1Research and Development Division, Pharmaceuticals Group, Nippon Kayaku, Co., Lid., 31-12, 3-chome, Shimo, Kita-ku, Tokyo 115, ²Institute of Molecular and Cellular Biosciences, The University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113, Japan

Shuttle vectors, pR4C10 and pR4C11, composed of Streptomyces cosmid and Escherichia coli plasmid were constructed. With both plasmids, Streptomyces lividans and Escherichia coli K12 strains were transformed at high frequencies, comparable with the original plasmids. A vector, pR4C11, was shown to be encapsidated into particles of actinophage R4 in vivo, and transferred to Streptomyces lividans.

N. Ando, T. Umeyama, K. Ueda and S. Horinouchi

Department of Biotechnology, Graduate School of Agriculture and Life Sciences, The University of Tokyo, Bunkyo-ku, Tokyo l13-8657, Japan

The nucleotide sequence of a 12-kilobase region covering afsA encoding a key enzyme for A-factor biosynthesis in Streptomyces griseus was determined and open reading frames were deduced by means of G+C plot analysis. Although this region about150-kb from one end of the linear chromosome is known to be readily deleted, open reading frames crowd the region in a total of eleven, including afsA and sgaA involved in healthy growth on medium of high osmolality. These also include a pair of a sensor histidine kinase and a regulator which comprises a typical prokaryotic two-component regulatory system, proteins similar to O-methyltransferase, LysR-type regulator, 3-ketoacyl-acyl carrier protein reductase, and NADP-dependent alcohol dehydrogenase. In contrast to afsA-like genes in other Streptomyces strains, such as farX locating closely to the gene far the virginiae butanolide receptor in Streptomyces virginiae and farX locating closely to the IM-2-receptor gene in a Streptomyces sp., no open reading frames in this region in S. griseus show homology with the A-factor receptor protein, which suggests that the gene organization of receptor genes for g-butyrolactone autoregulators differs from strain to strain.

A. Matsumoto, Y. Takahashi, M. Mochizuki, A. Seino, Y. Iwai and S. Omura

Research Center For Biological Function, The Kitasato Institute, 5-9-1, Shirokane, Minato-ku, Tokyo 108-8642, Japan

We tried to isolate actinomycetes from fallen leaves by using three methods: the topping, the agar layered and the extraction methods. A large number of rare actinomycetes were isolated from fallen leaves. The genus Microbispora was high frequency in the samples of bamboo (Sasa and Pleioblastus) and loosestrife (Lythrum ).

22nd SAJ Colloquium
Takeda Chemical Industries LTD. Training Institute, Osaka, Japan
December 5, 1997

M. Nasu

Faculty of Pharmaceutical Science, Osaka University, Suita-shi, Osaka 565-0871, Japan

In nature, there are a large number of microorganisms whose isolation is extremely difficult. Recent advanced methodology to detect these micro-organisms, mainly Gram-negative bacteria, by fluorescence staining was presented.

M. Taniguchi

Faculty of science, Osaka City University, Sumiyosi-ku, Osaka 558-0022, Japan

New antimycin congeners which are much less toxic to mammal than antimycin were isolated and characterized from new Streptomyces sp. The bitter taste dialdehyde compound polygodial isolated from plants for spice was discussed from the view point of antifungal activities.

M. Arai

Facu1ty of Agriculture, Osaka Prefecture University, Sakai-shi, Osaka 591~032, Japan)

The history of studies about amylase inhibitors and properties of human amylases were at first summarized and structures and functions of amylase inhibitors from microbial origins were presented in details.

23rd SAJ Colloquium

Kitasato Main Building, Kitasato University, Minato-ku, Tokyo, Japan

February 12, 1998

S. Okamoto and K. Ochi

National Food Rcsea4 Institute, Kannondai, Tsukuba-shi, Ibaraki 305-0856, Japan

The obg gene, which encodes a GTP-binding protein, was cloned from S. griseus IFO13189 and S. coelicolor A3(2) and its possible involvement in differentiation in these organisms was culture and submerged spore formation in liquid culture, indicating that Obg is involved in the regulation of the onset of morphological differentiation in this organism.

