Localization, cloning, and sequence determination of the conjugative plasmid ColB2 pilin gene.

ColB2 is a colicin-producing, 96-kilobase plasmid which encodes a conjugative system that is similar, but not identical, to F. A restriction map of this plasmid was generated, and DNA homology studies between F and ColB2 plasmids revealed homology only between their transfer operons. The locations of the ColB2 transfer operon and ColB2 pilin gene were localized on this restriction map. The gene encoding ColB2 pilin, traA, was cloned and sequenced. The pilin protein of ColB2 is identical to F, except at the amino terminus, where ala-gln of ColB2 pilin corresponds to Ala-Gly-Ser-Ser of F pilin. This is due to a 6-base-pair deletion in the ColB2 pilin gene. Biochemical studies on tryptic peptides derived from ColB2 pilin demonstrate the location of this gene to be correct. There is a putative signal peptidase cleavage site after the sequence Ala-Met-Ala, giving a signal peptide of 51 amino acids and a mature pilin protein of 68 amino acids (7,000 daltons). The amino terminus is blocked, probably with an acetyl group. A chimera containing the ColB2 pilin gene was able to complement an F traA mutant, demonstrating that the pilus assembly proteins of F can utilize the ColB2 pilin protein to form a pilus.     

A receptor-binding site as revealed by the crystal structure of CfaE, the colonization factor antigen I fimbrial adhesin of enterotoxigenic Escherichia coli

CfaE is the minor, tip-localized adhesive subunit of colonization factor antigen I fimbriae (CFA/I) of enterotoxigenic Escherichia coli and is thought to be essential for the attachment of enterotoxigenic E. coli to the human small intestine early in diarrhea pathogenesis. The crystal structure of an in cis donor strand complemented CfaE was determined, providing the first atomic view of a fimbrial subunit assembled by the alternate chaperone pathway. The in cis donor strand complemented variant of CfaE structure consists of an N-terminal adhesin domain and a C-terminal pilin domain of similar size, each featuring a variable immunoglobulin-like fold. Extensive interactions exist between the two domains and appear to rigidify the molecule. The upper surface of the adhesin domain distal to the pilin domain reveals a depression consisting of conserved residues including Arg(181), previously shown to be necessary for erythrocyte adhesion. Mutational analysis revealed a cluster of conserved, positively charged residues that are required for CFA/I-mediated hemagglutination, implicating this as the receptor-binding pocket. Mutations in a few subclass-specific residues that surround the cluster displayed differential effects on the two red cell species used in hemagglutination, suggesting that these residues play a role in host or cell specificity. The C-terminal donor strand derived from the major subunit CfaB is folded as a beta-strand and fits into a hydrophobic groove in the pilin domain to complete the immunoglobulin fold. The location of this well ordered donor strand suggests the positioning and orientation of the subjacent major fimbrial subunit CfaB in the native assembly of CFA/I fimbriae.     

Major antigenic determinants of F and ColB2 pili.

F-like conjugative pili are expressed by plasmids with closely related transfer systems. They are tubular filaments that are composed of repeating pilin subunits arranged in a helical array. Both F and ColB2 pilin have nearly identical protein sequences, and both contain an acetylated amino-terminal alanine residue. However, they differ by a few amino acid residues at their amino termini. Rabbit antisera raised against purified F and ColB2 pili are immunologically cross-reactive by only 25%, as measured by a competition enzyme-linked immunosorbent assay (ELISA). A tryptic peptide corresponding to the first 15 amino acid residues of ColB2 pilin was isolated and found to remove nearly 80% of ColB2 pilus-directed rabbit antibodies. The corresponding tryptic peptide from F pilin, which reacted with anti-F pilus antibodies to remove 80%, was less than 20% reactive with anti-ColB2 pilus antiserum. Cleavage of these peptides with cyanogen bromide (at a methionine residue approximately in the middle of the peptide) did not affect the antigenicity of these peptides. Synthetic N alpha-acetylated peptides corresponding to the first eight amino acids of F pilin (Ac-Ala-Gly-Ser-Ser-Gly-Gln-Asp-Leu-COOH) and the first six amino acids of ColB2 pilin (Ac-Ala-Gln-Gly-Gln-Asp-Leu-COOH) were prepared and tested by competition ELISA with homologous and heterologous anti-pilus antisera. The F peptide F(1-8) inhibited the interaction of F pili and anti-F pilus antiserum to 80%, while the ColB2 peptide ColB2(1-6) inhibited anti-ColB2 pilus antiserum reacting with ColB2 pili by greater than 60%. The two peptides F(1-8) and ColB2(1-6) were inactive by competition ELISAs with heterologous antisera. These results suggest that the major antigenic determinant of both F and ColB2 pili is at the amino terminus of the pilin subunit and that 80% of antibodies raised against these pili are specific for this region of the pilin molecule.     

