Russell P W, Orndorff P E
Department of Microbiology, Pathology, and Parasitology, North Carolina State University College of Veterinary Medicine, Raleigh 27606.
J Bacteriol. 1992 Sep;174(18):5923-35. doi: 10.1128/jb.174.18.5923-5935.1992.
We describe the characterization of two genes, fimF and fimG (also called pilD), that encode two minor components of type 1 pili in Escherichia coli. Defined, in-frame deletion mutations were generated in vitro in each of these two genes. A double mutation that had deletions identical to both single lesions was also constructed. Examination of minicell transcription and translation products of parental and mutant plasmids revealed that, as predicted from the nucleotide sequence and previous reports, the fimF gene product was a protein of ca. 16 kDa and that the fimG gene product was a protein of ca. 14 kDa. Each of the constructions was introduced, via homologous recombination, into the E. coli chromosome. All three of the resulting mutants produced type 1 pili and exhibited hemagglutination of guinea pig erythrocytes. The latter property was also exhibited by partially purified pili isolated from each of the mutants. Electron microscopic examination revealed that the fimF mutant had markedly reduced numbers of pili per cell, whereas the fimG mutant had very long pili. The double mutant displayed the characteristics of both single mutants. However, pili in the double mutant were even longer than those seen in the fimG mutant, and the numbers of pili were even fewer than those displayed by the fimF mutant. All three mutants could be complemented in trans with a single-copy-number plasmid bearing the appropriate parental gene or genes to give near-normal parental piliation. On the basis of the phenotypes exhibited by the single and double mutants, we believe that the fimF gene product may aid in initiating pilus assembly and that the fimG product may act as an inhibitor of pilus polymerization. In contrast to previous studies, we found that neither gene product was required for type 1 pilus receptor binding.
我们描述了两个基因fimF和fimG（也称为pilD）的特征，它们编码大肠杆菌1型菌毛的两个次要成分。在体外针对这两个基因分别产生了明确的框内缺失突变。还构建了一个与两个单突变缺失相同的双突变体。对亲本和突变体质粒的小细胞转录和翻译产物的检测表明，正如从核苷酸序列和先前报告所预测的那样，fimF基因产物是一种约16 kDa的蛋白质，而fimG基因产物是一种约14 kDa的蛋白质。通过同源重组将每个构建体引入大肠杆菌染色体。所有三个所得突变体均产生1型菌毛，并表现出豚鼠红细胞的血凝作用。从每个突变体中分离出的部分纯化菌毛也表现出后一种特性。电子显微镜检查显示，fimF突变体细胞表面的菌毛数量明显减少，而fimG突变体的菌毛非常长。双突变体表现出两个单突变体的特征。然而，双突变体中的菌毛比fimG突变体中的更长，菌毛数量比fimF突变体中的更少。所有三个突变体都可以用携带适当亲本基因的单拷贝数质粒进行反式互补，以产生接近正常亲本的菌毛形成。基于单突变体和双突变体表现出的表型，我们认为fimF基因产物可能有助于启动菌毛组装，而fimG产物可能作为菌毛聚合反应的抑制剂。与先前的研究相反，我们发现1型菌毛受体结合不需要这两种基因产物。
