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ACTINOMYCETOLOGICA
Published by the Society for Actinomycetes, Japan ABSTRACTS OF PAPERS
Code Number:AC98004 DEVELOPMENT OF A TRANSFORMATION SYSTEM IN STREPTOMYCES VIRGINIAE R. Kawachi, T. Nihira and Y. Yamada Department of Biotechnology. Graduate School of Engineering, Osaka University, 2-1 Yamadaoka, Suita, Osaka 565, Japan Actinomycetologica, 11: 46-53, 1997 Streptomyces virginiae is one of the most difficult Streptomyces strains to transform because of its strong restriction barrier. We succeeded in transforming S. virginiae by combining heat treatment of protoplasts and in vitro methylation of plasmids. In order to avoid the host restriction barrier, plasmids were modified by methylase AluI and S. virginiae protoplasts were treated at 42 C for 15 min prior to transformation. Under optimal conditions plasmids pIJ486, pKC1064 and pUWL-KS isolated from Streptomyces lividans were introduced into S. virginiae at a frequency of 1.4x10^3, 7.0x10^2 and 1.2x10^3 transformants per ug DNA respectively. Plasmids pKC1064 and pUWL-KS, isolated from Escherichia coli DH5a, could not transform S. virginiae, but those isolated from E. coli JM110 (dam, dcm) could transform at a frequency of 1.0x10^2 and 2.4x10^2 transformants per ug DNA, indicating the presence of a methylation-specific restriction system in S. virginiae. Authors' Abstract
DISTRIBUTION OF THE ACTINOMYCETES IN THE REPUBLIC OF SOUTH AFRICA INVESTIGATED USING A NEWLY DEVELOPED ISOLATION METHOD M. Kizuka, R. Enokita, K. Takahashi and T. Okazaki Tsukuba Research Laboratories, Sankyo Co., Ltd., 33, Miyukigaoka, Tsukuba- shi, Ibaraki, 305 Japan Actinomycetologica, 11: 54-58, 1997 A total of 93 soil samples were collected at 3 sites in the southern, at 4 sites in the north-western and at 2 sites in the north-eastern regions of the Republic of South Africa. When these samples were subjected to the conventional dilution plate method for the isolation of actinomycetes, the genus Streptomyces was found to be dominant and, of 135 total isolates, 53 (39 %) were rare actinomycetes. However, the newly developed high-speed centrifugation method enabled us to isolate 335 isolates, of which 295 (88%) were rare actinomycete strains. These included the genera Planomonospora and Planobispora, which have been known to have a very low population density in soils. These two genera were detected only in soil samples collected from the extremely arid area, called "Namaqualand", in the north-western region of the Republic of South Africa. Authors' Abstract CLONING OF A DNA FRAGMENT FROM RHODOCOCCUS RHODOCHROUS WHICH SUPPRESSES ITS MUCOIDAL MORPHOLOGY N. Iwabuchi, M. Sunairi and M. Nakajima Laboratory of Molecular Microbiology, Department of Applied Biological Science, College of Bioresource Sciences, Nihon University, Fujisawa, Kanagawa 252, Japan. Actinomycetologica, 11: 59-63, 1997 A DNA fragment, which suppressed formation of mucoidal colonies, was isolated from Rhodococcus rhodochrous rough strain R-2. This fragment contained two ORFs in the same orientation, and their deduced amino acid sequences showed no significant homology to reported proteins. The essential region for suppression of mucoidal morphology was determined by subsequent subcloning. Authors' Abstract
A PROLYL ENDOPEPTIDASE INHIBITOR, PROPEPTIN PRODUCTION IN THE VARIOUS MICROBISPORA SP. K. Kimura, F. Kanou and M. Yoshihama Research Institute of Life Science, Snow Brand Milk Products Co., Ltd., Ishibashi-machi, Shimotsuga-gun, Tochigi 329-05, Japan Actinomycetologica, 11: 64-68, 1997 We examined the propeptin production in 10 mesophilic species of Microbispora. Three strains which have close DNA homology to Microbispora rosea, M. rosea (IFO 14044), M. rosea subsp. nonnitritogenes (IFO 14045) and M. indica (IFO 14879) produced propeptin, other 7 strains did not. The results suggest some relationship between secondary metabolite production and taxonomy. Authors' Abstract
STUDIES ON THE BASIS AND APPLICATION OF ANTIBIOTIC COSYNTHESIS T. Furumai Biotechnology Research Center, Toyama Prefectural University, 5180 Kurokawa, Kosugi-machi Toyama 939-03, Japan
Actinomycetologica, 11: 69-79, 1997 No abstract provided. The paper deals with the biosynthesis of platenomycin and of butirosin, the modification of butirosins, the biosynthesis of pradimicins and the microbial modification of pradimicin A.
