A mutation in the amino terminus of a hybrid TrpC-TonB protein relieves overproduction lethality and results in cytoplasmic accumulation.
A mutation in the amino terminus of a hybrid TrpC-TonB protein relieves overproduction lethality and results in cytoplasmic accumulation.
复制标题
杂合 TrpC-TonB 蛋白氨基末端的突变可减轻过度生产致死性并导致细胞质积累。
DOI：
10.1128/jb.171.8.4442-4447.1989
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发表时间：
1989
影响因子：
3.2
通讯作者：
Postle,K
中科院分区：
文献类型：
--
作者：
Skare,JT;Roof,SK;Postle,K
我们利用质粒编码的 TrpC-TonB 杂合蛋白的特性，开发了 trpC-tonB 基因融合突变的选择。 TrpC-TonB 杂合蛋白由正常细胞质蛋白 TrpC 的氨基酸 1 至 25 与 TonB 的氨基酸 12 至 239 融合。它由 trp 启动子表达，并受 trpR 基因和色氨酸存在与否的调节。在色氨酸存在的抑制条件下，trpC-tonB 基因可以恢复对 tonB 缺失突变体的 phi 80 敏感性，这表明 TrpC-TonB 可以输出并且具有功能。在色氨酸不存在的情况下，TrpC-TonB 蛋白的高水平表达导致携带 trpC-tonB 质粒的菌株几乎立即停止生长。通过选择诱导生长抑制（过度生产致死）的幸存者，我们分离出了多种突变。正如预期的那样，许多突变降低了 TrpC-TonB 蛋白的表达。此外，表达正常水平的TrpC-TonB蛋白的三个独立分离的突变体导致TonB的疏水性氨基末端内的Gly----Asp取代。突变蛋白被命名为TrpC-TonBG26D。这些突变受到 prlA 等位基因的抑制，已知该等位基因可以抑制输出（信号序列）突变。携带Gly----Asp取代的TrpC-TonB蛋白在细胞质中积累。我们得出结论，Gly----Asp 替换是一种输出突变。 TrpC-TonBG26D 蛋白已被纯化并用于产生特异性识别 TrpC-TonB 蛋白和野生型 TonB 蛋白的多克隆抗体。
We have developed a selection for mutations in a trpC-tonB gene fusion that takes advantage of the properties of the plasmid-encoded TrpC-TonB hybrid protein. The TrpC-TonB hybrid protein consists of amino acids 1 through 25 of the normally cytoplasmic protein, TrpC, fused to amino acids 12 through 239 of TonB. It is expressed from the trp promoter and is regulated by the trpR gene and the presence or absence of tryptophan. Under repressing conditions in the presence of tryptophan, the trpC-tonB gene can restore phi 80 sensitivity to a tonB deletion mutant, which indicates that TrpC-TonB can be exported and is functional. High-level expression of TrpC-TonB protein in the absence of tryptophan results in virtually immediate cessation of growth for strains carrying the trpC-tonB plasmid. By selecting for survivors of the induced growth inhibition (overproduction lethality), we have isolated a variety of mutations. Many of the mutations decrease expression of the TrpC-TonB protein, as expected. In addition, three independently isolated mutants expressing normal levels of TrpC-TonB protein result in a Gly----Asp substitution within the hydrophobic amino terminus of TonB. The mutant proteins are designated TrpC-TonBG26D. The mutations are suppressed by prlA alleles, known to suppress export (signal sequence) mutations. TrpC-TonB proteins carrying the Gly----Asp substitution accumulate in the cytoplasm. We conclude that the Gly----Asp substitution is an export mutation. TrpC-TonBG26D protein has been purified and used to raise polyclonal antibodies that specifically recognize both TrpC-TonB protein and wild-type TonB protein.