PilB and PilT Are ATPases Acting Antagonistically in Type IV Pilus Function inMyxococcus xanthus

Abstract

Type IV pili (T4P) are dynamic surface structures that undergo cycles of extension and retraction. T4P dynamics center on the PilB and PilT proteins, which are members of the secretion ATPase superfamily of proteins. Here, we show that PilB and PilT of the T4P system in Myxococcus xanthus have ATPase activity in vitro. Using a structure-guided approach, we systematically mutagenized PilB and PilT to resolve whether both ATP binding and hydrolysis are important for PilB and PilT function in vivo. PilB as well as PilT ATPase activity was abolished in vitro by replacement of conserved residues in the Walker A and Walker B boxes that are involved in ATP binding and hydrolysis, respectively. PilB proteins containing mutant Walker A or Walker B boxes were nonfunctional in vivo and unable to support T4P extension. PilT proteins containing mutant Walker A or Walker B boxes were also nonfunctional in vivo and unable to support T4P retraction. These data provide genetic evidence that both ATP binding and hydrolysis by PilB are essential for T4P extension and that both ATP binding and hydrolysis by PilT are essential for T4P retraction. Thus, PilB and PilT are ATPases that act at distinct steps in the T4P extension/retraction cycle in vivo.Type IV pili (T4P) are versatile, filamentous surface structures found in many gram-negative bacteria. In Myxococcus xanthus, Pseudomonas aeruginosa, and Neisseria gonorrhoeae T4P mediate surface motility (27). T4P also mediate attachment and microcolony formation by human pathogens such as Escherichia coli, N. gonorrhoeae, P. aeruginosa, and Vibrio cholerae on eukaryotic host cells (6). Moreover, T4P have important functions in biofilm formation (22,34) and DNA uptake by natural transformation (9). A hallmark of T4P compared to other filamentous surface structures is their dynamic nature; i.e., T4P undergo cycles of extension and retraction, and it is during the retraction step that a force sufficiently large to pull a bacterial cell forward is generated (29, 51, 52).T4P are thin (5-to 8-nm), flexible, helical filaments several micrometers in length, with high tensile strength (Ͼ100 pN) and typically composed only of the PilA pilin subunit (6). The protein machinery required for T4P biogenesis and function is highly conserved and encompasses 17 proteins as defined for T4P in Neisseria meningitidis (4). These proteins localize to the cytoplasm, inner membrane, periplasm, and outer membrane (35). In vitro analyses and genetic analyses of T4P in N. meningitidis suggest that these proteins interact extensively and form a transenvelope complex (4). Many of the proteins involved in T4P biogenesis and function share similarity with proteins found in type II secretion systems (T2SS) and archaeal flagellum systems (35). Several of the proteins are phylogenetically related, suggesting that the three machineries may share functional characteristics (35). Indeed, overexpression of pseudopilins from the T2SS in Klebsiella oxytoca, Xanthomonas campestris, and P. aeruginosa results in the formation of...

Alanine 32 in PilA is important for PilA stability and type IV pili function in Myxococcus xanthus

“…(2) The alterations in PilA affect its interaction with the retraction motor PilT. PilT forms hexameric polymers (Satyshur et al, 2007) that are essential for TFP retraction, which is powered by the ATPase activity of PilT (Jakovljevic et al, 2008). The structural changes in the mutated TFP may prevent correct interaction of PilA with PilT or affect the force between TFP and the PilT machinery necessary for retraction.…”

“…TFP are composed of several thousand subunits of PilA or pilin. The cycles of TFP extension and retraction are achieved by assembling PilA into polar filaments with the assistance of the ATPase PilB (Jakovljevic et al, 2008), followed by disassembling the pili into single subunits during retraction, a process that is mediated by the ATPase PilT (Jakovljevic et al, 2008). PilA monomers form a membrane-associated pool which can be recycled to form new TFP (Yang et al, 2010).…”

Gliding Motility Revisited: How Do the Myxobacteria Move without Flagella?

“…4A). In contrast, TFP retraction and disassembly are mediated by PilT, a PilB homolog with an opposing function (49). The molecular mechanisms by which PilB and PilT extend and retract the pili are unknown.…”

“…PilA monomers are anchored to the inner membrane through their 25 hydrophobic N-terminal residues. Prior to assembly, the PilA monomers are processed by the prepilin peptidase, PilD, which cleaves off the N-terminal leader sequence of the protein (49). The processed PilA monomers are polymerized by PilB, a cytosolic hexameric ATPase, and translocated through the outer membrane by the PilQ secretin and the Tgl lipoprotein (Fig.…”

DNA-uptake machinery of naturally competent Vibrio cholerae

“…S5A). We therefore decided to test whether ATP hydrolysis is important to PilB's role in DNA uptake/natural transformation, as for a very different phenotype, the Tfp-dependent gliding motility of Myxococcus xanthus (24). We first cloned a plasmid carrying a pilB with a site-directed mutation (E394A) and tested the variant in a complementation assay with natural transformation as the readout.…”