The Pseudomonas aeruginosa type IV pilin receptor binding domain functions as an adhesin for both biotic and abiotic surfaces

Pseudomonas aeruginosa readily binds to stainless steel and other abiotic surfaces, causing major problems in both the medical and food industries. In this study, we show that P. aeruginosa binds to abiotic surfaces in a concentration-dependent, saturable manner during the initial stages of biofilm formation. P. aeruginosa type IV pili mediate binding to stainless steel as a pilus-deficient strain does not bind to steel, purified type IV pili bound in a concentration-dependent, saturable manner, and purified pili competitively inhibited whole cell binding. PAK pili can also bind polystyrene and polyvinylchloride in a concentration-dependant and saturable manner. As an antibody specific for the C-terminal pilin receptor binding domain inhibited adherence to abiotic surfaces, the role of the C-terminal receptor binding domain in mediating binding to steel surfaces was examined. A synthetic peptide of the PAK pilin epithelial cell receptor binding domain [PAK(128-144)ox] bound directly to steel with high affinity. The interaction of pili with steel was specifically inhibited by this peptide with an apparent Ki of approximately 0.2 nM and effectively inhibited the binding of viable homologous and heterologous P. aeruginosa strains to steel with an apparent Ki of approximately 4 nM. A single point mutation (K130I) in the PAO receptor binding domain was observed to abolish binding to stainless steel while binding to human buccal epithelial cells was enhanced. Therefore, the C-terminal receptor binding domain appears to have evolved for binding a variety of surfaces.     

Type IV pilus genes pilA and pilC of Pseudomonas stutzeri are required for natural genetic transformation, and pilA can be replaced by corresponding genes from nontransformable species

Pseudomonas stutzeri lives in terrestrial and aquatic habitats and is capable of natural genetic transformation. After transposon mutagenesis, transformation-deficient mutants were isolated from a P. stutzeri JM300 strain. In one of them a gene which coded for a protein with 75% amino acid sequence identity to PilC of Pseudomonas aeruginosa, an accessory protein for type IV pilus biogenesis, was inactivated. The presence of type IV pili was demonstrated by susceptibility to the type IV pilus-dependent phage PO4, by occurrence of twitching motility, and by electron microscopy. The pilC mutant had no pili and was defective in twitching motility. Further sequencing revealed that pilC is clustered in an operon with genes homologous to pilB and pilD of P. aeruginosa, which are also involved in pilus formation. Next to these genes but transcribed in the opposite orientation a pilA gene encoding a protein with high amino acid sequence identity to pilin, the structural component of type IV pili, was identified. Insertional inactivation of pilA abolished pilus formation, PO4 plating, twitching motility, and natural transformation. The amounts of (3)H-labeled P. stutzeri DNA that were bound to competent parental cells and taken up were strongly reduced in the pilC and pilA mutants. Remarkably, the cloned pilA genes from nontransformable organisms like Dichelobacter nodosus and the PAK and PAO strains of P. aeruginosa fully restored pilus formation and transformability of the P. stutzeri pilA mutant (along with PO4 plating and twitching motility). It is concluded that the type IV pili of the soil bacterium P. stutzeri function in DNA uptake for transformation and that their role in this process is not confined to the species-specific pilin.     

Structure of a pilin monomer from Pseudomonas aeruginosa: implications for the assembly of pili

Type IV pilin monomers assemble to form fibers called pili that are required for a variety of bacterial functions. Pilin monomers oligomerize due to the interaction of part of their hydrophobic N-terminal alpha-helix. Engineering of a truncated pilin from Pseudomonas aeruginosa strain K122-4, where the first 28 residues are removed from the N terminus, yields a soluble, monomeric protein. This truncated pilin is shown to bind to its receptor and to decrease morbidity and mortality in mice upon administration 15 min before challenge with a heterologous strain of Pseudomonas. The structure of this truncated pilin reveals an alpha-helix at the N terminus that lies across a 4-stranded antiparallel beta-sheet. A model for a pilus is proposed that takes into account both electrostatic and hydrophobic interactions of pilin subunits as well as previously published x-ray fiber diffraction data. Our model indicates that DNA or RNA cannot pass through the center of the pilus, however, the possibility exists for small organic molecules to pass through indicating a potential mechanism for signal transduction.     

