Department of Oral Craniofacial Molecular Biology, Virginia Commonwealth University, Richmond, Virginia, USA.
Department of Microbiology and Immunology, Virginia Commonwealth University, Richmond, Virginia, USA.
J Bacteriol. 2022 Sep 20;204(9):e0024822. doi: 10.1128/jb.00248-22. Epub 2022 Aug 31.
FliA (also known as σ), a member of the bacterial σ family of transcription factors, directs RNA polymerase to flagellar late (class 3) promoters and initiates transcription. FliA has been studied in several bacteria, yet its role in spirochetes has not been established. In this report, we identify and functionally characterize a FliA homolog (TDE2683) in the oral spirochete Treponema denticola. Computational, genetic, and biochemical analyses demonstrated that TDE2683 has a structure similar to that of the σ of Escherichia coli, binds to σ-dependent promoters, and can functionally replace the σ of E. coli. However, unlike its counterparts from other bacteria, cannot be deleted, suggesting its essential role in the survival of T. denticola. site-directed mutagenesis revealed that E221 and V231, two conserved residues in the σ region of σ, are indispensable for the binding activity of TDE2683 to the σ-dependent promoter. We then mutated these two residues in and found that the mutations impair the expression of flagellin and chemotaxis genes and bacterial motility as well. Cryo-electron tomography analysis further revealed that the mutations disrupt the flagellar symmetry (i.e., number and placement) of T. denticola. Collectively, these results indicate that TDE2683 is a σ transcription factor that regulates the class 3 gene expression and controls the flagellar symmetry of T. denticola. To the best of our knowledge, this is the first report establishing the functionality of FliA in spirochetes. Spirochetes are a group of medically important but understudied bacteria. One of the unique aspects of spirochetes is that they have periplasmic flagella (PF, also known as endoflagella) which give rise to their unique spiral shape and distinct swimming behaviors and play a critical role in the pathophysiology of spirochetes. PF are structurally similar to external flagella, but the underpinning mechanism that regulates PF biosynthesis and assembly remains largely unknown. By using the oral spirochete Treponema denticola as a model, this report provides several lines of evidence that FliA, a σ transcriptional factor, regulates the late flagellin gene (class 3) expression, PF assembly, and flagellar symmetry as well, which provides insights into flagellar regulation and opens an avenue to investigate the role of σ in spirochetes.
FliA（也称为σ）是细菌σ家族转录因子的成员，它指导 RNA 聚合酶识别鞭毛晚期（第 3 类）启动子并启动转录。FliA 在几种细菌中进行了研究，但在螺旋体中的作用尚未确定。在本报告中，我们鉴定并功能表征了口腔螺旋体牙龈卟啉单胞菌中的 FliA 同源物（TDE2683）。计算、遗传和生化分析表明，TDE2683 的结构类似于大肠杆菌的σ，可与σ依赖性启动子结合，并可在功能上替代大肠杆菌的σ。然而，与其他细菌的对应物不同，不能被删除，这表明它在牙龈卟啉单胞菌的生存中起着至关重要的作用。定点突变显示，σ区域中的两个保守残基 E221 和 V231 对于 TDE2683 与σ依赖性启动子的结合活性是必不可少的。然后我们在突变中改变了这两个残基，发现这些突变会损害鞭毛蛋白和趋化性基因的表达以及细菌的运动性。低温电子断层摄影术分析进一步揭示了这些突变破坏了牙龈卟啉单胞菌的鞭毛对称性（即数量和位置）。总的来说，这些结果表明 TDE2683 是一种σ转录因子，可调节第 3 类基因的表达并控制牙龈卟啉单胞菌的鞭毛对称性。据我们所知，这是第一个在螺旋体中建立 FliA 功能的报告。螺旋体是一组具有重要医学意义但研究不足的细菌。螺旋体的一个独特方面是它们具有周质鞭毛（PF，也称为内鞭毛），这赋予了它们独特的螺旋形状和独特的游动行为，并在螺旋体的病理生理学中发挥着关键作用。PF 在结构上与外部鞭毛相似，但调节 PF 生物合成和组装的基础机制在很大程度上仍然未知。本报告以口腔螺旋体牙龈卟啉单胞菌为模型，提供了几条证据表明，σ转录因子 FliA 调节晚期鞭毛蛋白基因（第 3 类）的表达、PF 组装和鞭毛对称性，这为鞭毛调节提供了深入了解，并为研究σ在螺旋体中的作用开辟了途径。
