Pseudomonas aeruginosa Minor Pilins Are Incorporated into Type IV Pili

Type IV pili are long filamentous appendages required for both adhesion and a unique form of motility known as twitching. Twitching motility involves the extension and retraction of the pilus and requires a number of gene products, including five conserved pilin-like proteins of unknown function (FimU, PilV, PilW, PilX, and PilE in Pseudomonas aeruginosa), termed ‘minor’ pilins. Maintenance of a specific stoichiometric ratio among the minor pilins was important for function, as loss or overexpression of any component impaired motility. Disruption of individual minor pilin genes, or of the AlgR positive regulator of minor pilin operon expression in a strain where pilus retraction was blocked by inactivation of the PilT retraction ATPase, revealed that pili were produced, although levels of piliation were reduced relative to pilT positive control. Differences in the levels of piliation of complemented strains pointed to specific roles for each protein in the assembly process, with FimU and PilX being implicated as key promoters of pilus assembly on the cell surface. Using specific antibodies for each protein, we showed that the minor pilins FimU, PilV, PilW, PilX, and PilE were processed by the pre-pilin peptidase PilD and incorporated throughout the growing pilus filament. This is the first study to demonstrate that the minor pilins, conserved among bacteria expressing type IVa pili, are incorporated into the fiber and support a role for them in the initiation, but not termination, of pilus assembly.     

Type IV pilin structures: insights on shared architecture, fiber assembly, receptor binding and type II secretion

Type IV pili are long, flexible filaments that extend from the surface of Gram-negative bacteria and are formed by the polymerization of pilin subunits. This review focuses on the structural information available for each pilin subclass, type IVa and type IVb, highlighting the contributions crystal and nuclear magnetic resonance structures have made in understanding pilus function and assembly. In addition, the type II secretion pseudopilus subunit structure and helical assembly is compared to that of the type IV pilus. The pilin subunits adopt an alphabeta-roll fold formed by the hydrophobic packing of the C-terminal half of a long alpha-helix against an antiparallel beta-sheet. The conserved N-terminal half of the same alpha-helix, as well as two sequence- and structurally-variable regions, protrude from this globular head domain. Filament models have a hydrophobic core formed by the signature long alpha-helices, with variable regions at the filament surface.     

Structural and Functional Studies of the Pseudomonas aeruginosa Minor Pilin,PilE

Many bacterial pathogens, including Pseudomonas aeruginosa, use type IVa pili (T4aP) for attachment and twitching motility. T4aP are composed primarily of major pilin subunits, which are repeatedly assembled and disassembled to mediate function. A group of pilin-like proteins, the minor pilins FimU and PilVWXE, prime pilus assembly and are incorporated into the pilus. We showed previously that minor pilin PilE depends on the putative priming subcomplex PilVWX and the non-pilin protein PilY1 for incorporation into pili, and that with FimU, PilE may couple the priming subcomplex to the major pilin PilA, allowing for efficient pilus assembly. Here we provide further support for this model, showing interaction of PilE with other minor pilins and the major pilin. A 1.25 Å crystal structure of PilE_Δ1–28 shows a typical type IV pilin fold, demonstrating how it may be incorporated into the pilus. Despite limited sequence identity, PilE is structurally similar to Neisseria meningitidis minor pilins PilX_Nm and PilV_Nm, recently suggested via characterization of mCherry fusions to modulate pilus assembly from within the periplasm. A P. aeruginosa PilE-mCherry fusion failed to complement twitching motility or piliation of a pilE mutant. However, in a retraction-deficient strain where surface piliation depends solely on PilE, the fusion construct restored some surface piliation. PilE-mCherry was present in sheared surface fractions, suggesting that it was incorporated into pili. Together, these data provide evidence that PilE, the sole P. aeruginosa equivalent of PilX_Nm and PilV_Nm, likely connects a priming subcomplex to the major pilin, promoting efficient assembly of T4aP.     

