Vol. 6 No. 2,1992

Published by the Society for Actinomycetes, Japan

Code Number: AC93004

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MOLECULAR GENETICS AND BIOCHEMISTRY OF TYPE II POLYKETIDE SYNTHASES FROM THE PERSPECTIVE OF TETRACENOMYCIN C BIOSYNTHESIS IN STREPTOMYCES GLAUCESCENS

C. Richard Hutchinson^1,2, H.Decker^1, H.Motamedi^3, B.Shen^1, R.G.Summers^1, E.Wendt-Pienkowski^1 and W.L. Wessel^1

^1 School of Pharmacy and ^2 Department of Bacteriology, University of Wisconsin, Madison, WI 53706, USA; ^3 Process Genetics and Molecular Biology, Merck Research Laboratories, Rahway, NJ 07065, USA

Actinomycetologica, 6: 49-68, 1992

The genetics and biochemistry of type II polyketide synthesases (PKS) were examined with reference to tetracenomycin C (Tcm C) biosynthesis in Streptomyces glaucescens. Mutations in the strain that block Tcm C production, but do not affect either Tcm C resistance or S.glaucescens morphogenesis, allowed the sequence of steps in the pathway to be established. PKS gene mutants do not accumulate diffusible intermediates and are universal converter strains.

Characteristics of Tcm PKS genes were determined by gene cloning and sequencing. Analysis of subclones showed that a 1.8 kb BgeII DNA segment restores Tcm C production when introduced into a TcmI mutant.

The enzymology of Tcm PKS was investigated and its prospects for the future are discussed.

R.L.

QUANTITATIVE ANALYSIS FOR MADUROSE AND OTHER SUGARS IN A SMALL AMOUNT OF ACTINOMYCETE WHOLE CELLS BY GAS-LIQUID CHROMATOGRAPHY

Y.Takahashi, H.Egusa, B.Deng, H.Kiyohara, H.Yamada, Y.Iwai and S.Omura

The Kitasato Institute, 5-9-1, Shirokane, Minatoku, Tokyo 108, Japan

Actinomycetologica, 6: 69-78, 1992

A gas-liquid chromatographic (GLC) method for the detection of small amounts of madurose (3-O-methylgalactose) and of other sugars of taxonomic interest, present in actinomycete whole cells, is proposed. Following hydrolysis (1 mg dried cells) with trifluoroacetic acid, by GLC, using a capillary column, a good separation of alditol acetates derived from madurose as well as of other neutral sugars (rhamnose, arabinose, xylose, 3-O-methylmannose, mannose, galactose and glucose) was obtained.

Madurose was detected in cells of Kitasatosporia setae KM-6054, K.phosalacinea KA-338 and K.griseola AM-9660, as well as in Actinomadura madurae IFM 78 and in Streptosporangium roseum JCM-3005, but not in Streptomyces griseus IF0- 12875 .

Abridged Authors' Abstract, R.L.

TAXONOMIC SIGNIFICANCE OF ARABINOSE IN THE FAMILY PSEUDONOCARDIACEAE

M.Takeuchi, T.Nishii and A.Yokota

Institute for Fermentation, Osaka, 17-85, Jusohonmachi 2-chome, Yodogawa-ku, Osaka 532, Japan

Actinomycetologica, 6: 79-90, 1992

Sugar composition of whole cells and cell wall preparations of the family Pseudonocardiaceae was investigated. Members of the genera Actinopolyspora, Saccharopolyspora and Amycolata were confirmed to belong to chemotype IV/A, species of Pseudonocardia, Amycolatopsis, Saccharomonospora, Pseudoamycolata, Actinokineospora and Kibdelosporangium were found to be of type III/A and those of Saccharothrix of type III/A or III/C. Therefore the description of family Pseudonocardiaceae, proposed for closely related genera on the basis of 16S rRNA sequencing studies and considered to include organisms with wall chemotypes III and IV, should be emended.

Abridged Authors' Abstract, R.L.

MICROBIOLOGICAL CHARACTERISTICS OF GENUS MICROMONOSPORA

I.Kawamoto

Tokyo Research Laboratories, Kyowa Hakko Kogyo Co., Ltd., 3-6-6, Asahi-machi, Machida-shi, Tokyo 194, Japan

Actinomycetologica, 6: 91-104, 1992

Organisms of the genus Micromonospora show peculiarities in their metabolites, spore structure, cell wall and growth conditions. Various types of antibiotics and enzyme inhibitors were found in a screening program, suggesting that the organisms could be promising sources of useful drugs. More than 50% of the single spores on vegetative mycelia survived 20 min of moist heating at 60 C, and more than 10% survived after 30 min treatment at 30 C with solutions containing 60% polar solvents. The cell wall consists of a peptide subunit glycyl-D-glutaminyl-meso-diaminopimelyl-D-alanine, a direct cross-linkage between D-alanine and mesodiaminopimelic acid, N-glycolylmuramic acids, and ribitol teichoic acids containing arabinose and xylose. These characteristics, together with some physiological properties, are discussed in comparison with those of other genera of Actinomycetales.

