Actinomycetes University of Udine, Mycology Department ISSN: 0732-0574 Vol. 6, Num. 3, 1995

ACTINOMYCETOLOGICA

Vol. 9, No. 1, 1995

Published by the Society for Actinomycetes, Japan

ABSTRACTS OF PAPERS

Code Number: AC95016
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Establishment of a Host-Vector System for Micromonospora griseorubida and Characterization of Mycinamicin Biosynthetic Genes

M.Inouye and S.Horinouchi^*

Inst. for Life Science Research, Asahi Chem.Ind. Co., Ltd., 2- 1 Samejima, Fuji-shi, Shizuoka 416, and *Dept. of Biotechnology, University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113, Japan

Actinomycetologica, 9: 1-12, 1995

Mycinamicin is a 16-membered macrolide antibiotic produced by Micromonospora griseorubida.

Conditions for protoplasting the mycelium of the mycinamicin producer and regeneration of protoplasts were established. A shuttle cosmid vector, constructed from a cryptic plasmid of M. griseorubida and E.coli cosmid pJB8, was useful for manipulation of a long DNA sequence. The host vector system established in this way allowed the identification of a gene (mycG) encoding a P-450-1ike protein probably responsible for both steps of hydroxylation and epoxidization of the lactone ring of mycinamicin.

The mycG gene was located near myrB encoding a 23S rRNA methyltransferase as a self-resistance determinant and mycF encoding mycinamicin III O-methyltransferase.

Abridged Authors' Abstract

n-Alkane-Utilization by Oligocarbophilic Actinomycete Strains from Oil-Polluted Kuwaiti Desert Soil

G.Barabas, N.A.Sorkhoh*, F.Fardoon* and S.S.Radwan*

Inst. of Biology, University Medical School, H-4012 Debrecen, Hungary and *Dept. of Botany and Microbiology, Faculty of Science, Kuwait University, P.O. Box 5969, Safat 13060, Kuwait

Actinomycetologica, 9: 13-18, 1995

Among some 50 actinomycete strains with Streptomyces- like morphology isolated from oil polluted Kuwaiti desert soil, four arbitrarily selected strains were characterized for n-alkane utilization. The isolates were oligocarbophilic and grew on inorganic media. Growth was enhanced when n- hexadecane, n-octadecane or crude oil was added at the concentration of 1% (w/v). By gas-liquid chromatographic analysis it was possible to ascertain that the strains could utilize n-hexadecane and n-octadecane, typical oil constituents. Analysis of fatty acids showed that the incubation with n-alkanes resulted in an increase of the compounds with chain lengths equivalent to those of the alkane substrates. It was concluded that these oligocarbophilic strains are capable of utilising n-alkanes and therefore could be of value in bioremediation technology.

Abridged Authors' Abstract

Transfer of Staurosporine-Producing Strain Streptomyces staurosporeus AM-2282 to the Genus Saccharothrix as Saccharothrix aerocolonigenes (Labeda 1986) subsp. staurosporeus subsp. nov.

Y.Takahashi, M.Shinose, A.Seino, Y.Iwai and S.Omura

Research Center for Biological Function, The Kitasato Institute, 5-9-1, Shirokane, Minato-ku, Tokyo 108, Japan

Actinomycetologica, 9: 19-26, 1995

On the basis of chemotaxonomic characteristics Streptomyces staurosporeus is transferred to the genus Saccharothrix as Saccharothrix aerocolonigenes (Labeda, 1986) subsp. staurosporeus subsp.nov. A description of the subspecies is provided.

The type strain of the subspecies is AM-2282 (NRRL 11184).

R.L.