DNA sequence of the F traALE region that includes the gene for F pilin

The complete sequence of a 1.4-kilobase PstI fragment containing the F transfer genes traA, -L, and -E is presented. The traA reading frame has been located both genetically and by comparing the primary structure of F pilin (the traA product) predicted by the DNA sequence to the amino acid composition and sequence of N- and C-terminal peptides isolated from purified F pilin. Taken together, these data show that there is a leader peptide of 51 amino acids and that F pilin contains 70 amino acids, giving molecular weights of 13,200 for F propilin and 7,200 for mature F pilin. Secondary structure predictions for F pilin revealed a reverse turn that precedes the sequence Ala-Met-Ala51, a classic signal peptidase cleavage site. The N-terminal alanine residue is blocked by an acetyl group as determined by 1H-nuclear magnetic resonance spectroscopy. The traL and traE genes encode proteins of molecular weights 10,350 and 21,200, respectively. According to DNA sequence predictions, these proteins do not contain signal peptide leader sequences. Secondary structure predictions for these proteins are in accord with traLp and traEp being membrane proteins in which hydrophobic regions capable of spanning the membrane are linked by sequences that form turns and carry positively charged residues capable of interacting with the membrane surface.     

Adaptor function of PapF depends on donor strand exchange in P-pilus biogenesis of Escherichia coli

P-pilus biogenesis occurs via the highly conserved chaperone-usher pathway and involves the strict coordination of multiple subunit proteins. All nonadhesin structural P-pilus subunits possess the same topology, consisting of two domains: an incomplete immunoglobulin-like fold (pilin body) and an N-terminal extension. Pilus subunits form interactions with one another through donor strand exchange, occurring at the usher, in which the N-terminal extension of an incoming subunit completes the pilin body of the preceding subunit, allowing the incorporation of the subunit into the pilus fiber. In this study, pilus subunits in which the N-terminal extension was either deleted or swapped with that of another subunit were used to examine the role of each domain of PapF in functions involving donor strand exchange and hierarchical assembly. We found that the N-terminal extension of PapF is required to adapt the PapG adhesin to the tip of the fiber. The pilin body of PapF is required to efficiently initiate assembly of the remainder of the pilus, with the assistance of the N-terminal extension. Thus, distinct functions were assigned to each region of the PapF subunit. In conclusion, all pilin subunits possess the same overall architectural topology; however, each N-terminal extension and pilin body has specific functions in pilus biogenesis.     

The Structure of the PapD-PapGII Pilin Complex Reveals an Open and Flexible P5 Pocket

P pili are hairlike polymeric structures that mediate binding of uropathogenic Escherichia coli to the surface of the kidney via the PapG adhesin at their tips. PapG is composed of two domains: a lectin domain at the tip of the pilus followed by a pilin domain that comprises the initial polymerizing subunit of the 1,000-plus-subunit heteropolymeric pilus fiber. Prior to assembly, periplasmic pilin domains bind to a chaperone, PapD. PapD mediates donor strand complementation, in which a beta strand of PapD temporarily completes the pilin domain''s fold, preventing premature, nonproductive interactions with other pilin subunits and facilitating subunit folding. Chaperone-subunit complexes are delivered to the outer membrane usher where donor strand exchange (DSE) replaces PapD''s donated beta strand with an amino-terminal extension on the next incoming pilin subunit. This occurs via a zip-in–zip-out mechanism that initiates at a relatively accessible hydrophobic space termed the P5 pocket on the terminally incorporated pilus subunit. Here, we solve the structure of PapD in complex with the pilin domain of isoform II of PapG (PapGIIp). Our data revealed that PapGIIp adopts an immunoglobulin fold with a missing seventh strand, complemented in parallel by the G1 PapD strand, typical of pilin subunits. Comparisons with other chaperone-pilin complexes indicated that the interactive surfaces are highly conserved. Interestingly, the PapGIIp P5 pocket was in an open conformation, which, as molecular dynamics simulations revealed, switches between an open and a closed conformation due to the flexibility of the surrounding loops. Our study reveals the structural details of the DSE mechanism.     

