Discovery of PilU as a second Type IV pilus retraction motor in Myxococcus xanthus

Type IV pili (T4P) drive social (S) motility in Myxococcus xanthus through cycles of extension and retraction powered by the ATPases PilB and PilT. Although the canonical retraction ATPase PilT is essential for force generation, M. xanthus encodes four PilT-like paralogs whose contributions to motility remain unclear. Here, we identify MXAN_1995 as the long-sought PilU protein that serves as a second T4P retraction motor. A frameshift mutation or deletion of pilU abolishes S-motility, while preserving pilus assembly and exopolysaccharide (EPS) production, phenocoping the pilT mutant. Single-cell analyses revealed that {Delta}pilU mutants exhibit rare, low-force movements, consistent with a role for PilU in force generation. Fluorescence microscopy showed PilU localizes predominantly to cell poles, similar to PilT; this localization is independent of PilT but partially dependent on core T4P assembly proteins. Notably, calcium differentially modulates motility, enhancing movement in wild-type cells while suppressing it in {Delta}pilU mutants, indicating a role for PilU under varying environmental conditions. Structural modeling, together with an intragenic suppressor, highlights a regulatory function for the intrinsically disordered C-terminal region of PilU. Together, our findings establish PilU as a secondary retraction ATPase and uncover a dual-motor retraction system that is environmentally responsive and mechanically tunable. ImportanceT4P are widespread motility and adhesion systems that enable bacteria to move, interact, and form multicellular communities. While the primary retraction ATPase PilT is well characterized, the function of additional PilT-like proteins remains unclear in many species. This work provides the first mechanistic characterization of PilU in Myxococcus xanthus, a model for multicellular behavior and T4P biology. We show that PilU is essential for productive T4P retraction, functioning as an accessory motor that enhances or stabilizes PilT-driven force generation. We further reveal that PilU activity is modulated by environmental calcium and depends on a flexible C-terminal region that influences motor complex dynamics. These findings uncover a dual-motor architecture that enables adaptive control of T4P retraction in response to environmental cues.