Biogenesis of Type V pili

Pili or fimbriae, which are filamentous structures present on the surface of bacteria, were purified from a periodontal pathogen, Porphyromonas gingivalis, in 1980s. The protein component of pili (stalk pilin), which is its major component, was named FimA; it has a molecular weight of approximately 41 kDa. Because the molecular weight of the pilin from P. gingivalis is twice that of pilins from other bacterial pili, the P. gingivalis Fim pili were suggested to be formed via a novel mechanism. In earlier studies, we reported that the FimA pilin is secreted on the cell surface as a lipoprotein precursor, and the subsequent N-terminal processing of the FimA precursor by arginine-specific proteases is necessary for Fim pili formation. The crystal structures of FimA and its related proteins were determined recently, which show that Fim pili are formed by a protease-mediated strand-exchange mechanism. The most recent study conducted by us, wherein we performed cryoelectron microscopy of the pilus structure, provided evidence in support of this mechanism. As the P. gingivalis Fim pili are formed through novel transport and assembly mechanisms, such pili are now designated as Type V pili. Surface lipoproteins, including the anchor pilin FimB of Fim pili that are present on the outer membrane, have been detected in certain Gram-negative bacteria. Here, we describe the assembly mechanisms of pili, including those of Type V and other pili, as well as the lipoprotein transport mechanisms.     

New tools in an old trade: CS1 pilus morphogenesis

CS1 pili serve as the prototype for a large class of serologically distinct pili associated with enterotoxigenic Escherichia coli that cause diarrhoea in humans. The four genes essential for CS1 pilus morphogenesis, cooB, A, C and D, are arranged in an operon and encode structural and assembly proteins unlike those of other pilus systems commonly associated with Gram-negative bacteria. CS1 pili are composed primarily of the major pilin subunit, CooA, which determines the serological type of the pilus. The major pilin subunit is assembled into pili by the proteins CooB, CooC and CooD. CooD is both a minor component found at the pilus tip and an essential assembly protein, whereas CooC is an outer membrane protein thought to be involved in pilin transport. CooB is a novel periplasmic chaperone-like protein that forms intermolecular complexes with and stabilizes the major and minor pilins. Unlike other pilin chaperones, CooB also stabilizes the outer membrane component of the assembly system, CooC. The proteins of CS1 pili have no significant homology to those of the well-characterized Pap (pyelonephritis-associated) pili and related systems, although most of the features of pilus morphogenesis are similar. Therefore, these appear to be among the rare cases of convergent evolution. Thus, for CS1 pili, enterotoxigenic E. coli use new protein 'tools' in the old 'trade' of forming functional pili.     

Structure of the Vibrio cholerae Type IVb Pilus and stability comparison with the Neisseria gonorrhoeae type IVa pilus

Type IV pili are multifunctional filaments displayed on many bacterial pathogens. Members of the Type IVa pilus subclass are found on a diverse group of human pathogens, whereas Type IVb pili are found almost exclusively on enteric bacteria. The Type IVa and IVb subclasses are distinguished by differences in the pilin subunits, including the fold of the globular domain. To understand the implications of the distinct pilin folds, we compared the stabilities of pilin subunits and pilus filaments for the Type IVa GC pilus from Neisseria gonorrhoeae and the Type IVb toxin-coregulated pilus (TCP) from Vibrio cholerae. We show that while recombinant TCP pilin is more stable than GC pilin, the GC pili are more resistant to proteolysis, heat and chemical denaturation than TCP, remaining intact in 8?M urea. To understand these differences, we determined the TCP structure by electron microscopy and three-dimensional image reconstruction. TCP have an architecture similar to that of GC pili, with subunits arranged in a right-handed 1-start helix and related by an 8.4-? axial rise and a 96.8° azimuthal rotation. However, the TCP subunits are not as tightly packed as GC pilins, and the distinct Type IVb pilin fold exposes a segment of the α-helical core of TCP. Hydrophobic interactions dominate for both pilus subtypes, but base stacking by aromatic residues conserved among the Type IVa pilins may contribute to GC pilus stability. The extraordinary stability of GC pili may represent an adaptation of the Type IVa pili to harsh environments and the need to retract against external forces.     