BIOCHEMICAL AND GENETIC STUDIES ON SECONDARY METABOLISM O. Nimi Hiroshima Bunkyo Women's college, 1-2- 1, Kabehigashi, Asakita-ku, Hiroshima 73 1-02, Japan Actinomycetologica, 11: 80-85, 1997 Main studies on Streptomyces, especially S. griseus, during about thirty years of the author's career are outlined. 1. Studies on streptomycin biosynthesis using resting mycelium-suspension culture of S. griseus revealed the following: 1) streptomycin 6- phosphate is the final precursor, which is converted to streptomycin by a specific alkaline phosphatase, 2) streptomycin 6-phosphotransferase contributes to self-protection from a toxic effect of streptomycin and 3) suppression of the phospholipid cycle of cell wall synthesis results in the accumulation of glucosamine 6-phosphate in the resting mycelium and the enhancement of streptomycin production. 2. Genetic studies provided the following: 1) S. griseus has two genes contributing to self- protection against streptomycin, 2) pleiotropic mutants were obtained at a high frequency by incubating mycelium under stress condition for growth and 3) a physical macrorestriction map of S. griseus 2247 strain was constructed. Authors' Abstract
SAJ REGULAR COLLOQUIA: 20th SAJ Colloquium Institute of Physical and Chemical Research (RIKEN), Wako-shi, Saitama, October 16, 1997 POTENTIAL SOURCE FAR NOVEL BIOACTIVE METABOLITES: AMINO ACID ANALOGUE RESISTANT ACTINOMYCETES K. Hotta National Institute of Infectious Diseases, 1-23-1, Toyama, Shinjuku-ku, Tokyo 162, Japan Actinomycetologica, 11: S1, 1997 The probability of discovering novel bioactive compounds of microbial origin by the screening is declining as the number of known compounds increases. Subsequently, establishing innovative approaches is required to enhance the probability. A critical point in the screening is what kinds of organisms should be targeted as the screening source. My colleagues and I have chosen actinomycetes with resistance to amino acid analogues on the basis of the following. For the production of secondary metabolites, microorganisms require primary metabolites as precursors. In this regard, metabolism of amino acids should be a key factor, since they are involved in the biosynthesis of wide varieties of secondary metabolites. It seems, therefore, likely that differences in the regulation of amino acid metabolism may result in differences in the secondary metabolite profile. It has been known in bacteria that amino acid formation undergoes feedback regulation and a variety of amino acid fermentations were successfully established by obtaining amino acid analogue (AAA) resistant mutants which acquired insensitivity to feedback regulation. Therefore, we aimed at natural AAA resistance of actinomycetes. First we examined strains that were known to produce amino acid related metabolites. It turned out that they showed different profiles of AAA resistance. Subsequently, we established an AAA cross gradient agar plate method by which wide varieties of AAA resistant actinomycete strains were efficiently isolated from soils. Then they were screened for the production of secondary metabolites. About 1,200 strains with amino acid analogue (AAA) resistance were examined for their productivity of bioactive metabolites using a variety of assays. About 40% of them showed antibiotic productivity and strains regarded as b- lactam producers were found at a rate of 10%. Novel compounds were discovered at a rate of 1/100-700. They include isoxazol-4-carboxylic acid with herbicidal activity, a depsipeptide with antimycoplasma activity, a diketopiperazine which was found by a neutrophil stimulation screen, and a cyclic peptide with a fatty acid side chain which was screened by a PCR inhibition assay. These structures suggested that their biosynthesis involved amino acid metabolism. It seems thus conclusive that the AAA resistance based approach is advantageous in searching for amino acid related secondary metabolites including novel ones. Author's Abstract
ISOLATION OF ACTINOMYCETES IN NATURAL PRODUCTS SCREENING S. Matsukuma and J. Watanabe Nippon Roche Research Center, Japan Actinomycetologica, 11: S3, 1997 The diversity is most important far supplying microorganisms and their metabolites in natural products screening. We compared the number of isolates to the number of petri dishes and media, and the collection sites for the efficient isolation of actinomycetes. Although it was possible to obtain many strains since we utilized a sufficient number of petri dishes inoculated under the some condition, and utilized different media for the various soil samples, the frequency of duplicates increased in proportion to the number of petri dishes or the number of media. It was also possible to obtain many strains from soil samples collected at sites near by. However, after the elimination of duplicates among them, we could non obtain so many different isolates. In addition, we demonstrated that plant material gave us totally different actinomycete strains, depending on the degree of the decomposition of the samples. Such comparative studies with the statistical analysis showed that these combinations are important far obtaining efficiently actinomycetes that we could subject to our microbial screening assays. We have already established the RAPD method for the efficient selection of similar strains which are difficult to select by empirically. Using this method, we analyzed similarities of 147 hygroscopic strains in the genus Streptomyces collected from different area, only eight strains were duplicate in 147 strains. This supports the fact that hygroscopic strains are a diverse group. In order to perform a high quality natural products screening, we have to isolate diverse actinomycetes. For the isolation, we have to take into account the number of media and petri dishes, and the collection sites and samples, as well as the methods for the selective isolation, so that we can reduce duplicate strains. Authors' Abstract
