臺灣博碩士論文加值系統

第六型蛋白質分泌系統廣泛存在於變形菌門（Proteobacteria），參與細菌與細菌間、或細菌與真核宿主之間的交互作用。Azorhizobium caulinodans ORS571是一株屬於α-變形菌綱 (α-proteobacteria) 的根瘤菌，根據序列比對分析結果，推測其基因體上應有一組包含impA~impL 基因群的第六型分泌系統基因座。然而初期實驗並未能偵測到分泌指標蛋白 Hcp (hemolysin-coregulated protein) 的外泌活性。為了探討第六型分泌系統在 A. caulinodans ORS571 的生物功能及調控機制，本研究使用菌落轉移免疫檢測法要從轉位子突變株庫中篩檢出具有Hcp外泌活性的突變株。突變株庫的建構是利用接合作用將帶有 mini-Tn5 轉位子的質體送入 ORS571 細胞，再藉由不可逆的 Tn5 轉位作用造成隨機插入(基因體) 突變。結果顯示若將接合後的菌株立即保存在低溫狀態（3~11℃）2天~ 4週，則突變效率可提升3~20倍。由於突變效率提升，在適當稀釋倍率下即可得到高密度、發育完全的突變株庫，便於直接將菌落轉移至硝化纖維膜，再經室溫28oC過夜培養後促使外泌物質累積，利於免疫檢測。為減少因菌落附著造成的背景訊號干擾，在進行免疫檢測之前，施以適量的過碘酸鈉 (NaIO4) 分散劑並輔以渦流震盪可有效降低偵測極限至約6.3 ng的Hcp蛋白。本研究共篩檢了大約68,000突變株，從中獲得5株標的菌株： 4株為Hcp 外泌突變株，1 株為Hcp高表現量突變株。根據表型觀察及遺傳分析的結果，推測外泌突變株 Tn-A3的 Hcp 釋出表型及細胞存活率迅速衰退現象可能導因於噬菌體相關基因座 Azc_2137-2139 的過量表現。外泌突變株Tn-A2與Tn-h91 則因丙氨酸消旋酶 Azc_2065 的羧端結構被破壞，經由imp以外的途徑分泌/釋出Hcp，並呈現異常低落的細胞生長活性。而外泌突變株Tn-A4 的插入突變位於假定基因 Azc_0044，暫時不予探討。另一方面，Hcp高表現量菌株 Tn-A1為高黏附性、磷酸恆定相關 (Azc_4037) 突變株，而磷酸恆定是否參與 T6SS調控仍待驗證。根據此趨近飽和的隨機突變株篩選結果，推測A. caulinodans ORS571可能具有別於既知第六型分泌系統的Hcp 蛋白分泌/釋出途徑，而此機制尚有待闡明。

Type VI secretion system (T6SS) is molecular machine that is widespread among phylum Proteobacteria to mediate interactions with neighboring eukaryotic and prokaryotic cells. The legume symbiont, Azorhizobium caulinodans ORS571 (α-proteobacteria), possess one deduced T6SS cluster harboring impA~impL genes on the genome. However, the T6SS hallmark protein, Hcp (hemolysin-coregulated protein) is not detectable in the extracellular matrix of ORS571 under current culture conditions. To facilitate the study of the role and regulatory pathway of T6SS in ORS571 life cycle, I screened for T6SS-active mutants from a transposon mutant library using colony lift immunoassay. The genome-wide mutant library was constructed by conjugative transfer of mini-Tn5 vector (pFAJ1819) into ORS571 cells followed by irreversible random Tn5 insertions into the genome. Efficiency of the transposon-based mutagenesis was increased 3~20 fold by refrigerating (3~11oC) the post-conjugation suspension for 2 days ~ 4 weeks before spreading on antibiotic selection plates. The improved mutation efficiency enabled colony development at high density, and these mutant colonies were directly transferred onto membrane and incubated overnight for accumulation of secreted Hcp protein. Background noise caused by adherent colonies was effectively reduced by using the dispersing chemical NaIO4 followed by a thorough vortex step. Accordingly, the detection limit was improved down to 6.3 ng Hcp protein. Approximately 68,000 mutants were analyzed, and 4 Hcp-secreting mutants and 1 Hcp-overexpression mutant were identified. Among the 4 Hcp-secreting mutants, strain Tn-A3 displayed decreased viability and imp-independent Hcp release caused by overexpression of phage-related loci (Azc_2137-2139); strain Tn-A2 and Tn-h91 showed compromised vitality and imp-independent Hcp secretion/release caused by C-terminal disruption of alanine racemase (alr, Azc_2065); strain Tn-A4 was disrupted in hypothetical gene Azc_0044, so it was pending further investigation. On the other hand, Hcp-overexpression strain Tn-A1 was unexpectedly identified by its phenotype of hyper-adherence presumably due to phosphate dyshomeostasis (pstS, Azc_4037). Further genetic verifications are required to assert the regulatory role of phosphate homeostasis on T6SS. Overall, the near-saturation screen suggests pathway(s) other than the deduced T6SS (Azc_2586-2605) participating in Hcp secretion/ release in A. caulinodans ORS571, and the mechanism(s) await future elucidation.

Contents
中文摘要 I
Abstract III
Introduction 1
Materials and methods 10
Results 24
Discussion 42
References 51
Supplementary data 58

List of Tables
Table 1 Bacterial strains used in this study 19
Table 2 Plasmids used in this study 21
Table 3 Primers used in this study 22

List of Figures
Figure 1 Estimation of near-saturation screening 25
Figure 2 Challenges in initial design 27
Figure 3 Cold-incubation improved conjugational Tn5 mutagenesis 30
Figure 4 Combine NaIO4 and agitation to remove adherent colonies 32
Figure 5 Three-round screening for extracellular Hcp. 34
Figure 6 Tn5-insertion site mapping 37
Figure 7 Identify the causative elements underlying Hcp secretion/release. 41
Figure 8 Structural prediction and analyses of Azc_2065. 46

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