Investigation of the role of type IV Aeromonas pilus (Tap) in the pathogenesis of Aeromonas gastrointestinal infection.

Although there is substantial evidence that type IV pili purified from diarrhea-associated Aeromonas species (designated Bfp for bundle-forming pilus) are intestinal colonization factors (S. M. Kirov, L. A. O'Donovan, and K. Sanderson, Infect. Immun. 67:5447-5454, 1999), nothing is known regarding the function of a second family of Aeromonas type IV pili (designated Tap for type IV Aeromonas pilus), identified following the cloning of a pilus biogenesis gene cluster tapABCD. Related pilus gene clusters are widely conserved among gram-negative bacteria, but their significance for virulence has been controversial. To investigate the role of Tap pili in Aeromonas pathogenesis, mutants of Aeromonas strains (a fish isolate of A. hydrophila and a human dysenteric isolate of A. veronii bv. sobria) were prepared by insertional inactivation of the tapA gene which encodes the type IV pilus subunit protein, TapA. Exotoxic activities were unaffected by the mutation in tapA. Inactivation of tapA had no effect on the bacterial adherence of these two isolates to HEp-2 cells. For the A. veronii bv. sobria isolate, adhesion to Henle 407 intestinal cells and to human intestinal tissue was also unaffected. There was no significant effect on the duration of colonization or incidence of diarrhea when the A. veronii bv. sobria strain was tested in the removable intestinal tie adult rabbit diarrhea model or on its ability to colonize infant mice. Evidence was obtained that demonstrated that TapA was expressed by both Aeromonas species and was present on the cell surface, although if assembled into pili this pilus type appears to be an uncommon one under standard bacterial growth conditions. Further studies into factors which may influence Tap expression are required, but the present study suggests that Tap pili may not be as significant as Bfp pili for Aeromonas intestinal colonization.

Epidemiology and pathogenesis of Vibrio vulnificus.

Vibrio vulnificus is capable of causing severe and often fatal infections in susceptible individuals. It causes two distinct disease syndromes, a primary septicemia and necrotizing wound infections. This review discusses the interaction of environmental conditions, host factors, and bacterial virulence determinants that contribute to the epidemiology and pathogenesis of V. vulnificus.

Description and characterization of IS994, a putative IS3 family insertion sequence from the salmon pathogen, Renibacterium salmoninarum.

Renibacterium salmoninarum, a slowly growing, Gram-positive bacterium, is responsible for bacterial kidney disease in salmonid fishes world-wide. To date, no mobile genetic elements have been reported for this pathogen. Here, we describe the first insertion sequence (IS) identified from R. salmoninarum. This element, IS994, has a significant predicted amino acid sequence homology (64.8 and 71.9%) to the two open reading frames encoding the transposase of IS6110 of Mycobacterium tuberculosis. Protein parsimony and protein distance matrix analyses show that IS994 is a member of group IS51 of the IS3 family. From a conservative estimate, there are at least 17 chromosomal insertions of IS994 or closely related elements. Sequence analysis of seven of these loci reveals single nucleotide polymorphisms throughout the element (including the terminal inverted repeats), a 15bp insertion in three of the seven loci, and an absence of flanking direct repeats or conserved insertion site. Restriction fragment length polymorphism analysis of XbaI-digested chromosomal DNA shows variations among European and North American isolates, indicating that IS994 may be a useful molecular marker for epizootiological studies.

Differential expression of the virulence-associated protein p57 and characterization of its duplicated gene msa in virulent and attenuated strains of Renibacterium salmoninarum.

Virulence mechanisms utilized by the salmonid fish pathogen Renibacterium salmoninarum are poorly understood. One potential virulence factor is p57 (also designated MSA for major soluble antigen), an abundant 57 kDa soluble protein that is predominately localized on the bacterial cell surface with significant levels released into the extracellular milieu. Previous studies of an attenuated strain, MT 239, indicated that it differs from virulent strains in the amount of surface-associated p57. In this report, we show overall expression of p57 in R. salmoninarum MT 239 is considerably reduced as compared to a virulent strain, ATCC 33209. The amount of cell-associated p57 is decreased while the level of p57 in the culture supernatant is nearly equivalent between the strains. To determine if the lowered amount of cell-associated p57 was due to a sequence defect in p57, a genetic comparison was performed. Two copies of the gene encoding p57 (msa1 and msa2) were found in 33209 and MT 239, as well as in several other virulent isolates. Both copies from 33209 and MT 239 were cloned and sequenced and found to be identical to each other, and identical between the 2 strains. A comparison of msa1 and msa2 within each strain showed that their sequences diverge 40 base pairs 5' to the open reading frame, while sequences 3' to the open reading frame are essentially identical for at least 225 base pairs. Northern blot analysis showed no difference in steady state levels of msa mRNA between the 2 strains. These data suggest that while cell-surface localization of p57 may be important for R. salmoninarum virulence, the differences in localization and total p57 expression between 33209 and MT 239 are not due to differences in msa sequence or differences in steady state transcript levels.

