Rahman, M.; Subedi, K.; Harms, A.; Wall, D.; Treuner-Lange, A.

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.