Insertion Mutations in pilE Differentially Alter Gonococcal Pilin Antigenic Variation

Pilus antigenic variation in Neisseria gonorrhoeae occurs by the high-frequency, unidirectional transfer of DNA sequences from one of several silent pilin loci (pilS) into the expressed pilin gene (pilE), resulting in a change in the primary pilin protein sequence. Previously, we investigated the effects of large or small heterologous insertions in conserved and variable portions of a pilS copy on antigenic variation. We observed differential effects on pilin recombination by the various insertions, and the severity of the defect correlated with the disruption or displacement of a conserved pilin DNA sequence called cys2. In this study, we show that disruption or displacement of the pilE cys2 sequence by the same insertions or a deletion also affects pilin recombination. However, in contrast to the insertions in pilS, the analogous insertions in pilE impaired, but did not block, recombination of the flanking pilin sequences. These results, the change in the spectrum of donor silent copies used during variation, and our previous results with pilS mutations show that the donor pilS and recipient pilE play different roles in antigenic variation. We conclude that when high-frequency recombination mechanisms are blocked, alternative mechanisms are operative.     

The Escherichia coli K-12 F plasmid gene traX is required for acetylation of F pilin.

The Escherichia coli F plasmid gene required for amino-terminal acetylation of F-pilin subunits was identified. Using Western blots (immunoblots), we assayed the reaction of monoclonal antibodies with F-pilin polypeptides in inner membrane preparations from various F mutant strains. It was known that JEL92 recognizes an internal pilin epitope and JEL93 recognizes the acetylated amino-terminal sequence (L.S. Frost, J.S. Lee, D.G. Scraba, and W. Paranchych, J. Bacteriol. 168:192-198, 1986). As expected, neither antibody reacted with inner membranes from F- cells or Flac derivatives that do not synthesize pilin. Mutations that affected the individual activities of F tra genes traA, -B, -C, -D, -E, -F, -G, -H, -I, -J, -K, -L, -M, -N, -P, -R, -U, -V and -W or trb genes trbA, -B, -C, -D, -E, -G, -H, and -I did not prevent JEL92 or JEL93 recognition of membrane pilin. However, Hfr deletion mutants that lacked the most-distal transfer region genes did not express pilin that reacted with JEL93. Nevertheless, all strains that retained traA and traQ did express JEL92-reactive pilin polypeptides. Analysis of strains expressing cloned tra segments showed that traA and traQ suffice for synthesis of JEL92-reactive pilin, but synthesis of JEL93-reactive pilin is additionally dependent on traX. We concluded that the traX product is required for acetylation of F pilin. Interestingly, our data also showed that TraA+ TraQ+ cells synthesize two forms of pilin which migrate at approximately 7 and 8 kDa. In TraX+ cells, both become acetylated and react with JEL93. Preparations of wild-type F-pilus filaments contain both types of subunits.     

Promiscuous DNA transfer system of Agrobacterium tumefaciens: role of the virB operon in sex pilus assembly and synthesis

Conjugative transfer of DNA that occurs between bacteria also operates between bacteria and higher organisms. The transfer of DNA between Gram-negative bacteria requires initial contact by a sex pilus followed by DNA traversing four membranes (donor plus recipient) using a transmembrane pore. Accumulating evidence suggests that transfer of the T-DNA from Agrobacterium tumefaciens to plants may also occur via a conjugative mechanism. The virB operon of the Ti plasmid exhibits close homologies to genes that are known to encode the pilin subunits and pilin assembly proteins. The proteins encoded by the PilW operon of IncW plasmid R388 share strong similarities (average similarity=50.8%) with VirB proteins. Similarly, the TraA, TraL and TraC proteins of IncF plasmid F have similarities to VirB2, VirB3 and VirB4 respectively (average similarity = 45.3%). VirB2 protein (12.3 kDa) contains a signal peptidase-I cleavage sequence that generates a polypeptide of 7.2 kDa. Likewise, the 12.8 kDa propilin protein TraA of plasmid F also possesses a peptidase-I cleavage site that generates the 7.2 kDa pilin structural protein. Similar amino acid sequences of the conjugative transfer genes of F, R388 as well as plasmid RP4 and the genes of the ptl operon of Bortedella pertussis suggest the existence of a superfamily of transmembrane proteins adapted to the promiscuous transfer of DNA-protein complexes.     

