Summary of Research Project Results Under the JSPS FY2000
"Research for the future Program"



1.Research Institution University of Tokyo
 
2.Research Area Integrated Fields
 
3.Research Field Production of Novel Useful Substances through Integration of Life Sciences and Chemistry
 
4.Term of Project FY1996〜FY2000
 
5.Project Number 96I00303
 
6.Title of Project Molecular analysis of the initiation of secondary metabolite formation and its application to molecular breeding of producers

7.Projetct Leader
Name Institution,Department Title of Position
Horinouchi, Sueharu Univ. of Tokyo, Graduate School of Agricultural & Life Sciences Professor

8.Core Members

Names Institution,Department Title of Position
Yamada, Yasuhiro Osaka Univ., Graduate School of Engineering Professor
Nihira, Takuya Osaka Univ., Graduate School of Engineering Assoc. Professor
Beppu, Teruhiko Nihon Univ., College of Bioresource Sciences Professor

9.Cooperating Researchers

Names Institution,Department Title of Position
Ohnishi, Yasuo Univ. of Tokyo, Graduate School of Agricultural & Life Sciences Assistant
Ueda, Kenji Nihon Univ., College of Bioresource Sciences Assistant
Ihara, Fumio Osaka Univ., Graduate School of Engineering Assistant

10.Summary of Research Results

(1) The A-factor-specific receptor served as a repressor-type regulator for adpA encoding a transcriptional activator of many genes required for secondary metabolism and morphological differentiation. The regulatory cascade leading to streptomycin biosynthesis has been elucidated. An A-factor-dependent sigma factor of RNA polymerase essential for morphogenesis was also identified. Additional regulatory systems including eukaryotic-type protein phosphorylation, cAMP, and calmodulin were found to control secondary metabolism and morphogenesis and cross-talk with the A-factor regulatory cascade.
(2) Virginiae butanolide-specific receptor protein (BarA) and IM-2 specific receptor protein (FarA) were purified from Streptomyces virginiae and Streptomyces lavendulae FRI-5, respectively. With the corresponding genes, recombinant receptors were overexpressed in E. coli, and in vitro analyses using the purified receptor revealed the high affinity (Kd= 〜lnM) and the high ligand specificity of the receptors. Phenotypic analyses of null mutants of the receptor genes as well as the functional analyses of genes surrounding the VB receptor gene (barA) clarified the detailed regulation mechanism on the VB-mediated expression of antibiotic resistance together with the overall signalling cascade leading to the antibiotic biosynthesis.
(3) As environmental factors affecting the initiation of secondary metabolism and morphological differentiation in Streptomyces, we characterized not only the involvement of carbon-sources and copper, but also the interspecific stimulation of those events mediated by putative diffusible signal substances, which is widely distributed among Streptomyces. As for the former subject, we cloned related genes and generated knockdown mutants, which revealed the role of those genes. For the latter, we purified several chemical substances that possibly work as the interspecific signals. Furthermore, we identified and inactivated other novel regulatory genes for secondary metabolism or morphogenesis. All results enabled us to establish a fundamental standpoint for studying the effect by various environmental factors on secondary metabolism and cellular differentiation in Streptomyces.

11.Key Words

(1)Streptomyces、(2)autoregulatory factors、(3)antibiotic production
(4)morphogenesis、(5)control of gene expression、(6)microbial hormone
(7)A-factor

12.References

[Reference Articles]
Author Title of Article
Horinouchi, S. Streptomyces genes involved in aerial mycelium formation.
Journal Volume Year Pages Concerned
FEMS Microbiol. Lett. 141 1996 1-9

Author Title of Article
Onaka, H. DNA binding activity of the A-factor receptor protein in Streptomyces griseus.
Journal Volume Year Pages Concerned
Annu. Rep. IC Biotech. 19 1996 586-592

Author Title of Article
Ueda, K. The aerial mycelium-defective phenotype of Streptomyces griseus resulting from A-factor deficiency is suppressed by a Ser/Thr kinase of S. coelicolor A3(2).
Journal Volume Year Pages Concerned
Gene 169 1996 91-95

Author Title of Article
Umeyama, T. Expression of the Streptomyces coelicolor A3(2) ptpA gene encoding a phosphotyrosine protein phosphatase leads to overproduction of secondary metabolites in S. lividans.
Journal Volume Year Pages Concerned
FEMS Microbiol. Lett. 144 1996 177-184

Author Title of Article
Ando, N. Involvement of AfsA in A-factor biosynthesis as a key enzyme.
Journal Volume Year Pages Concerned
J. Antibiot. 50 1997 847-852

