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

Abstract

FimV is a Pseudomonas aeruginosa inner membrane protein that regulates intracellular cyclic AMP (cAMP) levels-and thus type IV pilus (T4P)-mediated twitching motility and type II secretion (T2S)-by activating the adenylate cyclase CyaB. Its cytoplasmic domain contains three predicted tetratricopeptide repeat (TPR) motifs separated by an unstructured region: two proximal to the inner membrane and one within the "FimV C-terminal domain," which is highly conserved across diverse homologs. Here, we present the crystal structure of the FimV C terminus, FimV 861-919 , containing a TPR motif decorated with solvent-exposed, charged side chains, plus a C-terminal capping helix. FimV 689 , a truncated form lacking this C-terminal motif, did not restore wild-type levels of twitching or surface piliation compared to the full-length protein. FimV 689 failed to restore wild-type levels of the T4P motor ATPase PilU or T2S, suggesting that it was unable to activate cAMP synthesis. Bacterial two-hybrid analysis showed that TPR3 interacts directly with the CyaB activator, FimL. However, FimV 689 failed to restore wild-type motility in a fimV mutant expressing a constitutively active CyaB (fimV cyaB-R456L), suggesting that the C-terminal motif is also involved in cAMP-independent functions of FimV. The data show that the highly conserved TPR-containing C-terminal domain of FimV is critical for its cAMP-dependent and -independent functions. IMPORTANCEFimV is important for twitching motility and cAMP-dependent virulence gene expression in P. aeruginosa. FimV homologs have been identified in several human pathogens, and their functions are not limited to T4P expression. The C terminus of FimV is remarkably conserved among otherwise very diverse family members, but its role is unknown. We provide here biological evidence for the importance of the C-terminal domain in both cAMP-dependent (through FimL) and -independent functions of FimV. We present X-ray crystal structures of the conserved C-terminal domain and identify a consensus sequence for the C-terminal TPR within the conserved domain. Our data extend our knowledge of FimV's functionally important domains, and the structures and consensus sequences provide a foundation for studies of FimV and its homologs. T ype IV pili (T4P) are filamentous surface appendages produced by a wide range of bacteria and archaea (1, 2), where they assist in DNA uptake, surface attachment, and twitching motility (3-5). There are two major subfamilies of T4P: T4aP and T4bP. T4aP are typically associated with twitching (2), a process in which pili undergo repeated cycles of extension, adhesion, and retraction, thus acting as molecular grappling hooks to pull cells along a surface.The T4aP machinery is composed of four structural subcomplexes (6). In the model bacterium Pseudomonas aeruginosa, an inner membrane motor subcomplex consisting of the platform protein PilC and three hexameric ATPases-PilB, PilT, and PilU-provide energy for T4aP extension and retraction (7-9). A second inner membrane al...

Cyclic AMP-Independent Control of Twitching Motility in Pseudomonas aeruginosa

“…However, transcription of the pilMNOPQ operon is Vfr dependent and thus cAMP dependent (21). To determine if any of these phenotypes are independent of cAMP, we examined levels of PilMNOPQ and PilU and twitching motility in a fimV mutant, a fimV LysM mutant encoding only the cytoplasmic domain of FimV (fimV 1194 ), and a fimV cyaB-R456L double mutant that expresses constitutively active CyaB (25,36).…”

“…Taken together, the data show that the cAMP-independent role(s) of FimV requires both its periplasmic and cytoplasmic domains. The levels of PilU (a proxy for intracellular cAMP levels) (25) and assembly machinery components PilMNOPQ were assessed by Western blotting in various fimV mutant backgrounds complemented with the empty vector or with full-length fimV; a representative blot is shown. Surface piliation and twitching motility are shown in the bottom two rows.…”

“…HubP from Shewanella putrefaciens is responsible for localization of the chemotactic machinery but not the flagellar system (27). Yamaichi et al (28) showed that the polar localization of V. cholerae HubP is dependent on the conserved LysM motif (25). Wehbi et al (29) showed that P. aeruginosa fimV mutants have decreased levels of the T4aP alignment subcomplex proteins PilMNOP, while in-frame deletion of FimV's LysM motif resulted in fewer PilQ multimers, suggesting that PG binding is important for optimal secretin formation.…”

“…For example, provision of exogenous cAMP to mutants lacking PilG restored piliation but not motility (17), and a mutant expressing a constitutively active form of CyaB but lacking FimV failed to twitch (25). FimL was proposed to link PilG to the C-terminal TPR motif of FimV, leading to CyaB activation, and FimV localized both FimL and PilG to cell poles (22,25).…”

“…Its periplasmic domain contains a lysin (LysM) motif that binds peptidoglycan (PG) (23), and its cytoplasmic domain contains three discontinuous tetratricopeptide repeat (TPR) motifs involved in proteinprotein interactions (24). FimV homologs have been identified in other T4P-producing bacteria (10), although their overall sequence identity is low, with the most conserved features being the LysM motif (COG3170), the single transmembrane segment, and a highly conserved cytoplasmic "FimV C-terminal domain"-TIGR03504 -encompassing a single TPR and capping helix (25).…”