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首页> 外文期刊>Journal of bacteriology >A mutation in the amino terminus of a hybrid TrpC-TonB protein relieves overproduction lethality and results in cytoplasmic accumulation.
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A mutation in the amino terminus of a hybrid TrpC-TonB protein relieves overproduction lethality and results in cytoplasmic accumulation.

机译：杂色TrpC-TonB蛋白氨基末端的突变可缓解生产过剩的杀伤力，并导致细胞质积累。

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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.
机译：我们已经开发了利用质粒编码的TrpC-TonB杂合蛋白特性的trpC-tonB基因融合中的突变选择。 TrpC-TonB杂合蛋白由正常细胞质蛋白TrpC的1至25位氨基酸组成，与TonB的12至239位氨基酸融合。它由trp启动子表达，并受trpR基因和色氨酸存在与否的调节。在色氨酸存在的阻遏条件下，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。在不存在色氨酸的情况下，TrpC-TonB蛋白的高水平表达实际上导致携带trpC-tonB质粒的菌株的生长立即停止。通过选择诱导生长抑制（过度生产致死率）的幸存者，我们分离了各种突变。如预期的那样，许多突变都会降低TrpC-TonB蛋白的表达。另外，表达正常水平的TrpC-TonB蛋白的三个独立分离的突变体在TonB的疏水氨基末端内导致了Gly-Asp取代。突变蛋白被命名为TrpC-TonBG26D。该突变被已知抑制输出（信号序列）突变的prIA等位基因抑制。携带Gly ---- Asp取代的TrpC-TonB蛋白在细胞质中积累。我们得出的结论是，Gly ---- Asp替代是出口突变。 TrpC-TonBG26D蛋白已经纯化，并用于产生可特异性识别TrpC-TonB蛋白和野生型TonB蛋白的多克隆抗体。

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