Minnich S A, Ohta N, Taylor N, Newton A
Department of Molecular Biology, Princeton University, New Jersey 08544.
J Bacteriol. 1988 Sep;170(9):3953-60. doi: 10.1128/jb.170.9.3953-3960.1988.
Caulobacter crescentus incorporates two distinct, but related proteins into the polar flagellar filament: a 27-kilodalton (kDa) flagellin is assembled proximal to the hook and a 25-kDa flagellin forms the distal end of the filament. These two proteins and a third, related flagellin protein of 29 kDa are encoded by three tandem genes (alpha-flagellin cluster) in the flaEY gene cluster (S.A. Minnich and A. Newton, Proc. Natl. Acad. Sci. USA 84: 1142-1146, 1987). Since point mutations in flagellin genes had not been isolated their requirement for flagellum function and fla gene expression was not known. To address these questions, we developed a gene replacement protocol that uses cloned flagellin genes mutagenized by either Tn5 transposons in vivo or the replacement of specific DNA fragments in vitro by the antibiotic resistance omega cassette. Analysis of gene replacement mutants constructed by this procedure led to several conclusions. (i) Mutations in any of the three flagellin genes do not cause complete loss of motility. (ii) Tn5 insertions in the 27-kDa flagellin gene and a deletion mutant of this gene do not synthesize the 27-kDa flagellin, but they do synthesize wild-type levels of the 25-kDa flagellin, which implies that the 27-kDa flagellin is not required for expression and assembly of the 25-kDa flagellin; these mutants show slightly impaired motility on swarm plates. (iii) Mutant PC7810, which is deleted for the three flagellin genes in the flaEY cluster, does not synthesize the 27- or 29-kDa flagellin, and it is significantly more impaired for motility on swarm plates than mutants with defects in only the 27-kDa flagellin gene. The synthesis of essentially normal levels of 25-kDa flagellin by strain PC7810 confirms that additional copies of the 25-kDa flagellin map outside the flaEY cluster (beta-flagellin cluster) and that these flagellin genes are active. Thus, while the 29- and 27-kDa flagellins are not absolutely essential for motility in C. crescentus, their assembly into the flagellar structure is necessary for normal flagellar function.
一种27千道尔顿（kDa）的鞭毛蛋白在钩近端组装，一种25-kDa的鞭毛蛋白形成鞭毛丝的远端。这两种蛋白质以及第三种相关的29-kDa鞭毛蛋白由flaEY基因簇中的三个串联基因（α-鞭毛蛋白簇）编码（S.A. Minnich和A. Newton，《美国国家科学院院刊》84：1142 - 1146，1987）。由于尚未分离到鞭毛蛋白基因中的点突变，它们对鞭毛功能和fla基因表达的需求尚不清楚。为了解决这些问题，我们开发了一种基因替换方案，该方案使用通过体内Tn5转座子诱变的克隆鞭毛蛋白基因，或通过抗生素抗性ω盒体外替换特定DNA片段。对通过该程序构建的基因替换突变体的分析得出了几个结论。（i）三个鞭毛蛋白基因中任何一个的突变都不会导致运动能力完全丧失。（ii）27-kDa鞭毛蛋白基因中的Tn5插入以及该基因的缺失突变体不合成27-kDa鞭毛蛋白，但它们确实合成野生型水平的25-kDa鞭毛蛋白，这意味着27-kDa鞭毛蛋白对于25-kDa鞭毛蛋白的表达和组装不是必需的；这些突变体在群体平板上的运动能力略有受损。（iii）突变体PC7810缺失了flaEY簇中的三个鞭毛蛋白基因，不合成27-或29-kDa鞭毛蛋白，并且在群体平板上的运动能力比仅27-kDa鞭毛蛋白基因有缺陷的突变体受损更严重。PC7810菌株合成基本正常水平的25-kDa鞭毛蛋白，这证实了25-kDa鞭毛蛋白的额外拷贝位于flaEY簇（β-鞭毛蛋白簇）之外，并且这些鞭毛蛋白基因是活跃的。因此，虽然29-和27-kDa鞭毛蛋白对于新月柄杆菌的运动不是绝对必需的，但它们组装到鞭毛结构中对于正常的鞭毛功能是必要的。
