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.     

Processing and maturation of the pilin of the type IV secretion system encoded within the gonococcal genetic island

The type IV secretion system (T4SS) encoded within the gonococcal genetic island (GGI) of Neisseria gonorrhoeae has homology to the T4SS encoded on the F plasmid. The GGI encodes the putative pilin protein TraA and a serine protease TrbI, which is homologous to the TraF protein of the RP4 plasmid involved in circularization of pilin subunits of P-type pili. TraA was processed to a 68-amino acid long circular peptide by leader peptidase and TrbI. Processing occurred after co-translational membrane insertion and was independent of other proteins. Circularization occurred after removal of three C-terminal amino acids. Mutational analysis of TraA revealed limited flexibility at the cleavage and joining sites. Mutagenesis of TrbI showed that the conserved Lys-93 and Asp-155 are essential, whereas mutagenesis of Ser-52, the putative catalytic serine did not influence circularization. Further mutagenesis of other serine residues did not identify a catalytic serine, indicating that TrbI either contains redundant catalytic serine residues or does not function via a serine-lysine dyad mechanism. In vitro studies revealed that circularization occurs via a covalent intermediate between the C terminus of TraA and TrbI. The intermediate is processed to the circular form after cleavage of the N-terminal signal sequence. This is the first demonstration of a covalent intermediate in the circularization mechanism of conjugative pili.     

Nucleotide sequence of the tra YALE region from IncFV plasmid pED208

The pED208 plasmid is a 90-kilobase conjugative plasmid which is the derepressed form of Fo lac plasmid (IncFV). A 3.3-kilobase HindIII-PstI fragment from the pED208 plasmid was cloned and sequenced and was found to contain four open reading frames which were highly homologous to the traA, traL, traE, and traY gene products of the F plasmid. The pED208 traA propilin protein was 119 amino acids in length, consisting of a leader sequence of 55 amino acids and a mature pilin subunit of 64 residues. The leader sequence contained a hydrophobic region followed by a classic signal peptidase cleavage site (Ala-Ser-Ala-55). F and pED208 pilin proteins shared 27 conserved residues and had similar predicted secondary structures. The pED208 traA and traL genes were separated by a single base pair, and no ribosome binding site preceded the traL gene. The pED208 traY gene contained an IS2 insertion element in orientation II 180 nucleotides (60 residues) upstream of the traY stop codon. This insertion of IS2 resulted in a predicted fusion peptide of 69 residues for traY which may provide the observed traY activity. Since IS2 is absent in the wild-type plasmid, Fo lac, derepression and concomitant multipiliation may be due to the insertion of IS2 providing constitutive expression of the pED208 tra operon.     

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.     

Role of the propilin leader peptide in the maturation of F pilin.

F-pilin maturation and translocation result in the cleavage of a 51-amino-acid leader sequence from propilin and require LepB and TraQ but not the SecA-SecY secretion pathway. The unusual propilin leader peptide and the dependence of its cleavage on TraQ suggested that TraQ recognition may be specific for the leader peptide. An in vitro propilin cleavage assay yielded propilin (13 kDa), the pilin polypeptide (7 kDa), and a 5.5-kDa protein as the traA products. The 5.5-kDa protein comigrates with the full-length 51-amino-acid leader peptide, and [14C]proline labeling confirmed its identity since the only proline residues of propilin are found within the leader peptide. The in vitro and in vivo propilin-processing reactions proceed similarly in a single polypeptide cleavage step. Furthermore, TraQ dependence is a property of F-pilin maturation specifically rather than a property of the leader peptide. A propilin derivative with an amino-terminal signal sequence generated by deleting codons 2 to 28 required TraQ for processing in vivo. On the other hand, a chimeric protein with the propilin wild-type leader peptide fused to the mature portion of beta-lactamase was processed in a TraQ-independent manner. Thus, despite its unusual length, the propilin leader peptide seems to perform a function similar to that of the typical amino-terminal signal sequence. This work suggests that TraQ is not necessary for the proteolysis of propilin and therefore is likely to act as a chaperone-like protein that promotes the translocation of propilin.     

Membrane location of the Ti plasmid VirB proteins involved in the biosynthesis of a pilin-like conjugative structure on Agrobacterium tumefaciens

Abstract The virB operon of the Agrobacterium tumefaciens Ti plasmid encodes 11 proteins. Specific antisera to VirB2, VirB3 and VirB9 were used to locate these virulence proteins in the A. tumefaciens cell. Immunoblot analysis located VirB2 protein to the inner and outer membranes; VirB3 and VirB9 were likewise associated with both membranes, but mainly in the outer membrane. VirB2 is processed from a 12.3-kDa protein into a 7.2-kDa polypeptide. Such sized protein results from cleavage at residue Ala⁴⁷, upstream of which two additional alanine residues Ala⁴⁵-Ala⁴⁶ are contained and bearing resemblance to a signal peptide peptidase-I cleavage sequence. VirB2 and VirB3 sequences are strikingly similar to the pilin biosynthetic proteins TraA and TraL encoded by the tra operon of F and R1-19 plasmids. Since traA encodes a propilin that is cleaved into a 7.2-kDa conjugative pilin product and since this cleavage site is present in both TraA and VirB2, we propose that virB2 encodes a pilin-like protein which together with VirB3 and VirB9 as well as other VirB proteins may be used for interkingdom T-DNA transfer between bacteria and plants.     

