The Peptidoglycan-Binding Protein FimV Promotes Assembly of the Pseudomonas aeruginosa Type IV Pilus Secretin

Abstract

The Pseudomonas aeruginosa inner membrane protein FimV is among several proteins of unknown function required for type IV pilus-mediated twitching motility, arising from extension and retraction of pili from their site of assembly in the inner membrane. The pili transit the periplasm and peptidoglycan (PG) layer, ultimately exiting the cell through the PilQ secretin. Although fimV mutants are nonmotile, they are susceptible to killing by pilus-specific bacteriophage, a hallmark of retractable surface pili. Here we show that levels of recoverable surface pili were markedly decreased in fimV pilT retraction-deficient mutants compared with levels in the pilT control, demonstrating that FimV acts at the level of pilus assembly. Levels of inner membrane assembly subcomplex proteins PilM/N/O/P were decreased in fimV mutants, but supplementation of these components in trans did not restore pilus assembly or motility. Loss of FimV dramatically reduced the levels of the PilQ secretin multimer through which pili exit the cell, in part due to decreased levels of PilQ monomers, while PilF pilotin levels were unchanged. Expression of pilQ in trans in the wild type or fimV mutants increased total PilQ monomer levels but did not alter secretin multimer levels or motility. PG pulldown assays showed that the N terminus of FimV bound PG in a LysM motif-dependent manner, and a mutant with an in-frame chromosomal deletion of the LysM motif had reduced motility, secretin levels, and surface piliation. Together, our data show that FimV's role in pilus assembly is to promote secretin formation and that this function depends upon its PG-binding domain.Type IV pili (T4P) are thin, long, flexible, and retractable protein filaments. The broad distribution of T4P among bacterial genera correlates with their ability to mediate a wide range of functions, including attachment to surfaces, DNA uptake, and twitching motility. During twitching motility, pili extend from the cell and attach to a surface, and pilus retraction occurs, moving the bacteria forward toward the point of adhesion (41,51). T4P have been classified into two distinct subtypes, T4aP and T4bP, based on differences in structure of the major pilin subunit and in architecture of the assembly systems (3,16,43). T4aP have been characterized extensively in species such as Pseudomonas aeruginosa, Neisseria spp., and Myxococcus xanthus (10,22,23,39,40,46,49,52). T4bP are found predominately in enteric pathogens, where they promote adhesion (37,58) and bacterial aggregation (6).In P. aeruginosa, a large number of genes involved in the regulation, assembly, and dynamics of T4aP function have been identified. These include the major pilin subunit PilA, as well as a conserved set of minor pilin-like proteins (21); the N-methyltransferase/peptidase PilD, essential for processing prepilins into their mature form (53); an inner membrane assembly subcomplex composed of PilM/N/O/P (4, 47); an outer membrane PilQ secretin pore oligomerized with the assistance of the lipoprotein PilF (7, 29); a...

A multidomain hub anchors the chromosome segregation and chemotactic machinery to the bacterial pole

“…Outside of the Vibrionaceae, homologs are found principally in the g-proteobacteria, where they are significantly less conserved, but many appear to be encoded within a similar chromosomal neighborhood. Some of these proteins (e.g., FimV of Pseudomonas aeruginosa) have been found to be associated with the assembly of type IV pili and pilus-associated twitching motility (Semmler et al 2000;Wehbi et al 2011). HubP is directed to the V. cholerae pole and mid-cell by its periplasmic domain Given the effect of HubP deficiency on subcellular targeting of polar proteins, we assessed whether HubP itself is present at the V. cholerae cell poles.…”

Esx Systems and the Mycobacterial Cell Envelope: What's the Connection?

“…In the Neisseria gonorrhoeae type IVa pilus system, a peptidoglycan-binding protein anchors the apparatus in the membrane (153). In the Pseudomonas aeruginosa type IV pilus system FimV, an inner membrane protein that binds peptidoglycan is required for multimerization of PilQ (outer membrane component of the type VI pilus [154]). …”

The Conserved Tetratricopeptide Repeat-Containing C-Terminal Domain of Pseudomonas aeruginosa FimV Is Required for Its Cyclic AMP-Dependent and -Independent Functions

“…In addition to controlling cAMP production and related phenotypes, FimV functions in a cAMP-independent manner, promoting multimerization of the PilQ secretin (18). Based on recent work in other species, FimV may be a hub protein, similar to HubP of Vibrio cholerae and Shewanella putrefaciens, that coordinates interactions of a number of landmark proteins involved in motility and chromosome segregation at the poles of rod-shaped bacteria (27,28).…”

“…Also implicated in regulation of T4aP assembly and function is the inner membrane protein, FimV (17,18). FimV is involved in a number of virulence mechanisms, including twitching motility, type II secretion (T2S) of lipases and proteases, and regulation of cAMP production, and thus Vfr function (15,17,19).…”

“…Based on recent work in other species, FimV may be a hub protein, similar to HubP of Vibrio cholerae and Shewanella putrefaciens, that coordinates interactions of a number of landmark proteins involved in motility and chromosome segregation at the poles of rod-shaped bacteria (27,28). These large proteins have very limited sequence identity aside from their LysM motifs, single transmembrane segment, and a highly conserved "FimV C-terminal sequence" of ϳ50 residues (NCBI Conserved Domain Database designation TIGR03504) (18) predicted to include a single TPR motif. FimV's domain architecture and C-terminal sequence are shown in Fig.…”