Human buccal epithelial cell receptors of Pseudomonas aeruginosa: identification of glycoproteins with pilus binding activity

Adherence of Pseudomonas aeruginosa to a patient's epithelial surface is thought to be an important first step in the infection process. Pseudomonas aeruginosa is capable of attaching to epithelial cells via its pili, yet little is known about the epithelial receptors of this adhesin. Using nitrocellulose replicas of polyacrylamide gels of solubilized human buccal epithelial cells (BECs), glycoproteins (Mz: 82,000, and four bands between 40,000 and 50,000) that bound purified pili from P. aeruginosa strain K (PAK) were identified by immunoblotting with a pilus-specific monoclonal antibody that does not affect pilus binding to BECs (PK3B). All pilus-binding glycoproteins were surface localized, as determined by surface radioiodination of intact BECs. Binding of pili to all of the glycoproteins was inhibited by Fab fragments of monoclonal antibody PK99H, which inhibits PAK pili binding to BECs by binding to or near the binding domain of the pilus, but not by Fab fragments of monoclonal antibody PK41C, which binds to PAK pilin but does not inhibit pili binding to BECs, demonstrating that pilus binding to these glycoproteins is likely via the same region of the pilus that binds to intact BECs. Periodate oxidation of the blot eliminated pili binding to all glycoproteins, indicating that a carbohydrate moiety is an important determinant for pilus-binding activity. However, not all of the glycoproteins exhibited the same degree of sensitivity to periodate oxidation. Furthermore, monosaccharide inhibition of pilus binding to BECs implicated L-fucose and N-acetylneuraminic acid as receptor moieties.     

Mapping the surface regions of Pseudomonas aeruginosa PAK pilin: the importance of the C-terminal region for adherence to human buccal epithelial cells

The adherence of non-mucoid Pseudomonas aeruginosa strains is believed to be mediated by the pilus, which consists of a single protein subunit of 15,000 Daltons called pilin. Ten antipeptide antisera were raised to map the surface regions of pilin from P. aeruginosa strain K (PAK). Only one of the antipeptide antisera to the eight predicted surface regions failed to react with PAK pili in direct ELISA. Five out of eight synthetic peptides representing the eight predicted surface regions reacted with anti-PAK pilus antiserum, indicating their surface exposure. Combining the antipeptide and antipilus antisera results, all eight predicted surface regions were demonstrated to be surface-exposed. The PAK 128-144-OH peptide produced the best binding antiserum to PAK pili. Only antipeptide Fab fragments directed against the disulphide bridged C-terminal region of PAK pilin blocked the adherence of pili to human buccal epithelial cells, which suggests that this region contains the receptor-binding domain of the PAK pilus.     

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.     

Products of three accessory genes, pilB, pilC, and pilD, are required for biogenesis of Pseudomonas aeruginosa pili.

The polar pili of Pseudomonas aeruginosa are composed of monomers of the pilin structural subunits. The biogenesis of pili involves the synthesis of pilin precursor, cleavage of a six-amino-acid leader peptide, membrane translocation, and assembly of monomers into a filamentous structure extending from the bacterial surface. This report describes three novel genes necessary for the formation of pili. DNA sequences adjacent to pilA, the pilin structural gene, were cloned and mutagenized with transposon Tn5. Each of the insertions were introduced into the chromosome of P. aeruginosa PAK by gene replacement. The effect of the Tn5 insertions in the bacterial chromosome on pilus assembly was assessed by electron microscopy and sensitivity of mutants to a pilus-specific bacteriophage. The resultant mutants were also tested for synthesis and membrane localization of the pilin antigen in order to define the genes required for maturation, export, and assembly of pilin. A 4.0-kilobase-pair region of DNA adjacent to the pilin structural gene was found to be essential for formation of pili. This region was sequenced and found to contain three open reading frames coding for 62-, 38- to 45-, and 28- to 32-kilodalton proteins (pilB, pilC, and pilD, respectively). Three proteins of similar molecular weight were expressed in Escherichia coli from the 4.0-kilobase-pair fragment flanking pilA with use of a T7 promoter-polymerase expression system. The results of the analyses of the three genes and the implications for pilin assembly and maturation are discussed.     

