臺灣博碩士論文加值系統

革蘭氏陰性的 Xanthomonas campestris pv. campestris 為十字花科蔬菜黑腐病 (black rot) 的植物病原菌，此菌所產生之大量胞外多醣 （Exopolysaccharide，亦稱為xanthan gum） 及胞外酵素為本菌之重要致病因子。 目前只知 Clp （cAMP receptor protein-like protein） 為一 global transcription factor ，參與調控胞外酵素以及胞外多醣之產生與分泌。 本實驗室前人將 X. campestris pv. cmpestris 已知的 Clp-binding sequences (CBSs) 加以彙整找出其 genome 中 46 個可能受 Clp 調控的基因。 其中屬於第 III 類基因 （macromolecule metabolism）的 XCC0637 被預測為一 DNA binding protein。 因為其具有 H-NS conserved domain，且與 Stenotrophomonas maltophilia、Xylella fastidiosa、Rhodopseudomonas palustris 以及 Escherichia coli 的 H-NS 之相同度 (identity) 分別為 72%、62%、46% 以及 35%，因此將之命名為 hns。 已知 H-NS 為一 global transcription regulator，負向調控一些基因的表現；不過，也有少數基因可受其正向調控。 在 E. coli，H-NS 與 CRP各自獨立調控一些基因的表現，但並沒有 CRP 可調控 hns 基因表現的相關報導。 由於 Xanthomonas 的 H-NS 還沒有被報導過，本研究利用 Clp 與 hns 上游片段之 gel retardation 試驗，以及透過 Phns-lacZ transcriptional fusion construct 分析野生株 Xc17 以及 clp 突變株 TC817 的 hns 上游片段之啟動子活性，確認 Clp 可經由與 hns 上游區域結合而正向調控 hns 基因表現。 另外構築 hns 突變株 HNSM，進行其特性分析，結果顯示 hns 的缺失造成 Xc17 之胞外酵素分泌能力、胞外多醣產量略為下降，swarming motility 下降 0.5 ㎝，感染十字花科植物時，病徵的出現較野生株晚約一天。 最後，透過 promoter-lacZ transcriptional fusion construct，分析野生株 Xc17 以及 hns 突變株 HNSM 的 hns、prt1、engA、pelB 以及 xpsE 上游片段之啟動子活性，確定 H-NS 可負向調控 hns、pelB 以及 xpsE，正向調控 engA，但並不會對 prt1 基因施以調控。

The gram-negative plant pathogenic Xanthomonas campestris pv. campestris causes black rot in crucifers. The virulence of this bacterium is mainly depending on its ability to produce large mounts of an exopolysaccharide (EPS, also called xanthan gum) and several extracellular enzymes, the virulence factors. It is known that the global transcription factor Clp (cAMP receptor protein-like protein) is involved in the production and secretion of these virulence factors. Using the consensus Clp-binding sequences (CBS) predicted in our laboratory for genome-wide search, 46 genes with at least one CBS located in the upstream region, suggesting possible regulation by Clp, were found in X. campestris pv. campestris strain 17. One of them was XCC0637, which was predicted to be a DNA-binding protein genes belonging to functional category Ⅲ possibly involved in macromolecule metabolism. It was designated as an X. campestris pv. campestris H-NS, because it possesses an H-NS conserved domain and shares 72, 62, 46 and 35% identities with the global regulator H-NS from Stenotrophomonas maltophilia, Xylella fastidiosa, Rhodopseudomonas palustris and Escherichia coli, respectively. In enterobacteria, H-NS regulates gene expression primarily as a transcriptional repressor, with only a few examples required for a direct positive control. In addition, H-NS and CRP in E. coli regulate gene expression independently. In this study, gel retardation and Phns-lacZ transcriptional fusion assays show that Clp positively regulates expression of hns by direct binding to the promoter. An hns mutan was constructed by insertional mutation. Testing with this mutant indicated that, relative to the wild-type, i) production of EPS and extracellular enzymes was slightly reduced, ii) appearance of the black rot symptom was slightly delayed, by about one day slower, iii) swarming motility was drastically reduced. Finally, transcriptional fusion assays also demonstrated that the X. campestris pv. campestris H-NS exerts negative control on the expression of hns, pelB and xpsE and positive control on the expression of engA.

