Pseudomonas syringae pv. tomato DC3000 PSPTO_3482 基因操作組之特性分析

Characterization of the PSPTO_3482 operon in Pseudomonas syringae pv. tomato DC3000

張雅文(Ya-Wen Chang)

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(2021年)

https://doi.org/10.6342/NTU202101677

摘要

革蘭氏陰性細菌可以利用第六型分泌系統 (type VI secretion system, T6SS) 將效應蛋白運送到真核或原核細胞中，參與在其致病力、養分獲取或細菌間的競爭之中。實驗室之前的研究中發現，模式植物病原細菌 Pseudomonas syringae pv. tomato (Pst) DC3000具有 HSI-I和 HSI-II 兩套第六型分泌系統基因叢集 (gene cluster)；此外，為了找到由特定 VgrG 攜帶的效應蛋白，搜尋到基因體序列中帶有七個 vgrG 同源基因，且從 vgrG3 啟動子的高表現量推測其可能在Pst DC3000的生理活性上具有功能。因此本研究便從第六型分泌系統的角度切入，針對 VgrG3 操作組 (operon) 上的基因進行深入的探討，以了解 VgrG3 對Pst DC3000的重要性。透過生物資訊學分析，VgrG3 座落於一由PSPTO_3482-3485四個基因所組成的操作組之中，除了 PSPTO_3482的基因產物被預測為VgrG 之外，其下游的 PSPTO_3483產物為帶有DUF4123結構域的蛋白質，PSPTO_3485推測為具有 lipase 活性的效應蛋白，而 PSPTO_3484 無特殊結構域，但根據其位於 vgrG 操作組中且位於可能為效應蛋白的 PSPTO_3485 上游，故推測其可能為 PSPTO_3485 之免疫蛋白。利用半定量反轉錄聚合酶連鎖反應確定了各基因在不同醣類培養基中培養時均會表現但表現量會有不同。透過競爭試驗發現，PSPTO_3485突變株抑制 P. savastanoi pv. phaseolicola (Psph) 1448a 及 Escherichia coli MG1655 生長的能力下降，且此現象為 contact-dependent；而缺失PSPTO_3482、PSPTO_3483 與 PSPTO_3484也同樣會影響Pst DC3000對此二菌的競爭現象。將PSPTO_3485表達在Psph 1448a 及 E. coli MG1655 細胞質中，並不會對細菌生長造成影響；但如果透過加上 signal peptide 使其表達於 E. coli BL21(DE3) 的周質時，會發現 E. coli 生長被抑制，但同時表達 PSPTO_3484和 PSPTO_3485 可以緩減 PSPTO_3485 對 E. coli BL21(DE3) 的毒性。此外，若在 Psph 1448a 中表現 PSPTO_3484 時，可以緩減 Pst DC3000 對其造成的生長抑制。本研究結果顯示 PSPTO_3485 在 Pst DC3000 與其他細菌的競爭作用上是重要的，且其作用位點應該是在目標細胞的周質中；PSPTO_3484 的出現則具有保護作用，推測其可能具有免疫蛋白的功能。但由於無法確認 PSPTO_3485 是否會分泌至胞外，故尚無法確認其為 Pst DC3000 第六型分泌系統的效應蛋白。

關鍵字

第六型分泌系統 ； 細菌間競爭 ； VgrG ； 效應蛋白 ； 免疫蛋白

並列摘要

The type VI secretion system (T6SS) is used by many Gram-negative bacteria as a molecular weapon to inject effector proteins into target prokaryotic or eukaryotic cells, and participates in virulence, nutrient acquisition or bacterial competition. Previous studies showed that the model phytopathogenic bacterium Pseudomonas syringae pv. tomato (Pst) DC3000 contains two T6SS clusters, namely HSI-I and HSI-II. In order to identify T6SS effectors in Pst DC3000, previous approach searching for cargo effectors linked to VgrG homologs revealed that there are seven vgrGs in Pst DC3000, among which vgrG3 (PSPTO_3482) exhibited the highest expression in a GUS activity assay, suggesting it might have T6SS-related functions. In this study, investigation of the role of VgrG3 in Pst DC3000 was carried out. Bioinformatics analysis uncovered that vgrG3 is located in a small operon, containing four genes, PSPTO_3482 to PSPTO_3485. In addition to PSPTO_3482, predicted to be VgrG, PSPTO_3483 is a domain of unknown function (DUF) 4123-containing protein, and PSPTO_3485 showed homology toa family of lipase effectors. Although no special domain or motif can be detected in PSPTO_3484, the fact that it localizes in the vgrG operon and upstream of a putative effector PSPTO_3485, suggests PSPTO_3484 is the corresponding immunity protein of PSPTO_3485. The expression of each gene under different carbon sources was determined using semi-quantitative RT-PCR. Deletion of PSPTO_3485 reduces the ability of Pst DC3000 to inhibit growth of P. savastanoi pv. phaseolicola (Psph) 1448a and Escherichia coli MG1655 growth in an interbacterial competition assay. Growth inhibition of Psph 1448a and E. coli MG1655 by PSPTO_3485 is a contact-dependent manner. Moreover, PSPTO_3482, PSPTO_3483 and PSPTO_3484 are also involved in such inhibitory effect. Expression of PSPTO_3485 in the cytoplasm of Psph 1448a and E. coli MG1655 does not affect bacterial growth. However, periplasmic PSPTO_3485 obtained by addition of PelB singal peptide at the N-terminus of PSPTO_3485 can inhibit growth of E. coli BL21(DE3), and this suppression can be alleviated by expression of PSPTO_3484 simuotaneously. Furthermore, PSPTO_3484 can protect Psph 1448a from attack by Pst DC3000. According to the results shown here, PSPTO_3485 participates in bacterial competition of Pst DC3000 and PSPTO_3484 can reduce the toxicity caused by PSPTO_3485. Unfortunately, secretion of PSPTO_3485 could not be detected, and thus determining whether PSPTO_3485 is a T6SS effector protein of Pst DC3000 can not be confirmed yet.

並列關鍵字

Type VI secretion system (T6SS) ； interbacterial competition ； VgrG ； effector ； immunity protein

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