Abridged Author's Abstract. R.L.

STUDIES ON THE C-P BOND FORMATION MECHANISMS IN BIALAPHOS

T Hidaka and H.Seto

Institute of Applied Microbiology, The University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113. Japan

Actinomycetologica, 6: 105-112, 1992

Bialaphos, a metabolite produced by Streptomyces hygroscopicus, is a strong inhibitor of glutamate synthetase and so is used as an herbicide.

The involvement of the P-methylation enzyme in bialaphos biosynthetic pathway is demonstrated.

R.L.

TOGETHER WITH STREPTOMYCES FOR THIRTY-FIVE YEARS

R.Nomi

Junior College Division, Hiroshima Bunkyo Women's College, 1-2-1, Kabehigashi, Asakitaku, Hiroshima 731-02, Japan

Actinomycetologica, 6: 113-118, 1992

Discoveries concerning Streptomyces during the author's 35-years study of the organisms are outlined.

On the basis of morphological characteristics of aerial mycelia and sporiferous hyphae, the genus Streptomyces was divided into eight morphological types. Four of them were further subdivided by the morphological features of spirals.

Streptomycin 6-phosphate was produced in the cells of Streptomyces griseus. This compound had no antibiotic activity and was transformed to streptomycin by dephosphorylation with the specific alkaline phosphatase at the periplasmic space or outside of the cell.

S.griseus produced streptomycin-6-kinase in the cell. This enzyme contributed to self-protection of the streptomycin producer by inactivating streptomycin and liberating ribosome-bound streptomycin .

Suppression of the phospholipid cycle of cell wall synthesis enhanced streptomycin production.

Abridged Author's Abstract, R.L.

Abstracts of the SAJ Tokyo Colloquium

Kitasato Institute, Tokyo, Oct. 16,1992

CLONING OF TYLOSIN ACYLTRANSFERASE GENES FROM STREPTOMYCES THERMOTOLERANS AND DIRECT FERMENTATIVE PRODUCTION OF ACYLTYLOSINS

A.Arisawa, H.Tsunekawa, K. Okamura and R.Okamoto

Central Research Laboratories, Mercian Co.

Actinomycetologica, 6: S2, 1992

Genes involved in the acylation of macrolide antibiotics were cloned from the carbomycin biosynthetic gene cluster of S.thermotolerans. The macrolide 4"-O-acyltransferase activity was found within a 2.7 kb region comprising two genes (acyB1 and acyB2). S.lividans, transformed by plasmids carrying the DNA region, could convert supplemented tylosin into 4"-O-acyltylosins.

One of the genes (acyB1) was the same as carE, reported as encoding the putative 4"-O-mycarosyl isovaleryl-CoA transferase activity (Epp, J.K. et al., Gene, 85: 293-301, 1989). The second (acyB2) transcribed divergently to acyB1 and, on the basis of expression at transcriptional level and northern blot analysis, was assumed to encode a novel regulatory protein that activates acyB1.

Macrolide 3-O-acyltransferase gene (acyA), located at least 40 kb from 3' end of acyB1, was also cloned and sequenced. The amino acid sequence of acyA showed significant homology to that of acyB 1. Tylosin producer S.fradiae transformed by plasmids containing acyA and/or acyB1-acyB2 produced acyltylosins such as 3-O-acetyltylosin (3-AT) and 3-O-acetyl-4"-O-isovaleryltylosin (AIV).

Abridged Author's Abstract, R.L.

ARYLSULFATASES FROM ACTINOMYCETES

T.Ueki, S.Yamamoto, Y.Sawada, Y.Fukagawa and T.Oki

Bristol-Myers Squibb Research Institute

Actinomycetologica, 6: S2-S3, 1992

In the course of clinical treatment of tumours with etoposide, a potent antitumour antibiotic, the tumour antibody: enzyme conjugate was assumed to be promising, since etoposide sulphate was found to be less cytotoxic in vivo. A search for sulphatase, highly specific to etoposide sulphate, was carried out in bacteria, fungi, yeasts and actinomycetes by using nitrophenylsulphate as the primary substrate. Two extracellular sulphatases designated Es-l and Es-2 were found in Streptomyces griseorubiginosus S980-14 and Streptomyces sp. T109-3, respectively. Both enzymes seem uncommon since, during fermentation, the presence of high concentrations of inorganic salts, such as NaCl, or tap water suppresses their production. Es-l sulphatase was purified by Butyl-Toyoperal column chromatography, chromatofocusing and Cellulofine GC-700-m column chromatography, and Es-2 sulphatase by Sulphate-Cellulofine and DEAE-cellulose column chromatography. The two enzymes, both with a molecular weight of 45,000 showed isoelectric points of 4.95 and 5.65 respectively. Ca++ was found to be essential for enzyme activity. With reference to known arylsulphatases, only these two enzymes attack etoposide sulphate, resulting in regeneration of cytotoxic etoposide.