Streptomyces kasugaensis sp.nov.: A New Species of Genus Streptomyces

M.Hamada, N.Kinoshita, S.Hattori, A.Yoshida, Y.Okami, K.Higashide*, N.Sakata* and M.Hori*

Inst. of Microbial Chemistry, 14-23, Kamiosaki 3-chome, Shinagawa-ku, Tokyo 141, and *Showa College of Pharmaceutical Science, 3-3165 Higashi Tamagawagakuen, Machida-shi, Tokyo 194, Japan

Actinomycetologica, 9: 27-36, 1995

Although the name Streptomyces kasugaensis has already been used for a kasugamycin producer strain MB273-C4 that is approved as a Streptomyces host in the Japanese guideline for recombinant DNA experiments, the name has not been recognized taxonomically. We examined two kasugamycin producers, strains M338-M1 and MB273-C4, for their taxonomic properties including not only morphology and physiology, but also structures of some cell components and DNA. The results indicated that the two strains should belong together to a new species of the genus Streptomyces. We officially propose the name Streptomyces kasugaensis, the type strain being strain M338-Ml (= ATCC 15714). The new species is characterized by spiral spore chains, smooth spore surfaces, grey aerial mass, yellow to brownish soluble pigments, LL-diaminopimelic acid in cell walls, type PII phospholipids, lack of mycolic acids, MK-9 (H6,H8,H4) menaquinones, fatty acid components of ai- 15:0, 16:0, ai-17:0 and i- 16:0, and a G+C content of 70.4 to 70.9 mol %.

Authors' Abstract

Involvement of a Small ORF Downstream of the afsR Gene in the Regulation of Secondary Metabolism in Streptomyces coelicolor A3(2)

A.Matsumoto^1, H.Ishizuka^2, T.Beppu^3 and S.Horinouchi

Dept. of Biotechnology, University of Tokyo, Bunkyo-ku, Tokyo 113, Japan, ^1Pharmaceutical Lab. Kirin Brewery Co., Ltd., Maebashi-shi, Gunma 371, Japan, ^2Dept. of Experimental Pediatrics MD Anderson Cancer Center, The University of Texas, Houston, Texas 77030, USA and ^3Dept. of Applied Biological Sciences, Nihon University, Fujisawa-shi, Kanagawa 252, Japan

Actinomycetologica, 9: 37-43, 1995

The afsR gene encoding a regulatory protein and the afsK gene encoding a protein serine/threonine kinase constitute a protein phosphorylation system controlling secondary metabolism in Streptomyces coelicolor A3(2). The region between these two genes conferred the production of A-factor and the pigmented antibiotics actinorhodin and undecylprodigiosin on Streptomyces lividans, when it was carried on a high copy number plasmid. The nucleotide sequence between afsR and afsK revealed the presence of two small open reading frames named ORF-B of 176 amino acids and ORF-C of 63 amino acids. These ORFs showed no homology with proteins registered in the databases. ORF-B with the same orientation as AfsK contained three tandem repeats of Gly-Ser-Gly-Gly-Ser/Gly. ORF-C with the same orientation as AfsR contained three repeats of Thr-(X)2-Asp-Asn-His-Met-Pro- (X)2-Pro-Ala (X represents a non conserved amino acid). Subcloning experiments showed that overexpression of ORF-C conferred pigment and A-factor production on S.lividans. The gene encoding ORF-C was therefore named afsS. It is thus apparent that afsS encoding a protein of 63 amino acids is involved in the regulation of secondary metabolism in S.coelicolor A3(2).

Authors' Abstract

Transfer Function of spi Gene in Plasmid pSAl.l of Streptomyces azureus ATCC 14921

K.Doi, E.Yokoyama, Y.Nakano, S.Tokunaga and S.Ogata

Microbial Genetics Division, Institute of Genetic Resources, Faculty of Agriculture, Kyushu University, Fukuoka 812, Japan

Actinomycetologica, 9: 44-48, 1995

Four derivative plasmids (pSAK1, pSAK52, pSAK61 and pSAK- S9) of pSA1.1, a conjugative plasmid of Streptomyces azureus, were constructed to examine the transfer ability of the spi gene.

From the results obtained the authors suggest that the spi gene product is incorporated in the host cell membrane and promotes the plasmid transfer to neighbouring cells.

R.L.

