Phase Variation of Flagella and Toxins in Clostridioides difficile is Mediated by Selective Rho-dependent Termination

• Published in: Journal of molecular biology 2024-03, Vol.436 (6), p.168456, Article 168456
• Main Authors: Warren Norris, Mercedes A.H., Plaskon, Dylan M., Tamayo, Rita
• Format: Article
• Language: English
• Subjects: Bacterial Proteins - genetics; Bacterial Proteins - metabolism; Bacterial Toxins - genetics; Bacterial Toxins - metabolism; Clostridioides difficile - genetics; Clostridioides difficile - metabolism; Flagella - genetics; Flagella - metabolism; flagellar switch; Gene Expression Regulation, Bacterial; gene regulation; heterogeneity; motility; Phase Variation; RNA, Messenger - metabolism; transcription termination
• ISSN: 0022-2836; 1089-8638; 1089-8638
• DOI: 10.1016/j.jmb.2024.168456

[Display omitted] •How does Rho termination mediate flagellar phase variation in C. difficile?•Rho binds the left inverted repeat region of flg-OFF but not flg-ON mRNA.•The N-terminal insertion domain is required for efficient flg-OFF termination in vivo.•The RNA binding domain discriminates between flg-ON and flg-OFF RNA.•Broadens our understanding of Rho mediated termination in AT-rich organisms. Clostridioides difficile is an intestinal pathogen that exhibits phase variation of flagella and toxins through inversion of the flagellar (flg) switch controlling flagellar and toxin gene expression. The transcription termination factor Rho preferentially inhibits swimming motility of bacteria with the ‘flg-OFF’ switch sequence. How C. difficile Rho mediates this selectivity was unknown. C. difficile Rho contains an N-terminal insertion domain (NID) which is found in a subset of Rho orthologues and confers diverse functions. Here we determined how Rho distinguishes between flg-ON and -OFF mRNAs and the roles of the NID and other domains of C. difficile Rho. Using in vitro ATPase assays, we determined that Rho specifically binds a region containing the left inverted repeat of the flg switch, but only of flg-OFF mRNA, indicating that differential termination is mediated by selective Rho binding. Using a suite of in vivo and in vitro assays in C. difficile, we determined that the NID is essential for Rho termination of flg-OFF mRNA, likely by influencing the ability to form stable hexamers, and the RNA binding domain is critical for flg-OFF specific termination. This work gives insight into the novel mechanism by which Rho interacts with flg mRNA to mediate phase variation of flagella and toxins in C. difficile and broadens our understanding of Rho-mediated termination in an organism with an AT-rich genome.