- Volume 136, Issue 9, 1990
Volume 136, Issue 9, 1990
- Genetics And Molecular Biology
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DNA homology between Saccharopolyspora strains and other erythromycin-producing actinomycetes
More LessThe macrolide antibiotic erythromycin is produced by different genera of the Actinomycetales, including strains of Saccharopolyspora, Arthrobacter, Micromonospora and possibly Streptomyces. Erythromycin is also produced by several unclassified strains, four of which could be assigned to the genus Saccharopolyspora on the basis of bacteriophage host specificity. We used cloned erythromycin biosynthetic and resistance genes from Saccharopolyspora erythraea as hybridization probes to determine if the DNA sequences encoding these genes are conserved in different genera. Our results indicated strong hybridization to genomic restriction fragments from strains that were assigned to the genus Saccharopolyspora, and weak hybridization to DNA from strains which produce erythromycin or other macrolides but belong to other genera.
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- Pathogenicity And Medical Microbiology
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Purification of El Tor cholera enterotoxins and comparisons with classical toxin
More LessIn 55 clinical isolates of Vibrio cholerae biotype El Tor, cholera toxin (CT) production was higher after growth in liquid medium first under relatively anaerobic conditions followed by excessive aeration (AKI conditions) as compared with growth under the optimal conditions for CT production from V. cholerae of classical biotype (median toxin level being 400 ng ml−1 and 1 ng ml−1 respectively, for the two different growth conditions). Large growth volumes further enhanced El Tor toxin production to levels at or above 3–5 μg ml−1 from several strains, which allowed for easy purification of toxin by salt precipitation, aluminium hydroxide adsorption and/or GM1 ganglioside affinity chromatography. However, such purified El Tor CT completely lacked the A subunit when examined by SDS-PAGE or by monoclonal anti-A subunit antibody GM1-ELISA. In contrast, when El Tor CT was prepared from bacteria grown in the presence of specific antiserum against soluble haemagglutinin/protease it contained the A subunit (unnicked) in the same proportion to the B subunit (1A:5B) as classical CT. Immunodiffusion-in-gel tests revealed that the B subunits of El Tor and classical CTs share major epitopes but also have one or more weaker biotype-specific epitopes. The two types of toxin were practically indistinguishable in various GM1-ELISA tests, and antisera raised against El Tor and classical CT, respectively, could also completely neutralize the heterologous as well as the homologous toxin activity in vivo. The results indicate that CTs from El Tor and classical V. cholerae, despite demonstrable epitope differences, are predominantly cross-reactive and give rise to antisera with strong cross-neutralizing activity.
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Purification and characterization of a protein antigen from Leptospira interrogans serovar hardjo, common to a wide range of bacteria
More LessA protein with a molecular mass of 64 kDa (P64) from Leptospira interrogans serovar hardjo was partially purified by using successively, phase partitioning with Triton X-114, ion-exchange chromatography and sucrose gradient centrifugation. Purification to homogeneity was obtained by electroelution of P64 from SDS-polyacrylamide gels. Monospecific rabbit antiserum (RαP64) was prepared using the purified protein preparation. P64 had a native molecular mass of > 670 kDa and was recognized by RαP64 as well as by human antisera. Western blotting of leptospiral serovars and 18 other bacterial species with RαP64 showed that P64 was cross-reactive with an equivalent antigen in a wide range of bacteria, indicating that it belongs to a family of antigens previously designated ‘common antigen’. This putative common antigen from Leptospira appears to have a sub-surface location, but its function is not yet known.
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- Physiology And Growth
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Culture parameters regulating stalk formation and growth rate of Gallionella ferruginea
More LessThe growth of Gallionella ferruginea in a mineral salts solution with carbon dioxide and iron sulphide was studied by acridine orange stained direct count and most probable number techniques. G. ferruginea grew to 2 × 106 cells ml−1 with a generation time of 8·3 h under aerobic gradient conditions. The optimum temperature for growth was 20 °C. No growth was obtained under anaerobic conditions, or without carbon dioxide. A method was developed for measuring the length of the stalks formed by G. ferruginea. When growing exponentially, the bacterium was freeliving, without stalks, and motile with one polar flagellum. A net production of stalk per cell began when the cell number exceeded 6 × 105 ml−1 if the pH exceeded 6. This occurred when growth entered the stationary phase. The stalk length increased from 3 × 103 μm ml−1 (detection limit) to 1·8 × 108 μm ml−1, during a 400 h growth experiment. There was no stalk formation at growth conditions where ferrous iron was stable, suggesting that stalk formation may be a protection mechanism against an increasing reducing capacity of ferrous iron as it becomes unstable in an environment that becomes oxidized. The results indicate that favourable growth conditions for G. ferruginea may be those present in reduced ground waters, rather than those in ditches, drainage tubes, wells, etc., where stalk-forming G. ferruginea can usually be found.
