Anti-s²⁸ Factor FlgM Regulates Flagellin Gene Expression and Flagellar Polarity of Treponema denticola

FlgM, an antagonist of FliA (also known as s²⁸), inhibits transcription of bacterial class 3 flagellar genes. It does so primarily through binding to free s²⁸ to prevent it from forming a complex with core RNA polymerase. We recently identified an FliA homolog (FliA_Td) in the oral spirochete Treponema denticola; however, its antagonist FlgM remained uncharacterized. Herein, we provide several lines of evidence that TDE0201 functions as an antagonist of FliA_Td. TDE0201 is structurally similar to FlgM proteins, although its sequence is not conserved. Heterologous expression of TDE0201 in Escherichia coli inhibits its flagellin gene expression and motility. Biochemical and mutational analyses demonstrate that TDE0201 binds to FliA_Td and prevents it from binding to the s²⁸-dependent promoter. Deletions of flgM genes typically enhance bacterial class 3 flagellar gene expression; however, deletion of TDE0201 has an opposite effect (e.g., the mutant has a reduced level of flagellins). Follow-up studies revealed that deletion of TDE0201 leads to FliA_Td turnover, which in turn impairs the expression of flagellin genes. Swimming plate, cell tracking, and cryo-electron tomography analyses further disclosed that deletion of TDE0201 impairs spirochete motility and alters flagellar number and polarity: i.e., instead of having bipolar flagella, the mutant has flagella only at one end of cells. Collectively, these results indicate that TDE0201 is a FlgM homolog but acts differently from its counterparts in other bacteria.