Substrate Specificity and Signal Transduction Pathways in the Glucose-Specific Enzyme II (EII(Glc)) Component of the Escherichia coli Phosphotransferase System

Escherichia coli adapted to glucose-limited chemostats contained mutations in ptsG resulting in V12G, V12F, and G13C substitutions in glucose-specific enzyme II (EII(Glc)) and resulting in increased transport of glucose and methyl-α-glucoside. The mutations also resulted in faster growth on mannose and glucosamine in a PtsG-dependent manner. By use of enhanced growth on glucosamine for selection, four further sites were identified where substitutions caused broadened substrate specificity (G176D, A288V, G320S, and P384R). The altered amino acids include residues previously identified as changing the uptake of ribose, fructose, and mannitol. The mutations belonged to two classes. First, at two sites, changes affected transmembrane residues (A288V and G320S), probably altering sugar selectivity directly. More remarkably, the five other specificity mutations affected residues unlikely to be in transmembrane segments and were additionally associated with increased ptsG transcription in the absence of glucose. Increased expression of wild-type EII(Glc) was not by itself sufficient for growth with other sugars. A model is proposed in which the protein conformation determining sugar accessibility is linked to transcriptional signal transduction in EII(Glc). The conformation of EII(Glc) elicited by either glucose transport in the wild-type protein or permanently altered conformation in the second category of mutants results in altered signal transduction and interaction with a regulator, probably Mlc, controlling the transcription of pts genes.

适应葡萄糖限制恒化器（glucose-limited chemostats）的大肠杆菌（Escherichia coli），其ptsG基因发生突变，导致葡萄糖特异性酶II（EII(Glc)）出现V12G、V12F及G13C氨基酸替换，同时可提升葡萄糖与甲基-α-葡萄糖苷（methyl-α-glucoside）的转运效率。此类突变还可使菌株以依赖ptsG的方式，在甘露糖（mannose）与葡萄糖胺（glucosamine）上实现更快生长。通过以葡萄糖胺上的生长增强表型作为筛选标记，研究人员又鉴定出4个可引发底物特异性拓宽的位点，其对应的氨基酸替换分别为G176D、A288V、G320S与P384R。这些发生改变的氨基酸残基，涵盖了此前被报道可影响核糖、果糖与甘露醇摄取的位点。上述突变可分为两类：其一，有2个位点的突变影响跨膜残基（transmembrane residues），即A288V与G320S，这类突变可能直接改变糖类选择性；更为值得关注的是，其余5个特异性突变所影响的残基并不位于跨膜区段内，且这类突变在葡萄糖缺失条件下，会伴随ptsG转录水平的上调。仅提升野生型EII(Glc)的表达量，本身并不足以使菌株利用其他糖类生长。本研究提出了一个模型：决定糖类可及性的蛋白质构象，与EII(Glc)中的转录信号转导（transcriptional signal transduction）存在关联。野生型蛋白经葡萄糖转运所诱导的EII(Glc)构象，或是第二类突变体中永久改变的构象，均可改变信号转导过程，并与调控因子（大概率为Mlc）发生相互作用，进而调控pts基因的转录。