DNA sequence analysis of point mutations in traA, the F pilin gene, reveal two domains involved in F-specific bacteriophage attachment

Summary Six missense point mutations in traA (WPFL43,44,45,46,47 and 51), the gene encoding F pilin in the transfer region of the F plasmid, have been characterized for their effect on the transfer ability, bacteriophage (R17, QB and fl) sensitivity and levels of piliation expressed by the plasmid. The sequence analysis of the first five of these mutations revealed two domains in the F pilin subunit exposed on the surface of the F pilus which mediate phage attachment. These two domains include residues 14–17 (approximately) and the last few residues at the carboxy-terminus of the pilin protein. One of these mutants had a pleiotropic affect on pilus function and was thought to have affected pilus assembly. The sixthe point mutant (WPFL51), previously thought to be in traA, was complemented by chimeric plasmids carrying the traG gene of the F transfer region, which may be involved in the acetylation of the pilin subunit. A traA nonsense mutant (JCFL1) carried an amber mutation near the amino-terminus which is well suppressed in SuI⁺ (supD) and SuIII⁺ (supF) strains. Neither the antigenicity of the pilin nor the efficiency of plating of F-specific bacteriophages were affected when this plasmid was harbored by either suppressor strain. A second amber mutant (JCFL25) which is not suppressible, carried its mutation in the codon for the single tryptophan in F pilin, suggesting that this residue is important in subunit interactions during pilus assembly. Two other point mutants (JCFL32 and 44) carried missense mutations in the leader sequence (positions 9 and 13) which affected the number of pili per cell presumably by altering the processing of propilin to pilin.     

Synthesis of F pilin.

Transfer of the Escherichia coli fertility plasmid, F, is dependent on expression of F pili. Synthesis of F-pilin subunits is known to involve three F plasmid transfer (tra) region products: traA encodes the 13-kDa precursor protein, TraQ permits this to be processed to the 7-kDa pilin polypeptide, and TraX catalyzes acetylation of the pilin amino terminus. Using cloned tra sequences, we performed a series of pulse-chase experiments to investigate the effect of TraQ and TraX on the fate of the traA product. In TraQ- cells, the traA gene product was found to be very unstable. While traA polypeptides of various sizes were detected early in the chase period, almost all were degraded within 5 min. Rapid traA product degradation was also observed in TraX+ cells, although an increased percentage of these products persisted during the chase. In TraQ+ cells, most of the traA product was processed to the 7-kDa pilin polypeptide within the 1-min pulse period; this product [7(Q)] was not degraded but was increasingly converted to an 8-kDa form [8(Q)] as the chase continued, suggesting that host enzymes can modify the pilin polypeptide. Similar results were observed in TraQ+ TraX+ cells, but the primary 7-kDa product appeared to be N-acetylated pilin (Ac-7). An 8-kDa product (Ac-8) was also detected, but this band did not increase in intensity during the chase. We suggest a pathway in which TraQ prevents the traA product from folding to a readily degradable conformation and assists its entry into the membrane, Leader peptidase I cleaves the traA product signal sequence, and a subset of the pilin polypeptides becomes modified by host enzymes; TraX then acetylates the N terminal of both the modified and unmodified pilin polypeptides.     

Characterization and sequence analysis of pilin from F-like plasmids.

Conjugative pili are expressed by derepressed plasmids and initiate cell-to-cell contact during bacterial conjugation. They are also the site of attachment for pilus-specific phages (f1, f2, and QB). In this study, the number of pili per cell and their ability to retract in the presence of cyanide was estimated for 13 derepressed plasmids. Selected pilus types were further characterized for reactivity with anti-F and anti-ColB2 pilus antisera as well as two F pilus-specific monoclonal antibodies, one of which is specific for a sequence common to most F-like pilin types (JEL92) and one which is specific for the amino terminus of F pilin (JEL93). The pilin genes from eight of these plasmids were cloned and sequenced, and the results were compared with information on F, ColB2, and pED208 pilin. Six pilus groups were defined: I, was F-like [F, pED202(R386), ColV2-K94, and ColVBtrp]; IIA was ColB2-like in sequence but had a lowered sensitivity to f1 phage due to its decreased ability for pilus retraction [pED236(ColB2) and pED203(ColB4)]; IIB was ColB2-like but retained f1 sensitivity [pED200(R124) and pED207(R538-1)]; III contained R1-19, which had a ColB2-like amino terminus but had an additional lysine residue at its carboxy terminus which may affect its phage sensitivity pattern and its antigenicity; IV was R100-1-like [R100-1 and presumably pED241(R136) and pED204(R6)] which had a unique amino-terminal sequence combined with a carboxy terminus similar to that of F. pED208(Folac) formed group V, which was multipiliated and exhibited poor pilus retraction although it retained full sensitivity to f1 phage. The pED208 pilin gene could not be cloned at this time since it shared no homology with the pilin gene of the F plasmid.     