中文摘要 i
英文摘要 ii
目次 iv
表目次 ix
圖目次 x
縮寫字對照表 xii
前言 1
材料 5
一、 菌種、噬菌體及質體 5
二、 藥品 5
三、 純化、透析及濃縮蛋白設備 5
四、 引子 (primers) 5
五、 培養基 5
(一) 液體培養基 5
(二) 固體培養基 6
六、 抗生素使用之最後濃度 6
七、 試劑與緩衝溶液 6
(一) 抽取質體 DNA試劑 6
(二) 抽取染色體之試劑 6
(三) 一般DNA電泳試劑 7
(四) SDS-PAGE 電泳試劑 7
(五) gel retardation 之緩衝液 7
(六) β-galactosidase 活性測試之試劑 7
(七) 純化 PelB 變性蛋白之緩衝液 8
(八) 以 electroelution 回收 PelB 變性蛋白之緩衝液 8
(九) Western blot之緩衝液 8
(十) 純化 H-NS 蛋白之緩衝液 8
(十一) 透析 H-NS 蛋白之緩衝液 9
方法 10
一、 細菌之培養和保存 10
二、 DNA之製備 10
(一) 小量質體之製備 10
(二) 細菌染色體 DNA 之抽取 10
三、 聚合酶連鎖反應 (Polymerase chain reaction, PCR) 11
四、 凝膠電泳分析 11
(一) 洋菜凝膠之製備 11
(二) SDS-聚丙烯醯胺凝膠之製備 11
(三) 聚丙烯醯胺凝膠之製備 12
五、 質體選殖 (Cloning) 12
(一) PCR 增幅片段之選殖 12
(二) 限制酶切割 ( Restriction Enzyme digestion) 12
(三) DNA 片段回收 12
(四) DNA 粘接反應(ligation) 12
六、 細菌之轉型作用 (transformation) 12
(一) Escherichia coli 之轉形作用 13
(二) X. campestris pv. campestris 之轉形作用 13
七、 蛋白質含量之測定 14
八、 Gel retardation 14
(一) DNA模版之生物素標定 14
(二) 結合反應 14
(三) 電泳分析與轉漬 14
(四) 偵測 14
九、 啟動子之活性分析 15
(一) 培養方式 15
(二) β-galactosidase 之活性測試 15
十、 點測試法 (Spot test) 15
十一、 致病性測試 15
十二、 不同 XOL plate 上之生長 16
十三、 不同 media 之生長 16
十四、 胞外酵素分泌能力測試 16
(一) Polygalacturonases 分泌測試 16
(二) Proteases 分泌測試 16
(三) α-amylase 分泌測試 16
(四) Cellulases 分泌測試 16
十五、 Motility assay 17
(一) swimming motility assay 17
(二) swarming motility assay 17
十六、 胞外多醣含量之測定 17
(一) 胞外多醣含量之測定 17
(二) 蛋白質含量之測定 18
十七、 純化 PelB 變性蛋白 18
(一) BL21(DE3)(pET30b-pelB ) 之培養、收菌及破菌 18
(二) His-bind column 之製備與 PelB 變性蛋白之純化 18
(三) 以 electroelution 回收 PelB 變性蛋白 18
十八、 胞內及胞外蛋白之收取 19
(一) 胞內蛋白之收取 19
(二) 胞外蛋白之收取 19
十九、 西方墨點法分析 (Western blot) 19
(一) 蛋白質轉漬 20
(二) 轉漬膜呈色 20
二十、 純化 H-NS 蛋白 20
(一) BL21(DE3)(pET30b-hns ) 之培養、收菌及破菌 20
(二) His-bind column 之製備與 H-NS 蛋白之純化及透析 20
結果與討論 22
一、 Clp 對於 hns 之調控 22
(一) hns 上游片段之 gel retardation 試驗 22
(二) hns 上游片段 -153/+27 在 LB 之啟動子活性 22
(三) hns 上游片段 -424/+76 在 LB 之啟動子活性 23
(四) hns 上游片段 -424/+76 在含不同碳源的　XOLN 之啟動子活性 24
(五) hns 上游片段 -424/+76 在比較貧乏的液體培養基 XVM2，以及比較營養的液體培養基 TYG 之啟動子活性 25
二、 hns 基因特性之探討 26
(一) hns 突變株 HNSM 之構築 26
1. 質體 pOK-HNSM::Gmr 之構築 26
2. 進行同質性互換 26
3. hns 突變株 HNSM 以 PCR 進行分析確認 26
(二) spot test 測試 hns 突變株 HNSM 對噬菌體 ψL7 的敏感性 26
(三) hns 突變株 HNSM 之致病性 26
(四) 野生株 Xc17 與 hns 突變株 HNSM 在 XOL plate 上之生長差異 26
(五) 野生株 Xc17 與 hns 突變株 HNSM 在不同 media 之生長 27
(六) 野生株 Xc17 與 hns 突變株 HNSM 的胞外酵素分泌能力 28
(七) 野生株 Xc17 與 hns 突變株 HNSM 的 motility 28
(八) 野生株 Xc17 與 hns 突變株 HNSM 之胞外多醣產量 29
三. H-NS 對於 hns、prt1、engA、pelB 以及 xpsE 之調控 30
(一) Phns-lacZ transcriptional fusion construct pFYhns-424+76 之啟動子活性 30
(二) Pprt1-lacZ transcriptional fusion construct pFYprt1-591-80 之啟動子活性 31
(三) PengA-lacZ transcriptional fusion construct pFYengA-568-23 之啟動子活性 32
(四) PpelB-lacZ transcriptional fusion construct pFYpelB-705-46 之啟動子活性 33
(五) PxpsE-lacZ transcriptional fusion construct pFYxpsE-119-3 之啟動子活性 34
(六) 以 Western blot 偵測 Xcc 的不同菌株之胞內以及胞外 PelB
蛋白量 35
1. Xc17、hns 突變株 HNSM、clp 突變株 TC817 及 rpfF 突變株
Xc17(rpfF::Gmr) 胞內之 PelB 蛋白表現量 35

