Pseudomonas kribbensis XP1-6 第六型分泌系統效應蛋白特性之初探

Abstract

Pseudomonas kribbensis XP1-6 為一株從番茄根部分離出的促進植物生長根棲細菌，其基因體中有兩套第六型分泌系統基因叢集，分別為 X1-T6SS 與 X2-T6SS，而 X2-T6SS 基因叢集參與 P. kribbensis XP1-6 與 Escherichia coli MG1655 與 Pseudomonas sp. TY108 之間的競爭能力上。為了更加了解 P. kribbensis XP1-6第六型分泌系統的作用機制，本研究進一步探討第六型分泌系統在 P. kribbensis XP1-6 各生長階段之表現情形，並透過生物資訊學及分泌體學的方式找尋第六型分泌系統的效應蛋白。搜尋 P. kribbensis XP1-6的基因體時，在兩套第六型分泌系統基因叢集的序列之外，還可找到 10 個包含 hcp 及/或 vgrG 同源基因的操作子，並推論出位於 X2-T6SS 基因叢集及這 10 個操作子內可能為第六型分泌系統效應蛋白的基因。利用半定量反轉錄聚合酶鏈鎖反應得知分別位於 vgrG1_3/hcpA1 操作子, vgrG1_4/hcpA2 操作子, vgrG1_5/hcpA3 操作子, vgrG1_6/hcpA4 操作子及 vgrG1_10/hcpA5 操作子的 PK_0660、PK_1102、PK_2134 (rhsC_4)、PK_2458 (rhsC_5) 及 PK_3762 (rhsC_7) 在營養充足的環境下有較高的表現量。後續發現刪除 tssM2 會降低 P. kribbensis XP1-6 毒殺 P. syringae pv. tomato (Pst) DC3000 以及抑制 Ralstonia solanacearum Pss4 生長的能力，顯示 X2-T6SS 基因叢集也會參與 P. kribbensis XP1-6 與番茄病原菌的競爭關係中。接著挑選其中表現量較高的 PK_1102 進行驗證，結果顯示其會參與毒殺 Pst DC3000 以及抑制 R. solanacearum Pss4 的生長。除此之外，蛋白質體學分析出會透過 X2-T6SS 分泌之蛋白質，除了 X2-T6SS 中的 PK_5712 及 VgrG1_11 外，還有推測的效應蛋白 RhsC_4、RhsC_5、RhsC_7。VgrG1_5、VgrG1_10 及 HcpA5 也會透過 X2-T6SS 分泌，而因為 HcpA2、HcpA3 及 HcpA4 具有相同的胺基酸序列，難以利用質譜分析得知分泌至胞外的 究竟是 HcpA2、HcpA3 或 HcpA4。未來可持續研究 vgrG1_5/hcpA3、vgrG1_10/hcpA5 操作子與 X2-T6SS 基因叢集之間的關係，以及 RhsC_4、RhsC_7、PK_5712 是否參與 X2-T6SS 所主導的抗菌能力，以對 P. kribbensis XP1-6 第六型分泌系統的作用機制及功能有更多了解。

Pseudomonas kribbensis XP1-6 is a plant growth-promoting rhizobacterium (PGPR) isolated from tomato roots. It possesses two type VI secretion system (T6SS) gene clusters, namely X1-T6SS and X2-T6SS, and X2-T6SS gene cluster contributes to its interbacterial competition ability with Escherichia coli MG1655 and Pseudomonas sp. TY108. In order to understand more about the mechanism of T6SS in P. kribbensis XP1-6, this study aimed to further investigate the gene expression pattern of X2-T6SS during vegetative growth and identify T6SS effector using bioinformatic and secretome approaches. In addition to two T6SS clusters, there are 10 operons containing hcp and/or vgrG homologous genes in the P. kribbensis XP1-6 genome, and putative T6SS effectors were identified within X2-T6SS gene cluster and these 10 operons in this study. Among them, PK_0660, PK_1102, PK_2134 (rhsC_4), PK_2458 (rhsC_5) and PK_3762 (rhsC_7) located in vgrG1_3/hcpA1 operon, vgrG1_4/hcpA2 operon, vgrG1_5/hcpA3 operon, vgrG1_6/hcpA4 operon and vgrG1_10/hcpA5 operon, respectively, exhibited higher expression levels under nutrient-rich conditions using semi-quantitative RT-PCR. Moreover, deletion of tssM2 reduced the ability of P. kribbensis XP1-6 to kill P. syringae pv. tomato (Pst) DC3000 and inhibit the growth of Ralstonia solanacearum Pss4, indicating that X2-T6SS was also involved in the competiton of P. kribbensis XP1-6 against tomato pathogens. PK_1102, which expresses at a higher level, was selected to verify the results of bioinformatic analysis, and the data indicated that it participates in the killing of Pst DC3000 and the inhibition of R. solanacearum Pss4. Furthermore, Hcps, VgrGs, and predicted effectors secreted by X2-T6SS were identified from proteomics analysis. PK_5712 and VgrG1_11 in X2-T6SS, along with the predicted effectors RhsC_4, RhsC_5, and RhsC_7, were found to be translocated by X2-T6SS. VgrG1_5, VgrG1_10, and HcpA5 were also secreted by X2-T6SS. However, due to identical amino acid sequences of HcpA2, HcpA3, and HcpA4, it was difficult to differentiate whether the secreted Hcps were HcpA2, HcpA3, or HcpA4. Further research can focus on the relationship between vgrG1_5/hcpA3, vgrG1_10/hcpA5 operons and X2-T6SS gene cluster, as well as the role of RhsC_4, RhsC_7, and PK_5712 in X2-T6SS-mediated interbacterial competition to increase our understanding of the mechanisms and functions of T6SS in P. kribbensis XP1-6.