Characterization of protein glycosylation in Francisella tularensis subsp. holarctica: identification of a novel glycosylated lipoprotein required for virulence

FTH_0069 is a previously uncharacterized strongly immunoreactive protein that has been proposed to be a novel virulence factor in Francisella tularensis. Here, the glycan structure modifying two C-terminal peptides of FTH_0069 was identified utilizing high resolution, high mass accuracy mass spectrometry, combined with in-source CID tandem MS experiments. The glycan observed at m/z 1156 was determined to be a hexasaccharide, consisting of two hexoses, three N-acetylhexosamines, and an unknown monosaccharide containing a phosphate group. The monosaccharide sequence of the glycan is tentatively proposed as X-P-HexNAc-HexNAc-Hex-Hex-HexNAc, where X denotes the unknown monosaccharide. The glycan is identical to that of DsbA glycoprotein, as well as to one of the multiple glycan structures modifying the type IV pilin PilA, suggesting a common biosynthetic pathway for the protein modification. Here, we demonstrate that the glycosylation of FTH_0069, DsbA, and PilA was affected in an isogenic mutant with a disrupted wbtDEF gene cluster encoding O-antigen synthesis and in a mutant with a deleted pglA gene encoding pilin oligosaccharyltransferase PglA. Based on our findings, we propose that PglA is involved in both pilin and general F. tularensis protein glycosylation, and we further suggest an inter-relationship between the O-antigen and the glycan synthesis in the early steps in their biosynthetic pathways.     

Two monoclonal antibodies specific for different epitopes within the amino-terminal region of F pilin.

Two murine monoclonal antibodies (JEL 92 and 93) specific for adjacent epitopes on F pilin were purified and characterized. JEL 93 immunoglobulin G (IgG) and its Fab fragments were specific for the amino-terminal region and were completely reactive with a synthetic peptide representing the first eight amino acids of F pilin. The acetyl group was demonstrated to be an important part of the epitope, since an unacetylated version of the amino-terminal peptide was 100-fold less reactive with JEL 93 IgG. JEL 92 IgG reacted with the region of F pilin surrounding Met-9, represented by a tryptic peptide derived from the first 17 amino acids. This reactivity was completely abolished by cleavage of the peptide with cyanogen bromide. As shown by electron microscopy, both monoclonal antibodies bound to a vesiclelike structure at one end of purified free pili and did not bind to the sides of the pili, nor did they appear to bind to the tip. When sonication was used to break pili into shorter fragments, the number of binding sites for JEL 92 but not JEL 93 IgG increased as measured by a competitive enzyme-linked immunosorbent assay.     

Comparative Biochemical Studies on F and EDP208 Conjugative Pili

EDP208 pili are encoded by a derepressed derivative of a naturally occurring lac plasmid, F(0)lac (incompatibility group FV), originally isolated from Salmonella typhi. EDP208 pili are serologically unrelated to F pili and do not promote infection by F-specific ribonucleic acid bacteriophages. However, they do confer sensitivity to the F-specific filamentous deoxyribonucleic acid phages. EDP208-containing cells are multi-piliated and contain approximately 20 pili per cell. These pili contain a single polypeptide subunit of 11,500 daltons. EDP208-specific RNA phages were readily isolated from local sewage. These phages were somewhat smaller in diameter than the F-specific ribonucleic acid phages and absorbed relatively weakly to EDP208 pili. Comparing EDP208 pilin to F, it was found that both contain the equivalent of two to three hexose units per subunit as well as blocked N-termini. EDP208 pilin contains one covalently linked phosphate residue per subunit, whereas the F pilin subunit contains two such residues. Although notable differences were found in the case of three or four amino acids, the overall amino acid compositions of F and EDP208 were very similar. Moreover, the tryptic peptide maps of the two proteins contained seven peptides with similar mobilities, suggesting considerable homology in their amino acid sequences. Substantial similarities were also noted in the secondary structures of F and EDP208 pilin on the basis of circular dichroism studies. The alpha-helix content of both proteins was calculated to be 65 to 70%. X-ray fiber diffraction studies have indicated that the arrangements of subunits in F and EDP208 pili are also similar. It was concluded that F and EDP208 pili are closely related structures.     

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.     

