RssA-RssB訊息傳遞控制Serratia marcescens早期多細胞發育

Abstract

單細胞的原核生物之多細胞行為，係指細菌可透過化學分子來感應其群體密度進而調控基因表現及特定行為甚至進行細胞分化，以強化其生存優勢。近年來科學界已普遍認為多數細菌具有不同的多細胞行為；多細胞行為不僅增強細菌存活率、提高病源菌的致病性、也對共生菌與宿主的互動有莫大的影響，其背後的調控機制也被廣泛地深入研究中。實驗室的研究重心之一著重於腸內菌Serratia marcescens在洋菜基表面移動的多細胞行為其背後的分子機制。 Swarming為細菌重要多細胞行為，已被研究數十年，但許多重要的調控機制仍未明朗。目前已知的調控機制包括：鞭毛系統、quorum sensing、chemotaxis等。在數年前，實驗室為進一步了解其中機制，以transposon mutagenesis大規模篩選具有過度移行能力的菌株，其中篩選出來的基因產物分布從細胞膜到細胞質內，功能亦是多樣化，構成一條可能的訊息傳遞途徑，RssA (一個two-component system的sensor kinase)亦在其中。 Two-component system為細菌用以感應外界環境的機制之一，主要組成有兩個蛋白質，其中sensor位於細胞內膜上，受到刺激後則會自體磷酸化，接著磷酸化下游的response regulator，藉以調控下游基因表現或蛋白質活性。在實驗室之前的研究中已證明RssA能夠磷酸化其對應的response regulator RssB，並且藉由這樣的訊息傳遞調控swarming。此外，EMSA、RT-PCR及primer extension的結果指出磷酸化後的RssB能夠直接與自身的啟動子結合並進行負向調控。 在此論文中，我進一步探討了RssA-RssB的訊息傳遞調控swarming的分子機制，並且建立了簡單的方式去即時觀察RssA-RssB在不同環境中個別細胞的活化情形，同時以in vitro的方式釣取RssB可能調控的基因。此論文的實驗結果提供了一個新的分子模式解釋RssA-RssB訊息傳遞在細菌多細胞行為中扮演的角色。

Bacterial swarming is a cell-density dependent multicellular surface migration behavior comprising at least swarming lag and actively swarming phases. How the initiation of swarming is regulated remains unknown. Previously we had identified a Serratia marcescens RssA-RssB two-component system regulating swarming. Herein we address the RssA-RssB signaling in swarming development. Activation of RssA-RssB signaling prohibits swarming and once signaling is deactivated, swarming is initiated. RssA-RssB signaling results in phosphorylation of RssB (RssB~P), followed by dissociating itself from the cognate inner membrane sensor kinase RssA and moving into the cytoplasm. RssB~P binds to the -35 region of the rssB promoter, supporting auto-inhibition of RssA-RssB signaling. The downstream genes regulated by RssA-RssB were subsequently identified, including those involved in DNA modification, iron acquisition, sugar transportation, assembly of flagellum and two-component signal transduction. Expression of these genes is coordinately regulated during swarming under RssA-RssB control. We propose that RssA-RssB signaling determines the time when swarming will be initiated and acts as an important temporal regulator in early swarming development.