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Transport of neutral amino acids and penicillin formation in Penicillium chrysogenum
More LessThe production of penicillin was inhibited by neutral amino acids in a resting cell system of Penicillium chrysogenum with cycloheximide. The inhibitory action was prevented by preincubation with glutathione, even though this stimulated uptake of the neutral amino acid glutamine. Chromatographic analysis of extracts obtained from cells incubated with labelled glutamine revealed that radioactivity was taken up through the formation of two intermediates; l-γ-glutamyl-l-glutamine and δ-(l-α-aminoadipyl)-l-glutamine. In a resting cell system prepared from cultures previously grown in the presence of 50 mm-l-lysine, a condition which restrains α-aminoadipate and penicillin formation, uptake of glutamine was 80% inhibited. We propose that the penicillin precursor δ-(l-α-aminoadipyl)-l-cysteinyl-d-valine, which is structurally related to glutathione, is also utilized in the uptake of neutral amino acids.
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Second messenger involvement in differentiation of the entomopathogenic fungus Metarhizium anisopliae
More LessThe conidial germling of Metarhizium anisopliae produces an appressorium upon contact with a hard hydrophobic surface. We have conducted an investigation into how this entomopathogen mediates intracellularly the inductive signal to shift from polarized germ-tube growth to non-polarized appressorial growth. During sporulation, conidia accumulated 45Ca2+ but there was no evidence for a gradient of Ca2+ in the spore which could establish the initial polarity. Calmodulin, however, was localized at the poles of the conidia, near the site of germ-tube emergence. Exposing conidia to Ca2+ deprivation or calmodulin antagonists inhibited germination and polar growth. Disruption of Ca2+ gradients by ionophoresis did not prevent germination but caused the multiple emergence of branched germ-tubes from conidia. These findings indicate that Ca2+ plays a fundamental role in establishing the dominance of apical growth. Although an external source of Ca2+ is not required for appressorium formation, germlings producing appressoria took up 45Ca2+ when available. The 45Ca2+ accumulated in the cell wall, plasma membrane and organelles suggesting that these may function as Ca2+ stores for Ca2+-stimulated exocytosis of cell-wall materials. Mitochondria and vacuoles sequestered 45Ca2+ indicating that they play a role in maintaining low cytoplasmic concentrations of 45Ca2+ consistent with the reorganization of the cytoskeleton required for appressorial growth. Several Ca2+-binding proteins in appressoria may provide an energy-independent component of Ca2+ buffering in the cytoplasm. The results indicate that the apical Ca2+ gradient is disrupted during differentiation and subsequent differential Ca2+ redistribution in the cell enlargement zone coincides with germ-tube swelling. cAMP may also be involved by potentiating the effects of small changes in Ca2+ concentration and stimulating exocytosis of mucus components required for adhesion, which is a prerequisite for differentiation.
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Acquisition of iron from citrate by Pseudomonas aeruginosa
More LessTransport of [14C]citrate, ferric [14C]citrate and [55Fe]ferric citrate into Pseudomonas aeruginosa grown in synthetic media containing citrate, succinate, or succinate and citrate as carbon and energy sources was measured. Cells grown in citrate-containing medium transported radiolabeled citrate and iron, whereas the succinate-grown cells transported iron but not citrate. Binding studies revealed that isolated outer and inner membranes of citrate-grown cells contain a citrate receptor, absent from membranes of succinate-grown cells. [55Fe]Ferric citrate bound to the isolated outer membranes of each cell type. The failure of citrate to compete with this binding suggests the presence of a ferric citrate receptor on the outer membranes of each cell type. Citrate induced the synthesis of two outer-membrane proteins of 41 and 19 kDa. A third protein of 17 kDa was more dominant in citrate-grown cells than in succinate-grown cells.
