Goldfarb D S, Rodriguez R L, Doi R H
Proc Natl Acad Sci U S A. 1982 Oct;79(19):5886-90. doi: 10.1073/pnas.79.19.5886.
The Gram-negative product-encoding Tn9-derived chloramphenicol-resistance (Cmr) gene can be cloned but not phenotypically expressed in Bacillus subtilis. We show that, even when transcribed from B. subtilis promoters, the ribosomal binding site for the Cmr gene does not function well in B. subtilis. The Cmr gene product, chloramphenicol acetyltransferase (CmAcTase; acetyl-CoA:chloramphenicol 3-O-acetyltransferase, EC 2.3.1.28), is detected in B. subtilis when the promoters, ribosomal binding sites, and initiation codons of B. subtilis genes are fused to the Cmr gene. These gene fusions lead to the in vivo production of mRNAs containing B. subtilis translation start signals followed in an open reading frame by the translation start site normally used by Escherichia coli to initiate translation of Cmr mRNA. Both fusion and native CmAcTase proteins are produced in E. coli, but only fusion CmAcTase is produced in B. subtilis. We conclude that the absence of native CmAcTase in B. subtilis is due to inability of the E. coli ribosomal binding site to function well in B. subtilis. Since fusion CmAcTase polypeptides are produced in E. coli, we conclude that these particular B. subtilis regulatory elements function heterologously in E. coli. The absence of a suitable binding site on the Cmr gene for B. subtilis ribosomes is consistent with reports that many E. coli genes are not expressed in B. subtilis and that E. coli mRNA functions poorly in B. subtilis in vitro translation systems. The functioning of B. subtilis regulatory sequences in E. coli is consistent with in vivo and in vitro data showing the expression of B. subtilis genes in E. coli. To confirm the hypothesis that the large CmAcTase proteins are NH2-terminal fusions of native CmAcTase we partially determined the sequence of one CmAcTase fusion protein.
源自Tn9的革兰氏阴性产物编码氯霉素抗性（Cmr）基因能够在枯草芽孢杆菌中克隆，但无法表型表达。我们发现，即便从枯草芽孢杆菌启动子转录，Cmr基因的核糖体结合位点在枯草芽孢杆菌中也不能很好地发挥作用。当枯草芽孢杆菌基因的启动子、核糖体结合位点和起始密码子与Cmr基因融合时，在枯草芽孢杆菌中可检测到Cmr基因产物氯霉素乙酰转移酶（CmAcTase；乙酰辅酶A：氯霉素3 - O - 乙酰转移酶，EC 2.3.1.28）。这些基因融合导致体内产生包含枯草芽孢杆菌翻译起始信号的mRNA，其开放阅读框中接着是大肠杆菌通常用于启动Cmr mRNA翻译的翻译起始位点。融合型和天然型CmAcTase蛋白在大肠杆菌中均有产生，但在枯草芽孢杆菌中仅产生融合型CmAcTase。我们得出结论，枯草芽孢杆菌中缺乏天然CmAcTase是由于大肠杆菌核糖体结合位点在枯草芽孢杆菌中不能很好地发挥作用。由于融合型CmAcTase多肽在大肠杆菌中产生，我们得出结论，这些特定的枯草芽孢杆菌调控元件在大肠杆菌中具有异源功能。Cmr基因上缺乏适合枯草芽孢杆菌核糖体的结合位点，这与许多大肠杆菌基因在枯草芽孢杆菌中不表达以及大肠杆菌mRNA在枯草芽孢杆菌体外翻译系统中功能不佳的报道一致。枯草芽孢杆菌调控序列在大肠杆菌中的功能与显示枯草芽孢杆菌基因在大肠杆菌中表达的体内和体外数据一致。为了证实大的CmAcTase蛋白是天然CmAcTase的NH2末端融合体这一假说，我们部分测定了一种CmAcTase融合蛋白的序列。
