Jack Bravo (@jpkbravo.bsky.social)

Temperate phages enhance host fitness via RNA-guided flagellar remodeling Bacterial flagella drive motility and chemotaxis while also playing critical roles in host-pathogen interactions, as their oligomeric subunit, flagellin, is specifically recognized by the mammalian immune system and flagellotropic bacteriophages. We recently discovered a family of phage-encoded, RNA-guided transcription factors known as TldR that regulate flagellin expression. However, the biological significance for this regulation, particularly in the context of host fitness, remained unknown. By focusing on a human clinical Enterobacter isolate that encodes a Flagellin Remodeling prophage (FRφ), here we show that FRφ exploits the combined action of TldR and its flagellin isoform to dramatically alter the flagellar composition and phenotypic properties of its host. This transformation has striking biological consequences, enhancing bacterial motility and mammalian immune evasion, and structural studies by cryo-EM of host- and prophage-encoded filaments reveal distinct architectures underlying these physiological changes. Moreover, we find that FRφ improves colonization in the murine gut, illustrating the beneficial effect of prophage-mediated flagellar remodeling in a host-associated environment. Remarkably, flagellin-regulating TldR homologs emerged multiple times independently, further highlighting the strong selective pressures that drove evolution of RNA-guided flagellin control. Collectively, our results reveal how RNA-guided transcription factors emerged in a parallel evolutionary path to CRISPR-Cas and were co-opted by phages to remodel the flagellar apparatus and enhance host fitness. ### Competing Interest Statement M.W.G.W. is a co-founder of Can9 Bioengineering. S.H.S. is a co-founder and scientific advisor to Dahlia Biosciences, a scientific advisor to CrisprBits and Prime Medicine, and an equity holder in Dahlia Biosciences and CrisprBits. A patent application has been filed related to this work (PCT Patent Application No. PCT/US24/40027). The remaining authors declare no competing interests. National Science Foundation, https://ror.org/021nxhr62, DGE-2036197, CAREER 2239685 National Institutes of Health, NIH DP2AI177904, R01CA259634, U24GM129539 Spain Ministry of Science Innovation and Universities Grant, PID2023-147463NB-I00 Simons Foundation, SF349247 Kinship Foundation Pew Biomedical Scholarship Irma T. Hirschl Career Scientist Award Howard Hughes Medical Institute Columbia University Irving Medical Center Deans Office Vagelos Precision Medicine Fund https://www.biorxiv.org/content/10.1101/2025.07.22.666180v1