The S. coelicolor obg gene was identified by Southern hybridization using S. griseus obg gene investigated.

obg gene of S. griseus was cloned by PCR using primers which are designed based on the conserved amino acid sequences found within the Obg/Gtp1 GTPase family. Introduction of multiple copies of obg into wild-type S. griseus suppressed aerial mycelium development in solid as a probe. Gene disruption experiment indicated that the obg gene product was essential for growth. Propagation of obg gene with multicopy plasmid suppressed aerial mycelium formation of S. coelicolor as was the case of S. griseus. To clarify whether the Obg protein regulates cell differentiation throughout its ability to bind GTP, six single amino acid substitutions were introduced within the consensus GTP-binding regions. Strikingly, propagation of one of the mutant obg gene (P168V) exerted more accentuated suppressive effect on aerial mycelium formation than did the wild-type obg gene. In contrast propagation of another mutant obg gene (GI71A) resulted in an enhancement of aerial mycelium formation. Moreover, this transformant revealed an increased actinorhodin production. No such phenotypic changes were detected by introduction of other four mutant obg genes (K172N, S173N, N2821, and D285A).

According to these results, we proposed that the Obg protein plays a crucial rote in regulating cell differentiation of actinomycetes.

H. Nakajima

Department of Biotechnology, The University of Tokyo, Bunkyo-ku, Tokyo 113 -0032, Japan

Organic solvents can be toxic to microorganisms, depending on both the inherent toxicity of the solvent and the intrinsic tolerance of the bacterial species and strain. Toxicity of an organic solvent is inversely correlated with the logarithm of the partition in n-octanol-water (log Pow value) of the solvent. The mechanisms of organic solvent tolerant toxicity is not well understood.

We have isolated a series of solvent-tolerant mutants of E. coli. These mutants were also found to be resistant to structurally unrelated antibiotics in a pattern that resembled the multiple antibiotic resistance (Mar) phenotype. Biochemical analysis showed that the cell surfaces of organic solvent-tolerant mutants have low hydrophobicity and that organic solvent molecules bind to E. coli cells in response to the polarity of file solvents and the hydrophobicity of the cells.

Genetic analysis revealed that overexpression of several stress response genes soxS, robA, and marA increases the organic solvent tolerance level of host E. coli strains. Overexpression of the stress response genes also increase the resistance to numerous hydrophobic antibiotics but not highly hydrophilic antibiotics. It is suggested that stress response system including drug efflux pumps plays a major rote in the organic solvent tolerance phenotype of E. coli.

We also isolated a cholesterol-converting strain Pseudomonas sp. ST-200, which specifically oxidize the C-3 and -6 positions of cholesterol. This organism showed remarkably effective oxidization of cholesterol dissolved in an organic solvent, but not the substrate suspended in the aqueous medium.

M. Yohda, M. Odaka and I. Endo

Biochetùca1 Systens Laboratory, The Institute of Physical and Chemical Reseaxh (RIKEN) Wako-shi, Saitama 351-0198, Japan

Nitrile hydratase (NHase; EC4.2.1.84) catalyzes hydration of various nitriles to the corresponding amides. The NHase of Rhodococcus sp. N771 shows a unique photoreactivity. In vivo, the NHase is inactivated by aerobic incubation in the dark, but the activity is almost completely recovered by light irradiation. This photoreactivity of the NHase is intrinsic to the enzyme because inactive NHase purified from the dark-inactivated cells is also reactivated by light irradiation. We have been studying this photoreactivity, and recently succeeded in revealing the molecular mechanism of it.

In the dark, nitric oxide (NO) produced in R. sp. N771 binds to the non-heme iron centre of NHase and inactivates it The inactivated NHase is reactivated by the photodissociation of the Fe-NO bond. The nitrosyl non-heme iron centre is located at the cysteine cluster in a-subunit, and its structure was determined by X-ray crystallography and mass spectrometry. The ligands to the non-heme iron atom are sulfur atoms of the three cysteine residues, two main chain amide nitrogen atoms and NO. Two of cysteine residues were post-translationaly oxidized. Two oxygen atoms or modified cysteines and Og of aSer113 protruded from the plane containing the iron atom like claws, and held an NO molecule at the center of them. This structure is named as "claw setting". The photoreactivity of the NHase is likely to be explained by this structure.

