沙門氏菌metE基因表現受到FnrS之調控
Other Title
Regulation of Salmonella typhimurium metE Gene Expression by FnrS
Journal
臺灣農業化學與食品科學
Journal Volume
53
Journal Issue
6
Start Page
259
End Page
267
ISSN
1605-2471
Date Issued
2015
Author(s)
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甲硫胺酸合成酵素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，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。在豐富培養基添加甲硫胺酸，透過β-galactosidase活性測試及西方轉漬法試驗，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。而RT-PCR的試驗亦呈現與西方轉漬法活性測試相符的結果；透過本研究對metE基因之調控有更進一步的探討，幫助鼠傷寒沙門氏菌之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 as a precursor or an intermediate is critical for a variety of metabolic processes in an organism. As an essential amino acid, methionine is not synthesized de novo in humans, and it must be taken from 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 methionine synthase (MetE). We used 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 a metE::lacZ fusion gene, and established the plasmids which carry the complete metE. We investigated the relations between metE and fnrS, through comparison among 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-regulated metE gene expression in an anaerobic condition. Accordingly, fnr and hfq down regulate metE indirectly. The results of RT-PCR were also consistant with the results from Western blot 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 the regulation of other gene expressions, which have a similar regulatory mechanism.
Subjects
甲硫胺酸合成酶
沙門氏菌
基因調控
Gene regulation
Methionine synthase
Salmonella typhimurium
Type
journal article