Author Title of Article
Ando, N. A Streptomyces griseus gene (sgaA) suppresses the growth disturbance caused by high osmolality and a high concentration of A-factor during early growth.
Journal Volume Year Pages Concerned
Microbiology 143 1997 2715-2723

Author Title of Article
Lezhava, A. Chromosomal deletions in Streptomyces griseus that remove the afsA locus.
Journal Volume Year Pages Concerned
Mol. Gen. Genet. 253 1997 478-483

Author Title of Article
Onaka, H. DNA-binding activity of the A-factor receptor protein and its recognition DNA sequences.
Journal Volume Year Pages Concerned
Mol. Microbiol. 24 1997 991-1000

Author Title of Article
Onaka, H. A mutation at proline-115 in the A-factor receptor protein of Streptomyces griseus abolishes DNA-binding ability but not ligand-binding ability.
Journal Volume Year Pages Concerned
Mol. Microbiol. 179 1997 2748-2752

Author Title of Article
Ando, N. Gene organization in a 12-kilobase region covering afsA encoding an A-factor biosynthetic enzyme.
Journal Volume Year Pages Concerned
Actinomycetologica 12 1998 40-45

Author Title of Article
Hong, S. -K. Effects of protein kinase inhibitors on in vitro protein phosphorylation and on secondary metabolism and morphogenesis in Streptomyces coelicolor A3(2).
Journal Volume Year Pages Concerned
J. Microbiol. Biotechnol. 8 1998 325-332

Author Title of Article
Horinouchi, S. The A-factor regulatory cascade and protein phosphorylation that control antibiotic production and morphogenesis in Streptomyces.
Journal Volume Year Pages Concerned
Annu. Rep. IC Biotech. 21 1998 in press

Author Title of Article
Onaka, H. Involvement of two A-factor receptor homologues in Streptomyces coelicolor A3(2) in the regulation of secondary metabolism and morphogenesis.
Journal Volume Year Pages Concerned
Mol. Microbiol. 28 1998 743-753

Author Title of Article
Sugiyama, M. Site-directed mutagenesis of the A-factor receptor protein: Val-41 important for DNA-binding and Trp-119 important for ligand-binding.
Journal Volume Year Pages Concerned
Gene 222 1998 33-144

Author Title of Article
Ueda, K. Characterization of an A-factor-responsive repressor for amfR essential for onset of aerial mycelium formation in Streptomyces griseus.
Journal Volume Year Pages Concerned
J. Bacteriol. 180 1998 5085-5093

Author Title of Article
Funa, N. A new pathway for polyketide synthesis in microorganisms.
Journal Volume Year Pages Concerned
Nature 400 1999 897-899

Author Title of Article
Kang, D. -K. Possible involvement of cAMP in aerial mycelium formation and secondary metabolism in Streptomyces griseus.
Journal Volume Year Pages Concerned
Microbiology 145 1999 1161-1172

Author Title of Article
Ohnishi, Y. The A-factor regulatory cascade leading to streptomycin biosynthesis in Streptomyces griseus: identification of a target gene of the A-factor receptor.
Journal Volume Year Pages Concerned
Mol. Microbiol. 34 1999 102-111

Author Title of Article
Umeyama, T. An AfsK/AfsR system involved in the response of aerial mycelium formation to glucose in Streptomyces griseus.
Journal Volume Year Pages Concerned
Microbiology 145 1999 2281-2292

Author Title of Article
Yonekawa, T. Involvement of amfC in physiological and morphological development in Streptomyces coelicolor A3(2).
Journal Volume Year Pages Concerned
Microbiology 145 1999 2273-2280

Author Title of Article
Ohnishi, Y. Regulation of secondary metabolism and morphogenesis by microbiol hormones in Streptomyces (in Japanese)
Journal Volume Year Pages Concerned
Tanpakushitsu-Kokusan-Koso 44 1999 1552-1561

Author Title of Article
Horinouchi, S. The A-factor regulatory cascade and cAMP in the regulation of physiological and morphological development in Streptomyces griseus.
Journal Volume Year Pages Concerned
J. Ind. Microbiol. Biotechnol. 25 2000 in press

Author Title of Article
Horinouchi, S. Isolation of DNA fragments bound by transcriptional factors, AdpA and ArpA, in the A-factor regulatory cascade.
Journal Volume Year Pages Concerned
Actinomycetologica 14 2000 37-42

Author Title of Article
Ohnishi, Y. An oligoribonuclease gene in Streptomyces griseus.
Journal Volume Year Pages Concerned
J. Bacteriol. 182 2000 4647-4653

Author Title of Article
Umeyama, T. Genetic and biochemical characterization of a protein phosphatase with dual substrate specificity in Streptomyces coelicolor A3(2).
Journal Volume Year Pages Concerned
Gene 258 2000 55-62

