Serratia marcescens多細胞表面移行行為之調控: 二元系統RssA-RssB與跳躍子TnTIR之角色

Abstract

本篇論文主要是以分生及生化的方法，分別探討Serratia marcescens在洋菜基表面的多細胞移行行為之分子機制，包括在菌株CH-1的flagella-dependent swarming 現象與菌株SS-1的flagella-independent sliding 現象。Swarming為細菌重要多細胞行為，本實驗室為進一步了解其中機制，以transposon mutagenesis篩選負向調控因子，本論文的一部分即在證實其中RssA-RssB這套二元系統(two-component system)的生化功能。純化出的RssA蛋白質，證實可以自體磷酸化並磷酸化其對應之RssB蛋白質，並透過RssB與DNA結合的能力，調控下游基因表現。 Serratia marcescens SS-1擁有一套quorum sensing訊息調控機制---SpnIR系統。透過SpnI所產生的訊息分子---AHL，和其所對應的調控蛋白SpnR， S. marcescens的sliding，毒素分子及許多次級代謝物都被微妙地調控。此基因組位於一個Tn3 family的transposon中，並仍具有在不同的質體與染色體間傳遞的活性，此結果為quorum-sensing基因組位於transposon中的首例。而SpnR也證實會負向影響此transposon的跳躍頻率。其上的SpnT被大量表現時，S. marcescens SS-1的sliding與色素分泌都會被抑制，但實驗結果推測SpnT並不透過與SpnIR系統之互動調控上述表現型。

The main focus of this thesis is to explore underlying mechanism of multicellular surface translocation behavior in Serratia marcescens, including flagella-dependent swarming behavior in strain CH-1 and flagella-independent sliding behavior in strain SS-1. In our lab, we had screened several potential negatively regulators involved in the swarming behaviour through transposon mutagenesis. In part of my thesis, I had proved the biochemical function of a two-component pair—RssA and RssB. In vitro assay confirmed the auto-phosphorylation activity of RssA and its phospho-relay activity to RssB. After phosphorylated, RssB was proved to bind target DNA. The SpnIR quorum sensing system in Serratia marcescens strain SS-1 regulates flagellum-independent multi-cellular surface migration (sliding motility) and the production of nuclease, biosurfactant, and the red pigment, prodigiosin. spnTIR was found located on a Tn3 family transposon, TnTIR. TnTIR has been proved to transpose between plasmids and chromosomes. SpnIR functions in the new host and negatively regulates the TnTIR transposition frequency. It’s the first report of a quorum sensing system located on a transposon and do great contribution reveal the horizontal transfer and evolutionary mechanism of quorum-sensing genes and alter the way we perceive regulation of bacterial multicellular behaviour. SpnT may function as a negative regulator of surface-dependent migration and secondary metabolite production independent of SpnIR, and possibly through interfering with DNA replication and subsequently cell division.