Structure and assembly of the pseudopilin PulG

The pseudopilin PulG is one of several essential components of the type II pullulanase secretion machinery (the Pul secreton) of the Gram-negative bacterium Klebsiella oxytoca. The sequence of the N-terminal 25 amino acids of the PulG precursor is hydrophobic and very similar to the corresponding region of type IV pilins. The structure of a truncated PulG (lacking the homologous region), as determined by X-ray crystallography, was found to include part of the long N-terminal alpha-helix and the four internal anti-parallel beta-strands that characterize type IV pilins, but PulG lacks the highly variable loop region with a disulphide bond that is found in the latter. When overproduced, PulG forms flexible pili whose structural features, as visualized by electron microscopy, are similar to those of bacterial type IV pili. The average helical repeat comprises 17 PulG subunits and four helical turns. Electron microscopy and molecular modelling show that PulG probably assembles into left-handed helical pili with the long N-terminal alpha-helix tightly packed in the centre of the pilus. As in the type IV pilins, the hydrophobic N-terminal part of the PulG alpha-helix is necessary for its assembly. Subtle sequence variations within this highly conserved segment seem to determine whether or not a type IV pilin can be assembled into pili by the Pul secreton.     

Pseudomonas aeruginosa Minor Pilins Prime Type IVa Pilus Assembly and Promote Surface Display of the PilY1 Adhesin

Type IV pili (T4P) contain hundreds of major subunits, but minor subunits are also required for assembly and function. Here we show that Pseudomonas aeruginosa minor pilins prime pilus assembly and traffic the pilus-associated adhesin and anti-retraction protein, PilY1, to the cell surface. PilV, PilW, and PilX require PilY1 for inclusion in surface pili and vice versa, suggestive of complex formation. PilE requires PilVWXY1 for inclusion, suggesting that it binds a novel interface created by two or more components. FimU is incorporated independently of the others and is proposed to couple the putative minor pilin-PilY1 complex to the major subunit. The production of small amounts of T4P by a mutant lacking the minor pilin operon was traced to expression of minor pseudopilins from the P. aeruginosa type II secretion (T2S) system, showing that under retraction-deficient conditions, T2S minor subunits can prime T4P assembly. Deletion of all minor subunits abrogated pilus assembly. In a strain lacking the minor pseudopilins, PilVWXY1 and either FimU or PilE comprised the minimal set of components required for pilus assembly. Supporting functional conservation of T2S and T4P minor components, our 1.4 Å crystal structure of FimU revealed striking architectural similarity to its T2S ortholog GspH, despite minimal sequence identity. We propose that PilVWXY1 form a priming complex for assembly and that PilE and FimU together stably couple the complex to the major subunit. Trafficking of the anti-retraction factor PilY1 to the cell surface allows for production of pili of sufficient length to support adherence and motility.     

Type IV pilin structure and assembly: X-ray and EM analyses of Vibrio cholerae toxin-coregulated pilus and Pseudomonas aeruginosa PAK pilin

Pilin assembly into type IV pili is required for virulence by bacterial pathogens that cause diseases such as cholera, pneumonia, gonorrhea, and meningitis. Crystal structures of soluble, N-terminally truncated pilin from Vibrio cholera toxin-coregulated pilus (TCP) and full-length PAK pilin from Pseudomonas aeruginosa reveal a novel TCP fold, yet a shared architecture for the type IV pilins. In each pilin subunit a conserved, extended, N-terminal alpha helix wrapped by beta strands anchors the structurally variable globular head. Inside the assembled pilus, characterized by cryo-electron microscopy and crystallography, the extended hydrophobic alpha helices make multisubunit contacts to provide mechanical strength and flexibility. Outside, distinct interactions of adaptable heads contribute surface variation for specificity of pilus function in antigenicity, motility, adhesion, and colony formation.     

Crystal Structure of the Minor Pilin FctB Reveals Determinants of Group A Streptococcal Pilus Anchoring

Cell surface pili are polymeric protein assemblies that enable bacteria to adhere to surfaces and to specific host tissues. The pili expressed by Gram-positive bacteria constitute a unique paradigm in which sortase-mediated covalent linkages join successive pilin subunits like beads on a string. These pili are formed from two or three distinct types of pilin subunit, typically encoded in small gene clusters, often with their cognate sortases. In Group A streptococci (GAS), a major pilin forms the polymeric backbone, whereas two minor pilins are located at the tip and the base. Here, we report the 1.9-Å resolution crystal structure of the GAS basal pilin FctB, revealing an immunoglobulin (Ig)-like N-terminal domain with an extended proline-rich tail. Unexpected structural homology between the FctB Ig-like domain and the N-terminal domain of the GAS shaft pilin helps explain the use of the same sortase for polymerization of the shaft and its attachment to FctB. It also enabled the identification, from mass spectral data, of the lysine residue involved in the covalent linkage of FctB to the shaft. The proline-rich tail forms a polyproline-II helix that appears to be a common feature of the basal (cell wall-anchoring) pilins. Together, our results indicate distinct structural elements in the pilin proteins that play a role in selecting for the appropriate sortases and thereby help orchestrate the ordered assembly of the pilus.     

