植物病原細菌 Pseudomonas syringae pv. tomato DC3000 中 MvaT 同源蛋白 PSPTO_3103 之特性分析

Characterization of PSPTO_3103, an MvaT-like protein, in phytopathogen Pseudomonas syringae pv. tomato DC3000

宋立綱(Li Kang Sung)

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(2018年)

https://doi.org/10.6342/NTU201802190

摘要

水平基因轉移 (Horizontal gene transfer, HGT) 係指遺傳物質除了從親代傳至子代之外，於生物間移動的現象，為演化動力之一，可使生物獲取新性狀，以適應環境。然而，若未經適當調控，而一味表現這些外來基因，會給予宿主壓力，並可能導致自身適性下降。而為了因應外源基因可能造成的負面效應，細菌演化出一特別之轉錄抑制子，稱為外源基因沉默子 (xenogeneic silencer)，可結合上潛在之外源基因序列，以調控其表現。此外源基因沉默子可按其序列分成三大類：分別是 Proteobacteria、Mycobacteria 及 Pseudomonas 屬所特有的轉錄抑制子 H-NS、Lsr2 及 MvaT。番茄細菌性斑點病菌 Pseudomonas syringae pv. tomato (Pst) DC3000 是一桿狀、具鞭毛的革蘭氏陰性菌，可在自然環境下感染番茄，其致病力依賴細菌第三型分泌系統，而與其他細菌競爭時則有第六型分泌系統參與其中。第三型分泌系統組成蛋白的基因群座落於一典型、由三部分所組成的病原性小島 (pathogenicity island) 之 hrp/hrc 基因叢集中；HSI-I 和 HSI-II 基因叢集則負責編碼第六型分泌系統的組成蛋白，於該二分泌系統之基因叢集內均可發現轉位酶 (transposase) 的基因，暗示兩叢集有可能是經由水平基因轉移獲得的外來序列。本研究在 Pst DC3000 中找到 PSPTO_0281、PSPTO_3103、PSPTO_4315 和 PSPTO_4755 四個 MvaT 之同源基因，經突變株建構與細菌間競爭試驗發現，PSPTO_3103 會負向調控 Pst DC3000 之細菌間競爭能力。後經由半定量反轉錄聚合酶連鎖反應、GUS 活性測試及西方墨點法，發現 PSPTO_3103 可抑制第六型分泌系統基因叢集及第三型分泌系統相關基因之表現，並影響胞內 Hcp2 之生產。此外，PSPTO_3103 突變株的致病力較野生株更高，說明其對於致病力的影響。透過凝膠遷移試驗，亦證明 PSPTO_3103 可與第六型基因叢集中三個操作子、hrpL及 avrPto 之啟動子結合。總而言之， PSPTO_3103 應為 Pst DC3000 中重要之 MvaT 同源蛋白，負責調控其可能為外來序列之細菌第三型及第六型分泌系統基因表現，進而影響 Pst DC3000對番茄的致病性，以及和其他細菌間的競爭能力。

關鍵字

Pseudomonas syringae ； 第三型分泌系統 ； 第六型分泌系統 ； MvaT-like protein ； 外源基因沉默子

並列摘要

Horizontal gene transfer (HGT) is a movement of genetic materials between organisms except transmission of DNA from parents to offspring, and could serve as a driving force for evolution. HGT is thought to be used to acquire new phenotypes for bacteria to adapt to the changing environments. However, inappropriate regulation of foreign genes obtained by HGT may lead to reduction of bacterial fitness. To avoid potential problems caused by foreign DNA, bacteria have evolved the so-called “xenogeneic silencers” to regulate the expressions of those foreign genes. The xenogeneic silencers can be divided into three groups based on their sequences, which are histone-like nucleoid structuring (H-NS) protein in Proteobacteria, Lsr2 in mycobacteria, and MvaT-like protein in Pseudomonas spp. Pseudomonas syringae pv. tomato (Pst) DC3000 is a rod-shaped, Gram-negative bacterium with polar flagella, and a phytopathogen that can cause tomato speck disease. In Pst DC3000, ability to infect host plants requires its type III secretion system (T3SS) while competing with other bacteria depends on full functions of type VI secretion system (T6SS). T3SS is encoded by hrp (hypersensitive response and pathogenicity) and hrc (hrp and conserved) genes within the canonical tripartite pathogenicity islands (T-PAIs), and core components of T6SS are encoded by genes in Hcp secretion island (HSI)-I and HSI-II gene clusters. It is noteworthy that genes for transposases are within or flanked the gene clusters of hrp/hrc, HSI-I, and HSI-II, suggesting that they might be acquired by HGT, and could be putative targets of xenogeneic silencers. In this study, four MvaT orthologs in Pst DC3000, namely PSPTO_0281, PSPTO_3103, PSPTO_4315, and PSPTO_4755, were identified. By means of mutagenesis and an interbacterial competition assay, we revealed that PSPTO_3103 negatively controls the competition ability of Pst DC3000. Results from semiquantitative RT-PCR, the GUS promoter assay and Western blotting analysis demonstrated that PSPTO_3103 represses the expressions of HSI-II gene clusters, hrpL and avrPto as well as Hcp2 production. Deletion of PSPTO_3103 also increases the virulence of Pst DC3000. Furthermore, the gel shift assay indicated that PSPTO_3103 can bind to the promoter regions of operons in HSI-II gene cluster, hrpL and avrPto in a non-specific manner. Taking together, we conclude that PSPTO_3103 is the key MvaT ortholog responsible for modulating the expressions of putative foreign sequences, i.e. T3SS and T6SS-related genes, in Pst DC3000, further controlling its virulence and interbacterial competition ability.

並列關鍵字

Pseudomonas syringae ； type III secretion system (T3SS) ； type VI secretion system (T6SS) ； MvaT-like protein ； xenogeneic silencer

參考文獻

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