Author Title of Article
Yamazaki, H. An A-factor-dependent extracytoplasmic function sigma factor σAdsA that is essential for morphological development in Streptomyces griseus.
Journal Volume Year Pages Concerned
J. Bacteriol. 182 2000 4596-4605

Author Title of Article
Funa, N. A new pathway for polyketide biosynthesis: application of a new enzyme to bioengineering (in Japanese).
Journal Volume Year Pages Concerned
Kagaku-to-Seibutsu 38 2000 283-285

Author Title of Article
Funa, N. Combinatorial biosynthesis with a bacterial chalcone synthase (Japanese).
Journal Volume Year Pages Concerned
Bioscience and Industry 58 2000 568-569

Author Title of Article
Lee, H. -S. A σB-like factor responsible for carotenoid biosynthesis in Streptomyces griseus.
Journal Volume Year Pages Concerned
J. Mol. Microbiol. Biotechnol. 3 2001 95-101

Author Title of Article
Yonekawa, T. A calcium-binding protein with four EF-hand motifs in Streptomyces ambofaciens.
Journal Volume Year Pages Concerned
Biosci. Biotechnol. Biochem. 65 2001 56-160

Author Title of Article
Chun, H. -K. Biotransformation of phenanthrene and 1-methoxynaphthalene with Streptomyces lividans cells expressing a marine bacterial phenanthrene dioxygenase gene cluster.
Journal Volume Year Pages Concerned
Biosci. Biotechnol. Biochem.   2001 in press

Author Title of Article
Katayama, K. Gene organization in the ada-rplL region of Streptomyces virginiae.
Journal Volume Year Pages Concerned
Gene 171 1996 135-136

Author Title of Article
Lee, C-H. Stereospecific reduction of virginiamycin M1 as the virginiamycin resistance pathway in Streptomyces virginiae.
Journal Volume Year Pages Concerned
Antimicrob. Agents Chemother. 40 1996 595-601

Author Title of Article
Yang, Y. K. Maximum virginiamycin production by optimization of cultivation conditions in batch culture with autoregulator addition.
Journal Volume Year Pages Concerned
Biotech. Bioeng. 49 1996 437-444

Author Title of Article
Yang, Y. K. Image Analysis of mycelial morphology in virginiamycin production by batch culture of Streptomyces virginiae.
Journal Volume Year Pages Concerned
J. Ferment. Bioeng. 81 1996 7-12

Author Title of Article
Sy, A. Gene organization of the region involved in regulation of secondary metabolism of Streptomyces virginiae.
Journal Volume Year Pages Concerned
Annu. Rep. IC Biotech. 19 1996 184-190

Author Title of Article
Kawachi, R. Development of a transformation system in Streptomyces virginiae.
Journal Volume Year Pages Concerned
Actinomycetologica 11 1997 46-5

Author Title of Article
Kawabuchi, M. Production of butyrolactone autoregulators by Streptomyces coelicolor A3(2).
Journal Volume Year Pages Concerned
FEMS Microbiol. Lett. 157 1997 81-85

Author Title of Article
Kinoshita, H. Butyrolactone autoregulator receptor protein (BarA) as a transcriptional regulator in Streptomyces virginiae.
Journal Volume Year Pages Concerned
J. Bacteriol. 179 1997 6986-6993

Author Title of Article
Waki, M. Cloning and characterization of IM-2 receptor gene (farA) from Streptomyces sp. strain FRI-5.
Journal Volume Year Pages Concerned
J. Bacteriol. 179 1997 5131-5137

Author Title of Article
Wangchaisoonthorn, U. Sequencing and analysis of 5'-flanking region of barA.
Journal Volume Year Pages Concerned
Annu. Rep. IC Biotech. 20 1997 217-227

Author Title of Article
Suzuki, N. Purification and characterization of virginiamycin Ml reductase from Streptomyces virginiae.
Journal Volume Year Pages Concerned
Antimicrob. Agents Chemother. 42 1998 2985-2988

Author Title of Article
Nakano, H. Gene Replacement Analysis of the Streptomyces virginiae barA gene encoding the butyrolactone autoregulator receptor reveals that BarA acts as a repressor in virginiamycin biosynthesis.
Journal Volume Year Pages Concerned
J. Bacteriol. 180 1998 3317-3322

Author Title of Article
Kinoshita, H. Characterization of bindig sequence for butyrolactone-autoregulator- receptors in Streptomycetes.
Journal Volume Year Pages Concerned
J. Bacteriol. 181 1999 5075-5080