Crystallographic analysis of the Pseudomonas aeruginosa strain K122-4 monomeric pilin reveals a conserved receptor-binding architecture

Adherence of pathogens to host cells is critical for the initiation of infection and is thus an attractive target for anti-infective therapeutics and vaccines. In the opportunistic human pathogen Pseudomonas aeruginosa, host-cell adherence is achieved predominantly by type IV pili. Analysis of several clinical strains of P. aeruginosa reveals poor sequence conservation between pilin genes, including the residues in the receptor-binding site. Interestingly, the receptor-binding sites appear to retain a conserved surface epitope because all Pseudomonas type IV pili recognize the same receptor on the host cell and cross-reactive antibodies specific for the receptor-binding site exist. Here, we present the crystallographic analysis of two crystal forms of truncated pilin from P. aeruginosa strain K122-4 (DeltaK122-4) at 1.54 and 1.8 A resolution, respectively. The DeltaK122-4 structure is compared to other crystallographically determined type IV pilin structures and an NMR structure of DeltaK122-4 pilin. A comparison with the structure of the highly divergent P. aeruginosa strain K (DeltaPAK) pilin indicates that the receptor-binding loop in both pilins forms a shallow depression with a surface that is formed by main-chain atoms. Conservation of this putative binding site is independent of the sequence as long as the main-chain conformation is conserved and could therefore explain the shared receptor specificity and antibody cross reactivity of highly divergent Pseudomonas type IV pilins.     

Pseudomonas aeruginosa Type IV pilus expression in Neisseria gonorrhoeae: effects of pilin subunit composition on function and organelle dynamics

Type IV pili (TFP) play central roles in the expression of many phenotypes including motility, multicellular behavior, sensitivity to bacteriophages, natural genetic transformation, and adherence. In Neisseria gonorrhoeae, these properties require ancillary proteins that act in conjunction with TFP expression and influence organelle dynamics. Here, the intrinsic contributions of the pilin protein itself to TFP dynamics and associated phenotypes were examined by expressing the Pseudomonas aeruginosa PilA(PAK) pilin subunit in N. gonorrhoeae. We show here that, although PilA(PAK) pilin can be readily assembled into TFP in this background, steady-state levels of purifiable fibers are dramatically reduced relative those of endogenous pili. This defect is due to aberrant TFP dynamics as it is suppressed in the absence of the PilT pilus retraction ATPase. Functionally, PilA(PAK) pilin complements gonococcal adherence for human epithelial cells but only in a pilT background, and this property remains dependent on the coexpression of both the PilC adhesin and the PilV pilin-like protein. Since P. aeruginosa pilin only moderately supports neisserial sequence-specific transformation despite its assembly proficiency, these results together suggest that PilA(PAK) pilin functions suboptimally in this environment. This appears to be due to diminished compatibility with resident proteins essential for TFP function and dynamics. Despite this, PilA(PAK) pili support retractile force generation in this background equivalent to that reported for endogenous pili. Furthermore, PilA(PAK) pili are both necessary and sufficient for bacteriophage PO4 binding, although the strain remains phage resistant. Together, these findings have significant implications for TFP biology in both N. gonorrhoeae and P. aeruginosa.     

Crystal structure of Pseudomonas aeruginosa PAK pilin suggests a main-chain-dominated mode of receptor binding

Fibers of pilin monomers (pili) form the dominant adhesin of Pseudomonas aeruginosa, and they play an important role in infections by this opportunistic bacterial pathogen. Blocking adhesion is therefore a target for vaccine development. The receptor-binding site is located in a C-terminal disulphide-bonded loop of each pilin monomer, but functional binding sites are displayed only at the tip of the pilus. A factor complicating vaccination is that different bacterial strains produce distinct, and sometimes highly divergent, pilin variants. It is surprising that all strains still appear to bind a common receptor, asialo-GM1. Here, we present the 1.63 A crystal structure of pilin from P. aeruginosa strain PAK. The structure shows that the proposed receptor-binding site is formed by two beta-turns that create a surface dominated by main-chain atoms. Receptor specificity could therefore be maintained, whilst allowing side-chain variation, if the main-chain conformation is conserved. The location of the binding site relative to the proposed packing of the pilus fiber raises new issues and suggests that the current fiber model may have to be reconsidered. Finally, the structure of the C-terminal disulphide-bonded loop will provide the template for the structure-based design of a consensus sequence vaccine.     

