Identification and Characterization of the Alternative σ²⁸ Factor in Treponema denticola.

FliA (also known as σ²⁸), a member of the bacterial σ⁷⁰ 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 σ²⁸ of Escherichia coli, binds to σ²⁸-dependent promoters, and can functionally replace the σ²⁸ of E. coli. However, unlike its counterparts from other bacteria, <i>TDE2683</i> cannot be deleted, suggesting its essential role in the survival of T. denticola. <i>In vitro</i> site-directed mutagenesis revealed that E221 and V231, two conserved residues in the σ₄ region of σ²⁸, are indispensable for the binding activity of TDE2683 to the σ²⁸-dependent promoter. We then mutated these two residues in <i>T. denticola</i> 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 σ²⁸ 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. <b>IMPORTANCE</b> 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 σ²⁸ 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 σ²⁸ in spirochetes.