Characterization of MtfA, a Novel Regulatory Output Signal Protein of the Glucose-Phosphotransferase System in Escherichia coli K-12
Characterization of MtfA, a Novel Regulatory Output Signal Protein of the Glucose-Phosphotransferase System in Escherichia coli K-12
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DOI：
10.1128/jb.06387-11
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发表时间：
2012-03-01
影响因子：
3.2
通讯作者：
Jahreis, Knut
中科院分区：
文献类型：
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作者：
Goehler, Anna-Katharina;Staab, Ariane;Jahreis, Knut
大肠杆菌 K-12 中的葡萄糖磷酸转移酶系统 (PTS) 是一个复杂的感觉和调节系统。除了在葡萄糖摄取中发挥核心作用外，它还向其他全球监管网络通报碳水化合物的可用性和细胞的生理状态。编码葡萄糖-PTS转运蛋白EIICBGlc的ptsG基因的表达主要通过阻遏物Mlc来调节，其失活是葡萄糖依赖性的。在葡萄糖转运和 ElICB(Glc) 去磷酸化过程中，Mlc 与转运蛋白的 B 结构域结合，导致多个 Mlc 调节基因的去抑制。此外，Mlc还可以通过直接的蛋白质-蛋白质相互作用被细胞质蛋白MtfA失活。在本研究中，我们通过测量突变的 MtfAs 对 ptsG 表达的影响，确定了 MtfA 羧基末端区域中 Mlc 的结合位点。此外，我们通过体内滴定和凝胶位移测定证明了 MtfA 灭活 Mlc 超级阻遏物的能力，而 EIICBGlc 无法灭活 Mlc 超阻遏物。最后，我们通过监测氨基 4-硝基苯胺底物的裂解来表征纯化的 MtfA 的蛋白水解活性，并显示 Mlc 增强该活性的能力。根据我们的发现，我们提出了一种 MtfA 模型，作为由细胞质 Mlc 激活的葡萄糖调节肽酶。当培养物进入静止期时，其活性可能是培养物生长过程中所必需的。 Mlc 调节的 MtfA 蛋白水解活性构成了新识别的 PTS 输出信号，可响应环境条件的变化。
The glucose-phosphotransferase system (PTS) in Escherichia coli K-12 is a complex sensory and regulatory system. In addition to its central role in glucose uptake, it informs other global regulatory networks about carbohydrate availability and the physiological status of the cell. The expression of the ptsG gene encoding the glucose-PTS transporter EIICBGlc is primarily regulated via the repressor Mlc, whose inactivation is glucose dependent. During transport of glucose and dephosphorylation of ElICB(Glc), Mlc binds to the B domain of the transporter, resulting in derepression of several Mlc-regulated genes. In addition, Mlc can also be inactivated by the cytoplasmic protein MtfA in a direct protein-protein interaction. In this study, we identified the binding site for Mlc in the carboxy-terminal region of MtfA by measuring the effect of mutated MtfAs on ptsG expression. In addition, we demonstrated the ability of MtfA to inactivate an Mlc super-repressor, which cannot be inactivated by EIICBGlc, by using in vivo titration and gel shift assays. Finally, we characterized the proteolytic activity of purified MtfA by monitoring cleavage of amino 4-nitroanilide substrates and show Mlc's ability to enhance this activity. Based on our findings, we propose a model of MtfA as a glucose-regulated peptidase activated by cytoplasmic Mlc. Its activity may be necessary during the growth of cultures as they enter the stationary phase. This proteolytic activity of MtfA modulated by Mlc constitutes a newly identified PTS output signal that responds to changes in environmental conditions.