PEPTIDOGLYCAN ANALYSIS FOR ACTINOMYCETE TAXONOMY K. Suzuki Japan collection of Microorganisms, The Institute of Physical and Chemical Research (RIKEN), Wako-shi, Saitama 351-01, Japan Actinomycetologica, 11: S3-S4, 1997 Peptidoglycans contain various significant criteria for actinomycete taxonomy. Determination of diaminopimelic acid isomers by thin-layer chromatography is one of the most essential techniques for identification of actinomycetes. Amino acid composition of the hydrolysate of peptidoglycan is a helpful information to speculate the peptidoglycan structure. A high-performance liquid chromatography procedure which separates D- and L-isomers of amino acids was applied to the peptidoglycan analysis. The genera Agromyces, Clavibacter and Rathayibacter contain 2,4-diaminobutyric acid (DAB) in the peptidoglycan and are differentiated from each other principally by menaquinone profile. Their peptidoglycan was known to be common in structure, possessing D- and L- isomers of DAB equally as B2 by Schleifer and Kandler. The type strains of all the subspecies of Clavibacter michiganensis had D- DAB and L- DAB almost equally in the cell wall peptidoglycan as previously reported. In contrast, the type strains of Clavibacter toxicus, and all the valid species of the genera Agromyces and Rathayibacter had almost exclusively L-isomer of DAB. This characteristic showed a good accordance with the phylogenetic analysis based on 16S rDNA sequence and menaquinone profiles. On the basis of these results, we propose the transfer of Clavibacter toxicus to the genus Rathayibacter as Rathayibacter toxicus comb. nov. The isomer profile of DAB is shown to be a good taxonomic marker to differentiate these genera. Isolates of actinomycetes bearing motile spores showed "spore dome" morphology. Their chemotaxonomic features suggested that they belonged to the genus Kineosporia However, they showed variation in the ratio of diaminopimelic acid (DM) isomers in the cell wall. Analyses using biomass cultivated in shake-flask divide the isolates into three types based on the ratio, i.e., LL- plus meso-DAP type, LL-DAP type and meso-DAP type. Meso-DAP type was further divided into two: presence and absence of rhamnose. Meso-DM was enriched by collecting the spore-fraction. The isolates were classified into five species, each characterized by the ratio of LL-DAP. The phylogenetic analysis based on 16S ribosomal DNA sequences supported that these isolates be included in one genus, the genus Kineosporia Pagani and Parenti, 1978, emend. Itoh et al. ,1989. A possibility to contain a variable amount of LL-DAP in the whole cell hydrolysate should be taken into account in the identification of this group of actinomycetes. Introduction of 16S rRNA analysis for bacterial taxonomy has given a large influence to the evaluation of peptidoglycan analysis as one of the phenotypic characteristics for taxonomic criteria. Author's Abstract CREATION OF NEW TRANSFORMATION SYSTEM WITH PLASMID DNA FOR ACTINOMYCETES AND BACTERIA: APPROACH BY TRIAL AND ERROR EXPERIMENTS A. Okanishi Faculty of Agriculture, Tarnagawa University and Dept. of Microbial Technology, Institute of Microbial Chemistry, Japan Actinomycetologica, 11: S4, 1997 The sorts of antibiotic-producing strains, other industrial microorganisms, and pathogenic bacteria are distributed among very different microorganisms. In order to carry out recombinant DNA experiments using so different microorganisms as hosts, the creation of simple and generally usable new transformation system is necessary. Streptomyces lividans TK21 (tr^s, mel^-) as a host and pIJ702 (ts^r, mel^+) as plasmid DNA were used. Transformants with the plasmid were detected with two phenotypes of Ts^r and Mel^+. The steps of transformation experiments are (1) cultivation to induce the competent mycelium formation, (2) composition of reaction mixture for transformation, (3) reaction temperature, and (4) detection of transformant colonies. Critical problems are that even if competent cells can be formed, the competency is unable to detect in the first step and able to do it only after development of transformants in the final step. Therefore, the function of every step is required to be proper for transformation. As a result of many trial and error experiments, an initial system showing reproducible transformation was found, though the transformation frequency was only 3~4 per 50 ng of pIJ702. On the basis of this initial system, cultural conditions for competent mycelium formation in the presence of EDTA, combination of PEG, various salts and buffers in the reaction mixture and also reaction temperatures were investigated to increase the transformation frequency. In the finally established system, 150~170 transformants per 50 ng of pIJ702 were obtained. Comparable results were found with S. kasugaensis, S. acrimycini, S. parvullus, E. coli and B. subtilis. In these experiments, the transformation frequency of E. coli NIHJ JC-2 with pBR322 was very low. However, using the pBR322 extracted from the transformant colony obtained in the experiments, the transformation showed high frequency of 1500 per 50 ng plasmid. Results suggest the use of the new method for transforming various actinomycetes and bacteria. Author's Abstract
Copyright 1998 C.E.T.A., The International Centre for Theoretical and Applied Ecology, Gorizia |
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