S. Sakuda

Department of Applied Biological Chemistry, The University of Tokyo, Bunkyo-ku, Tokyo 113-0032, Japan

Aflatoxin is one of the most potent environmental carcinogens, which causes worldwide concern about contamination of agricultural products. To protect foods and feeds from aflatoxin contamination, we are searching for a specific inhibitor of aflatoxin production of Aspergillus parasiticus, which may depress aflatoxin contamination without rapid emergence of a resistant strain. During the course of the screening, Streptomyces sp. MRI142 was found to produce a strong inhibitor, named aflastatin A.

The structure of aflastatin A was determined by NMR and degradation experiments, which is a unique tetramic acid derivative with a highly oxygenated long alkyl chain. Absolute configuration at some carbons were also elucidated by analysis of degradation products. Recently, it was shown that the structure of blasticidin A is similar to aflastatin A. The incorporation experiments using ¹³C-labeled acetates, propionate, glucose and glycolate suggested that most of C₂ and C₃ units involved in the alkyl chain moiety of aflastatin A were biosynthesized from acetic and propionic acid, but five C₂ units in the alkyl chain originated from glycolic acid.

Aflastatin A inhibited aflatoxin production by A. parasiticus at the concentration of 0.5 g/ml completely without inhibiting the growth of the producer. It also inhibited production of norsolorinic acid at about the some concentration, which is an intermediate located at the early stage of aflatoxin biosynthesis. The effect of aflastatin A on production of 6-methylsalicylic acid by Penicillium griseoflavum was weak, but clear reduction of the amount of the compound produced was observed by addition of aflastatin A at the concentration of 50 g/ml.

S. Akinaga

Kyowa Hakko Kogyo Pharmaceutical Research Institute, Nagaizumi-cho, Shizuoka 411-0943,Japau

One of the most remarkable features of cancer cells is their ability to proliferate under conditions where normal cells do not, reflecting a defective or abrogated restriction point control of cell growth, Recent accumulating evidences show that this key characteristics of cancer cells are stem from the aberrations among cell cycle regulators, in particular, those regulating G1-phase progression and/or G1 to S phase transition. Among these aberrations, the Rb pathway is thought to be the most important one because it includes proto-oncogene products (Cyclin D, CDK4/6, Cyclin E and E2F) as well as tumor suppressors (p16/p15, p27 and Rb itself), and each of them could be deregulated by several molecular mechanisms. Under these circumstances, Rb pathway (G1-phase of cell cycle) is thought to be a rationale target for the development of tumor selective anti-cancer agents.

UCN-01 (7-hydroxyl-staurosporine) was originally isolated from the culture broth of Streptomyces sp. as a PKC selective inhibitor. Because of its unique preclinical data (potent antitumor activity against several tumor models and the synergistic combined effects with key anti-cancer drugs), UCN-01 is now in Phase 1 clinical trials in USA and Japan.

Although the precise mechanism(s) of action for its antitumor activity is still not fully understood, results from our laboratory and others have revealed that UCN-01 inhibits cell cycle progression from G1 to S phase in several mammalian cell lines. Recently UCN-01 was shown to inhibit CDK2 activity of the target cells through the direct and indirect inhibition of the kinase, and the latter effect was shown to be mediated through the induction of CDK inhibitor p21 and/or p27.

GE3 was originally isolated from the culture broth of Streptomyces sp. through the screening of new antitumor antibiotics. GE3 is a novel member of cyclic hexadepsipeptide which exhibits potent cytotoxic activity against human tumor cell lines in vitro. Further studies revealed that the compound exhibited significant growth inhibitory activity against chemo-resistant human pancreatic carcinoma PSN-1 cells transplanted in nude mice. More importantly, GE was shown to inhibit the transcriptional activity of E2F, which is a positive key regulator of mammalian cell-cycle progression from G1 to S phase. Flow cytometry analysis showed that GE3 accumulated the human epidermoid carcinoma A431 cells in G1 phase as was expected from its action mechanism.

Copyright 1998 C.E.T.A., The International Centre for Theoretical and Applied Ecology, Gorizia