Channel formation by FhaC, the outer membrane protein involved in the secretion of the Bordetella pertussis filamentous hemagglutinin

Many virulence factors of pathogenic microorganisms are presented at the cell surface. However, protein secretion across the outer membrane of Gram-negative bacteria remains poorly understood. Here we used the extremely efficient secretion of the Bordetella pertussis filamentous hemagglutinin (FHA) to decipher this process. FHA secretion requires a single specific accessory protein, FhaC, the prototype of a family of proteins necessary for the extracellular localization of various virulence proteins in Gram-negative bacteria. We show that FhaC is heat-modifiable and localized in the outer membrane. Circular dichroism spectra indicated that FhaC is rich in beta-strands, in agreement with structural predictions for this protein. We further demonstrated that FhaC forms pores in artificial membranes, as evidenced by single-channel conductance measurements through planar lipid bilayers, as well as by liposome swelling assays and patch-clamp experiments using proteoliposomes. Single-channel conductance appeared to fluctuate very fast, suggesting that the FhaC channels frequently assume a closed conformation. We thus propose that FhaC forms a specific beta-barrel channel in the outer membrane for the outward translocation of FHA.     

Channel properties of TpsB transporter FhaC point to two functional domains with a C-terminal protein-conducting pore

Integral outer membrane transporters of the Omp85/TpsB superfamily mediate the translocation of proteins across, or their integration into, the outer membranes of Gram-negative bacteria, chloroplasts, and mitochondria. The Bordetella pertussis FhaC/FHA couple serves as a model for the two-partner secretion pathway in Gram-negative bacteria, with the TpsB protein, FhaC, being the specific transporter of its TpsA partner, FHA, across the outer membrane. In this work, we have investigated the structure/function relationship of FhaC by analyzing the ion channel properties of the wild type protein and a collection of mutants with varied FHA secretion activities. We demonstrated that the channel is formed by the C-terminal two-thirds of FhaC most likely folding into a beta-barrel domain predicted to be conserved throughout the family. A C-proximal motif that represents the family signature appears essential for pore function. The N-terminal 200 residues of FhaC constitute a functionally distinct domain that modulates the pore properties and may participate in FHA recognition.     

Lack of functional complementation between Bordetella pertussis filamentous hemagglutinin and Proteus mirabilis HpmA hemolysin secretion machineries.

The gram-negative bacterium Bordetella pertussis has adapted specific secretion machineries for each of its major secretory proteins. In particular, the highly efficient secretion of filamentous hemagglutinin (FHA) is mediated by the accessory protein FhaC. FhaC belongs to a family of outer membrane proteins which are involved in the secretion of large adhesins or in the activation and secretion of Ca2+-independent hemolysins by several gram-negative bacteria. FHA shares with these hemolysins a 115-residue-long amino-proximal region essential for its secretion. To compare the secretory pathways of these hemolysins and FHA, we attempted functional transcomplementation between FhaC and the Proteus mirabilis hemolysin accessory protein HpmB. HpmB could not promote the secretion of FHA derivatives. Likewise, FhaC proved to be unable to mediate secretion and activation of HpmA, the cognate secretory partner of HpmB. In contrast, ShlB, the accessory protein of the closely related Serratia marcescens hemolysin, was able to activate and secrete HpmA. Two invariant asparagine residues lying in the region of homology shared by secretory proteins and shown to be essential for the secretion and activation of the hemolysins were replaced in FHA by site-directed mutagenesis. Replacements of these residues indicated that both are involved in, but only the first one is crucial to, FHA secretion. This slight discrepancy together with the lack of functional complementation demonstrates major differences between the hemolysins and FHA secretion machineries.     

Translocator proteins in the two-partner secretion family have multiple domains

The two-partner secretion pathway in Gram-negative bacteria consists of a TpsA exoprotein and a cognate TpsB outer membrane translocator protein. Previous work has demonstrated that the TpsB protein forms a beta-barrel structure with pore forming activity and facilitates translocation of the TpsA protein across the outer membrane. In this study, we characterized the functional domains of the Haemophilus influenzae HMW1B protein, a TpsB protein that interacts with the H. influenzae HMW1 adhesin. Using c-Myc epitope tag insertions and cysteine substitution mutagenesis, we discovered that HMW1B contains an N-terminal surface-localized domain, an internal periplasmic domain, and a C-terminal membrane anchor. Functional and biochemical analysis of the c-Myc epitope tag insertions and a series of HMW1B deletion constructs demonstrated that the periplasmic domain is required for secretion of HMW1 and that the C-terminal membrane anchor (HMW1B-(234-545)) is capable of oligomerization and pore formation. Similar to our observations with HMW1B, examination of a Bordetella pertussis TpsB protein called FhaC revealed that the C terminus of FhaC (FhaC-(232-585)) is capable of pore formation. We speculate that all TpsB proteins have a modular structure, with a periplasmic domain that interacts with the cognate TpsA protein and with pore forming activity contained within the C terminus.     

