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ACTINOMYCETOLOGICA Vol. 6 No. 1, 1992
Code Number: AC92009
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Published by the Society for Actinomycetes, JapanABSTRACTS OF PAPERS BIOSYNTHESIS AND RECEPTOR PROTEIN OF BUTYROLACTONE AUTOREGULATOR OF STREPTOMYCES VIRGINIAE Yamada,Y., T.Nihira and S.Sakuda Department of Biotechnology, Faculty of Engineering, Osaka University 2-1 Yamadaoka, Suita, Osaka 565, Japan Actinomycetologica, 6: 1-8, 1992 Autoregulators, having in common a butyrolactone skeleton, are widely distributed among Streptomyces spp. The biosynthetic pathway of one of these autoregulators, virginiae butanolide A, is proposed on the basis of isotope labelled precursor experiments. The identification of the receptor protein for butyrolactone autoregulators in Streptomyces virginiae and its purification, characterization and cloning are also reviewed. Authors' Abstract STREPTOMYCES SUBTILISIN INHIBITOR: GENETICAL CHARACTERIZATION AND ITS APPLICATION Taguchi, S. Department of Biological Science and Technology, Science University of Tokyo Nodashi, Chiba 278, Japan Actinomycetologica, 6: 9-20, 1992 Streptomyces subtilisin inhibitor (SSI). first isolated from the culture filtrate of Streptomyces albogriseolus S3253, is one of the few well characterized microbial protease inhibitors and has a narrow range of specificity inhibiting alkaline serine proteinases [subtilisin (STN) family]. SSI exists as a stable dimer protein consisting of identical subunits with a molecular weight of 11,500. Genetic studies revealed that the SSI gene possesses two tandemly arranged promoters, two terminators and also two translational initiation codons as well as two SD sequences. Taking into consideration the fact that S.albogriseolus can produce extracellular SSI protein in large amounts, these types of dual structures might play an important and unique role in the efficient and versatile expression of the SSI gene. Secretory and protease inhibitory characteristics of SSI and their use in expression vectors for the efficient production of two heterologous proteins, cADI and apidaecin were also examined. The proposed systems should prove useful for the efficient secretory production of other heterologous gene products and as models for heterologous gene expression in Streptomyces. R.L. MICROMONOSPORA CHERSINA SP.NOV. Tomita, K., Y.Hoshino, N.Ohkusa, T.Tsuno and T.Miyaki Bristol-Myers Squibb Research Institute, 2-9-3 Shimo-meguro, Meguro-ku, Tokyo 153, Japan Actinomycetologica, 6: 21-28, 1992 A dynemicin producer, strain M9561, is proposed as a new species of the genus Micromonospora. M. chersina is distinguished from previously described species by menaquinone, fatty acid and cell-wall diamino acid composition and by physiological characteristics. Description of Micromonospora chersina sp.nov.: (chersina, Gr.f. adj. khersinos; "living in dry land" referring to the savanna vegetation from which the organism was isolated). The vegetative mycelium is branched monopodially and forms single spores which are sessile or borne on short or long monopodial sporophores. The spores are spherical with short blunt spines (1.2 to 1.8 um). No aerial mycelium is formed but rudimental aerial hyphae are occasion- ally observed on organic media such as yeast extract-malt extract agar. Fragmentation of hyphae has not been observed. Colonies on yeast extract-malt extract agar (ISP medium No. 2) are colorless to pale yellow and turn to olive black after sporulation. A fluorescent yellow diffusible pigment is formed in Czapek's sucrose-nitrate agar and inorganic salts-starch agar (ISP medium No. 4). Nitrate reductase is formed, but tyrosinase is not. NaCl tolerance is 3% or less. The maximum temperature for growth is 49 C, with optimum growth occurring at 37 to 44 C. Melibiose is utilized as a carbon source, but D-ribose, Rhamnose, inositol and D-mannitol are not. Utilization of raffinose is positive in PG medium but negative in LD