Schmitz A, Josenhans C, Suerbaum S
Ruhr-Universität Bochum, Medizinische Mikrobiologie und Immunologie, Germany.
J Bacteriol. 1997 Feb;179(4):987-97. doi: 10.1128/jb.179.4.987-997.1997.
Flagellar motility has been shown to be an essential requirement for the ability of Helicobacter pylori to colonize the gastric mucosa. While some flagellar structural components have been studied in molecular detail, nothing was known about factors that play a role in the regulation of flagellar biogenesis. We have cloned and characterized an H. pylori homolog (named flbA) of the lcrD/flbF family of genes. Many proteins encoded by these genes are known to be involved in flagellar biogenesis or secretion of virulence-associated proteins via type III secretion systems. The H. pylori flbA gene (2,196 bp) is capable of coding for a predicted 732-amino-acid, 80.9-kDa protein that has marked sequence similarity with other known members of the LcrD/FlbF protein family. An isogenic strain with a mutation in the flbA gene was constructed by disruption of the gene with a kanamycin resistance cassette and electroporation-mediated allelic exchange mutagenesis. The mutant strain expressed neither the FlaA nor the FlaB flagellin protein. The expression of the FlgE hook protein was reduced in comparison with the wild-type strain, and the extent of this reduction was growth phase dependent. The flbA gene disruption was shown to downregulate the expression of these flagellar genes on the transcriptional level. The flbA mutants were aflagellate and completely nonmotile. Occasionally, assembled hook structures could be observed, indicating that export of axial flagellar filament components was still possible in the absence of the flbA gene product. The hydrophilic part of the FlbA protein was expressed in Escherichia coli, purified, and used to raise a polyclonal rabbit antiserum against the FlbA protein. Western blot experiments with this antiserum indicated that the FlbA protein is predominantly associated with the cytoplasmic membrane in H. pylori. The antiserum cross-reacted with two other proteins (97 and 43 kDa) whose expression was not affected by the flbA gene disruption and which might represent further H. pylori homologs of the LcrD/FlbF protein family.
鞭毛运动已被证明是幽门螺杆菌定殖于胃黏膜能力的一项基本要求。虽然一些鞭毛结构成分已在分子细节上得到研究，但对于在鞭毛生物合成调控中起作用的因素却一无所知。我们克隆并鉴定了lcrD/flbF基因家族的一个幽门螺杆菌同源基因（命名为flbA）。已知这些基因编码的许多蛋白质参与鞭毛生物合成或通过III型分泌系统分泌毒力相关蛋白。幽门螺杆菌flbA基因（2196 bp）能够编码一个预测的732个氨基酸、80.9 kDa的蛋白质，该蛋白质与LcrD/FlbF蛋白家族的其他已知成员具有显著的序列相似性。通过用卡那霉素抗性盒破坏该基因并用电穿孔介导的等位基因交换诱变构建了flbA基因突变的同基因菌株。突变菌株既不表达FlaA鞭毛蛋白也不表达FlaB鞭毛蛋白。与野生型菌株相比，FlgE钩蛋白的表达降低，且这种降低的程度取决于生长阶段。flbA基因破坏在转录水平上显示下调这些鞭毛基因的表达。flbA突变体无鞭毛且完全不运动。偶尔可观察到组装好的钩结构，这表明在没有flbA基因产物的情况下，轴向鞭毛丝成分的输出仍然是可能的。FlbA蛋白的亲水性部分在大肠杆菌中表达、纯化，并用于制备针对FlbA蛋白的兔多克隆抗血清。用该抗血清进行Western印迹实验表明，FlbA蛋白主要与幽门螺杆菌的细胞质膜相关。该抗血清与另外两种蛋白质（97 kDa和43 kDa）发生交叉反应，其表达不受flbA基因破坏的影响，这两种蛋白质可能代表LcrD/FlbF蛋白家族的其他幽门螺杆菌同源物。
