Abstract
Type IVa pili (T4P) are bacterial surface structures that enable motility, adhesion, biofilm formation and virulence. T4P are assembled by nanomachines that span the bacterial cell envelope. Cycles of T4P assembly and retraction, powered by the ATPases PilB and PilT, allow bacteria to attach to and pull themselves along surfaces, so-called “twitching motility”. These opposing ATPase activities must be coordinated and T4P assembly limited to one pole for bacteria to show directional movement. How this occurs is still incompletely understood. Herein, we show that the c-di-GMP binding protein FimX, which is required for T4P assembly in Pseudomonas aeruginosa, localizes to the leading pole of twitching bacteria. Polar FimX localization requires both the presence of T4P assembly machine proteins and the assembly ATPase PilB. PilB itself loses its polar localization pattern when FimX is absent. We use two different approaches to confirm that FimX and PilB interact in vivo and in vitro, and further show that point mutant alleles of FimX that do not bind c-di-GMP also do not interact with PilB. Lastly, we demonstrate that FimX positively regulates T4P assembly and twitching motility by promoting the activity of the PilB ATPase, and not by stabilizing assembled pili or by preventing PilT-mediated retraction. Mutated alleles of FimX that no longer bind c-di-GMP do not allow rapid T4P assembly in these assays. We propose that by virtue of its high-affinity for c-di-GMP, FimX can promote T4P assembly when intracellular levels of this cyclic nucleotide are low. As P. aeruginosa PilB is not itself a high-affinity c-di-GMP receptor, unlike many other assembly ATPases, FimX may play a key role in coupling T4P mediated motility and adhesion to levels of this second messenger.
Highlights
Type IV pili (T4P) are long (5–20 μm), flexible surface structures that perform a variety of functions—motility, adherence, and DNA uptake—in Gram-negative and -positive bacteria, as well as Archaea [1,2,3]
Type IV pili (T4P) are assembled on the surfaces of many bacterial pathogens and commensals through the action of specialized assembly machines whose components and structures are the subject of intense study
Biochemical and microscopic approaches we report for the first time that FimX interacts with PilB to promote pilus assembly in P. aeruginosa
Summary
Type IV pili (T4P) are long (5–20 μm), flexible surface structures that perform a variety of functions—motility, adherence, and DNA uptake—in Gram-negative and -positive bacteria, as well as Archaea [1,2,3]. Recurrent cycles of pilus assembly and disassembly, driven by distinct assembly and retraction ATPases, allow for directed bacterial movement across surfaces. This “twitching motility” is critical for bacterial colonization of surfaces and required for formation and maturation of biofilms by pathogens such as Pseudomonas aeruginosa [7]. Electron cryo-tomography structures show an inner membrane (IM) protein (Pa PilC) aligned with periplasmic rings assigned to Pa PilN, PilO and PilP; these three proteins are proposed to form a scaffold that links the outer membrane pore to cytoplasmic components, such as Pa PilM [13, 14]. A prepilin peptidase (Pa PilD) processes the major pilin (Pa PilA) and a number of minor pilins before they can be incorporated into the base of the growing T4P by an assembly ATPase (Pa PilB)
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