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

Abstract

19Bacterial type IV pili are critical for diverse biological processes including horizontal gene 20 transfer, surface sensing, biofilm formation, adherence, motility, and virulence. These dynamic 21 appendages extend and retract from the cell surface. In many type IVa pilus systems, extension 22 occurs through the action of an extension ATPase, often called PilB, while optimal retraction 23 requires the action of a retraction ATPase, PilT. Many type IVa systems also encode a homolog 24 of PilT called PilU. However, the function of this protein has remained unclear because pilU 25 mutants exhibit inconsistent phenotypes among type IV pilus systems and because it is 26 relatively understudied compared to PilT. Here, we study the type IVa competence pilus of 27Vibrio cholerae as a model system to define the role of PilU. We show that the ATPase activity 28 of PilU is critical for pilus retraction in PilT Walker A and/or Walker B mutants. PilU does not, 29 however, contribute to pilus retraction in ΔpilT strains. Thus, these data suggest that PilU is a 30 bona fide retraction ATPase that supports pilus retraction in a PilT-dependent manner. We also 31 found that a ΔpilU mutant exhibited a reduction in the force of retraction suggesting that PilU is 32 important for generating maximal retraction forces. Additional in vitro and in vivo data show that 33PilT and PilU act as independent homo-hexamers that may form a complex to facilitate pilus 34 retraction. Finally, we demonstrate that the role of PilU as a PilT-dependent retraction ATPase 35 is conserved in Acinetobacter baylyi, suggesting that the role of PilU described here may be 36 broadly applicable to other type IVa pilus systems. 37 38Author Summary 39 Almost all bacterial species use thin surface appendages called pili to interact with their 40 environments. These structures are critical for the virulence of many pathogens and represent 41 one major way that bacteria share DNA with one another, which contributes to the spread of 42 antibiotic resistance. To carry out their function, pili dynamically extend and retract from the 43 bacterial surface. Here, we show that retraction of pili in some systems is determined by the 44 combined activity of two motor ATPase proteins. 45 46Type IV pili are ubiquitous surface appendages in Gram-negative bacteria that promote diverse 48 activities including attachment, virulence, biofilm formation, horizontal gene transfer, and 49 twitching motility [1][2][3][4][5]. These structures can dynamically extend and retract from the cell 50 surface, which is often critical for their function. A detailed mechanistic understanding of pilus 51 dynamic activity, however, remains lacking. 52Type IV pili are composed almost exclusively of a single protein called the major pilin, which 53 forms a helical fiber that extends from the cell surface [6]. Extension and retraction of the pilus is 54 hypothesized to occur through the interaction of cytoplasmic hexameric ATPases with the inner 55 membrane platform of the pilus machine, whereby ATP ...

Pilus Production in Acinetobacter baumannii Is Growth Phase Dependent and Essential for Natural Transformation

“…3A). Interestingly, we and others recently demonstrated that V. cholerae's PilU works solely in conjunction with PilT instead of compensating for the lack of PilT (51,65), which suggests that it enhances the retraction force instead of behaving as an independent retraction enzyme, as was also suggested for P. aeruginosa (51,65,66).…”

Acinetobacter baylyi regulates type IV pilus synthesis by employing two extension motors and a motor protein inhibitor

“…Deletion of pilT prevents T4P retraction 16 and may thus reveal more subtle effects on pilus extension. T4P labeling in pilB, tfpB, and pilB tfpB mutants revealed that all three mutants were defective in T4P synthesis while pilT was intact (Figure 2c).…”

“…Mutants in A. baylyi were made using natural transformation as described previously 16 . Briefly, mutant constructs were made by splicing-by-overlap (SOE) PCR to stich 1) ~3 kb of the homologous region upstream of the gene of interest, 2) the mutation where appropriate (for deletion by allelic replacement with an antibiotic resistance cassette, or the fusion protein), and 3) the downstream region of homology.…”

Type IV pili share a conserved mechanism of motor-independent retraction that is an inherent property of the pilus filament

“…A detailed mechanistic understanding of the factors that promote pilus retraction, however, remains lacking in many T4P. For T4aP, pilus depolymerization is mediated by dedicated retraction ATPase motors commonly referred to as PilT and PilU (21, 22). However, even in the absence of these retraction motors, numerous T4aP retain the ability to retract (4, 21, 23, 24).…”