Evidence that a globular conformation is not compatible with FhaC-mediated secretion of the Bordetella pertussis filamentous haemagglutinin

The 220 kDa Bordetella pertussis filamentous haemagglutinin (FHA) is the major extracellular protein of this organism. It is exported using a signal peptide-dependent pathway, and its secretion depends on one specific outer membrane accessory protein, FhaC. In this work, we have investigated the influence of conformation on the FhaC-mediated secretion of FHA using an 80 kDa N-terminal FHA derivative, Fha44. In contrast to many signal peptide-dependent secretory proteins, no soluble periplasmic intermediate of Fha44 could be isolated. In addition, cell-associated Fha44 synthesized in the absence of FhaC did not remain competent for extracellular secretion upon delayed expression of FhaC, indicating that the translocation steps across the cytoplasmic and the outer membrane might be coupled. A chimeric protein, in which the globular B subunit of the cholera toxin, CtxB, was fused at the C-terminus of Fha44, was not secreted in B. pertussis or in Escherichia coli expressing FhaC. The hybrid protein was only secreted when both disulphide bond-forming cysteines of CtxB were replaced by serines or when it was produced in DsbA^?E. coli. The Fha44 portion of the secretion-incompetent hybrid protein was partly exposed on the cell surface. These results argue that the Fha44–CtxB hybrid protein transited through the periplasmic space, where disulphide bond formation is specifically catalysed, and that secretion across the outer membrane was initiated. The folded CtxB portion prevented extracellular release of the hybrid, in contrast to the more flexible CtxB domain devoid of cysteines. We propose a secretion model whereby Fha44 transits through the periplasmic space on its way to the cell surface and initiates its translocation through the outer membrane before being released from the cytoplasmic membrane. Coupling of Fha44 translocation across both membranes would delay the acquisition of its folded structure until the protein emerges from the outer membrane. Such a model would be consistent with the extensive intracellular proteolysis of FHA derivatives in B. pertussis.     

FhaC takes a bow to FHA in the two‐partner do‐si‐do

FhaC is an outer membrane transporter from Bordetella pertussis belonging to the t wo‐ p artner s ecretion (TPS) pathway with its primary role being the secretion of the virulence factor f ilamentous h aem a gglutinin (FHA). FhaC serves as a model transporter of the TPS pathway and significant work has been done to characterize the role of FhaC in FHA secretion. Recent studies characterized interactions between FHA and the POTRA domains of FhaC, suggesting that secretion may involve a successive translocation mechanism mediated by β‐augmentation and/or electrostatic interactions. Moreover, it was also shown that reconstituted FhaC is necessary and sufficient to transport FHA into proteoliposomes. While the crystal structure of FhaC clearly suggests a role in transport, the putative transport pore is plugged by an N‐terminal α‐helix (H1 helix) that occludes access by FHA. Therefore, it has been proposed that the H1 helix must be expelled from the pore in order for secretion of FHA to occur. However, this has yet to be shown experimentally. Guérin et al. (2014) report the first direct experimental evidence to show that the FhaC H1 helix is quite dynamic and exchanges between closed and open states upon interaction with FHA.     

The prodomain of the Bordetella two‐partner secretion pathway protein FhaB remains intracellular yet affects the conformation of the mature C‐terminal domain

Two‐partner secretion (TPS) systems use β‐barrel proteins of the Omp85‐TpsB superfamily to transport large exoproteins across the outer membranes of Gram‐negative bacteria. The Bordetella FHA/FhaC proteins are prototypical of TPS systems in which the exoprotein contains a large C‐terminal prodomain that is removed during translocation. Although it is known that the FhaB prodomain is required for FHA function in vivo, its role in FHA maturation has remained mysterious. We show here that the FhaB prodomain is required for the extracellularly located mature C‐terminal domain (MCD) of FHA to achieve its proper conformation. We show that the C‐terminus of the prodomain is retained intracellularly and that sequences within the N‐terminus of the prodomain are required for this intracellular localization. We also identify sequences at the C‐terminus of the MCD that are required for release of mature FHA from the cell surface. Our data support a model in which the intracellularly located prodomain affects the final conformation of the extracellularly located MCD. We hypothesize that maturation triggers cleavage and degradation of the prodomain.     

