Mechanism of gene expression cascade in TOL plasmid from Pseudomonas putida

1994 Fiscal Year Final Research Report Summary

• Project/Area Number: 05680595
• Research Category: Grant-in-Aid for General Scientific Research (C)
• Allocation Type: Single-year Grants
• Research Field: Molecular biology
• Research Institution: Yamaguchi University
• Principal Investigator: INOUYE Sachiye Yamaguchi University School of Medicine, Assistant Professor, 医学部, 講師 (60159978)
• Co-Investigator(Kenkyū-buntansha): GOMADA Manabu Yamaguchi University School of Medicine, Reserch Assosiate, 医学部, 助手 (80243632)
• Project Period (FY): 1993 – 1994
• Keywords: TOL Plasmid / Pseudomonas putida / sctivator protein / transcriptional regulation / foot-printing / DNA bend

Research Abstract

TOL plasmid from Pseudomonas putida encodes a series of degradative enzymes for xylenes (OP1, upper-pathway operon and OP2, lower-pathway operon) and two transcriptional activators (XylR,XylS) . In the presence of xylene, XylR activates OP1 as well as xylS gene. Induced amout of XylS activates OP2. In this process, toluate facilitates the OP2 activation by XylS.Therefore, the regulation of xyl genes expression involves a sort of cascade mechanism including amplification of activator protein.
To undertand the mechanism of the activation of OP2 by XylS,we performed the in vivo footprint analysis of the prmoter-regulatory region of OP2. When XylS protein under the control of tac promoter was induced by addition of IPTG,two regions on OP2 DNA were protected by dimethy sulfate treatment. These results indicates that these reagions could be the binding sites of XylS and/or RNA polymerase.
In order to purify XylS,the xylS gene was introduced into a baculovirus vector. When XylS is purified at large amount, we will investigete the interaction of XylS with RNA polymerase isolated from Pseudomonas putida on the regulatory region of OP2.
These observation is expected to clalify the cascade mechanisn of the transcriptional activation of xyl genes on TOL plasmid.

Research Products

• [Publications] M.Gomada: "Analysis of DNA bend structure of promoter regulatory regions of xylene-metabolizing genes on the Pseudomonas TOL plasmid." J.Biochem.116. 1096-1104 (1994)
  • Description: 「研究成果報告書概要(和文)」より
• [Publications] K.Miura: "Identification of the binding site of XylS activator protein on the second operon of the Pseudomonas TOL plasmid." (In preparation).
  • Description: 「研究成果報告書概要(和文)」より
• [Publications] Gomada, M., Imaishi, H., Miura, K., Inouye, S., Nakazawa, T.and Nakazawa, A.: "Analysis of DNA bend structure of promoter regulatory regions of xylene-metabolizing genes on the Pseudomonas TOL plasmid." J.Biochem.116. 1096-1104 (1994)
  • Description: 「研究成果報告書概要(欧文)」より
• [Publications] Miura, K., Gomada, M., Inouye, S.and Nakazawa, A.: "Identification of the binding site of XylS activator protein on the second operon of the Pseudomonas TOL plasmid." (In preparation).
  • Description: 「研究成果報告書概要(欧文)」より