medium. a-Galactosidase is produced, but b-xylosidase and a-mannosidase are not. The cell wall contains meso-diaminopimelic acid but not 3-hydroxydiaminopimelic acid. The whole-cell hydrolysate and purified cell wall contain glucose, mannose, xylose and arabi- nose. The major menaqumones are MK9(H4), MK-9(H6), MK- 10(H4) and MK10(H6) Type strain of the species is strain M956-1 (= ATCC 53710). Monotypic. Abridged Authors' Abstract FORMATION OF SPONTANEOUSLY DEVELOPING POCKS AND PRODUCTION OF PHAGE TAILLIKE PARTICLES IN THIOSTREPTON PRODUCING STREPTOMYCES LAURENTII ATCC 31255 Ogata, S. 1, H. Matsubara 1, Y.Harada 1 and A.Umeda 2 Microbial Genetics Division, Institute of Genetic Resources Faculty of Agriculture 1 and Department of Bacteriology, Faculty of Medicines 2, Kyushu University, Higashi-ku, Fukuoka 812, Japan Actinomycetologica, 6: 29-32, 1992 Streptomyces laurentii ATCC 31255, grown on rye flakes agar, forms colonies with spontaneously developing pocks. These appear during the growth of the organisms, and increase in number during subculturing. In the pocks, lysis of the aerial and sporulating hyphae results from the production of a large number of phage-like (defective) particles. Neither spore formation nor typical aerial hyphae formation is ob- served in the pocks, whereas formation of substrate hyphae is not inhibited. The pocks increase in number during sub- culturing. The eroded colonies, or lawns with pocks, temporarily disappear after subculturing into Bennett broth and reinoculation on rye flakes agar. The developed lawn shows normal aerial mycelium and sporulating hyphae. Cultures become again abnormal following serial subculturing. Electron microscopy reveals that most hyphae in the pocks are broken or lysed. Around the broken hyphae, there are many hexagonal ringlike particles (about 15 nm in diameter). These particles morphologically resemble the end structure of phage tails. Small tubelike particles (15 x 7 nm), resembling parts of tail cores, are also observed. In conclusion S. laurentii appears to produce phage tail-like particles similar to those formed by other species of Streptomyces and Streptoverticillium. R.L.
CROSS GRADIENT PLATE METHOD FOR DIFFERENTIAL ISOLATION OF ACTINOMYCETES WITH VARIED PHYSIOLOGICAL PROPERTIES Hotta, K., M.Ichihara, N.Saito and S.Mizuno National Institute of Health, 2-10-35 Kamiosaki, Shina-gawa, Tokyo 14 1, Japan Actinomycetologica, 6: 33-36, 1992 A cross gradient plate method for the isolation of antibiotic resistant actinomycetes, based on the gradient plate described by Szybalski and Bryson, is proposed. Half-strength ISP No. 4 medium; Difco) supplemented with cycloheximide (50 u.g/ml) and nystatin (25 u.g/ml) was used as the basal medium. One antibiotic is added to the basal medium which is then poured into a plastic dish, slanted just sufficiently to cover the entire bottom. After hardening, the plate is placed in the horizontal position and 40 ml of the basal medium supplemented with a second antibiotic is added. After hardening the agar plates are placed in a refrigerator overnight to let the added reagents diffuse so as to form a good concentration gradient. Colonies with different resistance could be isolated. Additionally the combination of antibiotic effect of pH was analyzed. The cross concentration and the pH gradient plate method show the following advantages: well localized colony growth is observed which allows strains with differences in physiology and/or resistance to be distinguished; because of the combined effect of two factors, dispersed and isolated colony growth can be obtained even from heavy inocula; various kinds of selective pressures can be analysed. With the method proposed, a wide variety of actinomycetes could be obtained, including producers of novel bioactive secondary metabolites such as isoxazole-4-carboxylic acid. In addition to ISP medium 4, different substrata could be used for the isolation of rare actinomycetes. R.L. Abstracts of the SAJ Osaka Colloquium Human Resources