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標題: Analysis of XKBLA, an ampicillin sensitive mutant of Xanthomonas campestris pv. campestris
十字花科蔬菜黑腐病菌的-lactam類抗生素敏感性突變株 XKBLA之分析
作者: 張昕淳
Jhang, Sin-Chun
關鍵字: Xanthomonas;十字花科蔬菜黑腐病菌;antibiotic resistance;shuttle vector;抗藥性;穿梭載體
出版社: 分子生物學研究所
為了在X. campestris中進行基因功能的研究,本論文中以穩定存在於 X. campestris pv. vesicatoria 64中的迷你質體pXV64為基礎,構築了一系列帶有不同抗藥性基因、方便選殖、可同時在X. campestris與E. coli中存活的穿梭載體及表現載體。為了測試質體的功能性,利用紅色螢光基因插入所構築之表現載體pXB64G。結果顯示經西方轉漬法進行偵測後證實此穿梭表現系統在E. coli及X. campestris中皆受緊密調控,且在適當條件下可進行目標蛋白的大量表現及純化。將Xanthomonas campestris pv. campestris 17 (Xc17) 以mini-Tn5進行隨機突變後,獲得一株對ampicillin敏感之突變株,命名為XKBLA。 經南方墨點分析,證實mini-Tn5插入破壞的位置並非表現 -lactamase的bla基因或其調控基因ampR。 將XKBLA染色體DNA中mini-Tn5插入位置附近之序列選殖分析後,發現突變的目標基因主導的胺基酸序列與許多菌種的AmpG相似。 AmpG為一細胞內膜蛋白,主要功能為將細胞壁代謝產物運送進入細胞內再利用,並可能藉此影響 -lactamase基因的誘導。將全長ampG基因從Xc17的基因組中選殖出來並定序完成。AmpG之全長由431氨基酸組成,其中包含高量疏水性氨基酸,構成12個可能的穿膜區域。利用single cross-over得到Xc17的ampG突變株,XKG。經抗生素敏感性測試實驗得知 XKG對 -lactam類抗生素亦具敏感性。將包含完整ampG基因的質體 pXEG-ampG 送入ampG突變株 XKBLA與XKG中進行互補試驗,結果顯示兩互補株的抗生素最小抑制濃度皆可回復到與 Xc17同等程度。 ampG突變株、互補株與Xc17野生株之生長曲線相似,顯示在X. campestris中,ampG基因的突變對菌體的生長並無影響,但對 -lactamase基因之表現卻相當重要。進一步在ampG突變株中測定bla與ampR的啟動子強度後,發現ampR啟動子之表現在ampG突變株、野生株以及互補株中無明顯差異,但是bla的啟動子在ampG突變株中則幾乎無活性。由上述實驗結果,我們推測AmpR存在有兩種形式,一為inactive form,另一為active form。Inactive AmpR無法啟動bla之表現,甚至可能抑制其啟動子活性;active AmpR則會啟動bla之表現。結合於active AmpR上的因子與經由AmpG運送至細胞內的細胞壁代謝產物有關。這些代謝產物結合AmpR的情形有兩種不同機制:(一)這些代謝產物結合AmpR後形成active AmpR,(二)這些代謝產物必須競爭去除原先結合在inactive AmpR上的因子,才能形成active AmpR。此細胞壁代謝產物為何?尚無法確定。

To study gene function and regulation in Xanthomonas, a series of novel shuttle vectors and an expression vector based on miniplasmid pXV64 were constructed. In addition, a DsRed gene was cloned to the downstream of PBAD promoter of an expression vector pXB64G to examine its functionality. The Western blot result showed that L-arabinose is able to induce significant production of recombinant DsRed in E.coli and X. campestris. DsRed can clearly be visualized microscopically in these organisms. Additionally, his-recombinant protein can be easily purified by his-tag column. XKBLA is an ampicillin sensitive mutant of Xanthomonas campestris pv. campestris 17 (Xc17), obtained by mini-Tn5 transpositional mutation. In order to investigate the target geneinserted by mini-Tn5 and resulted to ampicillin-sensitive in this mutant, we analyzed the genome of XKBLA by Southern blotting and found that only one copy of mini-Tn5 was present in XKBLA. After sequencing of the DNA around mini-Tn5, the results revealed that the target gene is ampG, which encodes an inner membrane-bound permease. This result was confirmed by creating an Xc17 ampG mutant through single cross-over. Further analysis showed that ampG is a monocistronic gene with constitutive low level expression and can not be induced by ampicillin. To know the relationship between ampG and -lactamase expression, plasmids carrying promoter of bla or ampR at the upstream of reporter gene lacZ were cloned into wild type and ampG mutant of X. campestris separately. The result appeared that the bla promoter expresses extremely low activity in ampG mutants while the ampR promoter showed no significant difference between wild type and ampG mutant, and the -lactamase activity in ampG mutant is nearly undetectable. It is suggested that AmpR alone acts as a negative regulator or a functionless activator but as a positive regulator if muropeptides exists, and AnhMurNAc-tripeptide is strongly suspected as a key ligand. Meanwhile, the defect of AmpG shows no effect on the growth of the mutant strain. These results indicated that the ampG, a non-inducible structural gene, is not significant in survival and growth in bacterium, but plays a crucial role in -lactamase induction mechanism.
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