Taxonomic Characterization of Streptomyces coelicolor A3(2) and Streptomyces lividans 66 by Analysis of Ribosomal Proteins

K.Ochi

National Food Research Institute, 2-1-2 Kannondai, Tsukuba, Ibaraki 305, Japan

Actinomycetologica, 9: 49-52, 1995

On the basis of ribosomal protein analysis Streptomyces coelicolor A3(2) should be identified as S.violaceoruber. S.lividans 66 is considered to be an allied (if not the same) species of S.violaceoruber. However the epithets "coelicolor A3(2)" and "lividans 66" could be maintained in genetics, considering the long time the terms have been used in the literature.

R.L.

Isolation of Two Giant Linear Plasmids from Streptomyces lavendulae MA406 A-l

H.Fuse, S.Yashima and S.Kawamoto*

Technology Development Labs., Takeda Chem. Ind. Ltd., 2-17-83 Honmachi, Jyuso, Yodogawa-ku, Osaka 532 and *Dept. of Applied Bioscience, Hokkaido University, Sapporo 060, Japan

Actinomycetologica, 9: 53-56, 1995

By pulsed-field gel electrophoresis (PFGE) the existence of two giant linear plasmids (pSL1 and pSL2) has been demonstrated in S.lavendulae MA406 A-1. The former plasmid is lost in most of the tested bald mutants obtained by curing with acridine orange and acriflavine. However no relationship between plasmid loss and altered phenotype in the mutants has been found. Any essential gene(s) for growth do not seem to be encoded either in pSL1 or in pSL2 since the mutants lacking either or both plasmids grow normally as the wild type strain A-l. By restriction analysis a difference in the Dra I fragmentation pattern of chromosomal DNA between the wild type A-l and its mutants, and between the mutants, has been observed.

This suggests that, in addition to plasmid loss, genome rearrangement might have occurred in S.lavendulae mutants.

R.L.

Butyrolactone Autoregulators, Inducers of Secondary Metabolites, in Streptomyces

Y.Yamada

Dept. of Biotechnology, Faculty of Engineering, Osaka University, 2-1 Yamadaoka, Suitashi, 565 Osaka, Japan

Actinomycetologica, 9: 57-65, 1995

Recent progress on inducers of secondary metabolites is reported.

Virginiae butanolide (VB) biosynthesis and characteristics of VB receptor proteins are described.

The importance of butyrolactone autoregulators, in basic and in applied science, as signal substances is emphasized.

R.L.

Establishment of the Genus Herbidospora and Some New Taxa of Actinomycetes

T.Kudo

Japan Collection of Microorganisms, Institute of Physical and Chemical Research (RIKEN), Wako-shi, Saitama 351-01, Japan

Actinomycetologica, 9: 66-74, 1995

The status of the genus Herbidospora, of Actinopolyspora mortivallis and of Nocardia seriolae is reviewed with reference to other related actinomycete taxa.

R.L.

Screening of New Antibiotics Produced by Actinomycetes and Their Production

E.Higashide

Faculty of Agriculture, Okayama University, 1-1, Tsushima- naka, Okayama 700, Japan

Actinomycetologica, 9: 75-82, 1995

I have studied actinomycetes and their antibiotics for over thirty-eight years. My colleagues and I have been successful in discovering twelve new species of actinomycetes and fourteen new antibiotics. In this review, I would like to describe rufomycins, enduracidins, validamycins, maridomycins, T-2636 antibiotics and ansamitocins.