2. Xc17、hns 突變株 HNSM、clp 突變株 TC817 及 rpfF 突變株
Xc17(rpfF::Gmr) 胞外之 PelB 蛋白表現量 35
(七) H-NS 蛋白之誘導表現與純化 36

1. C-terminally 6xHis-tagged H-NS 蛋白表現質體 pET30b-hns 之
構築 36
2. H-NS 蛋白的大量表現 37
3. 以 Ni-NTA column 純化 H-NS 蛋白 37
參考書目 39
表 1. 本實驗中所使用之菌種 50
表 2. 本實驗中所使用之質體 51
表 3. 本實驗中所使用之引子 54
表 4. 野生株 Xc17 與 hns 突變株 HNSM 之胞外多醣產量 55
圖 1. Xc17 的 XCC0637 及其鄰近基因在染色體上之 organization 56
圖 2. Xc17 的 XCC0637 蛋白之保守性斗門 (domain) 57
圖 3. hns 上游可能的 CBS (Clp-binding site)、RBS (ribosome binding site) 以及 -10 與 -35 啟動子區域 58
圖 4. 不同濃度之 Clp 與 hns 上游片段 之 gel retardation 試驗 59
圖 5. Phns-lacZ fusion constructs 之構築 60
圖 6. Phns-lacZ transcriptional fusion construct pFYhns-153+27 在 Xc17
及 TC817 之啟動子活性 61
圖 7. Phns-lacZ transcriptional fusion construct pFYhns-424+76 在 Xc17 及 TC817 之啟動子活性 62
圖 8. 含有 Phns-lacZ transcriptional fusion construct pFYhns-424+76 的 Xc17 及 TC817 培養於不同培養基之啟動子活性 63
圖 9. 質體 pOK-HNSM::Gmr 之構築流程圖 64
圖 10. hns 突變株 HNSM 之構築 65
圖 11. 以 spot test 測試 hns 突變株 HNSM 對噬菌體 ψL7 的敏感性 66
圖 12. hns 突變株 HNSM 之致病性測試 67
圖 13. 野生株 Xc17 與 hns 突變株 HNSM 在 XOL plate 上之生長差
異 68
圖 14. 野生株 Xc17 與 hns 突變株 HNSM 在不同 media 之生長 69
圖 15. 野生株 Xc17 與 hns 突變株 HNSM 的胞外酵素分泌能力 70
圖 16. 野生株 Xc17 與 hns 突變株 HNSM 的 motility assay 71
圖 17. 將含有 Phns-lacZ transcriptional fusion construct pFYhns-424+76 的 Xc17 及 hns 突變株 HNSM 培養於不同培養基中之啟動子活性 72
圖 18. 質體 pOK-HNSM::Cmr 之構築流程圖 73
圖 19. Pprt1-lacZ transcriptional fusion construct pFYprt1-591-80 在 Xc17 及 hns 突變株 HNSM 之啟動子活性 74
圖 20. PengA-lacZ transcriptional fusion construct pFYengA-568-23 在 Xc17 及 hns 突變株 HNSM 之啟動子活性 75
圖 21. PpelB-lacZ transcriptional fusion construct pFYpelB-705-46 在 Xc17 及 hns 突變株 HNSM 之啟動子活性 76
圖 22. PxpsE-lacZ transcriptional fusion construct pFYxpsE-119-3 在 Xc17 及 hns 突變株 HNSM 之啟動子活性 77
圖 23. Clp 以及 H-NS 對於 hns、prt1、engA、pelB 以及 xpsE 之調控關係圖 78

圖 24. 以 12％ mini SDS-PAGE 分離經 Ni-NTA column 純化的 PelB 蛋白 79
圖 25. 以 12％ mini SDS-PAGE 分離經 15％ large SDS-PAGE 分離並切割回收後之 PelB 蛋白 80
圖 26. 以 Western blot 偵測 Xc17、hns 突變株 HNSM、clp 突變株 TC817 及 rpfF 突變株 Xc17(rpfF::Gmr) 胞內之 PelB 蛋白量 81
圖 27. 以 Western blot 偵測 Xc17、hns 突變株 HNSM、clp 突變株 TC817 及 rpfF 突變株 Xc17(rpfF::Gmr) 胞外之 PelB 蛋白量 82
圖 28. C-terminally 6xHis-tagged H-NS 蛋白表現質體 pET30b-hns 構築之流程圖 83
圖 29. X. campestris pv. campestris 之 H-NS 蛋白的大量表現 84
圖 30. 以 15 ％ mini SDS-PAGE 分離經 Ni-NTA column 純化的 H-NS 蛋白 85
圖 31. N-terminally 6xHis-tagged H-NS 蛋白表現質體 pQE30-hns 構築
之流程圖 86

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