The Gram-negative bacterium Neisseria gonorrhoeae is the only causative agent of the sexually transmitted disease gonorrhea. The gonococcal (Gc) pilus is an important virulence factor of this human pathogen. Since Gc do not possess a Type II or Type III secretion apparatus, this Type IV pilus has an important role in host-pathogen interactions. The Gc pilus is involved in many aspects of gonococcal pathogenesis. First, the pilus functions in the initiating events of colonization by enhancing the ability of the bacterium to adhere to and interact with cells of the human host. Second, the pilus is involved in allowing the efficient transport of DNA into the bacterial cell for genetic transformation. Third, it is involved in twitching motility, a specialized form of locomotion expressed by all Type IV piliated bacteria. Much has been learned about the components that make up the Gc pilus assembly apparatus in the past several years, but how the pilus and its assembly apparatus act to promote adherence, DNA transformation, and twitching motility remains largely undefined. We propose a series of experiments to further our understanding of how the Gc pilus provides these functions for pathogenesis. In Aim 1 will utilize saturating mutagenesis to isolate loss-of-function and gain-of-function mutations in the secretin gene, PilQ. Loss-of-function mutations will define domains of PilQ required for expression, stability, and multimerization. Gain-of-function mutations will be screened for mutations that effect DNA transformation, antibiotic sensitivity, and twitching motility. Mutants will also be tested for changes in epithelial cell adherence. In Aim 2 will undertake a biochemical, yeast two-hybrid and biophysical analysis of three related predicted NTPases involved in orchestrating pilus function. These studies will provide new findings and insights into the structure and functions of the gonococcal pilus.