Manayan R, Tenn G, Yee H B, Desai J D, Yamada M, Saier M H
Department of Biology, University of California at San Diego, La Jolla 92093.
J Bacteriol. 1988 Mar;170(3):1290-6. doi: 10.1128/jb.170.3.1290-1296.1988.
Three positive selection procedures were developed for the isolation of plasmid-encoded mutants which were defective in the mannitol enzyme II (IIMtl) of the phosphotransferase system (mtlA mutants). The mutants were characterized with respect to the following properties: (i) fermentation, (ii) transport, (iii) phosphoenolpyruvate(PEP)-dependent phosphorylation, and (iv) mannitol-1-phosphate-dependent transphosphorylation of mannitol. Cell lysis in response to indole acrylic acid, which causes the lethal overexpression of the plasmid-encoded mtlA gene, was also scored. No correlation was noted between residual IIMtl activity in the mutants and sensitivity to the toxic effect of indole acrylic acid. Plasmid-encoded mutants were isolated with (i) total or partial loss of all activities assayed, (ii) nearly normal rates of transphosphorylation but reduced rates of PEP-dependent phosphorylation, (iii) nearly normal rates of PEP-dependent phosphorylation but reduced rates of transphosphorylation, and (iv) total loss of transport activity but substantial retention of both phosphorylation activities in vitro. A mutant of this fourth class was extensively characterized. The mutant IIMtl was shown to be more thermolabile than the wild-type enzyme, it exhibited altered kinetic behavior, and it was shown to arise by a single nucleotide substitution (G-895----A) in the mtlA gene, causing a single amino acyl substitution (Gly-253----Glu) in the permease. The results show that a single amino acyl substitution can abolish transport function without abolishing phosphorylation activity. This work serves to identify a site which is crucial to the transport function of the enzyme.
开发了三种阳性选择程序，用于分离磷酸转移酶系统（mtlA突变体）的甘露醇酶II（IIMtl）有缺陷的质粒编码突变体。对这些突变体进行了以下特性的表征：（i）发酵，（ii）转运，（iii）磷酸烯醇丙酮酸（PEP）依赖性磷酸化，以及（iv）甘露醇的甘露醇-1-磷酸依赖性转磷酸化。还对响应吲哚丙烯酸（其导致质粒编码的mtlA基因致死性过表达）的细胞裂解进行了评分。未观察到突变体中残余IIMtl活性与对吲哚丙烯酸毒性作用的敏感性之间存在相关性。分离出的质粒编码突变体具有以下情况：（i）所测定的所有活性全部或部分丧失，（ii）转磷酸化速率接近正常，但PEP依赖性磷酸化速率降低，（iii）PEP依赖性磷酸化速率接近正常，但转磷酸化速率降低，以及（iv）转运活性完全丧失，但体外两种磷酸化活性大量保留。对第四类的一个突变体进行了广泛表征。该突变体IIMtl比野生型酶更不耐热，表现出改变的动力学行为，并且显示是由mtlA基因中的单个核苷酸取代（G-895→A）引起的，导致通透酶中单个氨基酸酰基取代（Gly-253→Glu）。结果表明，单个氨基酸酰基取代可以消除转运功能而不消除磷酸化活性。这项工作有助于确定对该酶转运功能至关重要的一个位点。