Developing Center, Takeda Chemical Industries, Ltd., Osaka, Dec. 6,1991 SIGNIFICANCE OF SUGARS IN CHEMOTAXONOMY OF ACTINOMYCETES Yokota, A. Institute for Fermentation, Osaka Actinomycetologica, 6: $5, 1992 Taxonomic significance of whole-cell and cell-wall sugars in actinomycetes is discussed under the following headings. - Whole-cell sugar pattern and the chemotaxonomy of actinomycetes. The presence of madurose-containing genera among meso-diaminopimelic acid containing ones other than those with wall chemotype III/B, and of madurose containing species in Streptomyces and Kitasatosporia are described, and their taxonomic significance discussed. - Cell-wall sugars in the family pseudonocardiaceae (wall chemotype IV). Examination of cell-wall sugar pat- tern of the species belonging to this family indicated the presence of species with wall chemotypes III and IV. Relationship between cell-wall sugar pattern and phylogenetic position based on 16S rRNA sequencing is discussed. Cell-wall sugar compositions of actinobacteria and their taxonomic significance. Type of cell-wall polysaccharides, sugar composition and relationships between cell-wall sugar composition and DNA-DNA hybridization are described and discussed in relation to the taxonomy of actinobacteria. Abridged Author's Abstract ANTIBIOTICS PRODUCED BY ACTINOMYCETES ISOLATED FROM CHINESE SOILS Isono, K. Antibiotics Lab., RIKEN Institute Actinomycetologica, 6: $5, 1992 In collaboration with Shanghai Pesticide Research Institute, we have screened a number of actinomycetes isolated from soils collected in Southeastern China. Several new antibiotics have been discovered. For example, antifungal acyl peptide antibiotics albopeptins A and B were isolated from Streptomyces albofaciens and dialkyl meleic anhydride antibiotics tautomycin and tautomycetin were isolated from S.spiroverticillatus and S.griseochromogenes, respectively. Anti-Xanthomonas xanthostatin, a cyclic depsipeptide was also isolated from the tautomycin-pro- ducing strain. Beside antifungal activity, tautomycin induced a severe morphological change in human leukemia cells K562. Specific inhibition of protein phosphatases from animal cells was demonstrated. Abridged Author's Abstract Abstracts of the SAJ Tokyo Colloquium National Institute of Health, Tokyo, Jan. 28,1992. TAXONOMY OF THE FAMILY STREPTOSPORANGIACEAE AND NOVEL ACTINOMYCETE STRAINS CLASSIFIED IN THIS FAMILY. Kudo, T. Japan Collection of Microorganisms, RIKEN Actinomycetologica, 6: $6, 1992 The family Streptosporangiaceae was proposed by Goodfellow et al. in 1990 for "maduromycetes". This family is a homogeneous taxon in respect to chemotaxonomy and molecular evolution. Morphology is the sole criterion for dis- tinguishing the genera. Seven strains, with distinct morphology, were isolated from soil and plant samples. Chemotaxonomic characteristics of the isolates, with the exception of menaquinone profiles, coincided with those of members of the family Strepto- sporangiaceae. The phylogenetic analysis using 5S rRNA sequences supported their placement in the family. On the basis of morphological characteristics and menaquinone composition, the isolates should be classified in a new genus of the family Streptosporangiaceae. Abridged Author's Abstract ACTINOMYCETES WHICH PRODUCE TETRODOTOXIN Kogure, K. Ocean Research Institute, Univ. of Tokyo Actinomycetologica, 6: $6, 1992 Tetrodotoxin (TTX), known as "puffer fish toxin", is a potent neurotoxin which blocks sodium channelling in excitable membranes. TTX occurs not only in puffer fish, but also in various marine animals, especially benthic ones. Since some bacteria were found to produce TTX it was thought that bacteria could be the sole TTX-producers in nature. This hypothesis has been supported by the observation that marine sediments usually contain rather high concentration of TTX and TTX-producing bacteria are easily isolated from these environ- ments. In order to clarify the accumulation mechanism of TTX and the possible