Please use this identifier to cite or link to this item: http://hdl.handle.net/11455/21552
標題: Molecular Characterization of the Multiple -galactosidase Genes of Xanthomonas campestris
十字花科黑腐病菌多重B-半乳糖苷酶基因之分析
作者: 楊翠青
Yang, Tsuey-Ching
關鍵字: Xanthomonas campestris
十字花科黑腐病菌
galactosidase
半乳糖苷
出版社: 分子生物學研究所
摘要: Xanthomonas campestris pv. campestris 是黃色的革蘭氏陰性菌,能產生一種水溶性的胞外多醣(俗稱黃原膠)。此物被廣泛地應用於食品、石化工業上。 由於此菌無法利用乳糖為唯一碳源而生長,所以不能直接以乳清為原料來生產黃原膠。 本研究利用亞硝酸鈉誘變,由本地分離株 Xc17篩選出一株能利用乳糖的突變株 (Xc17L)。 亦即在不需引進任何外來 DNA 或抗藥基因的情形下,使能直接利用乳糖為唯一碳源。 相信 Xc17L 可作為以乳清為原料生產黃原膠的有用菌株。 在Xc17基因體中,共有三個基因被命名為 -galactosidase 基因,亦即galA、galB 及 galC。基因選殖實驗顯示galA 及 galB 無法有效表現其酵素活性,但經選殖的 galC 則有明顯-galactosidase活性。 經由插入突變、 啟動子轉錄分析及西方轉漬試驗,發現galC 基因與其上游基因形成操縱子,一起被轉錄。 由於 GalC 蛋白與 X. axonopodis pv. manihotis 的 Bga 蛋白有高相似度,故GalC 可能與 Bga 一樣具有較高的-1,3-linked galactosidase活性 ; 再加上galC 鄰近基因組成的情形,推測 galC 基因的主要功能可能不是參與乳糖的代謝,而是多醣的分解。 Xc17(pRKGalC) 為能表現大量 -galactosidase之菌株,但其仍無法在乳糖碳源下正常生長,可見 Xc17 似乎缺乏一有效率的乳糖運送系統。 有三個實驗結果說明了gaIA、galB及galC基因皆不是導致Xc17L -galactosidase 活性升高的原因。其一為Xc17 及Xc17L 的gaIA, galB及galC 基因加以定序及比較結果 其二為gaIA, galB及galC 基因突變株 -galactosidase活性定量 其三為Xc17 與 Xc17L galC 的西方轉漬法的比較。 經由跳躍子插入誘變 Xc17L,發現了第四個-galactosidase基因 (galD),且經由電腦比對分析,發現在 Xc17 基因體中有一個與galD 相似的第五個-galactosidase基因 (galE) 。 GalD 蛋白在 Xc17 中可被表現且有明顯的活性,而 GalE 蛋白則無 -galactosidase酵素活性。 由鄰近基因之組成及西方轉漬法的結果指出galD 基因與其上游二個基因一起被轉錄,且其主要功能與galC 相似,可能不是參與乳糖的代謝,而是多醣的分解。演化分析的結果上顯示,galD 及galE 基因在演化上形成一明顯的演化分支,與傳統所知的 GH35 family 的-galactosidase基因分列於不同的分支中。 Xc17L 的galD及galE 基因的定序結果顯示在Xc17及 Xc17L中所表現的GalD及GalE蛋白的序列完全相同。 但是, 由GalD 的西方轉漬法的結果可看出GalD蛋白在Xc17L的表現量為在Xc17中表現量的三倍,此差異的幅度恰好與 Xc17與Xc17L所表現的-galactosidase 活性的差異幅度吻合,故galD基因是導致Xc17L -galactosidase 活性升高的原因,且其影響主要是在GalD蛋白的表現量上。 利用親合性管柱分析法分離出 GalD 蛋白並且了解影響 GalD 活性表現的重要因素。 由 GalD 蛋白膠體內活性染色結果及親合性管柱所分離的 GalD 蛋白,推測 GalD 蛋白在 Xc17菌體中可能必須與另一蛋白型成複合體的型式才具有活性。 此外,應用定點突變的策略,針對 GalD 蛋白的七個胺基酸分別進行定點突變,並評估每一突變點對活性的影響。結果顯示,第一百一十個氨基酸(valine)、 第三百三十個氨基酸(serine)、第三百九十二個氨基酸 (leucine) 及第四百零七個氨基酸(glycine) ,為影響活性的氨基酸。
The yellow-pigmented gram-negative bacterium Xanthomonas campestris pv. campestris (Xc17) can produce a water-soluble extracellular polysaccharide (xanthan gum) that is extensively applied in the food and other industries. However, owing to its low -galactosidase activities, Xc17 can't grow well in the medium containing lactose as the sole carbon source, so it can''t directly use whey as a substrate for xanthan production. A lactose-utilizing strain of X. campestris strain (Xc17L) obtained by nitrous acid mutagenesis can use lactose directly for xanthan gum production without incorporation of any exotic DNA or antibiotic resistance gene is reported here. The results of characteristic analyses of Xc17L, including -galactosidase activity, optimal condition of enzymatic activity, strain stability and xanthan gum production, indicate that Xc17L is potentially useful for xanthan production from whey. In the genome of Xc17, three annotated -galactosidase genes (galA, galB and galC) are molecularly characterized. Opposite to GalA and GalB which show no detectable enzyme levels even after being cloned in-frame with the lacZ'' gene of vector, GalC displays significant -galactosidase activity. Based on the results of insertional mutation, transcriptional fusion assay and Western blotting, galC is co-transcribed with the upstream gene(s) and expressed constitutively. GalC is a homologue of X. axonopodis pv. manihotis Bga proposed to be involved in cleaving -1,3- and -1,4-linked galactose from the terminal position of polysaccharide chains available in the environment instead of lactose. The homology together with the genome organization suggest that galC is not responsible for lactose utilization, but polysaccharide degradation. Xc17 appears to be deficient in lactose uptake system, because Xc17 carrying cloned lacZY can grow on lactose but not those with cloned galC or lacZ only. Results of DNA sequencing of galA and galB, mutation in galA, galB and galC, and the Western blotting of galC indicate that the galA, galB and galC genes are not correlated to the elevation of -galactosidae activity in Xc17L. A novel -galactosidase gene, galD, is revealed by insertional mutagenesis, and its homology, galE, exists in the genome. GalD, indeed expressed in Xc17, exists obvious -galactosidase activity, but GalE shows no detectable enzymatic activity. Combined the genome organization and Western blotting, it is indicated that galD is co-transcribed with the upstream two genes. The results of phylogenetic analysis strongly suggest that galD and galE form a separately evolutional divergency which is significantly different from the traditionally well-known GH35 family. Comparing the amino acid sequences of galD and galE in Xc17 and Xc17L, no difference is observed. But, the result of Western blotting indicates that the amount of GalD expressed in Xc17L is as three times as that in Xc17 which shows a perfect consistence between the -galactosidase activity and the expressed amount of GalD. So, elevated -galactosidase activity in Xc17L results from amount of GalD. Using the strategy of affinity chromatography,GalD protein can be purified and a lot of key factors determining the enzyme activity of GalD are revealed. Seven amino acid residues of GalD are substituted by site-directed mutagenesis, separately, and effects of a.a. substitution on the enzyme activity of GalD are evaluated.
URI: http://hdl.handle.net/11455/21552
Appears in Collections:分子生物學研究所

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