沙門氏菌metE基因表現受到FnrS之調控

Abstract

甲硫胺酸在生物體中作為多種生長代謝過程中不可或缺的前驅物及中間產物，於人體中是需額外攝取的必需胺基酸之一，對微生物來說，則是可以透過複雜的生合成及利用調控機制自行生合成。其中，metE基因所編碼的蛋白質為催化其生合成最終步驟的關鍵酵素。本研究以Salmonella Typhimurium LT2為研究背景，探討一受到厭氧調控蛋白FNR影響，並於厭氧下誘導表現的非編碼小片段核糖核酸-FnrS與metE在微生物適應厭氧環境下所進行的調控作用。本研究藉由metE::lacZ之融合菌株的建立，以及metE與fnrS基因及其突變基因之選殖質體的建構，透過於fnrS及其相關調控因子缺失株在氧氣轉換時的表現差異，探討metE基因與FnrS之間的關係。在豐富培養基或限制培養基添加甲硫胺酸，透過β-galactosidase及西方轉漬法試驗，證實FnrS於厭氧狀態下，與metE基因間具有負向調控的關係。藉由其相關的調節蛋白FNR及Hfq，促使metE基因間接負向調節的表現結果，輔助理解FnrS於厭氧狀態下對metE基因的調控。接著，觀察到FnrS自身結構完整是必要的，且發現metE-5’mRNA之Shine-Dalgarno (SD) 序列的位置對FnrS結合是重要的；而RT-PCR的試驗亦呈現與β-galactosidase相符的結果；最後，觀察到metE基因本身於豐富或限制培養基添加甲硫胺酸之表現差異，顯示metE基因於豐富培養基中，應存在其他因子進行調控作用。透過本研究對metE基因之調控有更進一步的探討，幫助對鼠傷寒沙門氏菌之metE調控機制的瞭解，並期許能在未來作為類似基因研究之輔助，使之成為類似病原菌之對應的模式菌株。

Methionine is critical for the variety of metabolic processes in the organism, such as a precursor or intermediate for the final products. As an essential amino acid, methionine is not synthesized de novo in humans, and it must be ingested through food. In microorganisms, methionine is synthesized via a complicated biosynthetic pathway. The last step for the synthesis of methionine is the methylation of homocysteine, which is catalyzed by the MetE. We use the Salmonella Typhimurium LT2 as a model, to test the regulation of the metE gene by an anaerobically induced small non-coding RNA-fnrS, whose expression is strictly dependent on the anaerobic regulator-FNR. In this study, we constructed the metE::lacZ fusion gene, and established various plasmids which carry the complete metE or fnrS gene or their derivatives. We investigate the relations between metE and fnrS, through the wild-type, fnrS and/or fnr as well as hfq deficient strains under the oxygen-limited state. The results of the β-galactosidase assays and Western blotting analyses showed that FnrS down-regulate metE gene expression in an anaerobic condition. Accordingly, the fnr and hfq genes were down regulating the metE gene indirectly; we proved the fnrS-mediated metE expression. Furthermore, we observed that the secondary structure of fnrS is necessary for the regulation, and we also noted that the SD sequence of metE is important for the base-pairing with fnrS. Through the results of RT-PCR, we also found out the mRNA expressions were coincided with the results fromβ-galactosidase analyses. Additionally, the negative regulation of metE by fnrS was obviously observed under the unlimited-methionine condition. Through this study, it would be helpful for understanding of the regulation of other gene expressions, which have a similar regulatory mechanism.