Rufomycins, cyclic peptides with a specific activity against mycobacteria, were isolated from Streptomyces atratus. Enduracidins were screened as antibiotics with low toxicity and a wide antibacterial spectrum, including activity against drug-resistant streptococci. For aminocyclitol antibiotics, validamycins, new assay methods (reversed layer method and dendroid-test method) were established in order to develop industrial fermentations for their agricultural utilization. As to the T-2636 antibiotics with macrolactone ring, the esterase catalyzing the transformation of T-2636 C to T-2636 A was identified in the producing organism, S.rochei subsp.volubilis and then the industrial enzymatic transformation was established. Maridomycins, new macrolide antibiotics, were discovered as a complex of about twenty components isolated from S.hygroscopicus No. B-5050. Selective and improved production of maridomycin III was obtained by strain improvement through mutation of the regulation of amino acid metabolism, involved in maridomycin biosynthesis. Ansamitocins, new antitumor antibiotics, were discovered from a new species of Actinosynnema and turned out to be similar in structure to maytansine, present in tropical plants and characterised by strong antimitotic activity.

Abridged Author's Abstract

11th SAJ Colloquium

Held on November 11, 1994 at the Institute of Microbial Chemistry, Tokyo

Fungal polyketide biosynthesis - Enzymological and molecular genetic approach

I.Fujii, K.-X.Huang, Z.-G.Chen, N.Iwakami, Y.Ono, Y.Ebizuka and U.Sankawa

Faculty of Pharmaceutical Sciences, Univ. of Tokyo

Actinomycetologica, 9: S14-15, 1995

Fungi are among the most prolific sources of polyketide compounds along with streptomycetes. Polyketides produced by fungi vary from the simplest aromatic tetraketides, orsellinic acid and 6-methylsalicylic acid, to the highly modified aflatoxins.

Polyketide synthases (PKS) are enzymes which catalyze the formation of the specific "poly beta-ketone" intermediates and their cyclization. However, inherent lability of PKS made their biochemical studies difficult. Successful purification was only reported for 6-methylsalicylic acid synthase (MSAS) of Penicillium patulum.

Probing with the cloned PKS genes has been considered a powerful tool for screening related polyketide biosynthesis genes, as in Streptomyces PKS gene cloning studies. Therefore, Southern blot analysis of genomic DNA of polyketide producing fungi was carried out with MSAS gene as a probe. Interestingly, no homologous band was detected in P.cyclopium genomic DNA which produces orsellinic acid, indicating differences between MSAS and orsellinic acid synthase genes. In Aspergillus terreus, a strain known to produce (+)-geodin as a main metabolites, strong hybridizing band was observed with MSAS probe.

From the genomic DNA library of A. terreus, MSAS homologous gene was cloned and sequenced. Head-to-tail homology was observed with MSAS at amino acid level, but the actual function was not determined. Southern blot analysis using another fungal PKS gene AC71 from Colletotrichum lagenarium as a probe revealed the presence of homologous bands in all polyketide producing fungi so far examined, including orsellinic acid producing P.cyclopium.

This indicated the possible usefulness of AC71 gene as a probe for fungal PKS gene cloning as act I in Streptomyces PKS cloning studies.

Seco-anthraquinones are a class of compounds derived from anthraquinone with modified ring system. (+)-Geodin and asterric acid are examples of these class of compounds. Their biosynthesis have been extensively studied at enzyme level.

In anthraquinone biosynthesis, the direct product of PKS is considered to be emodinanthrone, which is then oxidized to anthraquinone, emodin. This anthrone oxygenase activity was first found in A.terreus cell-free extracts. Emodinanthrone oxygenase was purified and characterized as a novel class of internal monooxygenase with non-heme ferric iron co-factor. The key reaction in the structural conversion from anthraquinone to benzophenone is the Baeyer- Villiger type oxidative ring cleavage of anthraquinone. The enzyme, named questin oxygenase, was detected in the cell-free extracts of A.terreus, but further characterization was hindered by its lability.

Stereospecific intramolecular phenol oxidative coupling reaction to form unique spiro structure from benzophenone is involved in (+)geodin and asterric acid biosynthesis. The enzyme dihydrogeodin oxidase catalyzing this phenol oxidative coupling reaction was purified from A.terreus and found to be a complex type blue copper protein.

The genomic DNA and cDNA for this dihydrogeodin oxidase was carried out and the gene was expressed in P.frequentans.

