquid cultures were haploid. Homozygous diploid amoebae constructed from one strain grew less well than the haploid cells. Genetic analysis of crosses suggested that amoebae able to grow in liquid axenic medium fell into one major phenotypic class with respect to growth rate, and that mutation at only one or two loci was necessary to allow amoebae to grow in axenic medium. Diploid, heterozygous amoebae constructed by mating a mutant with a wild-type strain were unable to grow in axenic medium, indicating that at least one of the putative axe alleles was recessive.
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Structural and Functional Analysis of Ribosomal Subunits from Vegetative Mycelium and Spores of Streptomyces antibioticus
More LessThe structure and function of ribosomes from spores and vegetative mycelium of Streptomyces antibioticus were compared. Differences were observed in the sedimentation coefficient of ribosomes from spores (56·86S) and vegetative mycelium (69·77S). Reverse-phase high-performance liquid chromatography of ribosomal proteins of the 30S and 50S subunits revealed differences which included several polypeptides present in the vegetative ribosomes but absent from spore ribosomes. The latter were also defective in their ability to promote polyphenylalanine synthesis, the functional activity of both ribosomal subunits being affected. The soluble fraction of spores also showed decreased protein-synthesizing activity, compared to that of the vegetative mycelium. Recovery of normal ribosomal subunits and soluble fraction activity occurred early in the germination process, reaching activity values approaching those of the vegetative state during initiation of germination. It is suggested that regulation of cellular metabolism at the level of translation may be involved in the establishment of spore dormancy.
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Distribution of Modules among the Central Regions of the Genornes of Several Actinophages of Faenia and Saccharopolyspora
More LessThe central regions of the genomes of 𝜙FR 114, 𝜙FR113 and Mp 1, three temperate phages of the thermophilic actinomycete Faenia, are shown to differ mainly with respect to modules of about 35 kb, designated J-module (𝜙FR114) and N-module (𝜙FR113, Mp1). The distribution of J and N was observed amongst 22 phages of Faenia and Saccharopolyspora; J-module homology was found on six phage genomes, whereas homology to the N-module was detected on ten phage genomes.
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Isolation of Plasmid DNA Sequences That Complement Rhodobacter sphaeroides Mutants Deficient in the Capacity for CO2-dependent Growth
More LessMutants deficient in the proper regulation and derepression of ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBPC/O) in Rhodobacter sphaeroides were isolated by ethyl methanesulphonate (EMS) and Tn5 mutagenesis of a recA parental strain. Mutants were identified by their ability to grow under conditions where the organism requires basal levels of RuBPC/O for growth yet fail to grow under conditions which require derepression of the enzyme (Aut−). The newly isolated Aut− mutants exhibited phenotypes distinguishable from the previously isolated Aut− mutant, strain KW25/11. Rocket immunoelectrophoretic examination of RuBPC/O levels revealed marked variance in the ability of mutants to derepress form I and form II RuBPC/O in the absence of exogenous carbon. Evidence that some of the mutants possessed different mutations was substantiated by complementation of the EMS-generated mutants by entirely different genes isolated from a genomic library of R. sphaeroides constructed in the broad-host-range cosmid vector pVK102. Southern hybridization analysis of the complementing library isolates showed the complementing genes to be normally carried on the endogenous plasmids of R. sphaeroides. The gene complementing mutant strain KW25/11 was mapped by Tn5 insertional inactivation and the complementing region found to reside on a 1·5 kb PstI-BamHI fragment. Complemented strains were unable to match wild-type levels of RuBPC/O under conditions requiring derepression of the enzyme, except for mutant strain EMS45. The Aut− phenotype, represented by the mutants isolated in this study, stems from a deficiency in some aspect of photoautotrophic growth.
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Functioning of the Colicin A Lysis Protein is Affected by Triton X-100, Divalent Cations and EDTA
More LessThe colicin A lysis protein, Cal, is synthesized at the same time as colicin A by Escherichia coli harbouring plasmid pColA after induction by mitomycin C. Its function in the induced bacteria involves the release of colicin A, quasi-lysis, the death of the producing cells and the activation of the outer membrane phospholipase A. We have found that these various functions are affected differently by treatment of the induced cells with Triton X-100, divalent cations or EDTA. Triton X-100 and EDTA caused increased quasi-lysis and a higher level of mortality of the producing cells, but while Triton X-100 enhanced the release of colicin A, EDTA reduced it. Divalent cations protected the cells against both killing and quasi-lysis without greatly affecting colicin release. The effects of these agents were similar for both wild-type and phospholipase A mutants and depended only on the presence of a functional cal gene.
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Genetic Recombination by Spheroplast Fusion of Sterol-transforming Mycobacterium Strains
More LessWall-deficient forms of fast-growing mycobacteria were produced in growth medium containing vancomycin and glycine, and spheroplasts were prepared by lysozyme treatment of wall-deficient cells. Spheroplasts gave rise to recombinants with high frequency (2-6%) when they were fused using polyethylene glycol 6000. The results demonstrated that in vivo genetic recombination could be used to produce genetically modified Mycobacterium strains with applications in transformation of steroids. Useful intermediates of steroid drug synthesis and new degradation products were obtained from sterols by selected recombinant strains.
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Specific Neisseria gonorrhoeae DNA-Probes Derived from Ribosomal RNA
More LessEighteen sequences complementary to less-conserved regions within the 16S and 23S ribosomal ribonucleic acid (rRNA) of Neisseria gonorrhoeae were subcloned or chemically synthesized and used as probes in a dot-spot deoxyribonucleic acid (DNA): DNA hybridization format. Some of these probes exclusively detected Neisseria gonorrhoeae nucleic acid, whereas others also showed hybridization signals with nucleic acid from other bacterial species. Our results indicate that rRNA-derived DNA-probes can be used to differentiate between very closely related taxa without the use of Southern-blot analysis.
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