Structure and function of conjugative pili: monoclonal antibodies as probes for structural variants of F pili.

The lac-tra operon fusion plasmid pTG801 contains the known F plasmid DNA transfer (tra) genes required by Escherichia coli to elaborate functional F pili (T. Grossman and P. M. Silverman, J. Bacteriol. 171:650-656, 1989). Here, we show that these pili are actually structural variants of normal F pili and that the F plasmid must contain additional genes that affect pilus structure and function. We confirmed a previous report that two monoclonal antibodies that recognize epitopes at and near the amino terminus of F pilin do not decorate the sides of normal F pili, as determined by immunogold electron microscopy. However, both antibodies laterally decorated pTG801 pili. The epitope for one of the antibodies has been shown to include the amino-terminal acetyl group of F pilin, which must therefore also be present on pTG801 pilin. Normal antibody staining was restored to pTG801 pili when cells contained, in addition to pTG801, the compatible plasmid pRS31, which must therefore include at least one gene affecting F-pilus structure. One candidate, traD, was excluded as the sole such gene, since traD+ derivatives of a pTG801 strain still elaborated pili that could be laterally decorated with antibody. Moreover, although traD alone restored RNA bacteriophage R17 infectivity to pTG801 cells, as expected, it did not mimic pRS31 in restoring to pTG801 pili other characteristics of normal F pili. We conclude that pRS31 contains as yet uncharacterized genes required for elaboration of structurally normal F pili. Finally, we identified vesicular material, especially abundant in cultures of pTG801 transformants, that stained heavily with the anti-F-pilin monoclonal antibodies. This material may reflect the inner membrane pool of F pilin.     

The role of the pilus in recipient cell recognition during bacterial conjugation mediated by F-like plasmids

The effects of defined mutations In the lipopolysaccharide (LPS) and the outer membrane protein OmpA of the recipient cell on mating-pair formation in liquid media by the transfer systems of the F-Iike plasmids pOX38 (F), ColB2 and R100-1 were investigated. Transfer of all three plasmids was affected differently by mutations in the rfa (LPS) locus of the recipient cell, the F plasmid being most sensitive to mutations that affected rfaP gene expression which is responslbie for the addition of pyrophosphorylethanolamine (PPEA) to heptose I of the inner core of the LPS. CoIB2 transfer was more strongly affected by mutations in the heptose II-heptose III region of the LPS (rfaF) whereas R100-1 was not strongly affected by any of the rfa mutations tested. ompA but not rfa mutations further decreased the mating efficiency of an F plasmid carrying a mutation in the mating-pair stabilization protein TraN. An F derivative with a chloramphenicol acetyltransferase (CAT) cassette interrupting the traA pilin gene was constructed and pilin genes from F-like plasmids (F, ColB2, R100-1) were used to complement this mutation. Unexpectediy, the results suggested that the differences in the pilin sequences were not responsible for recognizing specific groups in the LPS, OmpA or the TraT surface exclusion protein. Other corroborating evidence is presented suggesting the presence of an adhesin at the F pilus tip.     

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.     

Insecticidal pilin subunit from the insect pathogen Xenorhabdus nematophila

Xenorhabdus nematophila is an insect pathogen and produces protein toxins which kill the larval host. Previously, we characterized an orally toxic, large, outer membrane-associated protein complex from the culture medium of X. nematophila. Here, we describe the cloning, expression, and characterization of a 17-kDa pilin subunit of X. nematophila isolated from that protein complex. The gene was amplified by PCR, cloned, and expressed in Escherichia coli. The recombinant protein was refolded in vitro in the absence of its cognate chaperone by using a urea gradient. The protein oligomerized during in vitro refolding, forming multimers. Point mutations in the conserved N-terminal residues of the pilin protein greatly destabilized its oligomeric organization, demonstrating the importance of the N terminus in refolding and oligomerization of the pilin subunit by donor strand complementation. The recombinant protein was cytotoxic to cultured Helicoverpa armigera larval hemocytes, causing agglutination and subsequent release of the cytoplasmic enzyme lactate dehydrogenase. The agglutination of larval cells by the 17-kDa protein was inhibited by several sugar derivatives. The biological activity of the purified recombinant protein indicated that it has a conformation similar to that of the native protein. The 17-kDa pilin subunit was found to be orally toxic to fourth- or fifth-instar larvae of an important crop pest, H. armigera, causing extensive damage to the midgut epithelial membrane. To our knowledge, this is first report describing an insecticidal pilin subunit of a bacterium.