On the Mechanism of Functional Expression of Rhizobial Type III Secretion System

Research Project

Project/Area Number	15K07003
Research Category	Grant-in-Aid for Scientific Research (C)
Allocation Type	Multi-year Fund
Section	一般
Research Field	Functional biochemistry
Research Institution	Nara Women's University
Principal Investigator	Saeki Kazuhiko 奈良女子大学, 自然科学系, 教授 (40201511)
Project Period (FY)	2015-04-01 – 2019-03-31
Project Status	Completed (Fiscal Year 2018)
Budget Amount *help	¥5,070,000 (Direct Cost: ¥3,900,000、Indirect Cost: ¥1,170,000) Fiscal Year 2017: ¥1,300,000 (Direct Cost: ¥1,000,000、Indirect Cost: ¥300,000) Fiscal Year 2016: ¥1,690,000 (Direct Cost: ¥1,300,000、Indirect Cost: ¥390,000) Fiscal Year 2015: ¥2,080,000 (Direct Cost: ¥1,600,000、Indirect Cost: ¥480,000)
Keywords	菌体外分泌系 / 転写活性化因子 / 根粒共生 / 転写因子 / 菌体外分泌装 / 広域宿主ベクター / ゲノム編集 / 菌体外分泌装置
Outline of Final Research Achievements	The Type III Secretion System (T3SS) was found as a toxin-injection system from pathogenic bacteria to mammalian cell. Genes for T3SS are identified not only in animal and plant pathogens but also in symbiotic bacteria such as rhizobia. Here, we have studied Mesorhizobial T3SS, which shows limited homology even to T3SSs in plant pathogens. We have investigated target genes of the T3SS transcriptional activator protein, TtsI, and found a few new binding sites. We also constructed reporter systems to access the secretion activity as well as transcriptional activation and utilized the systems to evaluate the necessity for secretion activity of the relatively small genes that situate around the main T3SS gene cluster. Simultaneously, we constructed new broad-host range vector-sets that are comprised of controllable promoter plasmid and temperature sensitive replication.
Academic Significance and Societal Importance of the Research Achievements	根粒菌の３型分泌系では、分泌装置の主要構成物（内膜リング構成物）においては動植物の病原菌の３型分泌系と類似性を示すものの、動物病原菌の系で知られている分泌シャペロンの相同物は見つかっておらず、分泌機能の発現や制御については知見が限られていた。本研究では、ミヤコグサ根粒菌を用いて３型分泌系の転写活性化因子であるTtsIの標的配列の検出と菌体外分泌活性を総合して、分泌活性に必須ではないもののTtsIにより発現する遺伝子の制御配列を同定した。これらに加えて、根粒菌以外のグラム陰性細菌でも利用可能な新しい発現プラスミド群を構築したことは、関連分野の進展に寄与するものと信ずる。