Molecular analysis of sucrose metabolism of Erwinia amylovora and influence on bacterial virulence
Molecular analysis of sucrose metabolism of Erwinia amylovora and influence on bacterial virulence
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DOI：
10.1128/jb.182.19.5351-5358.2000
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发表时间：
2000-10-01
影响因子：
3.2
通讯作者：
Geider, K
中科院分区：
文献类型：
--
作者：
Bogs, J;Geider, K
蔗糖是植物重要的储运糖，也是许多植物致病菌的能量来源。为分析火疫病病原菌淀粉Erwinia amylovora对蔗糖代谢的调控及其生化特性，克隆了大肠杆菌以蔗糖为唯一碳源的染色体片段，通过转座诱变对淀粉Erwinia amylovora的scr调控基因进行了标记，并确定了其核苷酸序列。5个开放阅读框，包含基因scrK， scrY；scrA、scrB和scrR与来自肺炎克雷伯菌和质粒pUR400的scr调控基因具有高度同源性。amylovora的scrB和scrR被融合到组氨酸标签和麦芽糖结合蛋白（MalE） off上。分别杆菌。ScrB （53 kDa）催化蔗糖的水解，K(m)为125 mM。凝胶迁移率变化表明，MalE-ScrR融合蛋白与scrYAB启动子片段结合。该复合物在果糖存在下解离，而在添加蔗糖后不解离。用scrYAB启动子-绿色荧光蛋白基因融合研究scr调控子的表达，并通过流式细胞术和荧光光谱法测定。该操纵子受分解代谢物抑制和蔗糖或果糖诱导的影响，基因诱导水平与植物组织中蔗糖浓度相关，流式细胞术显示，定点诱变产生的蔗糖突变体在苹果幼苗上没有产生明显的火枯病症状，这表明蔗糖代谢对淀粉样芽孢杆菌定植寄主植物的重要性。
Sucrose is an important storage and transport sugar of plants and an energy source for many phytopathogenic bacteria. To analyze regulation and biochemistry of sucrose metabolism of the fire blight pathogen Erwinia amylovora, a chromosomal fragment which enabled Escherichia coli to utilize sucrose as sole carbon source was cloned, By transposon mutagenesis, the scr regulon of E. amylovora was tagged, and its nucleotide sequence was determined. Five open reading frames, with the genes scrK, scrY; scrA, scrB, and scrR, had high homology to genes of the scr regulons from Klebsiella pneumoniae and plasmid pUR400. scrB and scrR of E. amylovora were fused to a histidine tag and to the maltose-binding protein (MalE) off. coli, respectively. ScrB (53 kDa) catalyzed the hydrolysis of sucrose with a K(m) of 125 mM. Binding of a MalE-ScrR fusion protein to an scrYAB promoter fragment was shown by gel mobility shifts. This complex dissociated in the presence of fructose but not after addition of sucrose, Expression of the scr regulon was studied with an scrYAB promoter-green fluorescent protein gene fusion and measured by flow cytometry and spectrofluorometry. The operon was affected by catabolite repression and induced by sucrose or fructose, The level of gene induction correlated to the sucrose concentration in plant tissue, as shown by flow cytometry, Sucrose mutants created by site-directed mutagenesis did not produce significant fire blight symptoms on apple seedlings, indicating the importance of sucrose metabolism for colonization of host plants by B. amylovora.