番茄細菌性斑點病菌 PSPTO_3283 之特性分析

Characterization of PSPTO_3283 in Pseudomonas syringae pv. tomato DC3000

呂亞容(Ya-Jung Lu)

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/NTU201803711

摘要

細菌會使用各種方式與周圍的環境進行交互作用，分泌系統便是其中一種重要的機制。透過分泌系統分泌出效應蛋白 (effector)，細菌得以自環境中獲得養分、克服非生物逆境及與真核或原核細胞作用，以利自身生存。迄今為止已發現九種分泌系統，其中，在 2006 年於 Vibrio cholera 及 Pseudomonas aeruginosa 中被定義出的第六型分泌系統 (Type VI secretion system, T6SS) 被認為在病原菌的致病力和細菌間競爭能力上扮演重要的角色。第六型分泌系統主要是由十三個核心成分所組成，而其中 Hcp 和 VgrG 負責組成胞外類似噬菌體尾絲的外管。番茄細菌性斑點病菌 Pseudomonas syringae pv. tomato (Pst) DC3000，是研究分子植物病理學的重要模式生物，先前的研究發現，其具有兩套 T6SS 基因叢集，分別為 HSI-I 和 HSI-II，但僅偵測過後者的表現情形。以液相層析串聯式質譜儀 (LC/MS/MS) 分析 Pst DC3000 野生株、ΔtssM2 和 ΔclpV2 突變株之分泌蛋白質體 (secretome)，找到了可能是經由 T6SS 分泌的效應蛋白。初篩後得到 PSPTO_3283 突變株對大腸桿菌及同種菌株 P. savastanoi pv. phaseolicola (Psph) 1448a的競爭能力較野生株低，且在 E. coli BL21 (DE3) 中異位表達 PSPTO_3283 也會降低大腸桿菌的生長。Phyre2 預測 PSPTO_3283 屬於 GntR 家族轉錄因子中的 FadR 同源蛋白，由於大腸桿菌的 FadR 與脂肪酸代謝調控有關，可讓大腸桿菌利用長鏈脂肪酸。然而在含有油酸的培養基上，表現 PSPTO_3283 的菌株代謝油酸的能力下降外，生長也有受到抑制的情形。綜合以上所述，Pst DC3000 中可能存在一種新的 T6SS 效應蛋白，能夠藉由調控競爭者脂肪酸代謝與合成之平衡，來影響其生長。

關鍵字

第六型分泌系統 ； 番茄細菌性斑點病菌 ； 第六型分泌系統效應蛋白 ； GntR 家族 ； FadR 轉錄因子

並列摘要

Bacterial cells use various strategies to communicate with their surrounding environments, one of which is secretion system. Using secretion systems, bacterial cells can secret effectors to obtain nutrient, overcome abiotic stress and interact with either eukaryotic or prokaryotic cells. Nine types of secretion systems have been found thus far, and type VI secretion system (T6SS) was first identified in Vibrio cholerae and in Pseudomonas aeruginosa in 2006. T6SS utilizes a one-step mechanism analogous bacteriophages to translocate effectors into eukaryotic and prokaryotic cells, and is involved in pathogenesis and interbacterial competition ability. It is composed of 13 core components, two of which, Hcp and VgrG, are assumed to be extracellular components of the secretion machinery related to bacteriophage tail structure. Pseudomonas syringae pv. tomato (Pst) DC3000 causes bacterial speck disease on tomato, and represents an important model organism for studying molecular plant pathology. Two T6SS gene clusters, HSI-I and HSI-II were found in the genome of Pst DC3000, but only the expression of HSI-II can be detected. In a previous study, T6SS-dependent secretome was analyzed using Pst DC3000 wild type and two T6SS mutants, ΔtssM2 and ΔclpV2 and LC-MS-MS. Among those putative T6SS effectors, PSPTO_3283,which is a transcription factor of GntR family, is of interest. This effector is involved in interbacterial competition with E. coli and P. savastanoi . pv. phaseolicola 1448a. Furthermore, ectopic expression of PSPTO_3283 in E. coli reduced growth of E. coli. Analyzed by Phyre2, PSPTO_3283 was predicted to be homologous to FadR, which is involved in regulation of fatty acid metabolism, allowing E. coli to utilize long-chain fatty acid. However, expression of PSPTO_3283 reduced E. coli ability to utilize oleate, a long-chain fatty acid, as well as bacterial growth. Taking together, these data suggest a novel type of T6SS effector present in Pst DC3000, which can inhibit competitor growth via interfere homeostasis of fatty acid metabolism.

並列關鍵字

Type VI secretion system ； Pseudomonas syringae pv. tomato DC3000 ； Type VI effector ； GntR family ； FadR transcription factor

參考文獻

Abdallah A. M., Gey van Pittius N. C, Champion P. A, Cox J., Luirink J., Vandenbroucke-Grauls C. M., Appelmelk B. J., Bitter W. (2007). Type VII secretion--mycobacteria show the way. Nat Rev Microbiol. Nov; 5(11), 883-91.

Aschtgen, M. S., Gavioli, M., Dessen, A., Lloubes, R., and Cascales, E. (2010). The SciZ protein anchors the enteroaggregative Escherichia coli type VI secretion system to the cell wall. Mol. Microbiol. 75, 886-899.

Alcoforado Diniz, J., Liu, Y. C., and Coulthurst, S. J. (2015). Molecular weaponry: diverse effectors delivered by the Type VI secretion system. Cell. Microbiol. 17, 1742–1751

Alcoforado D. J., and Coulthurst, S. J. (2015). Intraspecies competition in Serratia marcescens is mediated by type VI-secreted Rhs effectors and a conserved effector-associated accessory protein. J. Bacteriol. 197, 2350-2360.

Armshaw P, Pembroke, T, J. (2013). Control of expression of the ICE R391 encoded