murI 基因之功能探討

1. MurI 蛋白是否為生長必需蛋白

MurI 為催化 D-glutamate 產生的重要酵素。在大腸桿菌中若此蛋白缺失則會造 成細菌無法生長而致死(Baliko and Venetianer ,1993)；在 B. subtilis 中有兩個基因 racE、yrpC 轉譯 glutamase racemase，在營養豐富培養基中 racE 為必需表現蛋白，

但在營養貧瘠培養基中 racE 為非必需表現蛋白，而 yrpC 只在營養貧瘠培養基中 表現，racE 缺失株無法在營養豐富培養基中正常生長，至於基因必須表現是否和 D-glutamase 生合成有關，目前仍未知 (Kimura et al., 2004)。本論文實驗結果卻顯 示 murI 突變株在營養貧瘠液體和固體培養基上生長速度與野生型菌株無異(圖十 八、十九)，且野生型菌株和 murI 突變株在菸草 N. benthamiana 內的增生速度無異 (圖十八)，所以我們認為 murI 突變株生長能力正常；造成這些異於前人研究發現 的結果之原因可能有三種。首先，在青枯病菌中可能有D-amino acid transaminase 產生D-glutamate 使細菌正常細胞分裂、形成肽聚醣細胞壁，而可以正常生長。前 人發現若將D-amino acid transaminase 的基因互補到大腸桿菌 D-glutamate 營養缺 陷株WM335 (murI 基因缺失株)中，可以產生代償作用(Compensation)產生低量的 D-glutamate 而維持細胞生存，可見在革蘭氏陰性菌中只需少量的 D-glutamate 即可 使細菌存活(Liu et al., 1998)。但是目前的研究文獻顯示，由於革蘭氏陽性菌細胞壁 有 較 厚 的 肽 聚 醣 層 ， 需 要 較 多 的 D-glutamate ， 因 此 大 多 有 D-amino acid transaminase，革蘭氏陰性菌目前尚未發現有 D-amino acid transaminase (Lundqvist et

al., 2007)，所以屬於革蘭氏陰性菌的青枯病菌應該沒有 D-amino acid transaminase，

因此 murI 突變株應不是利用此酵素產生 D-glutamate 而生存。

其次，細菌中除了有兩個可以產生D-glutamate 的路徑外，還可利用消耗 ATP 將外來的L-glutamate 和 D-glutamate 運送至體內(Jacobs et al., 1995)，也許青枯病菌 突變株是利用此路徑來補充體內 D-glutamate 的不足，未來可以將青枯病菌 murI 突變株培養在以L-glutamate 為唯一碳源的培養基中，測試其生長情況是否與野生 型菌株一致，以確認 murI 突變株是否會利用外來的 D-glutamate 來補充體內 D-glutamate 的不足。

此外，推測可能因為本研究的突變株是插在 murI 中間，並未完全破壞此蛋白 功能，尤其並未破壞其N 端，由前人研究可知革蘭氏陰性菌大腸桿菌中 MurI 的 N 端有活化子UDP-MurNAc-L-Ala 的辨認區(Ho et al., 1995)，如果青枯病菌也需要活 化子 UDP-MurNAc-L-Ala 的活化才有酵素活性的話，此區域的完整性是非常性要 的。並且在大腸桿菌中，發現只要有MurI 的 N 端酵素就有 glutamate racemase 功 能，催化D-glutamate 的產生，雖然產量只有 MurI 全長酵素的 20%，但是由於革 蘭氏陰性細菌只須少量的D-glutamate 即可生存，因此不影響其生長(Ho et al., 1995;

Liu et al., 1998)。所以本研究的突變株可能仍然有部分 glutamate racemase 功能，製 造少量的 D-glutamate 而維持生存。未來可以利用基因置換策略構築 murI 剔除株 (murI deletion mutant)，確認 MurI 是否為青枯病菌生長必需蛋白。

2. 青枯病菌MurI在細胞型態與生理特性之功能

在大腸桿菌中若murI條件突變，可發現其細胞分裂受到影響，而有細胞成絲 狀延長的現象(Baliko and Venetianer , 1993)。本論文雖然尚未進行細菌細胞型態之 量化分析，利用穿透式電子顯微鏡觀察到murI突變株似乎較野生型長(圖二十一)，

推測本實驗murI突變株的glutamate racemase催化產生D-glutamate的能力應該有受 某種程度的到影響，至於其DNA gyrase抑制子能力是否有受影響目前仍未知，未 來可以測試murI突變株的DNA gyrase抑制子能力是否有受影響。

