Expression of type IV pili (Tfp) correlates with the ability of Neisseria gonorrhoeae to colonize the human host, as well as with adherence to human epithelial tissue, twitching motility, competence for natural transformation, and autoagglutination. N. gonorrhoeae PilF (required for Tfp biogenesis) and PilT (required for twitching motility and transformation) share significant identities with members of a family of putative ATPases involved in membrane trafficking of macromolecules. An open reading frame downstream of the pilT locus encoding a 408-amino-acid protein with 33% identity with the gonococcal PilT protein and 45% identity with the PilU protein in Pseudomonas aeruginosa was characterized, and the corresponding gene was designated pilU. Unlike N. gonorrhoeae pilT mutants, pilU mutants express twitching motility and are competent for DNA transformation. However, loss-of-function mutations in pilU increased bacterial adherence to ME-180 human epithelial cells eightfold and disrupted in vitro Tfp-associated autoagglutination. Comparative alignment of N. gonorrhoeae PilU with other members of the TrbB-like family of traffic ATPases revealed a conserved carboxyterminal domain unique to family members which are not essential for Tfp biogenesis but which specifically modify Tfp-associated phenotypes. Studies of the pilT-pilU locus by using Northern blotting, transcriptional fusions, and reverse transcription-PCR showed that the two genes encoding closely related proteins with dissimilar effects on Tfp phenotypes are transcribed from a single promoter.

The ability of bacteria to establish complex communities on surfaces is believed to require both bacterial–substratum and bacterial–bacterial interactions, and type IV pili appear to play a critical but incompletely defined role in both these processes. Using the human pathogen Neisseria gonorrhoeae, spontaneous mutants defective in bacterial self‐aggregative behaviour but quantitatively unaltered in pilus fibre expression were isolated by a unique selective scheme. The mutants, carrying single amino acid substitutions within the conserved amino‐terminal domain of the pilus fibre subunit, were reduced in the ability to adhere to a human epithelial cell line. Co‐expression of the altered alleles in the context of a wild‐type pilE gene confirmed that they were dominant negative with respect to aggregation and human cell adherence. Strains expressing two copies of the altered alleles produced twice as much purifiable pili but retained the aggregative and adherence defects. Finally, the defects in aggregative behaviour and adherence of each of the mutants were suppressed by a loss‐of‐function mutation in the twitching motility gene pilT. The correlations between self‐aggregation and the net capacity of the microbial population to adhere efficiently demonstrates the potential significance of bacterial cell–cell interactions to colonization.