Under static growth conditions, hyperpiliated, nontwitching pilT and pilU mutants of Pseudomonas aeruginosa formed dense biofilms, showing that adhesion, not twitching motility, is necessary for biofilm initiation. Under flow conditions, the pilT mutant formed mushroom-like structures larger than those of the wild type but the pilU mutant was defective in biofilm formation. Therefore, twitching motility affects the development of biofilm structure, possibly through modulation of detachment.Type IV pili (TFP) are the major virulence-associated adhesins of Pseudomonas aeruginosa (8). TFP mediate binding to host cells and generate a form of surface translocation called twitching motility (15). P. aeruginosa is a model organism for the study of biofilms, attached communities of microorganisms that are medically significant due to their increased tolerance of antimicrobials (3). Recent studies have given contrasting evidence on the role of twitching motility in microcolony formation and biofilm differentiation. O'Toole and Kolter (12) showed that pilB, pilC, and pilY1 mutants, which have been found by electron microscopy to lack TFP, did not form microcolonies, a deficit attributed to the loss of twitching-mediated cell aggregation in early (8-h) biofilms. Other adhesins (cupA) can promote biofilm formation under static growth conditions in the absence of TFP, suggesting that neither TFPmediated adhesion nor motility is absolutely necessary (18). Heydorn et al. (9) showed that a TFP-dependent chemosensory mutant (⌬pilHIJK), which has aberrant twitching motility, formed dense microcolonies in a mature (98-h) biofilm. P. aeruginosa variants with enhanced biofilm-forming abilities have been noted to have a hyperpiliated phenotype (5), but the specific TFP genes involved were not identified. These hyperpiliated and hyperadhesive variants had a small rough colony phenotype similar to that recently described for drug-resistant P. aeruginosa variants with enhanced biofilm-forming ability isolated from cystic fibrosis patients undergoing antibiotic therapy (6).In P. aeruginosa, both PilT and its homologue PilU are required for twitching motility (21,22). PilT is thought to be a pilin depolymerase that disassembles TFP into pilin subunits, resulting in filament retraction (11), while the function of PilU is currently unknown. Mutations in either pilT or pilU result in a nontwitching, hyperpiliated phenotype (22). In this study, we used pilT and pilU mutants to examine the role of twitching motility in biofilm initiation and development under both static and flowing growth conditions. Adhesion, not twitching motility, is important for biofilm formation. Strains used in this study are listed in Table 1. pUCP20Gm was constructed by linearizing pUCP20 (10) at a unique ScaI site within the bla gene, followed by insertion of a gentamicin resistance cassette released from pUCGm (14) with SmaI. Primers pilTup (5Ј-GGGATCCGCTCATCCGGT GTTTTCCTT-3Ј) and pilTdown (5Ј-GGGAAGCTTCTTGAA TCCTAGACGCAGTT-3Ј), based on the Pseudomonas genome seq...

The opportunistic pathogen Pseudomonas aeruginosa expresses polar type IV pili (TFP), which are responsible for adhesion to various materials and twitching motility on surfaces. Twitching occurs by alternate extension and retraction of TFP, which arise from assembly and disassembly of pilin subunits at the base of the pilus. The ATPase PilB promotes pilin assembly, while the ATPase PilT or PilU or both promote pilin dissociation. Fluorescent fusions to two of the three ATPases (PilT and PilU) were functional, as shown by complementation of the corresponding mutants. PilB and PilT fusions localized to both poles, while PilU fusions localized only to the piliated pole. To identify the portion of the ATPases required for localization, sequential C-terminal deletions of PilT and PilU were generated. The conserved His and Walker B boxes were dispensable for polar localization but were required for twitching motility, showing that localization and function could be Type IV pili (TFP) are expressed by more than 25 gramnegative species, including Pseudomonas aeruginosa, Neisseria gonorrhoeae, Moraxella bovis, Vibrio cholerae, and enteropathogenic Escherichia coli (EPEC), many of which use TFP in their pathogenic interactions with hosts ranging from fungi and plants to animals and humans (32). The genetics of the TFP system have been extensively studied in P. aeruginosa, a ubiquitous opportunistic pathogen that predominately affects immunocompromised and cystic fibrosis patients. In P. aeruginosa, over 50 gene products to date have been shown to be required for the biosynthesis and function of TFP (21, 32), which localize to the same pole as the monotrichous flagellum (15). Each pilus is approximately 6 nm in diameter and up to several micrometers long (38) and is composed of repeating pilin subunits arranged in a helical conformation, forming a filamentous polymeric surface structure (13,14). In addition to being an adhesive structure, the pilus exhibits a surface-associated, flagellum-independent motility termed twitching motility (5). Twitching motility operates via pilus extrusion, surface attachment of the pilus tip, and pilus retraction to convey the cell toward the point of adhesion (34,48). This process requires the function of TFP ATPases, which are thought to use ATP to generate the mechanical forces required for pilus assembly and disassembly (22,33,53). In P. aeruginosa, the PilB ATPase powers the extrusion of the pilus, while the PilT ATPase is responsible for pilus retraction (32,56). A third ATPase, PilU (a paralogue of PilT), is also required for twitching motility, although its exact function is unclear (57).TFP ATPases are members of the AAAϩ (ATPases associated with cellular activities) family of motor proteins which function as ring-shaped oligomers (22). AAAϩ ATPases can also be found in macromolecule transport systems, such as type II secretion (T2S) and type IV secretion (T4S) systems in gram-negative bacteria and DNA uptake systems in grampositive bacteria (40,41). These proteins are characterized...

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