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- Plant-Microbe Interactions
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Zeamatin, an antifungal protein from maize with membrane-permeabilizing activity
More LessA 22 kDa antifungal protein (zeamatin) was purified from Zea mays seeds. It was identified and assayed by its unusual property of acting synergistically with nikkomycin to inhibit growth of Candida albicans. Alone, it inhibited growth in suspension culture of C. albicans, Neurospora crassa and Trichoderma reesei. Zeamatin contained no detectable chitinase, 1,3-β-glucanase or ribosome-inactivating protein activity, enzymes present in a variety of plants that have been shown to have antifungal properties. At low concentrations zeamatin caused the rapid release of cytoplasmic material from C. albicans and N. crassa. This was confirmed microscopically by observing zeamatin-induced hyphal rupture of these fungi. These results suggest that zeamatin permeabilizes the fungal plasma membrane. We believe zeamatin to be a representative of a previously unrecognized class of plant antifungal proteins.
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- Systematics
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Evaluation of the stability of biochemical phenotypes of Escherichia coli upon subculturing and storage
M. Katouli, I. Khn and R. MllbyStability of the biotypic characters of 72 enteropathogenic Escherichia coli (EPEC) and 21 faecal E. coli strains was evaluated after storage and after subculturing using a computerized biochemical fingerprinting method. Sixteen (22%) EPEC strains and nine (43%) faecal strains exhibited changes in their biochemical reactions after subculturing. In contrast, strains stored at −70 °C and 4 °C did not show any measurable changes. Of 23 biochemical markers tested, eight were subject to changes in at least one of these strains. Change in lactulose fermentation was most frequent, occurring in 17 (18%) strains. A decrease or loss of activity in the fermentation of 5-ketogluconate, arbutin and methyl β-d-glucoside in six strains (6%), and an increase in the ability to ferment sucrose, raffinose, melibiose and d-arabinose in 20 strains (22%) were observed. Mean similarity of the strains, when compared pairwise before and after subculturing, was slightly affected by these changes, but the overall biochemical phenotypes of the strains remained constant.
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Small subunit ribosomal RNA evolution in the genus Acanthamoeba
More LessReverse transcription of small subunit ribosomal RNA (srRNA) was used to determine the partial nucleotide sequences of the srRNA of seven isolates of Acanthamoeba These seven sequences and the sequence of the corresponding region in an A. castellanii previously totally sequenced were compared in order to investigate evolution of the srRNA gene in Acanthamoeba. The results of the comparisons were consistent with the hypotheses that the genus is monophyletic, that the Pussard and Pons grouping system is valid, that the Acanthamoeba expansion segments in the srRNA gene evolve at a much faster rate than the rest of the gene, and that the extent of nucleotide sequence divergence in the genus Acanthamoeba is roughly similar to that differentiating vertebrates and invertebrates.
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A taxonomic review of the genus Microbispora and a proposal to transfer two species to the genus Actinomadura and to combine ten species into Microbispora rosea
More LessWe conducted a taxonomic review of the genus Microbispora using chemotaxonomic and DNA-DNA hybridization techniques, and reached the following conclusions: Microbispora viridis should be transferred to the genus Actinomadura as Actinomadura rugatobispora comb. nov., nom. nov. (type strain SF2240 = IFO 14382 = JCM 3366) and Microbispora echinospora should be transferred to the genus Actinomadura as Actinomadura echinospora comb. nov. (type strain JCM 3148 = ATCC 27300). We also propose that Microbispora rosea, Microbispora amethystogenes, Microbispora chromogenes, Microbispora diastatica, Microbispora indica, Microbispora karnatakensis and Microbispora parva should be combined into the species Microbispora rosea subsp. rosea (type strain JCM 3006 = ATCC 12950), and that Microbispora aerata, Microbispora thermodiastatica and Microbispora thermorosea should be combined and transferred to the new subspecies Microbispora rosea subsp. aerata comb. nov. (type strain IFO 12581 = ATCC 15448). Microbispora bispora clearly differs from these ten strains at the species level.
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Differentiation of Mycobacterium species by direct sequencing of amplified DNA
More LessNucleotide sequences specific for a range of Mycobacterium species were defined by computer-assisted sequence comparisons of small subunit ribosomal RNA. A polymerase chain reaction-based sequencing strategy was used to demonstrate that the 16S rRNA sequence can be used for the rapid identification of mycobacterial isolates. Identification at the species level can be obtained within 2 d, requiring < 10000 bacteria. This procedure reliably differentiates Mycobacterium spp. which are difficult to identify by classical methods, such as M. malmoense, M. szulgai and M. flavescens.
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