Department of Chemistry and Chemical Biology, Harvard University, Cambridge, MA, USA.
Department of Life Sciences, Ben-Gurion University of the Negev, Beer-Sheva, Israel.
Mol Syst Biol. 2021 Jun;17(6):e10200. doi: 10.15252/msb.202110200.
The relationship between sequence variation and phenotype is poorly understood. Here, we use metabolomic analysis to elucidate the molecular mechanism underlying the filamentous phenotype of E. coli strains that carry destabilizing mutations in dihydrofolate reductase (DHFR). We find that partial loss of DHFR activity causes reversible filamentation despite SOS response indicative of DNA damage, in contrast to thymineless death (TLD) achieved by complete inhibition of DHFR activity by high concentrations of antibiotic trimethoprim. This phenotype is triggered by a disproportionate drop in intracellular dTTP, which could not be explained by drop in dTMP based on the Michaelis-Menten-like in vitro activity curve of thymidylate kinase (Tmk), a downstream enzyme that phosphorylates dTMP to dTDP. Instead, we show that a highly cooperative (Hill coefficient 2.5) in vivo activity of Tmk is the cause of suboptimal dTTP levels. dTMP supplementation rescues filamentation and restores in vivo Tmk kinetics to Michaelis-Menten. Overall, this study highlights the important role of cellular environment in sculpting enzymatic kinetics with system-level implications for bacterial phenotype.
序列变异与表型之间的关系尚未完全阐明。在这里，我们使用代谢组学分析来阐明携带二氢叶酸还原酶（DHFR）不稳定突变的大肠杆菌菌株丝状表型的分子机制。我们发现，尽管存在 SOS 反应表明存在 DNA 损伤，但部分丧失 DHFR 活性会导致可逆的丝状化，这与通过高浓度抗生素甲氧苄啶完全抑制 DHFR 活性实现的胸腺嘧啶缺乏死亡（TLD）形成鲜明对比。这种表型是由细胞内 dTTP 不成比例下降触发的，根据胸苷酸激酶（Tmk）的米氏常数样体外活性曲线（下游将 dTMP 磷酸化为 dTDP 的酶），不能用 dTMP 的下降来解释。相反，我们表明 Tmk 的高度协同（Hill 系数为 2.5）体内活性是导致 dTTP 水平不佳的原因。dTMP 补充可挽救丝状化并使体内 Tmk 动力学恢复到米氏常数。总的来说，这项研究强调了细胞环境在塑造酶动力学方面的重要作用，对细菌表型具有系统水平的意义。
