PilT and PilU are homohexameric ATPases that coordinate to retract type IVa pili

Abstract

Bacterial type IV pili are critical for diverse biological processes including horizontal gene transfer, surface sensing, biofilm formation, adherence, motility, and virulence. These dynamic appendages extend and retract from the cell surface. In many type IVa pilus systems, extension occurs through the action of an extension ATPase, often called PilB, while optimal retraction requires the action of a retraction ATPase, PilT. Many type IVa systems also encode a homolog of PilT called PilU. However, the function of this protein has remained unclear because pilU mutants exhibit inconsistent phenotypes among type IV pilus systems and because it is relatively understudied compared to PilT. Here, we study the type IVa competence pilus of Vibrio cholerae as a model system to define the role of PilU. We show that the ATPase activity of PilU is critical for pilus retraction in PilT Walker A and/or Walker B mutants. PilU does not, however, contribute to pilus retraction in ΔpilT strains. Thus, these data suggest that PilU is a bona fide retraction ATPase that supports pilus retraction in a PilT-dependent manner. We also found that a ΔpilU mutant exhibited a reduction in the force of retraction suggesting that PilU is important for generating maximal retraction forces. Additional in vitro and in vivo data show that PilT and PilU act as independent homo-hexamers that may form a complex to facilitate pilus retraction. Finally, we demonstrate that the role of PilU as a PilT-dependent retraction ATPase is conserved in Acinetobacter baylyi, suggesting that the role of PilU described here may be broadly applicable to other type IVa pilus systems.

c-di-GMP modulates type IV MSHA pilus retraction and surface attachment in Vibrio cholerae

“…6), suggesting that PilU can only function as a retraction ATPase for the MSHA system in the presence of PilT. Similar results have recently been reported for MSHA and ChiRP pili for O1 El Tor strain A1552, as well as for ChiRP in strain E7946, suggesting that this PilT-dependent PilU function is similarly conserved across O1 El Tor isolates 37,38 . c-di-GMP levels mediate MSHA production and retraction.…”

Competitive binding of independent extension and retraction motors explains the quantitative dynamics of type IV pili

“…Our analysis confirms that PilU is indeed not needed for retraction but does affect retraction speed (Supplementary Figure 6). Recent studies also suggest that these motors may have more complicated interactions 38,39 , and in the future our model could help tease apart the specific contributions of different mutants to the extension and retraction cycle.…”

“…We also note that our model abstracts the extension and retraction motors. In P. aeruginosa , PilB is the only known extension motor, PilT is considered the primary retraction motor, and PilU has been shown to affect retraction 22,38 . Our analysis confirms that PilU is indeed not needed for retraction but does affect retraction speed (Supplementary Figure 6).…”

A modular chromosomally integrated toolkit for ectopic gene expression inVibrio cholerae

“…All of these strains also harbored Pconstitutive-tfoX and ∆luxO mutations, which rendered these strains constitutively competent. Strains were tested for natural transformation using chitin-independent transformation assays exactly as previously described (36). The transforming DNA using in these experiments was 100 ng of a 6 kb ∆VC1807::Erm R PCR product.…”

“…Pconstitutive-tfoX, a ∆luxO mutation, and a deletion of the retraction ATPase pilT (∆pilT). Strains were grown and labeled with Alexa fluor 488-maleimide exactly as previously described (36).…”