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標題: Characterization of hns Gene of Xanthomonas campestris pv. campestris
作者: 王清華
Wang, Ching-Hua
關鍵字: Xanthomonas campestris;十字花科蔬菜;Clp;H-NS;黑腐病;植物病原菌
出版社: 分子生物學研究所
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.

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。
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