Kloser A W, Reading J T, McDermott T, Stidham R, Misra R
Department of Microbiology, Arizona State University, Tempe, Arizona 85287, USA.
J Bacteriol. 2001 Jan;183(1):264-9. doi: 10.1128/JB.183.1.264-269.2001.
We employed two separate genetic approaches to examine the roles of various OmpF residues in assembly. In one approach, intragenic suppressors of a temperature-sensitive OmpF assembly mutant carrying a W214E substitution were sought at 42 degrees C, or at 37 degrees C in a genetic background lacking the periplasmic folding factor SurA. In the majority of cases (58 out of 61 revertants), the suppressors mapped either at the original site (position 214) or two residues downstream from it. In the remaining three revertants that were obtained in a surA background, an alteration of N230Y was located 16 residues away from the original site. The N230Y suppressor also corrected OmpF315 assembly at 42 degrees C in a surA(+) background, indicating that the two different physiological environments imposed similar assembly constraints. The specificity of N230Y was tested against five different residues at position 214 of mature OmpF. Clear specificity was displayed, with maximum suppression observed for the original substitution at position 214 (E214) against which the N230Y suppressor was isolated, and no negative effect on OmpF assembly was noted when the wild-type W214 residue was present. The mechanism of suppression may involve compensation for a specific conformational defect. The second approach involved the application of informational suppressors (Su-tRNA) in combination with ompF amber mutations to generate variant OmpF proteins. In this approach we targeted the Y40, Q66, W214, and Y231 residues of mature OmpF and replaced them with S, Q, L, and Y through the action of Su-tRNAs. Thus, a total of 16 variant OmpF proteins were generated, of which three were identical to the parental protein, and two variants carrying W214Q and Y231Q substitutions were similar to assembly-defective proteins isolated previously (R. Misra, J. Bacteriol. 175:5049-5056, 1993). The results obtained from these analyses provided useful information regarding the compatibility of various alterations in OmpF assembly.
我们采用了两种不同的遗传学方法来研究各种外膜蛋白F（OmpF）残基在组装过程中的作用。在一种方法中，在42℃下，或在缺乏周质折叠因子SurA的遗传背景中于37℃下，寻找携带W214E替代的温度敏感型OmpF组装突变体的基因内抑制子。在大多数情况下（61个回复突变体中的58个），抑制子定位于原始位点（第214位）或其下游两个残基处。在surA背景中获得的其余三个回复突变体中，N230Y的改变位于距原始位点16个残基处。N230Y抑制子在surA(+)背景中也能在42℃下校正OmpF315的组装，这表明两种不同的生理环境施加了相似的组装限制。针对成熟OmpF第214位的五个不同残基测试了N230Y的特异性。显示出明显的特异性，对于分离出N230Y抑制子的原始替代（E214），在第214位观察到最大抑制，并且当存在野生型W214残基时，未注意到对OmpF组装有负面影响。抑制机制可能涉及对特定构象缺陷的补偿。第二种方法涉及将信息抑制子（Su-tRNA）与ompF琥珀突变结合使用，以产生变体OmpF蛋白。在这种方法中，我们针对成熟OmpF的Y40、Q66、W214和Y231残基，并通过Su-tRNA的作用将它们替换为S、Q、L和Y。因此，总共产生了16种变体OmpF蛋白，其中三种与亲本蛋白相同，并且两种携带W214Q和Y231Q替代的变体类似于先前分离的组装缺陷蛋白（R. Misra，《细菌学杂志》175:5049 - 5056，1993）。从这些分析中获得的结果提供了关于OmpF组装中各种改变的兼容性的有用信息。