Substrate recognition by the POTRA domains of TpsB transporter FhaC

Widespread in Gram-negative bacteria, the two-partner secretion (TPS) pathway mediates the secretion of large, β-helical 'TpsA' proteins with various functions. TpsA proteins harbour a conserved, N-proximal TPS domain essential for secretion. TpsB transporters specifically recognize their TpsA partners in the periplasm and mediate their translocation across the outer membrane through a hydrophilic channel. The FHA/FhaC pair of Bordetella pertussis represents a model TPS system. FhaC is composed of a β barrel preceded by two periplasmic POTRA domains in tandem. Here we show that both POTRAs are involved in FHA recognition. Surface plasmon resonance analyses indicated an interaction of micromolar affinity between the POTRAs and the TPS domain with fast association and dissociation steps, consistent with the transient character of this interaction in vivo. Major interaction sites in POTRAs correspond to hydrophobic grooves formed by a β sheet edge and the flanking α helix, well-suited to accommodate extended, amphipathic strands of the substrate and consistent with β augmentation. The initial recruitment of the TPS domain to POTRAs appears to be facilitated by electrostatic attractions. A domain corresponding to the first part of the repeat-rich central region of FHA is also recognized by the POTRAs, suggesting successive interactions in the course of secretion.     

Role of DegP for two-partner secretion in Bordetella

Sorting of proteins destined to the surface or the extracellular milieu is mediated by specific machineries, which guide the protein substrates towards the proper route of secretion and determine the compartment in which folding occurs. In Gram-negative bacteria, the two-partner secretion (TPS) pathway is dedicated to the secretion of large proteins rich in β-helical structure. The secretion of the filamentous haemagglutinin (FHA), a 230 kDa adhesin of Bordetella pertussis , represents a model TPS system. FHA is exported by the Sec machinery and transits through the periplasm in an extended conformation. From there it is translocated across the outer membrane by its dedicated transporter FhaC to finally fold into a long β-helix at the cell surface in a progressive manner. In this work, we show that B. pertussis lacking the periplasmic chaperone/protease DegP has a strong growth defect at 37°C, and the integrity of its outer membrane is compromised. While both phenotypes are significantly aggravated by the presence of FHA, the chaperone activity of DegP markedly alleviates the periplasmic stress. In vitro , DegP binds to non-native FHA with high affinity. We propose that DegP chaperones the extended FHA polypeptide in the periplasm and is thus involved in the TPS pathway.     

Incorporation of outer membrane protein OmpG in lipid membranes: protein-lipid interactions and beta-barrel orientation

OmpG is an intermediate size, monomeric, outer membrane protein from Escherichia coli, with n beta = 14 beta-strands. It has a large pore that is amenable to modification by protein engineering. The stoichiometry ( N b = 20) and selectivity ( K r = 0.7-1.2) of lipid-protein interaction with OmpG incorporated in dimyristoyl phosphatidylcholine bilayer membranes was determined with various 14-position spin-labeled lipids by using EPR spectroscopy. The limited selectivity for different lipid species is consistent with the disposition of charged residues in the protein. The conformation and orientation (beta-strand tilt and beta-barrel order parameters) of OmpG in disaturated phosphatidylcholines of odd and even chain lengths from C(12:0) to C(17:0) was determined from polarized infrared spectroscopy of the amide I and amide II bands. A discontinuity in the protein orientation (deduced from the beta-barrel order parameters) is observed at the point of hydrophobic matching of the protein with lipid chain length. Compared with smaller (OmpA; n beta = 8) and larger (FhuA; n beta = 22) monomeric E. coli outer membrane proteins, the stoichiometry of motionally restricted lipids increases linearly with the number of beta-strands, the tilt (beta approximately 44 degrees ) of the beta-strands is comparable for the three proteins, and the order parameter of the beta-barrel increases regularly with n beta. These systematic features of the integration of monomeric beta-barrel proteins in lipid membranes could be useful for characterizing outer membrane proteins of unknown structure.     