Use of synthetic peptides to confirm that the Pseudomonas aeruginosa PAK pilus adhesin and the Candida albicans fimbrial adhesin possess a homologous receptor-binding domain

Pseudomonas aeruginosa PAK pili and Candida albicans fimbriae are adhesins present on the microbial cell surfaces which mediate binding to epithelial cell-surface receptors. The receptor-binding domain (adhesintope) of the PAK pilus adhesin has been shown previously to reside in the carboxy-terminal disulphide-bonded region of P. aeruginosa pilin (PAK128-144). The delineation of the C. albicans fimbrial adhesintope was investigated in these studies using synthetic peptides which correspond to the whole (PAK128-144) or part of (PAK134-140) adhesintope of the PAK pilus and their respective anti-peptide antisera and biotinylated PAK pili (Bt-PAK pili), fimbriae (Bt-fimbriae), P. aeruginosa whole cells (Bt- P. aeruginosa ) and C. albicans whole cells (Bt- C. albicans ). The results from these studies confirmed that a structurally conserved motif akin to the PAK(128-144) peptide sequence is present in C. albicans fimbrial adhesin and that the seven-amino-acid residue PAK(134-140) sequence plays an important role in forming the adhesintope for both P. aeruginosa PAK pilus and C. albicans fimbrial adhesins.     

Characterization of type IV pilus genes in plant growth-promoting Pseudomonas putida WCS358.

In a search for factors that could contribute to the ability of the plant growth-stimulating Pseudomonas putida WCS358 to colonize plant roots, the organism was analyzed for the presence of genes required for pilus biosynthesis. The pilD gene of Pseudomonas aeruginosa, which has also been designated xcpA, is involved in protein secretion and in the biogenesis of type IV pili. It encodes a peptidase that processes the precursors of the pilin subunits and of several components of the secretion apparatus. Prepilin processing activity could be demonstrated in P. putida WCS358, suggesting that this nonpathogenic strain may contain type IV pili as well. A DNA fragment containing the pilD (xcpA) gene of P. putida was cloned and found to complement a pilD (xcpA) mutation in P. aeruginosa. Nucleotide sequencing revealed, next to the pilD (xcpA) gene, the presence of two additional genes, pilA and pilC, that are highly homologous to genes involved in the biogenesis of type IV pili. The pilA gene encodes the pilin subunit, and pilC is an accessory gene, required for the assembly of the subunits into pili. In comparison with the pil gene cluster in P. aeruginosa, a gene homologous to pilB is lacking in the P. putida gene cluster. Pili were not detected on the cell surface of P. putida itself, not even when pilA was expressed from the tac promoter on a plasmid, indicating that not all the genes required for pilus biogenesis were expressed under the conditions tested. Expression of pilA of P. putida in P. aeruginosa resulted in the production of pili containing P. putida PilA subunits.     

The binding of Pseudomonas aeruginosa pili to glycosphingolipids is a tip-associated event involving the C-terminal region of the structural pilin subunit

