Masaki H, Akutsu A, Uozumi T, Ohta T
Department of Biotechnology, Faculty of Agriculture, University of Tokyo, Japan.
Gene. 1991 Oct 30;107(1):133-8. doi: 10.1016/0378-1119(91)90306-v.
Plasmid immunity to a nuclease-type colicin is defined by the specific binding of an immunity (or inhibitor) protein, Imm, to the C-terminal nuclease domain, T2A, of the colicin molecule. Whereas most regions of colicin operons exhibit extensive sequence identity, the small plasmid region encoding T2A and Imm is exceptionally varied. Since immunity is essential for the survival of the potentially lethal colicin plasmid (Col), we inferred that T2A and Imm must have co-evolved, retaining their mutual binding specificities. To evaluate this co-evolution model for the col and imm genes of ColE3 and ColE6, we attempted to obtain a stabilized clone from a plasmid which had been destabilized with a non-cognate immunity gene. A hybrid Col, in which the immE3 gene of the ColE3 was replaced with immE6 from ColE6, was lethal to the host cells upon SOS induction. From among this suicidal cell population, we isolated a stabilized, i.e., evolved, clone which produced colicin E3 (E3) stably and exhibited immunity to E3. This change arose from only a single mutation in ImmE6, from Trp48 to Cys, the same residue as in the ImmE3 sequence. In addition, we constructed a series of chimeric genes through homologous recombination between immE3 and immE6. Characterization of these chimeric immunity genes confirmed the above finding that colicins E3 and E6 are mostly distinguished by only Cys48 of the ImmE3 protein.
质粒对核酸酶型大肠杆菌素的免疫性是由免疫（或抑制）蛋白Imm与大肠杆菌素分子的C末端核酸酶结构域T2A的特异性结合来定义的。虽然大肠杆菌素操纵子的大多数区域表现出广泛的序列同一性，但编码T2A和Imm的小质粒区域却异常多样。由于免疫性对于潜在致死性大肠杆菌素质粒（Col）的存活至关重要，我们推断T2A和Imm必定共同进化，保留了它们相互的结合特异性。为了评估ColE3和ColE6的col和imm基因的这种共同进化模型，我们试图从一个已被非同源免疫基因使其不稳定的质粒中获得一个稳定的克隆。在一个杂种大肠杆菌素中，ColE3的immE3基因被ColE6的immE6取代，在SOS诱导时对宿主细胞具有致死性。从这个自杀性细胞群体中，我们分离出了一个稳定的，即进化的克隆，它能稳定产生大肠杆菌素E3（E3）并对E3具有免疫性。这种变化仅源于ImmE6中的一个单一突变，即从色氨酸48突变为半胱氨酸，与ImmE3序列中的残基相同。此外，我们通过immE3和immE6之间的同源重组构建了一系列嵌合基因。对这些嵌合免疫基因的表征证实了上述发现，即大肠杆菌素E3和E6的主要区别仅在于ImmE3蛋白的半胱氨酸48。