在逆境 EDTA(可抓住金屬離子)測試中發現，野生型菌株與 murI 突變株並無 差異，可見青枯病菌MurI 蛋白不須金屬離子當作輔酶幫助其催化活性，就如同其 他細菌中的 MurI 為不須輔酶(cofactor-independent)的 glutamate racemase (Mobitz and Bruice, 2004)。

3. 青枯病菌 MurI 影響致病力分析

由實驗結果圖二十二、二十七可知青枯病菌 murI 突變株的游動力(swimming motility)下降。游動力是青枯病菌早期入侵寄主的重要致病因子，若青枯病菌無法 順利游到寄主根部，則無法順利造成入侵感染。前人研究可知無法產生鞭毛的青 枯病菌突變株(fliC)澆灌番茄會造成致病力下降，但若直接用莖部穿刺感染番茄，

則可恢復其致病力(Tans-Kersten et al., 2001)。本研究發現青枯病菌 murI 突變株澆 灌番茄植株會造成致病力下降，但直接注入菸草 N. benthamiana 會產生嚴重壞死之 病徵，且實驗室前人利用莖部穿刺此突變株感染番茄，發現其恢復致病力而造成 番茄的萎凋(附錄七)。由此推測此突變株應該因早期致病因子(游動力)的缺失而造 成致病力下降，直接將病菌注入寄主植株則可恢復其致病力。在 Sinorhizobium meliloti 中發現鞭毛結構 rotor-stator 間有幾個重要且保留性高的 glutamate 胺基酸，

可以影響鞭毛的游動速度(Attmannspacher et al., 2005)。在 Pseudomonas aeruginosa 中若以L-glutamate 為單一氮源，會促使其 swarming motility(藉由鞭毛和纖毛在半 固體培養基上游動)增加(Kohler et al., 2000)。可見細菌體內的 glutamate 與其游動力 相關，但其中詳細機制目前仍未知，未來應該以定量RT-PCR 來檢測 murI 突變株 中的鞭毛相關基因是否表現正常。

趨化性(chemotaxis)對於生活在土中的青枯病菌是否能正確感應寄主植物根部 的位置進而順利的游到此部位是非常重要的。在 Rhodobacter sphaeroides 中發現細 菌需要運送glutamate 到細胞中及在體內有適當的 glutamate 代謝才能表現完整的趨 化性(Jacobs et al., 1995)，因此未來應該要測試青枯病菌 murI 突變株對植物寄主根 部的趨化性是否正常。

4. murI 為青枯病菌之關鍵致病基因

實驗室前人曾以完整的MurI蛋白(無基因前端啟動子)互補至Pss190背景下 murI突變株，發現可以稍稍恢復其對阿拉伯芥的致病力(附錄八)，但致病力恢復效 果不佳，並且無法恢復其對番茄的致病力(尚未發表)，推測可能因需要murI啟動子 才能成功互補其對番茄的致病力。因此本研究在進行互補實驗時，構築了不同大 小的互補株(附錄九)。前人曾對大腸桿菌 glutamate racemase突變株 JM109，進行 互補大腸桿菌的murI啟動子(murI前推上游500鹼基對)，發現無法成功互補其 glutamate racemase 表 現 ， 但 若 在 其 murI 前 添 加 人 工 的 核 醣 體 結 合 區 (ribosome-binding region)即可成功互補glutamate racemase表現(Liu et al., 1998)，推 測可能大腸桿菌中murI前端並無murI啟動子。本研究卻發現互補青枯病菌murI前推 要是參與檸檬酸循環(TCA cycle)中將malate催化成fumarate的酵素(Woods et al., 1988)，在fumarate形成後再經一連串的酵素催化可以代謝成2-oxoglutarate，而

結語：

由 R. solanacearum 所引起的青枯病係一全球重要之土壤傳播性維管束病害，

目前我們對於這個極為複雜且破壞力極強的病菌的了解仍是十分有限，且無有效的 防治方法。本論文藉由深入研究青枯病菌各個致病因子，首次發現植物病原菌中 LPS 生合成基因可同時參與第三型分泌系統的調控，並確認新穎蛋白 RSc0411 與 RSc1956 確實是青枯病菌之關鍵致病基因。希冀藉由全面性地了解青枯病菌的致病 機制，以期許能夠進而建立新的、具有潛力的策略，有效地應用於重要作物之防治 病害工作上。

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