Abridged Authors, Abstract, R.L.

ACTINOMYCETES IN AGRICULTURE

Y.Komoto and K.Yokoyama

National Institute of Agro-Environmental Sciences

Actinomycetologica, 6: S3, 1992

The importance of actinomycetes in agricultural production is summarised. Actinomycetes act as animal and plant pathogens and degraders of woods and other agricultural material. On the other hand, they have an important role as producers of agricultural drugs, agents of biological control of plant diseases and as plant growth enhancers. The positive role is more important since damage to the production by pathogens towards animals or the degradation is relative. In our department actinomycetes are investigated with the view of utilising them as agents of biological control against plant diseases caused by pathogenic actinomycetes. Some antagonistic strains isolated from natural soils control potato soil rot. The need for studies on actinomycetes as ordinary components of soil microflora is discussed.

Abridged Authors' Abstract, R.L.

STUDIES ON A SELECTIVE ISOLATION METHOD FOR SACCHAROPOLYSPORA AND ITS FERMENTATION PRODUCTS

K.Suzuki, K.Nagai, S.Miyazaki and M.Morioka

Bioscience Res. Lab. I, Central Res. Lab., Yamanouchi Pharmaceutical Co., Ltd.

Actinomycetologica, 6: S3-S4, 1992

Saccharopolyspora is one of the most important sources of antibiotics and physiologically active substances: sporaricins, destomycin, vanoxonin, and saccharocin have been isolated from organisms of this genus. The present report describes the results of an investigation on a selective isolation method.

Experiments were carried out using soil samples collected from the Southwest islands of Okinawa Prefecture, Japan .

MSA-2 a medium, which contains cabbage meal and sugarcane bagasse meal as C and/or N source and kanamycin and streptothricin as selective agents, was used. Saccharopolyspora was isolated efficiently on MSA-2 medium at 32 C after two month incubation.

Colonies were then transferred to screening media: KP (containing 20 ug/ml of kanamycin and penicillin), KPB (20 ug/ml of kanamycin, penicillin G and novobiocin respectively) and KPL (20 ug/ml of kanamycin and penicillin G and 100 ug/ml of lysozyme). Saccharopolyspora grew in all media, while most strains belonging to the other genera did not.

Two hundred and six strains belonging to the genus Saccharopolyspora were isolated. Strains were grouped into four types on the basis of cultural and physiological characteristics. Type 1 strains, identified as S.hirsuta, were found to produce sporaricin (aminoglycosides) while strains of type 2 (identified as S.erythraea) produced erythromycin (macrolides).

A new species of Saccharopolyspora was found in the course of the investigation.

Abridged Authors' Abstract, R.L.

BIOSYNTHESIS OF THE PRADIMICIN FAMILY OF ANTIBIOTICS

S.Kakinuma, T.Furumai and T.Oki

Bristol-Myers Squibb Research Institute

Actinomycetologica, 6: S3-S4, 1992

In order to elucidate the biosynthetic pathway of the pradimicin family of antibiotics, mutants of Actinomadura verrucosospora subsp. neohibisca E-40, a high pradimicin producer, were obtained by NTG and/or UV treatment. 37 non-producers were grouped into 9 classes on the basis of their activity in cosynthesis and bioconversion tests and metabolites were isolated for characterisation. Results show that mutants belonging to class III are true converters, mutants belonging to classes IV, V and VI are secretors producing putative biosynthetic intermediates of pradimicins and mutants belonging to classes VII, VIII and IX, are producers of shunt metabolites. On the basis of product analysis the following biosynthetic pathway of pradimicin A is proposed: 11-O-demethylpradinone II->11- O-dimethyl-7-methoxypradinone II-> 11-O-demethylpradinone I- >11-O-demethylpradimicinone I->pradimicinone I-> pradimicin B-> pradimicin A.

Sinefungin, a specific inhibitor of methyltransferase, appears to block the O-methylation step at C-7 position, causing an accumulation of shunt products, 11-O-demethylpradimicinone II and pradimicinone II.

A similar study with a converter derived from Actinomadura s. AB1236, a benanomicin A-producer, indicates that benanomicin A is biosynthesised in an similar way.

Abridged Authors' Abstract, R.L.

Copyright 1993 C.E.T.A.