Regulated,sequential processing by multiple proteases is required for proper maturation and release of Bordetella filamentous hemagglutinin

Filamentous hemagglutinin (FHA) is a critically important virulence factor produced by Bordetella species that cause respiratory infections in humans and other animals. It is also a prototypical member of the widespread two partner secretion (TPS) pathway family of proteins. First synthesized as a ~370 kDa protein called FhaB, its C‐terminal ~1,200 amino acid ‘prodomain’ is removed during translocation to the cell surface via the outer membrane channel FhaC. Here, we identify CtpA as a periplasmic protease that is responsible for the regulated degradation of the prodomain and for creation of an intermediate polypeptide that is cleaved by the autotransporter protease SphB1 to generate FHA. We show that the central prodomain region is required to initiate degradation of the prodomain and that CtpA degrades the prodomain after a third, unidentified protease (P3) first removes the extreme C‐terminus of the prodomain. Stepwise proteolysis by P3, CtpA and SphB1 is required for maturation of FhaB, release of FHA into the extracellular milieu, and full function in vivo. These data support a substantially updated model for the mechanism of secretion, maturation and function of this model TPS protein.     

Distinct roles of the N-terminal and C-terminal precursor domains in the biogenesis of the Bordetella pertussis filamentous hemagglutinin.

The 220-kDa Bordetella pertussis filamentous hemagglutinin (FHA) is the major exported protein found in culture supernatants. The structural gene of FHA has a coding potential for a 367-kDa protein, and the mature form constitutes the N-terminal 60% of the 367-kDa precursor. The C-terminal domain of the precursor was found to be important for the high-level secretion of full-length FHA but not of truncated analogs (80 kDa or less). The secretion of full-length and truncated FHA polypeptides requires the presence of the approximately 100-amino-acid N-terminal domain and the outer membrane protein FhaC, homologous to the N-terminal domains of the Serratia marcescens and Proteus mirabilis hemolysins and their accessory proteins, respectively. By analogy to these hemolysins, it is likely that the N-terminal domain of the FHA precursor interacts, directly or indirectly, with the accessory protein during FHA biogenesis. However, immunogenicity and antigenicity studies suggest that the N-terminal domain of FHA is masked by its C-terminal domain and therefore should not be available for its interactions with FhaC. These observations suggest a model in which the C-terminal domain of the FHA precursor may play a role as an intramolecular chaperone to prevent premature folding of the protein. Both heparin binding and hemagglutination are expressed by the N-terminal half of FHA, indicating that this domain contains important functional regions of the molecule.     

Secretion signal of the filamentous haemagglutinin, a model two-partner secretion substrate

The sorting of proteins to their proper subcellular compartment requires specific addressing signals that mediate interactions with ad hoc transport machineries. In Gram-negative bacteria, the widespread two-partner secretion (TPS) pathway is dedicated to the secretion of large, mostly virulence-related proteins. The secreted TpsA proteins carry a characteristic 250-residue-long N-terminal 'TPS domain' essential for secretion, while their TpsB transporters are pore-forming proteins that specifically recognize their respective TpsA partners and mediate their translocation across the outer membrane. However, the nature of the secretion signal has not been elucidated yet. The whooping cough agent Bordetella pertussis secretes its major adhesin filamentous haemagglutinin (FHA) via the TpsB transporter FhaC. In this work, we show specific interactions between an N-terminal fragment of FHA containing the TPS domain and FhaC by using two different techniques, an overlay assay and a pull-down of the complex. FhaC recognizes only non-native conformations of the TPS domain, corroborating the model that in vivo, periplasmic FHA is not yet folded. By generating single amino acid substitutions, we have identified interaction determinants forming the secretion signal. They are found unexpectedly far into the TPS domain and include both conserved and variable residues, which most likely explains the specificity of the TpsA-TpsB interaction. The N-terminal domain of FhaC is involved in the FHA-FhaC interaction, in agreement with its proposed function and periplasmic localization.     