The type IV leader peptidase/N-methyltransferase of Vibrio vulnificus controls factors required for adherence to HEp-2 cells and virulence in iron-overloaded mice.

Vibrio vulnificus expresses a number of potential virulence determinants that may contribute to its ability to cause a severe and rapidly disseminating septicemia in susceptible hosts. We have cloned and characterized two genes encoding products related to components of the type IV pilus biogenesis and general secretory (type II) pathways by complementation of a type IV peptidase/N-methyltransferase (PilD) mutant of Pseudomonas aeruginosa with a V. vulnificus genomic library. One of the genes (vvpD) encodes a protein homologous to PilD and other members of the type IV peptidase family that completely restores this activity in a P. aeruginosa mutant deficient in the expression of PilD. The other gene (vvpC) encodes a homolog of PilC from P. aeruginosa, where it is essential for assembly of type IV pili. Phenotypic characterization of a V. vulnificus vvpD mutant, constructed by allelic exchange, showed that VvpD is required for the expression of surface pili, suggesting that the pili observed on V. vulnificus are of the type IV class. This mutant was also unable to secrete at least three extracellular degradative enzymes, and the localization of one of these (the cytolysin/hemolysin) to the periplasmic space indicates that these proteins are normally exported via the type II secretion pathway. Loss of VvpD resulted in significant decreases in CHO cell cytotoxicity, adherence to HEp-2 cells, and virulence in a mouse model. Capsule formation and serum resistance were not affected in the vvpD mutant, indicating that in addition to capsule, virulence of V. vulnificus requires type IV pili and/or extracellular secretion of several exoenzymes.

Sensitive detection of Renibacterium salmoninarum in whole fry, blood, and other tissues of pacific salmon by reverse transcription-polymerase chain reaction.

A sensitive, reproducible assay for detecting Renibacterium salmoninarum in a variety of tissues, including blood, has been developed. This assay, based on reverse transcription-polymerase chain reaction (RT-PCR) of 16S ribosomal RNA, exhibited sensitivity to

Aeromonas spp. possess at least two distinct type IV pilus families.

Type IV pili have been purified from strains of most of the Aeromonas species associated with gastroenteritis (A. veronii biovar sobria, A. hydrophila, A. trota and A. caviae). They appear to be a related family (molecular mass of pilin 19 to 23 kDa) with a tendency to bundle-formation. Hence, we have designated them 'bundle-forming pili' (Bfp). A type IV pilus biogenesis gene cluster (tapABCD) recently cloned from a strain of A. hydrophila, however, encoded a 17 kDa pilin which differed significantly in its N-terminal amino acid sequence from the Bfp pilins. This paper describes the cloning of part (tapA and approximately 20% of tapB) of a homologous pilin gene cluster from a Bfp-positive strain of A. veronii biovar sobria, and presents evidence that the entire pilin gene cluster (tapABCD) is present in this strain. The predicted N-terminal amino acid sequence of the pilin encoded by the A. veronii biovar sobria tapA differed markedly from the corresponding sequence of its Bfp pilin, and those of the Bfp purified from other Aeromonas strains and species. Probing with tapA and tapD genes showed that these Bfp-positive Aeromonas strains also possessed the Tap gene cluster. TapA proteins of A. veronii biovar sobria and A. hydrophila shared 53% identity and 63% homology. We conclude that Aeromonas species are potentially able to express at least two distinct families of type IV pili (Bfp and Tap).

Structure-function relationship of type-IV prepilin peptidase of Pseudomonas aeruginosa--a review.