Structural characterization of CFA/III and Longus type IVb pili from enterotoxigenic Escherichia coli

The type IV pili are helical filaments found on many Gram-negative pathogenic bacteria, with multiple diverse roles in pathogenesis, including microcolony formation, adhesion, and twitching motility. Many pathogenic enterotoxigenic Escherichia coli (ETEC) isolates express one of two type IV pili belonging to the type IVb subclass: CFA/III or Longus. Here we show a direct correlation between CFA/III expression and ETEC aggregation, suggesting that these pili, like the Vibrio cholerae toxin-coregulated pili (TCP), mediate microcolony formation. We report a 1.26-Å resolution crystal structure of CofA, the major pilin subunit from CFA/III. CofA is very similar in structure to V. cholerae TcpA but possesses a 10-amino-acid insertion that replaces part of the α2-helix with an irregular loop containing a 3₁₀-helix. Homology modeling suggests a very similar structure for the Longus LngA pilin. A model for the CFA/III pilus filament was generated using the TCP electron microscopy reconstruction as a template. The unique 3₁₀-helix insert fits perfectly within the gap between CofA globular domains. This insert, together with differences in surface-exposed residues, produces a filament that is smoother and more negatively charged than TCP. To explore the specificity of the type IV pilus assembly apparatus, CofA was expressed heterologously in V. cholerae by replacing the tcpA gene with that of cofA within the tcp operon. Although CofA was synthesized and processed by V. cholerae, no CFA/III filaments were detected, suggesting that the components of the type IVb pilus assembly system are highly specific to their pilin substrates.     

Expression of multiple types of N-methyl Phe pili in Pseudomonas aeruginosa

The nature of pili synthesized by Pseudomonas aeruginosa when plasmid-borne genes of homologous pilins from Bacteroides nodosus are introduced as thermoregulated expression systems has been ascertained. Expression of B. nodosus pili inhibited the production of indigenous P. aeruginosa pili, and an organism harbouring pilin genes from two strains of B. nodosus produced two serologically distinct populations of pili on each cell. Simultaneous production of both indigenous and foreign pili was achieved by partial induction of expression. Homogeneity in pilus structure suggests either that there is an exclusive specificity of interaction between identical pilin subunits in pilus assembly, or that each pilus is produced from the translation products of a single messenger RNA molecule, with translation and pilus assembly closely coupled.     

The structure of an archaeal pilus

Bacterial pili are involved in a host of activities, including motility, adhesion, transformation, and immune escape. Structural studies of these pili have shown that several distinctly different classes exist, with no common origin. Remarkably, it is now known that the archaeal flagellar filament appears to have a common origin with the bacterial type IV pilus, and assembly in both systems involves hydrophobic N-terminal α-helices that form three-stranded coils in the center of these filaments. Recent work has identified further genes in archaea as being similar to bacterial type IV pilins, but the function or structures formed by such gene products was unknown. Using electron cryo-microscopy, we show that an archaeal pilus from Methanococcus maripaludis has a structure entirely different from that of any of the known bacterial pili. Two subunit packing arrangements were identified: one has rings of four subunits spaced by ∼ 44 Å and the other has a one-start helical symmetry with ∼ 2.6 subunits per turn of a ∼ 30 Å pitch helix. Remarkably, these schemes appear to coexist within the same filaments. For the segments composed of rings, the twist between adjacent rings is quite variable, while for the segments having a one-start helix there is a large variability in both the axial rise and the twist per subunit. Since this pilus appears to be assembled from a type IV pilin-like protein with a hydrophobic N-terminal helix, it provides yet another example of how different quaternary structures can be formed from similar building blocks. This result has many implications for understanding the evolutionary divergence of bacteria and archaea.     

Assembly of mutant pilins in Pseudomonas aeruginosa: formation of pili composed of heterologous subunits.

