Department of Agricultural Chemistry, College of Bio-Resource and Agriculture, National Taiwan University, Taipei, Taiwan, ROC.
Department of Agricultural Chemistry, College of Bio-Resource and Agriculture, National Taiwan University, Taipei, Taiwan, ROC.
Microbiol Res. 2019 Dec;229:126319. doi: 10.1016/j.micres.2019.126319. Epub 2019 Aug 24.
Methionine is critical for variety of metabolic processes in biological organisms, acting as a precursor or intermediate for many final products. The last step for the synthesis of methionine is the methylation of homocysteine, which is catalyzed by MetE. Here, we use Salmonella enterica serovar Typhimurium LT2 to study the regulation of the metE gene by an anaerobically induced small non-coding RNA-FnrS, the expression of which is strictly dependent on the anaerobic regulator-FNR. The MetE-HA protein was expressed at an increased level in the fnrS and hfq deficient strains under anaerobic conditions. The Hfq protein is predicted to stabilize the binding between small RNA(s) and their target mRNA(s). A transcriptional (op) and translational (pr) metE::lacZ fusion gene were separately constructed, with the metE-promoter fused to a lacZ reporter gene. In an anaerobic environment, the metE::lacZ (pr) fusion gene and reverse transcription-PCR identified that FnrS and/or FNR negatively regulate metE mRNA levels in the rich media. Analysis of FnrS revealed a sequence complementary to the 5' mRNA translational initiation region (TIR) of the metE gene. Mutation(s) predicted to disrupt base pairing between FnrS and metE TIR were constructed in fnrS, and most of those resulted in the loss of repressive activity. When compensatory mutation(s) were made in metE 5' TIR to restore base pairing with FnrS, the repressive regulation was completely restored. Therefore, in this study, we identified that in anaerobic phase, there is a repression of metE gene expression by FnrS and that base-paring, between both expressive transcripts, plays an important role for this negative regulation.
甲硫氨酸对生物体内多种代谢过程至关重要，作为许多最终产物的前体或中间产物。甲硫氨酸合成的最后一步是同型半胱氨酸的甲基化，这一步由 MetE 催化。在这里，我们使用沙门氏菌 Typhimurium LT2 来研究厌氧诱导的小非编码 RNA-FnrS 对 metE 基因的调节，其表达严格依赖于厌氧调节剂-FNR。在厌氧条件下，FnrS 和 hfq 缺失菌株中甲硫氨酸酶-HA 蛋白的表达水平增加。Hfq 蛋白被预测能稳定小 RNA(s)与其靶 mRNA(s)之间的结合。我们分别构建了转录（op）和翻译（pr）metE::lacZ 融合基因，其中 metE 启动子与 lacZ 报告基因融合。在厌氧环境下，metE::lacZ (pr) 融合基因和反转录-PCR 鉴定出 FnrS 和/或 FNR 负调控丰富培养基中 metE mRNA 水平。对 FnrS 的分析显示了一个与 metE 基因 5' mRNA 翻译起始区（TIR）互补的序列。构建了预测会破坏 FnrS 与 metE TIR 之间碱基配对的突变（s），其中大部分突变导致抑制活性丧失。当在 metE 5' TIR 中进行补偿突变以恢复与 FnrS 的碱基配对时，抑制调节完全恢复。因此，在本研究中，我们确定在厌氧阶段，FnrS 抑制 metE 基因的表达，并且两个表达转录物之间的碱基配对对于这种负调控起着重要作用。
