Type IV pili (T4P) are long adhesive surface filaments produced by bacteria and are a key virulence factor for many pathogens. T4P are produced by a dynamic intracellular nanomachine that facilitates the assembly (extension) and disassembly (retraction) of pili. Pilus dynamics are enabled by the motor subcomplex of the nanomachine, where cytoplasmic ATPases power pilus assembly (PilB) and disassembly (PilT and PilU). In many, but not all, T4P expressing bacteria – including our model organism Pseudomonas aeruginosa – two retraction ATPases are required for functional retraction, which can be assessed by measuring twitching motility. Deletion of pilT results in loss of twitching and phage susceptibility (another hallmark of pilus function) while deletion of pilU results in loss of twitching but retention of phage susceptibility, indicating pili can still be retracted. We hypothesized that PilU adds to the force of pilus retraction, facilitating disassembly when the fiber is under tension. We mutated ΔpilU and pilU::Tn5 strains with ethyl methanesulfonate and screened for gain-of-twitching mutants. Whole genome sequencing revealed multiple point mutations in the major pilin protein PilA or the pilus adhesin, PilY1. These point mutations were recapitulated in a ΔpilU strain and restored twitching to varying degrees. Complementation of pilA point mutants with pilU in trans influenced the twitching zone of only one mutant, and in trans expression of wild-type pilA resulted in a significant reduction in twitching in most. The contribution of PilU to the force of pilus retraction was further investigated by a polyacrylamide micropillar assay, where no pulling events could be detected for either ΔpilT or ΔpilU mutants. Exopolysaccharide production, a proxy for surface sensing, was uncoupled from twitching motility in the pilA point mutants. These results are a significant step forward to understanding what PilU does and, provides insight to the dynamics of the pilus fiber. / Thesis / Master of Science (MSc) / Pseudomonas aeruginosa is a bacterium that causes serious infections. P. aeruginosa uses adhesive, “grappling hook” filaments called Type IV pili (T4P) to stick to its hosts. T4P can be repeatedly extended and retracted, allowing the bacteria to crawl on surfaces (twitching) but making them susceptible to bacteriophages, viruses that attach to pili then kill the bacterial cells. The motor proteins PilT and PilU are required for twitching, but only PilT is essential for phage killing, implying that pili are retracted even when PilU is missing. Here we hypothesized that PilU is important for twitching because it helps generate force for retraction when pili are under tension. We isolated multiple mutations in pilus components that restored twitching in the absence of PilU, and propose that these mutations allow for easier retraction of pili. This information helps us understand how T4P help the bacteria to spread during infection.