Author Title of Article
Kitani, S. In vitro analysis of the butyrolactone autoregulator receptor protein (FarA) of Streptomyces lavendulae FRI-5 reveals that FarA acts as a DNA-binding transcriptional regulator that controls its own synthesis.
Journal Volume Year Pages Concerned
J. Bacteriol. 181 1999 5081-5084

Author Title of Article
Lee, C. -K. Identification and in vivo functional analysis of a virginiamycin S resistance gene (varS) from Streptomyces virginiae.
Journal Volume Year Pages Concerned
J. Bacteriol. 181 1999 3293-3297

Author Title of Article
Shikura, N. Identification and characterization of 6-dehydro VB-A reductase from Streptomyces antibioticus.
Journal Volume Year Pages Concerned
FEMS Microbiol. Lett. 171 1999 183-189

Author Title of Article
Kawachi, R. Idenification by gene deletion analysis of a regulator, VmsR, that controls virginiamycin biosynthesis in Streptomyces virginiae.
Journal Volume Year Pages Concerned
J. Bacteriol. 182 2000 6259-6263

Author Title of Article
Nakano, N. A null mutant of the Streptomyces virginiae barA gene encoding butyrolactone autoregulator receptor, and its pleiotropic and transcriptional analyses.
Journal Volume Year Pages Concerned
J. Biosci. Biotechnol. 90 2000 204-207

Author Title of Article
Kitani, S. Conjugal transfer of plasmid DNA from Escherichia coli to Streptomyces lavendulae FRI-5.
Journal Volume Year Pages Concerned
J. Microbiol. Biotechnol. 10 2000 535-538

Author Title of Article
Shikura, N. Identification of a plausible biosynthetic enzyme for the IM-2-type autoregulator in Streptomyces antibioticus.
Journal Volume Year Pages Concerned
Biochim. Biophys. Acta 1475 2000 329-336.

Author Title of Article
Takano, E.  
Journal Volume Year Pages Concerned
J. Biol. Chem. 275 2000 11010-11016

Author Title of Article
Kawachi, R. Identification of an AfsA homologue (BarX) from Streptomyces virginiae as a pleiotropic regulator controlling autoregulator biosynthesis, virginiamycin biosynthesis and virginiamycin resistance.
Journal Volume Year Pages Concerned
Mol. Microbiol. 36 2000 302-313

Author Title of Article
Namwat, W. Identification of the varR gene as a transcriptional repressor of virginiamycin S resistance in Streptomyces virginiae.
Journal Volume Year Pages Concerned
J. Bacteriol.   2001 in press.

Author Title of Article
Ueda, K. Regulation of cell differentiation by a regulatory gene amfR in Streptomyces.
Journal Volume Year Pages Concerned
Bioscience and Industry 55 2001 30-32

Author Title of Article
Ueda, K. Stimulatory effect of copper on antibiotic production and morphological differentiation in Streptomyces tanashiensis.
Journal Volume Year Pages Concerned
J. Antibiot. 50 1997 693-695

Author Title of Article
Ueda, K. A putative regulatory element for carbon-source-dependent differentiation in Streptomyces griseus.
Journal Volume Year Pages Concerned
Microbiology 145 1999 2265-2272

Author Title of Article
Ueda, K. Cloning and characterization of regulatory elements for the onset of differentiation in Streptomyces griseus.
Journal Volume Year Pages Concerned
Actinomycetologica 13 1999 89-93

Author Title of Article
Ueda, K. Wide distribution of interspecific stimulatory events on antibiotic production and sporulation among Streptomyces species.
Journal Volume Year Pages Concerned
J. Antibiot. 53 2000 979-982

Author Title of Article
Ueda, K. Carbon-source-dependent transcriptional control involved in the initiation of cellular differentiation in Streptomyces griseus.
Journal Volume Year Pages Concerned
Antonie van Leeuwenhoek   2000 in press

[Reference Books]
Author Title of Book
Horinouchi, S. Dynamic Aspects of Natural Products Chemistry: Molecular Biological Approaches
Publisher Year Pages
Kodansha and Harwood Academic Publishers 1997 165-186

Author Title of Book
Horinouchi, S. Proceedings of the International Symposium on Bioactive Compounds and Strain Development
Publisher Year Pages
The Korean Society for Applied Microbiology 1998 66-72

Author Title of Book
Horinouchi, S. Cell-Cell Signaling in Bacteria
Publisher Year Pages
American Society for Microbiology 1999 93-207

Author Title of Book
Yamada, Y. Biotechnology of Antibiotics
Publisher Year Pages
Marcel Dekker, Inc 1999 63-79

Author Title of Book
Yamada, Y. Comprehensive Natural Products Chemistry
Publisher Year Pages
Elsevier Science 1998 377-413


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