Proleome and transcriptome analyses of an Escherichia coli mutant defective in oxidative phosphorylation

氧化磷酸化缺陷的大肠杆菌突变体的蛋白质组和转录组分析

• 批准号: 13660072
• 负责人: YOKOTA Atsushi
• 金额: $ 2.3万
• 依托单位: HOKKAIDO UNIVERSITY
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (C)
• 财政年份: 2001
• 资助国家: 日本
• 起止时间: 2001 至 2002
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-13660072/
• 关键词: Escherichia coli; Proteome; Transcriptome; F_1-ATPase; Energy metabolism; Chemostat culture; ケモスタット培養; DNAアレイ

Effects of a defect in oxidative phosphorylation (F_1-ATPase defect) on Escherichia coli cells was investigated by proteome and transcriptome analyses.(1) Sapmple preparation : E. coli K-12 strain W1485 and its F_1-ATPase defective mutant ware cultured in glucose in limited chemostat and used as samples for the analyses.(2) Proteome analysis : All the expressed proteins were separated by 2 dimentional gel electrophoresis, and the protein spots that showed difference in their densities between the two strains were identified by matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) mass spectrometry.(3) Transcriptome analysis : Total RNA was extracted from both strains, and ^<30>P-labeled cDNA wsa prepared. DNA array was probed with the prepared cDNA, and the image analysis of the hybridized DNA array was conducted for the analysis of the difference in the gene expression between the two strains.[Results] Among enzymes involved in glycolysis, several enzymes (phosphoglycerate kinase, enolase, pyruvate kinase I, pyruvate dehydrogenase) showed increased expression both in the level of enzyme activities and in protein amounts (proteome analysis) in the mutant. However, transcriptome analysis showed only pyruvate dehydrogenase expression was increased in the mutant, and no difference was observed in the transcription of the rest of the glycolytic enzymes. Many TCA cycle enzymes (citrate synthase, succinyl-CoA synthetase, malate dehydrogenase, isocitrate lyase, malate synthase) were found to be down-regulated in tha mutant in the levels of enzyme activities, proteome and transcriptome analyses. However, succinate dehydrogenase and fumarate hydratase were found to be up-regulated only in the level of enzyme activities and in proteome analysis. In respiratory chain, expression of NADH dehydrogenase-2 and cytochrome bd oxidase were found to be up-regulated in transcriptome analysis.

应用蛋白质组学和转录组学方法研究了氧化磷酸化缺陷（F_1-ATP酶缺陷）对大肠杆菌细胞的影响。(1)样品制备：E.将大肠杆菌K-12菌株W1485及其F_1-ATPase缺陷突变株在有限恒化器中培养于葡萄糖中，作为分析样品。(2)蛋白质组分析：用双向凝胶电泳分离表达蛋白，用基质辅助激光解吸电离飞行时间质谱（MALDI-TOF）鉴定两株菌中密度不同的蛋白质点。(3)转录组分析：从两个菌株中提取总RNA，并制备13 <30>P标记的cDNA。用制备的cDNA探测DNA阵列，并对杂交的DNA阵列进行图像分析，以分析两种菌株之间基因表达的差异。【结果】在参与糖酵解的酶中，磷酸甘油酸激酶、烯醇化酶、丙酮酸激酶I、丙酮酸脱氢酶等几种酶在突变体中的表达水平和蛋白质含量（蛋白质组学分析）均有所提高。然而，转录组分析表明，只有丙酮酸脱氢酶的表达增加的突变体，并没有观察到差异，其余的糖酵解酶的转录。通过酶活性、蛋白质组学和转录组学分析，发现柠檬酸合成酶、琥珀酰辅酶A合成酶、苹果酸脱氢酶、异柠檬酸裂解酶、苹果酸合成酶等TCA循环酶在突变体中表达下调。然而，琥珀酸脱氢酶和富马酸水合酶被发现是上调，只有在酶的活性水平和蛋白质组分析。在呼吸链中，转录组分析发现NADH脱氢酶-2和细胞色素bd氧化酶的表达上调。

项目成果

YOKOTA Atsushi: "Mechanisms that enhance glucose metabolism under the conditions of enegy deficiency"Kagaku to Seibutsu. 40. 772-774 (2002)

横田敦：“在能量缺乏的情况下增强葡萄糖代谢的机制” Kagaku to Seibutsu。

横田 篤: "エネルギーが欠乏しても糖代謝活性は亢進するメカニズムとは?"化学と生物. 40・12. 772-774 (2002)

Atsushi Yokota：“即使在没有能量的情况下，葡萄糖代谢活性增强的机制是什么？” 40・12（2002）。

横田 篤: "エネルギーが欠乏しても糖代謝活性は亢進するメカニズムとは?"化学と生物. 40. 772-774 (2002)

Atsushi Yokota：“即使在没有能量的情况下，葡萄糖代谢活性增强的机制是什么？” 40. 772-774 (2002)。