Characterization of the essential transport function of the AIDA-I autotransporter and evidence supporting structural predictions

The current model for autodisplay suggests a mechanism that allows a passenger protein to be translocated across the outer membrane by coordinate action of a C-terminal beta-barrel and its preceding linking region. The passenger protein, linker, and beta-barrel are together termed the autotransporter, while the linker and beta-barrel are here referred to as the translocation unit (TU). We characterized the minimal TU necessary for autodisplay with the adhesin-involved-in-diffuse-adherence (AIDA-I) autotransporter. The assumed beta-barrel structure at the C terminus of the AIDA-I autotransporter was studied by constructing a set of seven AIDA-I-cholera toxin B subunit fusion proteins containing various portions of AIDA-I. Surface exposure of the cholera toxin B moiety was assessed by dot blot experiments and trypsin accessibility of the chimeric proteins expressed in Escherichia coli JK321 or UT5600. Export of cholera toxin B strictly depended on a complete predicted beta-barrel region. The absolute necessity for export of a linking region and its influence on expression as an integral part of the TU was also demonstrated. The different electrophoretic mobilities of native and denatured chimeras indicated that the proposed beta-barrel resides within the C-terminal 312 amino acids of AIDA-I. Together these data provide evidence for the predicted beta-barrel structure and support our formerly proposed model of membrane topology of the AIDA-I autotransporter.     

Functional expression in Escherichia coli and membrane topology of porin HopE, a member of a large family of conserved proteins in Helicobacter pylori

HopE is one of the smallest members of a family of 31 outer membrane proteins in Helicobacter pylori and has been shown to function as a porin. In this study it was cloned into Escherichia coli where it was expressed in the outer membrane, as confirmed by indirect immunofluorescence using HopE-specific antibodies. HopE purified from E. coli reconstituted channels in planar bilayer membranes that were the same size as those formed by HopE purified from H. pylori. A model of the membrane topology of HopE was constructed and indicated that this protein formed a beta-barrel with 16 transmembrane amphipathic beta-strands. The accuracy of this model was tested by linker insertion mutagenesis, assuming that, like other porins, amino acid insertions were not tolerated in the transmembrane beta-strands but were tolerated in the adjoining loop regions. Generally, the results obtained with a series of 12 insertions of the sequence RSKDV and two substitutions were consistent with the topological model. The preponderance of amino acids that were conserved in the extended family of HopE paralogs were predicted to be within the membrane and comprised 45% of all residues in the membrane.     

Topology of the outer membrane usher PapC determined by site-directed fluorescence labeling

In contrast to typical membrane proteins that span the lipid bilayer via transmembrane alpha-helices, bacterial outer membrane proteins adopt a beta-barrel architecture composed of antiparallel transmembrane beta-strands. The topology of outer membrane proteins is difficult to predict accurately using computer algorithms, and topology mapping protocols commonly used for alpha-helical membrane proteins do not work for beta-barrel proteins. We present here the topology of the PapC usher, an outer membrane protein required for assembly and secretion of P pili by the chaperone/usher pathway in uropathogenic Escherichia coli. An initial attempt to map PapC topology by insertion of protease cleavage sites was largely unsuccessful due to lack of cleavage at most sites and the requirement to disrupt the outer membrane to identify periplasmic sites. We therefore adapted a site-directed fluorescence labeling technique to permit topology mapping of outer membrane proteins using small molecule probes in intact bacteria. Using this method, we demonstrated that PapC has the potential to encode up to 32 transmembrane beta-strands. Based on experimental evidence, we propose that the usher consists of an N-terminal beta-barrel domain comprised of 26 beta-strands and that a distinct C-terminal domain is not inserted into the membrane but is located instead within the lumen of the N-terminal beta-barrel similar to the plug domains encoded by the outer membrane iron-siderophore uptake proteins.     

Two-partner secretion of gram-negative bacteria: a single β-barrel protein enables transport across the outer membrane

The mechanisms of protein secretion by pathogenic bacteria remain poorly understood. In gram-negative bacteria, the two-partner secretion pathway exports large, mostly virulence-related "TpsA" proteins across the outer membrane via their dedicated "TpsB" transporters. TpsB transporters belong to the ubiquitous Omp85 superfamily, whose members are involved in protein translocation across, or integration into, cellular membranes. The filamentous hemagglutinin/FhaC pair of Bordetella pertussis is a model two-partner secretion system. We have reconstituted the TpsB transporter FhaC into proteoliposomes and demonstrate that FhaC is the sole outer membrane protein required for translocation of its cognate TpsA protein. This is the first in vitro system for analyzing protein secretion across the outer membrane of gram-negative bacteria. Our data also provide clear evidence for the protein translocation function of Omp85 transporters.     