The bifunctional enzyme prepilin peptidase (PilD) from Pseudomonas aeruginosa is a key determinant in both type-IV pilus biogenesis and extracellular protein secretion, in its roles as a leader peptidase and MTase. It is responsible for endopeptidic cleavage of the unique leader peptides that characterize type-IV pilin precursors, as well as proteins with homologous leader sequences that are essential components of the general secretion pathway found in a variety of Gram-negative pathogens. Following removal of the leader peptides, the same enzyme is responsible for the second posttranslational modification that characterizes the type-IV pilins and their homologues, namely N-methylation of the newly exposed N-terminal amino acid residue. This review discusses some of the work begun in order to answer questions regarding the structure-function relationships of the active sites of this unique enzyme.

Cloning of an Aeromonas hydrophila type IV pilus biogenesis gene cluster: complementation of pilus assembly functions and characterization of a type IV leader peptidase/N-methyltransferase required for extracellular protein secretion.

Aeromonas hydrophila secretes several extracellular proteins that are associated with virulence including an enterotoxin, a protease, and the hole-forming toxin, aerolysin. These degradative enzymes and toxins are exported by a conserved pathway found in many Gram-negative bacteria. In Pseudomonas aeruginosa this export pathway and type IV pilus biogenesis are dependent on the product of the pilD gene. PilD is a bifunctional enzyme that processes components of the extracellular secretory pathway as well as a type IV prepilin. An A. hydrophila genomic library was transferred into a P. aeruginosa pilD mutant that is defective for type IV pilus biogenesis. The A. hydrophila pilD homologue, tapD, was identified by its ability to complement the pilD mutation in P. aeruginosa. Transconjugants containing tapD were sensitive to the type IV pilus-specific phage, PO4. Sequence data revealed that tapD is part of a cluster of genes (tapABCD) that are homologous to P. aeruginosa type IV pilus biogenesis genes (pilABCD). We showed that TapB and TapC are functionally homologous to P. aeruginosa PilB and PilC, the first such functional complementation of pilus assembly demonstrated between bacteria that express type IV pili. In vitro studies revealed that TapD has both endopeptidase and N-methyltransferase activities using P. aeruginosa prepilin as substrate. Furthermore, we show that tapD is required for extracellular secretion of aerolysin and protease, indicating that tapD may play an important role in the virulence of A. hydrophila.

Posttranslational processing of type IV prepilin and homologs by PilD of Pseudomonas aeruginosa.

We have described the characterization of a protein initially identified as having an essential function in biogenesis of polar pili of P. aeruginosa by processing precursors of pilin. Other findings have also expanded the range of substrates for PilD to include a set of proteins that are essential components of the extracellular secretion machinery. Direct demonstration of prepilin processing necessitates use of purified substrates and enzymes, and we present general protocols for purification of both enzymes and substrates, as well as an assay for prepilin peptidase activity. For a source of enzyme and substrates, mutants of P. aeruginosa defective in pilin processing as well as clones overexpressing the pilin gene and PilD were developed. These methods are applicable to other bacterial systems that express Type IV pili and/or possess the PilD-dependent machinery of extracellular protein secretion. PilD is a bifunctional enzyme, which carries out not only cleavage but also amino-terminal methylation of the mature pilin. Cleavage and N-methylation of the pilin-like Xcp proteins involved in extracellular protein secretion have also been shown to be dependent on PilD. The leader peptidase activity of PilD is inhibited by sulfhydryl blocking reagents such as NEM and PCMB, whereas the methyltransferase activity of the purified enzyme is dependent on reduction with dithiothreitol. The conserved region containing the cysteine residues lies within the largest hydrophilic domain of the protein as predicted from hydrophobicity analysis, and it is probably exposed to the cytoplasmic side of the cytoplasmic membrane. Identification of the active site residues involved in recognition of the substrates for processing and subsequent methylation is currently underway. Studies on substrate specificities of PilD, with respect to its leader peptidase and methyltransferase activity, may prove to be useful in designing inhibitors which would interfere with maturation of Type IV prepilins and components of the extracellular protein secretion machinery. In light of the fact that an increasing number of both mammalian and plant pathogens are being shown to have extracellular secretion pathways homologous to that seen for P. aeruginosa, such inhibitors may be useful tools in the study of the role these peptidases play in bacterial virulence.

Identification of active-site cysteines in the conserved domain of PilD, the bifunctional type IV pilin leader peptidase/N-methyltransferase of Pseudomonas aeruginosa.

