Effects of Overexpressed Transketolase Genes on Carbon Metabolism in Rhodopseudomonas palustris

轉酮醇酵素普遍存在各種生物中，其作用需要硫胺雙磷酸(thiamin diphosphate)當作輔酶，才能在戊醣磷酸途徑(pentose phosphate pathway, PPP)中當作一個重要的催化劑。此外，在可行光合作用的物種中，轉酮醇酵素在卡爾文循環(Calvin-Benson-Bassham, CBB) 中也扮演著重要的角色。紫色非硫光合細菌Rhodopseudomonas palustris具有兩型轉酮醇酵素，CbbT1和CbbT2，分別由cbbT1 (rpa4643)和cbbT2 (rpa0945)兩個基因所轉錄轉譯而來。本研究中，我們在二氧化碳為唯一碳源的光合自營條件下，大量表現紫色非硫光合菌的不同型轉酮醇酵素基因：cbbT1和cbbT2。我們整合寡核苷酸微陣列晶片分析、即時定量聚合酶連鎖反應、生物發光三磷酸腺苷 (ATP) 測定、吸收光譜以及免疫金標定蛋白質定位法來研究過量表現轉酮醇酵素對紫色非硫光合菌造成的影響。 由我們的微陣列晶片分析結果中可見，過量表現紫色非硫光合菌 cbbT1所造成的影響較過量表現cbbT2顯著。在過量表現CbbT1的菌株中，有較高的吸收光譜且與光合作用相關的基因也有較高的表現量。另一方面，過量表現CbbT2的菌株其糖解作用中ATP的生合成速率明顯較高。我們進一步利用免疫金標定具有血凝集素抗原片段的轉酮醇融合蛋白在細胞內的位置，結果發現CbbT1主要位在具有光合系統的胞質內膜系統上，而CbbT2則位在細胞質中，而且在cbbT2的菌株中，胞質內膜系統不及cbbT1的菌株來的發達。從我們的結果顯示紫色非硫光合菌不同型的轉酮醇酵素似乎參與不同的碳代謝途徑，因此對於細菌生理具有不同的影響。Transketolase is a thiamin diphosphate (ThDP)-dependent enzyme that functions as a key catalyst in the oxidative pentose phosphate pathway of virtually all organisms. It also plays an important role in the Calvin-Benson-Bassham (CBB) reductive pentose phosphate cycle of photosynthetic organisms. In Rhodopseudomonas palustris, a purple, non-sulfur photobacterium, there are two isoforms of transketolase, CbbT1 and CbbT2, encoded by transketolase genes, cbbT1 (rpa4643) and cbbT2 (rpa0945), respectively.n this study, we overexpressed cbbT1 and cbbT2 under photoautotrophic conditions, in which CO2 was the only carbon source. We integrated oligo microarray analysis, real-time quantitative PCR (qPCR), bioluminescent ATP assay and absorbance spectra to study the effects of overexpressed transketolase. From our microarray data, overexpressed cbbT1 had more effects on R. palustris than overexpressed cbbT2. In the T1 strain, an increase of the peak amplitudes in the absorption spectrum and increased expression of related photosynthetic genes were observed. On the other hand, we measured the glycolytic ATP synthetic activity by bioluminescent ATP assay and observed an increased amount of ATP in the T2 strain. Moreover, immunogold labeling of HA-tag fusion tranketolase reveals that CbbT1 is mainly located in the intracytoplasmic membrane systems, but CbbT2 in the cytosol and the ICM structures are not abundant in the T2 strain compared with the T1 strain. In this study, we suggest that the two isoforms of transketolase are likely involved in different carbon metabolic pathways and have distinct effects on bacterial physiology.摘要 IIIbstract IV. Introduction 1.1 Rhodopseudomonas palustris 1.2 Photosynthesis in Purple Non-sulfur Phototrophic Proteobacteria 2.2.1 Overview 2.2.2 Photosynthesis Gene Cluster 3.2.3 Intracytoplasmic Membrane System (ICM) 4.2.4 Compositions of the Photosynthetic Unit 4.2.5 Regulation of Photosystem Synthesis 7.3 Calvin-Benson-Bassham Cycle 9.4 Transketolase 10.4.1 Background 10.4.2 Sequences 11.4.3 Structures 12.4.4 Subcellular Localization 12.5 Motivation and Goals 13. Materials and Methods 15.1 Enrichment and Maintenance Procedures for Rhodopseudomonas palustris 15.2 Construction and Overexpression 15.2.1 Amplify cbbT1 and cbbT2 by Polymerase Chain Reaction (PCR) 15.2.2 Plasmid Constructions for cbbT1 and cbbT2 Overexpression 15.3 Transcriptome Analysis 16.3.1 Total RNA Isolation 16.3.2 Microarray 16.3.3 Data Analysis 17.4 Real-time Quantitative PCR 17.4.1 Reverse Transcription PCR 17.4.2 Real-time PCR 17.5 Transketolase Activity Assay (TKL Assay) 18.5.1 Protein Isolation and Quantification 18.5.2 Absorbance of Reduced Nicotinamide Adenine Dinucleotide (NADH) at A340 18.6 Measurement of the Cellular Adenosine Triphosphate (ATP) Synthetic Activity 19.7 Absorption Spectrum 20.8 Protein Localization 20.8.1 Western Blotting 20.8.2 Immunogold Electron Microscopy 21.8.2.1 Negative Stain 21.8.2.2 Liquid Chemical Fixation for LR White Embedding 21.8.2.3 Ultra-section 21.8.2.4 Double Staining 22.8.2.5 Immunogold Labeling 22.9 Phylogenetic Analysis of Transketolase 22.10 Homology Modeling of R. palustris Transketolase Structures 23. Results 24.1 Overexpression of cbbT1 and cbbT2 Genes in R. palustris 24.2 Global Expression Profiling in Overexpressed T1 and T2 Strains 24.3 Expression Profiles by Real-time qPCR 25.4 Absorption Spectra of Overexpressed T1 and T2 Strains. 26.5 Cellular ATP Synthetic Activity of Overexpressed T1 and T2 Strains. 27.6 Subcellular in situ Localization of Differentially Expressed Transketolase Genes 28.7 Phylogenetic Analysis of Transketolase 29.8 Homology Modeling of R. palustris Transketolase 30. Discussion 32. References 36. Figures 46. Tables 75. Appendices 89ppendix I. Ultra-section electron micrographs of photoautotrophic R. palustris cells in (A) WT and (B) NC strains. 89ppendix II. Non-specific immunogold labeling in the NC strain. 90ublications 9