Pili are one of the adhesins of Pseudomonas aeruginosa that mediate adherence to epithelial cell-surface receptors. The pili of P. aeruginosa strains PAK and PAO were examined and found to bind gangliotetraosyl ceramide (asialo-GM₁) and, to a lesser extend, ll³N-acetylneuraminosylgangliotetraosyl ceramide (GM₁) in solid-phase binding assays. Asialo-GM₁, but not GM₁, inhibited both PAK and PAK pili binding to immobilized asialo-GM₁ on the microtitre plate. PAO pili competitively inhibited PAK pili binding to asialo-GM₁, suggesting the presence of a structurally similar receptor-binding domain in both pilus types. The interaction between asialo-GM₁ and pili occurs at the pilus tip as asialo-GM₁ coated colloidal gold only decorates the tip of purified pili. Three sets of evidence suggest that the C-terminal disulphide-bonded region of the Pseudomonas pilin is exposed at the tip of the pilus: (i) immunocytochemical studies indicate that P. aeruginosa pili have a basal-tip structural differentiation where the monoclonal antibody (mAb) PK3B recognizes an antigenic epitope displayed only on the basal ends of pili (produced by shearing) while the mAb PK99H, whose antigenic epitope resides in residues 134–140 (Wong et al., 1992), binds only to the tip of PAK pili; (ii) synthetic peptides, PAK(128–144)^ox-OH and PAO(128–144)^ox-OH, which correspond to the C-terminal disulphide-bonded region of Pseudomonas pilin are able to bind to asialo-GM₁ and inhibit the binding of pili to the glycolipid; (iii) PK99H was shown to block PAK pilus binding to asialo-GM₁ Monoclonal antibody PK3B had no effect on PAK pili binding to asialo-GM₁ Thus, the adherence of the Pseudomonas pilus to glycosphingolipid receptors is a tip-associated phenomenon Involving a tip-exposed C-terminal region of the pilin structural subunit.     

The pili of Pseudomonas aeruginosa strains PAK and PAO bind specifically to the carbohydrate sequence βGalNAc(1–4)βGal found in glycosphingolipids asialo-GM1 and asialo-GM2

Pseudomonas aeruginosa employs pili to mediate adherence to epithelial cell surfaces. The pilus adhesin of P. aeruginosa strains PAK and PAO has been shown to bind to the glycolipid asialo-GM₁ (Lee et al., 1994 —accompanying article). PAK and PAO pili were examined for their abilities to bind to the synthetic βGalNAc(1–4)βGal (a minimal structural carbohydrate receptor sequence of asialo-GM₁ and asialo-GM₂ proposed by Krivan et al., 1988a) using solid-phase binding assays. Both pill specifically bound to βGalNAc(1–4)βGal. The binding of βGal-NAc(1–4)βGal-Biotin to the Immobilized PAK and PAO pili was inhibited by corresponding free pili. The receptor binding domain of the PAK pilus resides in the C-terminal disulphide-looped region (residues 128–144) of the pilin structural subunit (Irvin et al., 1989). Biotinylated synthetic peptides corresponding the C-terminal residues 128–144 of P. aeruginosa PAK and PAO pilin molecules were shown to bind to the βGalNAc(1–4)βGal-(bovine serum albumin (BSA)). The binding of biotinylated peptides to βGalNAc-(1–4)βGal-BSA was inhibited by PAK pili, Ac-KCTSDQDEOFIPKGCSK-OH (AcPAK(128–144)_ox-OH) and Ac-ACKSTQDPMFTPKGCDN-OH (AcPAO(128–144)_ox-OH) peptides. (In these peptides Ac denotes N^α -acetylation of the N-terminus, -OH means a peptide with a free a-carboxyl group at the C-terminus and the‘ox’denotes the oxidation of the sulphhydryl groups of Cys–129 and Cys–142.) Both acetylated peptides were also able to inhibit the binding of βGalNAc(1–4)βGal-biotin to the corresponding BSA-Peptide(128–144)_ox-OH conjugates. The βGlcNAc(1–3)βGal(1–4)βGlc-biotin conjugate was unable to specifically bind to either Immobilized PAK and PAO pili or the respective C-termlnal peptides. The data above demonstrated that the P. aeruginosa pili recognize asialo-GM₁ receptor analogue and that βGalNAc(1–4)βGal disaccharlde is sufficient for binding. Furthermore, the binding to βGalNAc(1–4)βGal was mediated by residues 128–144 of the pilin subunit.     

Expression of the Pseudomonas aeruginosa PAK pilin gene in Escherichia coli.

