Fresh extension ofVibrio choleraecompetence type IV pili predisposes them for motor-independent retraction

Abstract

Bacteria utilize dynamic appendages called type IV pili (T4P) to interact with their environment and mediate a wide variety of functions. Pilus extension is mediated by an extension ATPase motor, commonly called PilB, in all T4P. Pilus retraction, however, can either occur with the aid of an ATPase motor, or in the absence of a retraction motor. While much effort has been devoted to studying motor-dependent retraction, the mechanism and regulation of motor-independent retraction remains poorly characterized. We have previously demonstrated that Vibrio cholerae competence T4P undergo motor-independent retraction in the absence of the dedicated retraction ATPases PilT and PilU. Here, we utilize this model system to characterize the factors that influence motor-independent retraction. We find that freshly extended pili frequently undergo motor-independent retraction, but if these pili fail to retract immediately, they remain statically extended on the cell surface. Importantly, we show that these static pili can still undergo motor-dependent retraction via tightly regulated ectopic expression of PilT, suggesting that these T4P are not broken, but simply cannot undergo motor-independent retraction. Through additional genetic and biophysical characterization of pili, we suggest that pilus filaments undergo conformational changes during dynamic extension and retraction. We propose that only some conformations, like those adopted by freshly extended pili, are capable of undergoing motor-independent retraction. Together, these data highlight the versatile mechanisms that regulate T4P dynamic activity and provide additional support for the long-standing hypothesis that motor-independent retraction occurs via spontaneous depolymerization.

Pseudomonas aeruginosa differentiates substrate rigidity using retraction of type IV pili

“…In addition to the effect of force on retraction speed we measured using the optical trap, TFP also exhibit a force-induced reversible stretching under extensile loads likely due to a conformational change in the PilA monomers. This unfolding like feature was first demonstrated in gonococcal TFP but has also been shown for TFP in Vibrio cholerae and P. aeruginosa [38,[43][44][45][46]. As shown in Supplementary Fig.…”

Retraction ATPase motors from three orthologous type IVa pilus systems support promiscuous retraction of the Vibrio cholerae competence pilus

“…As a control, we also ectopically expressed V. cholerae pilT (pilTVc). Competence pili in V. cholerae promote DNA uptake during natural transformation, a function that requires both pilus extension and retraction (8,14). Thus, we functionally assessed competence pilus dynamic activity by performing natural transformation assays.…”

“…Chitin-independent natural transformation assays were performed exactly as previously described (8,14). Briefly, strains were grown overnight in 3 mL of LB broth containing 100 μM IPTG at 30 o C rolling.…”