PilD is a bifunctional enzyme responsible for cleavage of the leader peptides from the precursors of the type IV pilin and four proteins with type IV pilin-like amino termini that are required for extracellular protein secretion in Pseudomonas aeruginosa. Following cleavage, PilD also catalyzes the second major posttranslational modification of these proteins, namely the N-methylation of the amino-terminal phenylalanine residues of the mature polypeptides. In this report, we demonstrate that the enzymatic activities of PilD involve cysteine residues that lie within a cytoplasmic domain that shows a high degree of similarity to other proteins postulated to perform the same function in other bacterial species. Both activities are reduced in the presence of sulfhydryl-reactive reagents such as N-ethylmaleimide and p-chloromercuribenzoate. Mutagenesis of pilD resulting in specific amino acid substitutions in all of the Cys residues in PilD show that the 4 conserved cysteines in the cytoplasmic domain are required for full peptidase activity in vivo and for complete peptidase and methyltransferase activities in vitro. Conversely, substitution for a Cys residue in a membrane spanning domain had no effect on PilD activities in vivo or in vitro. Evidence suggests that the peptidase and methyltransferase sites of PilD are adjacent, with the Cys residues in the cytoplasmic domain important for methyl donor binding, as prior reaction of PilD with the S-adenosyl-L-methionine analogue sinefungin afforded complete protection of peptidase activity from inactivation with N-ethylmaleimide.

A single bifunctional enzyme, PilD, catalyzes cleavage and N-methylation of proteins belonging to the type IV pilin family.

Precursors of the type IV pilins of a number of bacterial pathogens, as well as related proteins involved in extracellular protein export and DNA uptake, are synthesized with short basic leader sequences. Maturation of these proteins involves two consecutive posttranslational modifications. The leader sequence is first proteolytically removed by specialized endopeptidases, of which the prototype is encoded by the pilD gene of Pseudomonas aeruginosa. Subsequently, the amino termini of these proteins are methylated. Here we demonstrate that PilD, in addition to cleaving the amino-terminal leader sequences of prepilin, also catalyzes N-methylation of the amino-terminal phenylalanine of the mature pilin, using S-adenosyl-L-methionine as a methyl donor. Thus, to our knowledge, PilD is the first characterized bacterial N-methyltransferase. Complete inhibition of N-methylation, but not peptide cleavage, by structural analogues of S-adenosyl-L-methionine suggests that PilD is a bifunctional enzyme with proteolytic and methylation activities carried out within two distinct active sites.

Structure-function and biogenesis of the type IV pili.

Type IV pili are adhesins expressed by a number of diverse gram-negative microorganisms. These pili are related through similarities in the primary amino acid sequences of the structural subunits, a conserved assembly machinery, and a similar mechanism of transcriptional regulation. Type IV pilus assembly is preceded by proteolytic processing and N-methylation of the pilin polypeptide. This process is carried out by a novel bifunctional enzyme PilD, first identified in Pseudomonas aeruginosa. Moreover, proteins homologous with type IV pilins have been shown to function in extracellular protein secretion in gram-negative bacteria and in transformation competence in gram-positive microorganisms. Like prepilin, these proteins are also processed and N-methylated by PilD. Transcription of the genes for type IV pilins is carried out by an RNA polymerase with a minor sigma factor, RpoN. In P. aeruginosa two other regulatory elements (PilS and PilR) are required for pilin expression. RpoN, but not PilS and PilR, is required for expression of a diverse set of bacterial genes. Therefore, regulation of synthesis and posttranslational modification and assembly of type IV pili serves as a useful model for a number of diverse biological processes in the bacterial cell.

Kinetics and sequence specificity of processing of prepilin by PilD, the type IV leader peptidase of Pseudomonas aeruginosa.