Recently, we reported the degree of N-terminal processing within the cytoplasmic membranes of three mutant pilins from Pseudomonas aeruginosa PAK with respect to leader peptide removal and the methylation of the N-terminal phenylalanine (B. L. Pasloske and W. Paranchych, Mol. Microbiol. 2:489-495, 1988). The results of those experiments showed that the deletion of 4 or 8 amino acids within the highly conserved N terminus greatly inhibited leader peptide removal. On the other hand, the mutation of the glutamate at position 5 to a lysine permitted leader peptide cleavage but inhibited transmethylase activity. In this report, we have examined the effects of these mutant pilins upon pilus assembly in a P. aeruginosa PAO host with or without the chromosomally encoded pilin gene present. Pilins with deletions of 4 or 8 amino acids in the N-terminal region were not incorporated into pili. Interestingly, pilin subunits containing the glutamate-to-lysine mutation were incorporated into compound pili together with PAO wild-type subunits. However, the mutant pilins were unable to polymerize as a homopolymer. When wild-type PAK and PAO pilin subunits were expressed in the same bacterial strain, the pilin subunits assembled into homopolymeric pili containing one or the other type of subunit.     

Neisseria meningitidis Type IV Pili Composed of Sequence Invariable Pilins Are Masked by Multisite Glycosylation

The ability of pathogens to cause disease depends on their aptitude to escape the immune system. Type IV pili are extracellular filamentous virulence factors composed of pilin monomers and frequently expressed by bacterial pathogens. As such they are major targets for the host immune system. In the human pathogen Neisseria meningitidis, strains expressing class I pilins contain a genetic recombination system that promotes variation of the pilin sequence and is thought to aid immune escape. However, numerous hypervirulent clinical isolates express class II pilins that lack this property. This raises the question of how they evade immunity targeting type IV pili. As glycosylation is a possible source of antigenic variation it was investigated using top-down mass spectrometry to provide the highest molecular precision on the modified proteins. Unlike class I pilins that carry a single glycan, we found that class II pilins display up to 5 glycosylation sites per monomer on the pilus surface. Swapping of pilin class and genetic background shows that the pilin primary structure determines multisite glycosylation while the genetic background determines the nature of the glycans. Absence of glycosylation in class II pilins affects pilus biogenesis or enhances pilus-dependent aggregation in a strain specific fashion highlighting the extensive functional impact of multisite glycosylation. Finally, molecular modeling shows that glycans cover the surface of class II pilins and strongly decrease antibody access to the polypeptide chain. This strongly supports a model where strains expressing class II pilins evade the immune system by changing their sugar structure rather than pilin primary structure. Overall these results show that sequence invariable class II pilins are cloaked in glycans with extensive functional and immunological consequences.     

Chaperone-independent folding of type 1 pilus domains

An elementary step in the assembly of adhesive type 1 pili of Escherichia coli is the folding of structural pilus subunits in the periplasm. The previously determined X-ray structure of the complex between the type 1 pilus adhesin FimH and the periplasmic pilus assembly chaperone FimC has shown that FimH consists of a N-terminal lectin domain and a C-terminal pilin domain, and that FimC exclusively interacts with the pilin domain. The pilin domain fold, which is common to all pilus subunits, is characterized by an incomplete beta-sheet that is completed by a donor strand from FimC in the FimC-FimH complex. This, together with unsuccessful attempts to refold isolated, urea-denatured FimH in vitro had suggested that folding of pilin domains strictly depends on sequence information provided by FimC. We have now analyzed in detail the folding of FimH and its two isolated domains in vitro. We find that not only the lectin domain, but also the pilin domain can fold autonomously and independently of FimC. However, the thermodynamic stability of the pilin domain is very low (8-10kJmol(-1)) so that a significant fraction of the domain is unfolded even in the absence of denaturant. This explains the high tendency of structural pilus subunits to aggregate non-specifically in the absence of stoichiometric amounts of FimC. Thus, pilus chaperones prevent non-specific aggregation of pilus subunits by native state stabilization after subunit folding.     

Expression of multiple types of N-methyl Phe pili in Pseudomonas aeruginosa

The nature of pili synthesized by Pseudomonas aeruginosa when plasmid-borne genes of homologous pilins from Bacteroides nodosus are Introduced as thermoregulated expression systems has been ascertained. Expression of B. nodosus pili inhibited the production of indigenous P. aeruginosa pili, and an organism harbouring pilin genes from two strains of B. nodosus produced two seroiogically distinct populations of pili on each cell. Simultaneous production of both Indigenous and foreign pili was achieved by partial induction of expression. Homogeneity in pilus structure suggests either that there is an exclusive specificity of Interaction between identical pilin subunits in pilus assembly, or that each pilus is produced from the translation products of a single messenger RNA molecule, with translation and pilus assembly closely coupled.     

