Pseudomonas syringae pv. tomato DC3000 生長競爭之研究

The study of the growth competition of Pseudomonas syringae pv. tomato DC3000

劉正寅(Cheng-Ying Liu)

指導教授 ： 林乃君

國立臺灣大學/生物資源暨農學院/農業化學研究所/碩士(2012年)

https://doi.org/10.6342/NTU.2012.10779

摘要

細菌為了爭奪生長空間、營養源或寄主,會運用各種方法互相競爭。最近新 定義出來的第六型分泌系統 (Type VI secretion system,T6SS),被證實是許多動 物病原菌用來進行競爭的工具,例如:Pseudomonas aeruginosa PAO1 及 Vibrio cholerae V52 都可藉由 T6SS 分泌功能蛋白以殺死競爭者或抑制其生長。然而, 植物病原菌是否亦會利用 T6SS 來達到競爭優勢而存活下來,仍待進一步研究探 討。Pseudomonas syringae pv. tomato (Pst) DC3000 為模式植物性病原菌之一,其 基因體中具有兩套可能的 T6SS 基因叢集,分別為 HSI-I 及 HSI-II。目前測試過 的條件下,均僅能觀察到 HSI-II 的表現且分泌關鍵構造 (作用) 蛋白 Hcp2。本 研究證明 PstDC3000 能藉由 HSI-II 與其他植物病原細菌或腸道菌進行生長競爭, 且此競爭作用必須仰賴細菌間的接觸 (contact-dependent);另外我們也證明核心 基因 icmF2、clpV2 以及 vasH2 均會影響 HSI-II 的分泌與競爭作用。此外,在細 菌間的競爭作用中,PstDC3000 是以抑制競爭菌生長而使其菌數下降,與大多數 細菌具殺死目標菌的能力不同。在本研究中亦發現,全域調控因子 GacA 會正向 調控 hcp2 及 HSI-II 的表現,故其突變株會有競爭力下降的情形發生;然而,受 到 GacA 調控的群數感應基因 psyI 對 HSI-II 有影響,但 psyR 則沒有相同的調控 能力。另外我們發現,於 Sucrose minimal medium (SMM) 中生長的 PstDC3000, 會在 pH 值升高時降低 Hcp2 的分泌,同時競爭能力會下降。最後,利用隨機突 變的策略,我們初步發現數個 T6SS 相關基因以外、能夠影響 PstDC3000 生長競 爭的基因,包括 PTO2954、avrE1 及 cfa6。本研究為首次對植物病原菌 PstDC3000 細菌間競爭作用進行較為深入的研究,也對日後相關研究提供了一些基礎。

關鍵字

第六型分泌系統 ； 全域調控因子 ； 細菌間競爭能力

並列摘要

Bacteria often compete against other microbes for living spaces, resources, or host niches. The newly defined type VI secretion system (T6SS) has been confirmed to provide competition abilities to some animal pathogens such as Pseudomonas aeruginosa PAO1 and Vibrio cholerae V52, which may utilize T6SS to secrete effector proteins to either kill or inhibit their competitors in order to grow better. However, in many plant pathogens bearing T6SS gene clusters, whether they would gain advantages in competition by expressing T6SS further investigation. Pseudomonas syringae pv. tomato (Pst) DC3000 is a plant pathogen, it has two putative T6SS gene clusters, named HSI-I and HSI-II. Under the lab conditions been tested, we can only detect the expression of HSI-II and secretion of the key structural effector protein Hcp2. Here we report that PstDC3000 can grow better in a mixed culture with other phytopathogenic bacteria and enterobacteria by expressing HSI-II in a contact-dependent manner. We also confirmed the core components icmF2, clpV2 and vasH2 could affect HSI-II-dependent secretion and interbacterial competition ability. Furthermore, we found that PstDC3000 uses T6SS to suppress the growth of their competitors instead of killing them, different from the mechanism used by some animal pathogens. In this study, we observed that global regulator GacA could positively controls the expression of hcp2 and HSI-II and also interbacterial competition abilities. Instead of GacA, the downstream regulator PsyI could only affect the expression of HSI-II but not competition, whereas the other regulator, PsyR, didn’t have any effect on both promoters. In addition, we found that when growing in sucrose minimal medium (SMM), Hcp2 secretion in PstDC3000 was lower at higher pH, leading to decreased ability in competition. By means of random mutagenesis, we identified several genes, including PTO2954, avrE1, cfa6, PTO1851/1850 and vasH2, whose mutation would cause decreased ability in interbacterial competition of PstDC3000. This is the first report to investigate the competition of PstDC3000, which could provide fundamental knowledge for the relevant researches afterwards.

並列關鍵字

type VI secretion system ； global regulator ； interbacterial competition ability

參考文獻

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