Pseudomonas aeruginosa is a piliated opportunistic pathogen. We have recently reported the cloning of the structural gene for the pilus protein, pilin, from P. aeruginosa PAK (B. L. Pasloske, B. B. Finlay, and W. Paranchych, FEBS Lett. 183:408-412, 1985), and in this paper we present evidence that this chimera (pBP001) expresses P. aeruginosa PAK pilin in Escherichia coli independent of a vector promoter. The strength of the promoter for the PAK pilin gene was assayed, and the cellular location of the pilin protein within E. coli was examined. This protein was present mainly in the inner membrane fraction both with and without its six-amino-acid leader sequence, but it was not assembled into pili.     

Type IV pili are involved in plant–microbe and fungus–microbe interactions

Adherence of bacteria to eukaryotic cells is essential for the initiation of infection in many animal and human pathogens, e.g. Neisseria gonorrhoeae and Pseudomonas aeruginosa . Adhesion-mediating type IV pili, filamentous surface appendages formed by pilin subunits, are crucial virulence factors. Here, we report that type IV pilus-dependent adhesion is also involved in plant–bacteria and fungus–bacteria interactions. Nitrogen-fixing, endophytic bacteria, Azoarcus sp., can infect the roots of rice and spread systemically into the shoot without causing symptoms of plant disease. Formation of pili on solid media was dependent on the pilAB locus. PilA encodes an unusually short (6.4 kDa) putative pilin precursor showing 100% homology to the conserved N-terminus of the Pseudomonas aeruginosa type IV pilin. PilB encodes for a 14.2 kDa polypeptide showing similarity to FimF, a component of type I fimbriae of Escherichia coli . It was found to be extruded beyond the cell surface by immunofluorescence studies, and it may, therefore, be part of a pilus assembly complex or the pilus itself. Both genes are involved in the establishment of bacteria on the root surface of rice seedlings, as detected by fluorescence microscopy. Moreover, both genes are necessary for bacterial adhesion to the mycelium of an ascomycete, which was isolated from the same rhizosphere as the bacteria. In co-culture with the fungus, Azoarcus sp. forms complex intracytoplasmic membranes, diazosomes, which are related to efficient nitrogen fixation. Adhesion to the mycelium appears to be crucial for this process, as diazosomes were absent and nitrogen fixation rates were decreased in pilAB mutants in co-culture.     

Uptake of extracellular DNA: Competence induced pili in natural transformation of Streptococcus pneumoniae

Transport of DNA across bacterial membranes involves complex DNA uptake systems. In Gram‐positive bacteria, the DNA uptake machinery shares fundamental similarities with type IV pili and type II secretion systems. Although dedicated pilus structures, such as type IV pili in Gram‐negative bacteria, are necessary for efficient DNA uptake, the role of similar structures in Gram‐positive bacteria is just beginning to emerge. Recently two essentially very different pilus structures composed of the same major pilin protein ComGC were proposed to be involved in transformation of the Gram‐positive bacterium Streptococcus pneumoniae – one is a long, thin, type IV pilus‐like fiber with DNA binding capacity and the other one is a pilus structure that was thicker, much shorter and not able to bind DNA. Here we discuss how competence induced pili, either by pilus retraction or by a transient pilus‐related opening in the cell wall, may mediate DNA uptake in S. pneumoniae.     

The ancillary protein 1 of Streptococcus pyogenes FCT-1 pili mediates cell adhesion and biofilm formation through heterophilic as well as homophilic interactions

Gram‐positive pili are known to play a role in bacterial adhesion to epithelial cells and in the formation of biofilm microbial communities. In the present study we undertook the functional characterization of the pilus ancillary protein 1 (AP1_M6) from Streptococcus pyogenes isolates expressing the FCT‐1 pilus variant, known to be strong biofilm formers. Cell binding and biofilm formation assays using S. pyogenes in‐frame deletion mutants, Lactococcus expressing heterologous FCT‐1 pili and purified recombinant AP1_M6, indicated that this pilin is a strong cell adhesin that is also involved in bacterial biofilm formation. Moreover, we show that AP1_M6 establishes homophilic interactions that mediate inter‐bacterial contact, possibly promoting bacterial colonization of target epithelial cells in the form of three‐dimensional microcolonies. Finally, AP1_M6 knockout mutants were less virulent in mice, indicating that this protein is also implicated in GAS systemic infection.     

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.