Authors' Abstract

Mechanism based screens for microbial products which induce DNA cleavable complex with mammalian DNA topoisomerases

Y.Yamashita and H.Nakano

Kyowa Hakko Kogyo Co. Ltd., Tokyo Research Laboratories, Machida, Tokyo 194

Actinomycetologica, 9: S15, 1995

DNA topoisomerases I and II are nuclear enzymes that catalyze the concerted breaking and rejoining of DNA strands, thereby controlling the topological states of DNA. In addition to their important functions in DNA metabolism, both topoisomerases I and II have generated extensive clinical interest in cancer chemotherapy. In the 1980's, topoisomerases have been shown to be the principal targets for a number of clinically important antitumor agents including m-AMSA, VP-16 and camptothecin. Despite their apparent structural diversity, these drugs have the common properties of stabilizing a key covalent reaction intermediate of topoisomerases, termed the cleavable complex, which upon exposure to denaturant results in the induction of DNA cleavage. This attractive model has led to a search for new agents which induce cleavable complex with topoisomerases.

We have established a simple biochemical assay system with topoisomerases I and II purified from calf thymus, and screened newly isolated actinomycetes and fungi for their ability to produce metabolites that induce topoisomerase mediated DNA cleavage in vitro. The early findings in the course of screening were the effects of ingredients of cultured media; 5 mM calcium ion suppressed completely the topoisomerase II mediated DNA cleavage induced by m- AMSA, and flavonoids like genistein induced topoisomerase II mediated DNA cleavage. Using fermentation media without the interfering factors, we have screened over 40,000 fermentation cultures and identified more than ten microbial metabolites as inducers of cleavable complex with topoisomerases. These active compounds include streptonigrin, UCE6, terpentecin and UCT4B produced by actinomycetes, and santopin, UCE1022, bulgarein and clerocidin from fungi. Although most of the active metabolites acted selectively on either topoisomerase I or topoisomerase II, santopin, a new antitumor antibiotic produced by Paecilomyces sp., showed a unique mechanism of action since it induced cleavable complex with both topoisomerases I and II. Structure-activity studies showed good correlation between cytotoxicity and ability to induce cleavable complex. These results indicate that the formation of cleavable complex with topoisomerases could be responsible for their antitumor activity.

Abridged Authors' Abstract

Isolation of actinomycetes from plants

S.Matsukuma, T.Okuda and J.Watanabe

Dept. of Microbiology and Taxonomy, Nippon Roche Res. Center, Kamakura 247

Actinomycetologica, 9: S15-16, 1995

Selective isolation of actinomycetes from plants is important for obtaining new strains and for studying the ecology. Although many reviews have detailed procedures for selective isolation of actinomycetes from soil, there are few reports on selective isolation of actinomycetes from plants.

For direct isolation, samples were cut into several pieces and placed onto 0.02% yeast extract agar and humic acid- vitamin agar. After incubation for 2 to 4 weeks at 27øC, colonies were picked up with a needle under a stereo microscope and transferred to new media.

Actinomycete colonies were often covered by rapidly growing fungi. Benlate (200æg/ml) was effective in reducing fungal colony diameter without inhibiting actinomycete growth.

For serial dilution plant samples were incubated in a 0.05% sodium dodecyl sulfate solution, containing 6% yeast extract, and plated on agar media supplemented with 20æg/ml of nalidixic acid.

Surface sterilization with 70% of ethanol and sodium hypochlorite (1% chlorine), was used for "endophytic actinomycetes". Although few "endophytic" strains were obtained from intact leaves, many saprophytes were isolated from dead plants. The ratio of isolates with meso- diaminopimelic acid was higher than that of untreated samples.

Isolates obtained from pine litter layers (L, F and H) were compared. A large number of isolates of the L and F layers overlapped, but not those of the H layer. This suggests that the substratum succession of actinomycetes is similar to that of fungi.

