Moore D, Hamilton C M, Maneewannakul K, Mintz Y, Frost L S, Ippen-Ihler K
Department of Medical Microbiology and Immunology, Texas A&M University, College Station 77843.
J Bacteriol. 1993 Mar;175(5):1375-83. doi: 10.1128/jb.175.5.1375-1383.1993.
The Escherichia coli F plasmid gene required for amino-terminal acetylation of F-pilin subunits was identified. Using Western blots (immunoblots), we assayed the reaction of monoclonal antibodies with F-pilin polypeptides in inner membrane preparations from various F mutant strains. It was known that JEL92 recognizes an internal pilin epitope and JEL93 recognizes the acetylated amino-terminal sequence (L.S. Frost, J.S. Lee, D.G. Scraba, and W. Paranchych, J. Bacteriol. 168:192-198, 1986). As expected, neither antibody reacted with inner membranes from F- cells or Flac derivatives that do not synthesize pilin. Mutations that affected the individual activities of F tra genes traA, -B, -C, -D, -E, -F, -G, -H, -I, -J, -K, -L, -M, -N, -P, -R, -U, -V and -W or trb genes trbA, -B, -C, -D, -E, -G, -H, and -I did not prevent JEL92 or JEL93 recognition of membrane pilin. However, Hfr deletion mutants that lacked the most-distal transfer region genes did not express pilin that reacted with JEL93. Nevertheless, all strains that retained traA and traQ did express JEL92-reactive pilin polypeptides. Analysis of strains expressing cloned tra segments showed that traA and traQ suffice for synthesis of JEL92-reactive pilin, but synthesis of JEL93-reactive pilin is additionally dependent on traX. We concluded that the traX product is required for acetylation of F pilin. Interestingly, our data also showed that TraA+ TraQ+ cells synthesize two forms of pilin which migrate at approximately 7 and 8 kDa. In TraX+ cells, both become acetylated and react with JEL93. Preparations of wild-type F-pilus filaments contain both types of subunits.
鉴定出了F菌毛亚基氨基末端乙酰化所需的大肠杆菌F质粒基因。我们使用蛋白质免疫印迹法（免疫印迹），检测了单克隆抗体与来自各种F突变菌株内膜制剂中F菌毛多肽的反应。已知JEL92识别菌毛内部表位，JEL93识别乙酰化的氨基末端序列（L.S.弗罗斯特、J.S.李、D.G.斯克拉巴和W.帕兰奇奇，《细菌学杂志》168:192 - 198，1986年）。正如预期的那样，两种抗体都不与不合成菌毛的F⁻细胞或Flac衍生物的内膜发生反应。影响F菌毛基因traA、-B、-C、-D、-E、-F、-G、-H、-I、-J、-K、-L、-M、-N、-P、-R、-U、-V和-W或trb基因trbA、-B、-C、-D、-E、-G、-H和-I个别活性的突变，并不妨碍JEL92或JEL93对膜菌毛的识别。然而，缺乏最远端转移区域基因的高频重组（Hfr）缺失突变体不表达与JEL93发生反应的菌毛。尽管如此，所有保留traA和traQ的菌株确实表达与JEL92发生反应的菌毛多肽。对表达克隆tra片段的菌株的分析表明，traA和traQ足以合成与JEL92发生反应的菌毛，但与JEL93发生反应的菌毛的合成还额外依赖于traX。我们得出结论，traX产物是F菌毛乙酰化所必需的。有趣的是，我们的数据还表明，TraA⁺TraQ⁺细胞合成两种形式的菌毛，其迁移率约为7和8 kDa。在TraX⁺细胞中，两者都被乙酰化并与JEL93发生反应。野生型F菌毛丝制剂包含这两种类型的亚基。