PilD, originally isolated as an essential component for the biogenesis of the type IV pili of Pseudomonas aeruginosa, is a unique endopeptidase responsible for processing the precursors of the P. aeruginosa pilin subunits. It is also required for the cleavage of the leader peptides from the Pdd proteins, which are essential components of an extracellular secretion pathway specific for the export of a number of P. aeruginosa hydrolytic enzymes and toxins. Substrates for PilD are initially synthesized with short, i.e., 6- to 8-amino-acid-long, leader peptides with a net basic charge and share a high degree of amino acid homology through the first 16 to 30 residues at the amino terminus. In addition, they all have a phenylalanine residue at the +1 site relative to the cleavage site, which is N methylated prior to assembly into the oligomeric structures. In this study, the kinetics of leader peptide cleavage from the precursor of the P. aeruginosa pilin subunit by PilD was determined in vitro. The rates of cleavage were compared for purified enzyme and substrate as well as for enzyme and substrate contained within total membranes extracted from P. aeruginosa strains overexpressing the cloned pilD or pilA genes. Optimal conditions were obtained only when both PilD and substrate were contained within total membranes. PilD catalysis of P. aeruginosa prepilin followed normal Michaelis-Menten kinetics, with a measured apparent Km of approximately 650 microM, and a kcat of 180 min-1. The kinetics of PilD processing of another type IV pilin precursor, that from Neisseria gonorrhoeae with a 7-amino-acid-long leader peptide, were essentially the same as that measured for wild-type P. aeruginosa prepilin. Quite different results were obtained for a number of prepilin substrates containing substitutions at the conserved phenylalanine at the +1 position relative to the cleavage site, which were previously shown to be well tolerated in vivo. Substitutions of methionine, serine, and cysteine for phenylalanine show that Km values remain close to that measured for wild-type substrate, while kcat and kcat/Km values were significantly decreased. This indicates that while the affinity of enzyme for substrate is relatively unaffected by the substitutions, the maximum rate of catalysis favors a phenylalanine at this position. Interesting, PilD cleavage of one mutated pillin (asparagine) resulted in a lower Km value of 52.5 microM, which indicates a higher affinity for the enzyme, as well as a lower kcat value of 6.1 min m(-1). This suggests that it may be feasible to design peptide inhibitors of PilD.

Multiple roles of the pilus biogenesis protein pilD: involvement of pilD in excretion of enzymes from Pseudomonas aeruginosa.

In Pseudomonas aeruginosa, the genes pilB, pilC, and pilD encode proteins necessary for posttranslational modification and assembly of pilin monomers into pilus organelles (D. Nunn, S. Bergman, and S. Lory, J. Bacteriol. 172:2911-2919, 1990). We show that PilD, encoding a putative pilin-specific leader peptidase, also controls export of alkaline phosphatase, phospholipase C, elastase, and exotoxin A. pilD mutants accumulate these proteins in the periplasmic space, while secretion of periplasmic and outer membrane proteins appears to be normal. The periplasmic form of exotoxin A was fully mature in size, contained all cysteines in disulfide bonds, and was toxic in a tissue culture cytotoxicity assay, suggesting that in pilD mutants, exotoxin A was folded into its native conformation. The function of the other two accessory proteins, PilB and PilC, appears to be restricted to pilus biogenesis, and strains carrying mutations in their respective genes do not show an export defect. These studies show that in addition to cleaving the leader sequence from prepilin, PilD has an additional role in secretion of proteins that are released from P. aeruginosa into the surrounding media. PilD most likely functions as a protease that is involved in processing and assembly of one or more components of the membrane machinery necessary for the later stages of protein extracellular localization.

Amino acid substitutions in pilin of Pseudomonas aeruginosa. Effect on leader peptide cleavage, amino-terminal methylation, and pilus assembly.

A total of 37 separate mutants containing single and multiple amino acid substitutions in the leader and amino-terminal conserved region of the Type IV pilin from Pseudomonas aeruginosa were generated by oligonucleotide-directed mutagenesis. The effect of these substitutions on the secretion, processing, and assembly of the pilin monomers into mature pili was examined. The majority of substitutions in the highly conserved amino-terminal region of the pilin monomer had no effect on piliation. Likewise, substitution of several of the residues within the six amino acid leader sequence did not affect secretion and leader cleavage (processing), including replacement of one or both of the positively charged lysine residues with uncharged or negatively charged amino acids. One characteristic of the Type IV pili is the presence of an amino-terminal phenylalanine after leader peptide cleavage which is N-methylated prior to assembly of pilin monomers into pili. Substitution of the amino-terminal phenylalanine with a number of other amino acids, including polar, hydrophobic, and charged residues, did not affect proper leader cleavage and subsequent assembly into pili. Amino-terminal sequencing showed that the majority of substitute residues were also methylated. Substitution of the glycine residue at the -1 position to the cleavage site resulted in the inability to cleave the prepilin monomers and blocked the subsequent assembly of monomers into pili. These results indicate that despite the high degree of conservation in the amino-terminal sequences of the Type IV pili, N-methylphenylalanine at the +1 position relative to the leader peptide cleavage site is not strictly required for pilin assembly. N-Methylation of the amino acids substituted for phenylalanine was shown to have taken place in four of the five mutants tested, but it remains unclear as to whether pilin assembly is dependent on this modification. Recognition and proper cleavage of the prepilin by the leader peptidase appears to be dependent only on the glycine residue at the -1 position. Cell fractionation experiments demonstrated that pilin isolated from mutants deficient in prepilin processing and/or assembly was found in both inner and outer membrane fractions, indistinguishable from the results seen with the wild type.