Abridged Authors' Abstract

Genetic strategies of bacteria for stationary-phase survival

A.Ishihama

National Inst. of Genetics, Dept. of Molecular Genetics, Mishima 411

Actinomycetologica, 9: S16-17, 1995

A set of genetic programs operates in E.coli for survival in the stationary growth phase, including repression of genes highly expressed in exponentially growing cells and induction of stationary phase-specific genes. Several lines of genetic and molecular biological studies on the mechanisms underlying the global gene regulation during transition from exponentially growing to stationary phase of E.coli indicated that modifications of both transcription and translation are involved in stationary survival.

RNA polymerase core enzyme is functionally differentiated in two steps: association with promoter recognition subunit s to form holoenzyme and association of the holoenzyme with one of transcription factors.

In parallel with RNA polymerase modifications, ribosome monomers are converted into dimeric forms after association with ribosome modulation factor.

RMF is a small basic protein of 55 amino acid residues encoded by the rmf gene at 21.8 min on the E. coli chromosome. RMF is one of the stationary phase-specific gene products, it is however synthesized even in log phase cells growing at slow rates in poor media. Both native and synthetic RMF associate with 70S ribosomes to convert into dimers and concomitantly the ribosomes become inactive in protein synthesis, implying that the ribosome dimers represent inactive storage forms.

Author's Abstract

12th SAJ Colloquium

Held on December 9, 1994 at Takeda Chem.Ind., Osaka

Molecular basis of self-incompatibility of Brassica

A.Isogai

Nara Inst. of Science and Technology

Actinomycetologica, 9: S17-18, 1995

Self-incompatibility is one of the well organized systems for plants to prevent inbreeding.

Recognition systems in Brassica involve the interaction of same signal molecules from pollen coat with a multicomponental receptor system composed of SRK and SLG on the plasma membrane of the papillae cells. Phosphorylation of intermediates then stimulates a localized response which inhibits pollen development. In addition to SLG and SRK, the third gene which is determinant of the phenotype of pollen should be at the S-locus. Thus several genes are present on the S-locus and the term "haplotype" instead of classic "allele" is recommended for designating genetic constitution at the complex S-locus.

Abridged Author's Abstract

Molecular ecology of marine microorganisms: Hyperthermophiles and oligotrophs

Y.Ishida, Y.Sako and I.Yoshinaga

Lab. of Microbiology, Dept. of Fisheries, Faculty of Agriculture, Kyoto University

Actinomycetologica, 9: S18-19, 1995

Work on hyperthermophilic and oligotrophic bacteria is reported.

Microorganisms not previously isolated (Aeropyrus pernix, a hyperthermophile from coastal vents of Kodakara island, and some obligate oligotrophs) are characterized.

R.L.

Spontaneously developing pck in Streptomyces: Its cHaracterization and RELation to episomal plasmid and morphological differentiation

S.Ogata

Microbial Genetics Division, Inst. of Genetic Resources, Faculty of Agriculture, Kyusyu Univ.

Actinomycetologica, 9: S19-21, 1995

The phenomenon of spontaneous developing (SP) pocks in S.azureus ATCC 14921 and S.laurentii ATCC 31255 is discussed.

It appears that the free form of conjugative plasmids, pSAl and pSLS, which have an episomal feature, participate in SP pock formation. Excision of these plasmids from their chromosomal integrated sequences is considered to be associated with morphological differentiation, as these processes only occur on the solid media. The SP pock- appearance is always accompanied by the production of defective phage particles. The induction of the defective phage replicon might be caused by the cooperation of the pSAl and pSLS replicons.

It is suggested that the free forms of pSAl and pSLS may promote the excision of the integrated defective phage replicon. Lysis of hyphae would be due to an endolysin that acts at the last stage of phage multiplication. These events might only occur in the aerial and sporulating hyphae. The expansion of the lysis zone might be promoted by the tra gene (and/or spd gene) located on the conjugative plasmid.

It is also suggested that SP pock formation in nature would represent a mechanism of self-restraint against excessive propagation of streptomycetes caused by an overproduction of spores.

Abridged Author's Abstract

Copyright 1995 C.E.T.A