Corynebacterium diphtheriae employs specific minor pilins to target human pharyngeal epithelial cells

Adherence to host tissues mediated by pili is pivotal in the establishment of infection by many bacterial pathogens. Corynebacterium diphtheriae assembles on its surface three distinct pilus structures. The function and the mechanism of how various pili mediate adherence, however, have remained poorly understood. Here we show that the SpaA-type pilus is sufficient for the specific adherence of corynebacteria to human pharyngeal epithelial cells. The deletion of the spaA gene, which encodes the major pilin forming the pilus shaft, abolishes pilus assembly but not adherence to pharyngeal cells. In contrast, adherence is greatly diminished when either minor pilin SpaB or SpaC is absent. Antibodies directed against either SpaB or SpaC block bacterial adherence. Consistent with a direct role of the minor pilins, latex beads coated with SpaB or SpaC protein bind specifically to pharyngeal cells. Therefore, tissue tropism of corynebacteria for pharyngeal cells is governed by specific minor pilins. Importantly, immunoelectron microscopy and immunofluorescence studies reveal clusters of minor pilins that are anchored to cell surface in the absence of a pilus shaft. Thus, the minor pilins may also be cell wall anchored in addition to their incorporation into pilus structures that could facilitate tight binding to host cells during bacterial infection.     

Ultrahigh resolution and full-length pilin structures with insights for filament assembly, pathogenic functions, and vaccine potential

Pilin proteins assemble into Type IV pili (T4P), surface-displayed bacterial filaments with virulence functions including motility, attachment, transformation, immune escape, and colony formation. However, challenges in crystallizing full-length fiber-forming and membrane protein pilins leave unanswered questions regarding pilin structures, assembly, functions, and vaccine potential. Here we report pilin structures of full-length DnFimA from the sheep pathogen Dichelobacter nodosus and FtPilE from the human pathogen Francisella tularensis at 2.3 and 1 ? resolution, respectively. The DnFimA structure reveals an extended kinked N-terminal α-helix, an unusual centrally located disulfide, conserved subdomains, and assembled epitopes informing serogroup vaccines. An interaction between the conserved Glu-5 carboxyl oxygen and the N-terminal amine of an adjacent subunit in the crystallographic dimer is consistent with the hypothesis of a salt bridge between these groups driving T4P assembly. The FtPilE structure identifies an authentic Type IV pilin and provides a framework for understanding the role of T4P in F. tularensis virulence. Combined results define a unified pilin architecture, specialized subdomain roles in pilus assembly and function, and potential therapeutic targets.     

Assembly of pili on the surface of Corynebacterium diphtheriae

Pili of Gram-negative pathogens are formed from pilin precursor molecules by non-covalent association within the outer membrane envelope. Gram-positive microbes employ the cell wall peptidoglycan as a surface organelle for the covalent attachment of proteins, however, an assembly pathway for pili has not yet been revealed. We show here that pili of Corynebacterium diphtheriae are composed of three pilin subunits, SpaA, SpaB and SpaC. SpaA, the major pilin protein, is distributed uniformly along the pilus shaft, whereas SpaB is observed at regular intervals and SpaC seems positioned at the pilus tip. Assembled pili are released from the bacterial surface by treatment with murein hydrolase, suggesting that the pilus fibres may be anchored to the cell wall envelope. All three pilin subunit proteins are synthesized as precursors carrying N-terminal signal peptides and C-terminal sorting signals. Some, but not all, of the six sortase genes encoded in the genome of C. diphtheriae are required for precursor processing, pilus assembly or cell wall envelope attachment. Pilus assembly is proposed to occur by a mechanism of ordered cross-linking, whereby pilin-specific sortase enzymes cleave precursor proteins at sorting signals and involve the side chain amino groups of pilin motif sequences to generate links between pilin subunits. This covalent tethering of adjacent pilin subunits appears to have evolved in many Gram-positive pathogens that encode sortase and pilin subunit genes with sorting signals and pilin motifs.