RssA-RssB Signaling Controls Early Development of Serratia marcescens Muticellularity

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。warming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。 wo-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。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.中文摘要 ibstract iiontents iiiigure contents viable contents viiihapter 1 Introduction 1hapter 2 Materials and methods 4.1 Bacterial strains 4.2 Plasmids 4.3 Primers 5.4 Enzymes and chemicals 7.5 Isolation of plasmid DNA 7.6 Preparation of bacterial chromosomal DNA 8.7 Extraction of DNA from agarose gels 9.8 Restriction enzyme digestion 9.9 Ligation reaction 10.10 Calcium chloride Transformation 10.11 Eletroporation 11.12 Blue-white screening for recombinant plasmids 12.13 Colony-PCR screening for recombinant plasmids 13.14 DNA sequencing 13.15 Plasmid transfer from E. coli S17-1 to S. marcescens by conjugation 15.16 Southern blot analysis 15.17 DIG-detection assay 16.18 Swarming and swimming motility assays 17.19 Primer extension and DNase I footprinting 17.20 Plasmids construction 18.21 Measurement of transcriptional fusion EGFP fluorescence 21.22 Modified chromatin immunoprecipitation assay 22.23 Protein expression 23.24 Protein electrophoresis 24.25 Protein purification 24.26 Western blot analysis 26.27 In vitro protein-protein pull-down assay 27.28 Fluorescent microscopy 27.29 In vitro protein-DNA pull-down assay 28.30 RNA extraction 29.31 Reverse transcription-PCR (RT-PCR) assay 29hapter 3 Results 31.1 RssA-RssB controls the duration of swarming lag period 31.2 RssB~P binds two rssB promoter regions 32.3 Activation of RssA-RssB signaling upon entering stationary phase in LB broth culture 34.4 RssB phosphorylation affects interaction between RssB and RssA 36.5 RssA-RssB signaling controls dynamic localization of EGFP-RssB 38.6 Activation of RssA-RssB signaling at swarming lag phase 40.7 RssB~P downstream target genes and their expression during swarming 42hapter 4 Concluding Discussion 47eference 54ppendix Itandard buffers, solutions 58ppendix IItandard buffers, solutions 61igure contentsig. 3-1 Construction of S. marcescens CH-1 ΔrssBA strain. 31ig. 3-2 RssA-RssB two-component system regulates swarming motility independent of growth and swimming. 32ig. 3-3 DNase I footprinting analysis of RssB~P binding site on the promoter of rssB. 33ig. 3-4 RssB~P binds and negatively regulates its own promoter upon entering stationary phase. 35ig. 3-5 Interaction between RssB and cRssA. 37ig. 3-6 Complementation of ΔrssBA precocious swarming behavior by pEGFP-RssBA::Sm. 38ig. 3-7 Directly image the localization of EGFP-RssB in LB broth culture. 39ig. 3-8 Directly image the localization of EGFP-RssB during swarming development. 41ig. 3-9 Schematic experimental design to identify promoters regulated by RssB~P. 43ig. 3-10 Experimental result of GST-RssB~P target DNA searching assay. 43ig. 3-11 The mRNA levels of genes regulated by RssA-RssB as measured by RT-PCR. 44ig. 3-12 The mRNA levels of genes regulated by RssA-RssB during swarming development as measured by RT-PCR. 45ig. 4-1 Schematic representation of the genomic organization of the target genes whose promoter regions is bound by RssB~P and regulated by RssA-RssB. 49ig. 4-2 Proposed mechanism on how RssA-RssB regulates multicellularity in S. marcescens. 53able contentsable 2-1 T Table 2-1 The bacteria strains used in this thesis 4able 2-2 The plasmids used in this thesis. 4able 2-3 The primers used in this thesis. 5able 3-1 S. marcescens genes regulated by RssA-RssB two-component system during swarming. 4