Pseudomonas syringae pv. tomato DC3000 第六型分泌系統基因調控之初步探討

Abstract

革蘭氏陰性菌具雙層膜，因而發展出多種分泌系統，其中第一型到第五型已被廣泛研究，第六型分泌系統 (Type VI secretion system, T6SS) 則是近年來才被確認之新型分泌系統，已被證實和病原菌的致病性有關。Pseudomonas syringae pv. tomato DC3000 (PstDC3000) 是一種番茄病原菌，具有兩套T6SS基因叢集 (gene clusters)，其內除了有分泌系統組裝蛋白的基因之外，還包含有具調控T6SS分泌蛋白Hcp功能的sigma 54轉錄活化因子的基因，在其基因體中的編碼分別為PSPTO_2549及PSPTO_5424。本研究透過該二基因突變株，利用GUS報導基因系統來探討其對第六型分泌系統相關基因表現之影響，結果發現PSPTO_5424對於hcp-2的調控扮演極為重要的角色，而PSPTO_2549可能會幫助PSPTO_5424調控hcp-2。此外，利用GUS報導基因系統也找到了在hcp-2上游240bp到360bp之間，具有調控hcp-2表現的重要序列。與第三型分泌系統不同的是，第六型分泌系統在 King’s B 培養基中，只要菌數夠多即可大量表現，不必完全以基本培養基配合 fructose或其他醣類才能誘導其表現。這或許可以解釋為何之前的結果顯示第六型分泌系統不若第三型分泌系統，在 PstDC3000於植物的病原性上具有極重要的功能。

Due to the complexity of the double membrane structure, Gram-negative bacteria develop a variety of secretion systems, including Type I to Type V secretion system (T1SS to T5SS ) to secret proteins into the surrounding environments. In 2006, a novel secretion system, termed Type VI secretion system (T6SS), was recognized as another important virulence factor of the bacterial pathogen. In tomato pathogen, Pseudomonas syringae pv. tomato DC3000 (PstDC3000), two T6SS gene clusters in addition to its secretion components genes, hcp1 and hcp2, was identified using in silico analysis. Each cluster contains one copy of vasH homolog (PSPTO_2549 and PSPTO_5424), the gene coding for the sigma 54-dependent transcriptional activator, which controls the expression of T6SS-secreted protein, Hcp. By means of PSPTO_2549 and PSPTO_5424 mutants in accompany with a GUS reporter system, we showed that PSPTO_5424 plays a very important role in regulation of hcp-2 expression and PSPTO_2549 may cooperates with PSPTO_5424 to control the hcp-2 expression. Moreover, we also found that 240bp to 360bp upstream of the hcp-2 ORF is required for hcp-2 expression. Unlike type III secretion systems, genes in the second T6SS gene cluster or hcp-2 were expressed when PstDC3000 were grown in King’s B medium in a cell density-dependent manner, and expression of these genes is not completely induced by sugars as that of type III effector genes. The different expression patterns between T3SS and T6SS may explain why T6SS is not as important as T3SS in the virulence activity of PstDC3000 in planta.