The amino-terminal region of the long-chain fatty acid transport protein FadL contains an externally exposed domain required for bacteriophage T2 binding

The fatty acid transport protein FadL from Escherichia coli is predicted to be rich in beta-structure and span the outer membrane multiple times to form a long-chain fatty acid specific channel. Proteolysis of FadL within whole cells, total membranes, and isolated outer membranes identified two trypsin-sensitive sites, both predicted to be in externally exposed loops of FadL. Amino acid sequence analysis of the proteolytic fragments determined that the first followed R93 and yielded a peptide beginning with 94S-L-K-A-D-N-I-A-P-T-A104 while the second followed R384 and yielded a peptide beginning with 385S-I-S-I-P-D-Q-D-R-F-W395. Proteolysis using trypsin eliminated the bacteriophage T2 binding activity associated with FadL, suggesting the T2 binding domain within FadL requires elements within one of these extracellular loops. A peptide corresponding to the amino-terminal region of FadL (FadL28-160) was purified and shown to inactivate bacteriophage T2 in a concentration-dependent manner, supporting the hypothesis that the amino-proximal extracellular loop of the protein confers T2 binding activity. Using an artificial neural network (NN) topology prediction method in combination with Gibbs motif sampling, a predicted topology of FadL within the outer membrane was developed. According to this model, FadL spans the outer membrane 20 times as antiparallel beta-strands. The 20 antiparallel beta-strands are presumed to form a beta-barrel specific for long-chain fatty acids. On the basis of our previous studies evaluating the function of FadL using site-specific mutagenesis of the fadL gene, proteolysis of FadL within outer membranes, and studies using the FadL28-160 peptide, the predicted extracellular regions between beta-strands 1 and 2 and beta-strands 3 and 4 are expected to contribute to a domain of the protein required for long-chain fatty acid and bacteriophage T2 binding. The first trypsin-sensitive site (R93) lies between predicted beta-strands 3 and 4 while the second (R384) is between beta-strands 17 and 18. The trypsin-resistant region of FadL is predicted to contain 13 antiparallel beta-strands and contribute to the long-chain fatty acid specific channel.     

Structural and Functional Roles of the Surface-Exposed Loops of the β-Barrel Membrane Protein OmpA from Escherichia coli

The N-terminal domain of the OmpA protein from Escherichia coli, consisting of 170 amino acid residues, is embedded in the outer membrane, in the form of an antiparallel beta-barrel whose eight transmembrane beta-strands are connected by three short periplasmic turns and four relatively large surface-exposed hydrophilic loops. This protein domain serves as a paradigm for the study of membrane assembly of integral beta-structured membrane proteins. In order to dissect the structural and functional roles of the surface-exposed loops, they were shortened separately and in all possible combinations. All 16 loop deletion mutants assembled into the outer membrane with high efficiency and adopted the wild-type membrane topology. This systematic approach proves the absence of topogenic signals (e.g., in the form of loop sizes or charge distributions) in these loops. The shortening of surface-exposed loops did not reduce the thermal stability of the protein. However, none of the mutant proteins, with the exception of the variant with the fourth loop shortened, served as a receptor for the OmpA-specific bacteriophage K3. Furthermore, all loops were necessary for the OmpA protein to function in the stabilization of mating aggregates during F conjugation. An OmpA deletion variant with all four loops shortened, consisting of only 135 amino acid residues, constitutes the smallest beta-structured integral membrane protein known to date. These results represent a further step toward the development of artificial outer membrane proteins.     

Bacterial outer membrane ushers contain distinct targeting and assembly domains for pilus biogenesis

Biogenesis of a superfamily of surface structures by gram-negative bacteria requires the chaperone/usher pathway, a terminal branch of the general secretory pathway. In this pathway a periplasmic chaperone works together with an outer membrane usher to direct substrate folding, assembly, and secretion to the cell surface. We analyzed the structure and function of the PapC usher required for P pilus biogenesis by uropathogenic Escherichia coli. Structural analysis indicated PapC folds as a beta-barrel with short extracellular loops and extensive periplasmic domains. Several periplasmic regions were localized, including two domains containing conserved cysteine pairs. Functional analysis of deletion mutants revealed that the PapC C terminus was not required for insertion of the usher into the outer membrane or for proper folding. The usher C terminus was not necessary for interaction with chaperone-subunit complexes in vitro but was required for pilus biogenesis in vivo. Interestingly, coexpression of PapC C-terminal truncation mutants with the chromosomal fim gene cluster coding for type 1 pili allowed P pilus biogenesis in vivo. These studies suggest that chaperone-subunit complexes target an N-terminal domain of the usher and that subunit assembly into pili depends on a subsequent function provided by the usher C terminus.