Evidence for plasmid-mediated toxin and bacteriocin production in Clostridium botulinum type G.

A single 81-megadalton plasmid was previously isolated from each of six toxigenic strains of Clostridium botulinum type G (M. S. Strom, M. W. Eklund, and F. T. Poysky, Appl. Environ. Microbiol. 48:956-963, 1984). In this study, nontoxigenic derivatives isolated from each of the toxigenic strains following consecutive daily transfers in Trypticase (BBL Microbiology Systems, Cockeysville, Md.)-yeast extract-glucose broth at 44 degrees C simultaneously ceased to produce type G neurotoxin and to harbor the resident 81-megadalton plasmid. The nontoxigenic derivatives also ceased to produce bacteriocin and lost their immunity to the bacteriocin produced by the toxigenic strains. In contrast, all of the toxigenic isolates continued to carry the resident plasmid and to produce both bacteriocin and type G neurotoxin. This is the first evidence suggesting that the production of neurotoxin and bacteriocin by C. botulinum is mediated by a plasmid.

Expression and secretion of the cloned Pseudomonas aeruginosa exotoxin A by Escherichia coli.

The exotoxin A gene from Pseudomonas aeruginosa PAK was expressed in Escherichia coli from recombinant plasmids when transcription was initiated from a promoter in the cloning vector. The exotoxin A polypeptide synthesized was found to have an electrophoretic mobility in sodium dodecyl sulfate-polyacrylamide gels of 66,000 daltons, identical in size to the mature exotoxin A made by P. aeruginosa. Analysis of the location of exotoxin A in various bacterial compartments by immunoblotting revealed that exotoxin A was exported by E. coli into its periplasmic space. Several functional assays, including analyses of disulfide bond formation, potentiation of ADP-ribosyltransferase activity, and HeLa cell cytotoxicity, were used to establish that the conformation of exotoxin A isolated from the E. coli periplasmic space is identical to that of exotoxin exported by P. aeruginosa to its extracellular space. Previous studies with recombinant plasmids expressing exotoxin A from P. aeruginosa PA103 (G. D. Gray, D. Smith, J. Baldridge, R. Markins, M. Vasil, E. Chen, and M. Heyneker, Proc. Natl. Acad. Sci. USA 81:2645-2649, 1984) showed a complete lack of processing and export of pre-exotoxin A in E. coli, differing from results reported here. These discrepancies may be explained by observed differences in the sequence of signal peptides encoded by the exotoxin A genes of PAK and PA103 strains of P. aeruginosa.

Mapping of export signals of Pseudomonas aeruginosa pilin with alkaline phosphatase fusions.

Pili of Pseudomonas aeruginosa are assembled from monomers of the structural subunit, pilin, after secretion of this protein across the bacterial membrane. These subunits are initally synthesized as precursors (prepilin) with a six-amino-acid leader peptide that is cleaved off during or after membrane traversal, followed by methylation of the amino-terminal phenylalanine residue. This report demonstrates that additional sequences from the N terminus of the mature protein are necessary for membrane translocation. Gene fusions were made between amino-terminal coding sequences of the cloned pilin gene (pilA) and the structural gene for Escherichia coli alkaline phosphatase (phoA) devoid of a signal sequence. Fusions between at least 45 amino acid residues of the mature pilin and alkaline phosphatase resulted in translocation of the fusion proteins across the cytoplasmic membranes of both P. aeruginosa and E. coli strains carrying recombinant plasmids, as measured by alkaline phosphatase activity and Western blotting. Fusion proteins constructed with the first 10 amino acids of prepilin (including the 6-amino-acid leader peptide) were not secreted, although they were detected in the cytoplasm. Therefore, unlike that of the majority of secreted proteins that are synthesized with transient signal sequences, the membrane traversal of pilin across the bacterial membrane requires the transient six-amino-acid leader peptide as well as sequences contained in the N-terminal region of the mature pilin protein.