Li T K, Panchenko Y A, Drolet M, Liu L F
Department of Pharmacology, Robert Wood Johnson Medical School, University of Medicine and Dentistry of New Jersey, Piscataway 08854, USA.
J Bacteriol. 1997 Sep;179(18):5789-94. doi: 10.1128/jb.179.18.5789-5794.1997.
The Escherichia coli rho-15 mutant (deficient in transcription termination) is known to be incompatible with pBR322 and other plasmids (J. S. Fassler, G. F. Arnold, and I. Tessman, Mol. Gen. Genet. 204:424-429, 1986). We show that failure of pBR322 to transform rho-15 is mediated by transcription from the tet promoter and readthrough from the tet gene into the rom region. Using an isopropyl-beta-D-thiogalactopyranoside-inducible promoter to replace the tet promoter, we have demonstrated that plasmid-specific transcription inhibits growth of the rho-15 host, possibly due to the expression of the Rom protein. The involvement of Rom protein in pBR322-rho-15 incompatibility is further indicated by the following two experiments. (i) Functional inactivation of the rom gene in pBR322 enabled plasmids to transform E. coli rho-15. (ii) Specific overexpression of the rom gene abolished plasmid transformation into E. coli rho-15. An rpoB8(Ts) mutant RNA polymerase which compensated for the termination defect in E. coli rho-15 also restored plasmid-host compatibility, suggesting that Rom-mediated plasmid-host incompatibility is linked to a defect in transcription termination.
已知大肠杆菌rho - 15突变体（转录终止缺陷）与pBR322及其他质粒不兼容（J. S. 法斯勒、G. F. 阿诺德和I. 特斯曼，《分子与普通遗传学》204:424 - 429，1986）。我们发现，pBR322无法转化rho - 15是由tet启动子转录以及tet基因转录通读至rom区域介导的。使用异丙基 - β - D - 硫代半乳糖苷诱导型启动子取代tet启动子，我们证明质粒特异性转录抑制rho - 15宿主的生长，这可能是由于Rom蛋白的表达。以下两个实验进一步表明了Rom蛋白参与pBR322 - rho - 15的不兼容性。（i）pBR322中rom基因的功能失活使质粒能够转化大肠杆菌rho - 15。（ii）rom基因的特异性过表达消除了质粒转化大肠杆菌rho - 15的能力。一种补偿大肠杆菌rho - 15终止缺陷的rpoB8（Ts）突变型RNA聚合酶也恢复了质粒 - 宿主兼容性，这表明Rom介导的质粒 - 宿主不兼容性与转录终止缺陷有关。