contribution of bacteria, we have isolated bacteria, including actinomycetes from marine sediments. Production of TTX was checked by using the tissue culture bioassay, HPLC and GC-MS. TTX-producing bacteria are not restricted to a particular taxonomical group. Nine out of ten actinomycete strains were found to produce TTX. Seven of them were identified as Streptomyces species. In general, actinomycetes produce rather high concentrations of TTX. Abridged Author's Abstract MOLECULAR CHARACTERIZATION OF STREPTOMYCES SSI GENE AND ITS APPLICATION Taguchi, S. Science Univ. of Tokyo Actinomycetologica, 6: $6-$7, 1992 Studies on the genetic characterization of Streptomyces subtilisin inhibitor (SSI) and on its application are pre- sented. The SSI gene possesses two tandemly arranged promoters, two terminators and also two translational ini- tiation signals. These kinds of functional dual structures might play an important and unique role in the efficient and versatile expression of the SSI gene. We have also exploited the secretory and protease inhibitory characteristics of SSI and examined their use in expression vectors for the efficient production of two heterologous proteins. The established systems should be use- ful for the efficient secretory production of other heterologous gene products and as models for heterologous gene expression in Streptomyces. Abridged Author's Abstract SCREENING OF MICROBIAL SECONDARY METABOLITES REGULATING MAMMALIAN CELL PROLIFERATION AND DIFFERENTIATION Osada, H. and K.Isono RIKEN, Inst. of Physic. Chem. Research Actinomycetologica, 6: $7, 1992 On the basis of recent knowledge of the molecular mechanism of the cell growth and differentiation, we have de- veloped new screening systems for microbial metabolites as regulators of mammalian cell growth. Epiderstatin, actiketal and reveromycin A were isolated as inhibitors of the epidermal growth factor (EGF). Epiderstatin and reveromycin A arrested the cell cycle of temperature sensitive Roux sarcoma virus transformed rat kidney cells at GO/Gl phase, and reversed the transformed phenotype of normal ones. A human leukemia cell line, K-562, is sensitive to the tumor promoter which induces the morphological change on the cell surface. We found that the morphological change was caused by the activation of protein kinases or the inactivation of protein phosphatases. New indolocarbazole compounds, staurosporine, RK-286C, D and RK-1409, were isolated as inhibitors of protein kinase C. Tautomycin was isolated as an inhibitor of protein phosphatase. These inhibitors are regarded as potential tools in the study of mammalian cell proliferation and differentiation. Abridged Authors 'Abstract TRENDS IN ANTIBIOTIC RESEARCH - WHAT IS CURRENT DEFINITION ? - Yagisawa, M. Japan Antibiotics Research Association Actinomycetologica, 6: $7, 1992 Analysis of 2,734 articles published in the Journal of Antibiotics during 19821991, gave some idea on the current definition of the term "antibiotic". Discoveries of new anti-bacterial antibiotics have decreased rapidly from an average of 41 to 11 compounds per year. Anti-fungal/anti-viral agents and anticancer substances have increased from 11 to 18 and from 18 to 27, respectively. A marked increase, 7 to 22, has been noted biologically active microbial products. Biologically active compounds, e.g., anti-inflammatory, hypotensive, hypocholestemia, antiobesity, tissue-damage protective, immuno-modulatory, and other various functional compounds, are the major target of the current research on "antibiotic". Waksman's original definition of the term "antibiotic" is difficult to apply to current research. Further analysis of the articles shows that the discovery of new microbial products is still of major interest. However, biochemical studies are still limited. It is concluded that "research on antibiotics" is quite dynamic and is very much dependent on clinical demand. Abridged Author's Abstract Copyright 1995 CETA.
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