Type IV pili (T4P) are long, thin, flexible surface appendages used by various bacteria for surface adhesion, cell-cell aggregation, DNA uptake, biofilm formation and motility. Pseudomonas aeruginosa is a Gram-negative opportunistic pathogen, and uses T4P as a key virulence factor to infect immunocompromised individuals. Four subcomplexes make up a functional T4P system in P. aeruginosa and the role of the alignment subcomplex is to physically connect the outer membrane pore with the inner membrane motor, allowing for efficient extrusion of the pilus fibre from the cell. Two alignment subcomplex proteins, PilN and PilO, form heterodimers and are required for proper function of the system. These proteins may be able to transduce signals between various T4P components to indicate extension and/or retraction of the pilus fibre. This thesis focused on characterization of the interaction interfaces between PilN and PilO, and on understanding the dynamics required for proper function of the system. We show that although PilN and PilO make extensive interaction contacts throughout their lengths, single point substitutions at key residues can successfully disrupt the function of the T4P system. Crosslinking PilN and PilO as homo- or heterodimers can disrupt motility and surface piliation, indicating that interfaces between these proteins must be dynamic to allow proper T4P function. A high resolution X-ray crystal structure of PilO was solved and exhibits new structural features previously unidentified. This work furthers our understanding of the structures and regions of interaction between PilN and PilO, as well as defining a role for these proteins in extension and retraction. / Dissertation / Doctor of Philosophy (PhD) / Pseudomonas aeruginosa is an opportunistic bacterium, able to infect individuals with weakened immune systems. It attaches to and moves along surfaces using long, thin, sticky, retractable fibres known as type IV pili. Similar to a grappling gun, a functional type IV pilus system requires four subcomplexes working in unison to allow for the extension, adherence, and retraction of pilus fibres, which pulls the cell forward towards the point of attachment. Two key proteins, PilN and PilO, are bound to each other and allow for efficient extension and retraction of the pilus fibre. This study focused on characterization of the interactions of PilN and PilO, and on understanding whether dynamic rearrangements of the interfaces between these proteins is required for proper function of the system. We show that although these proteins have extensive interaction interfaces, single residue substitutions in either of them can disrupt the ability of the bacteria to properly extend and/or retract their pili. This work furthers our understanding of the structures and regions of interaction between PilN and PilO, providing information that might allow disruption of these interfaces to block bacterial attachment or motility, both of which are important for infection.