Conjugative pili of IncP plasmids, and the Ti plasmid T pilus are composed of cyclic subunits.

TrbC propilin is the precursor of the pilin subunit TrbC of IncP conjugative pili in Escherichia coli. Likewise, its homologue, VirB2 propilin, is processed into T pilin of the Ti plasmid T pilus in Agrobacterium tumefaciens. TrbC and VirB2 propilin are truncated post-translationally at the N terminus by the removal of a 36/47-residue leader peptide, respectively. TrbC propilin undergoes a second processing step by the removal of 27 residues at the C terminus by host-encoded functions followed by the excision of four additional C-terminal residues by a plasmid-borne serine protease. The final product TrbC of 78 residues is cyclized via an intramolecular covalent head-to-tail peptide bond. The T pilin does not undergo additional truncation but is likewise cyclized. The circular structures of these pilins, as verified by mass spectrometry, represent novel primary configurations that conform and assemble into the conjugative apparatus.     

Processed VirB2 Is the Major Subunit of the Promiscuous Pilus of Agrobacterium tumefaciens

Previous studies have implicated the obligatory requirement for the vir regulon (or “virulon”) of the Ti plasmid for the transfer of oncogenes from Agrobacterium tumefaciens to plant cells. The machinery used in this horizontal gene transfer has been long thought to be a transformation or conjugative delivery system. Based on recent protein sequence comparisons, the proteins encoded by the virB operon are strikingly similar to proteins involved in the synthesis and assembly of conjugative pili such as the conjugative pilus of F plasmid in Escherichia coli. The F pilus is composed of TraA pilin subunits derived from TraA propilin. In the present study, evidence is provided showing that the counterpart of TraA is VirB2, which like TraA propilin is processed into a 7.2-kDa product that comprises the pilus subunit as demonstrated by biochemical and electron microscopic analyses. The processed VirB2 protein is present exocellularly on medium on which induced A. tumefaciens had grown and appears as thin filaments of 10 nm that react specifically to VirB2 antibody. Exocellular VirB2 is produced abundantly at 19°C as compared with 28°C, an observation that parallels the effect of low temperature on the production of vir gene-specific pili observed previously (K. J. Fullner, L. C. Lara, and E. W. Nester, Science 273:1107–1109, 1996). Export of the processed VirB2 requires other virB genes since mutations in these genes cause the loss of VirB2 pilus formation and result in processed VirB2 accumulation in the cell. The presence of exocellular processed VirB2 is directly correlated with the formation of pili, and it appears as the major protein in the purified pilus preparation. The evidence provides a compelling argument for VirB2 as the propilin whose 7.2-kDa processed product is the pilin subunit of the promiscuous conjugative pilus, hereafter called the “T pilus” of A. tumefaciens.     

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.     

VirB2 is a processed pilin-like protein encoded by the Agrobacterium tumefaciens Ti plasmid.

The mechanism of DNA transmission between distinct organisms has remained a subject of long-standing interest. Agrobacterium tumefaciens mediates the transfer of plant oncogenes in the form of a 25-kb T-DNA sector of a resident Ti plasmid. A growing body of evidence leading to the elucidation of the mechanism involved in T-DNA transfer comes from studies on the vir genes contained in six major operons that are required for the T-DNA transfer process. Recent comparative amino acid sequence studies of the products of these vir genes have revealed interesting similarities between Tra proteins of Escherichia coli F factor, which are involved in the biosynthesis and assembly of a conjugative pilus, and VirB proteins encoded by genes of the virB operon of A. tumefaciens pTiC58. We have previously identified VirB2 as a pilin-like protein with processing features similar to those of TraA of the F plasmid and have shown that VirB2 is required for the biosynthesis of pilin on a flagella-free Agrobacterium strain. In the present work, VirB2 is found to be processed and localized primarily to the cytoplasmic membrane in E. coli. Cleavage of VirB2 was predicted previously to occur between alanine and glutamine in the sequence -Pro-Ala-Ala-Ala-Glu-Ser-. This peptidase cleavage sequence was mutated by an amino acid substitution for one of the alanine residues (D for A at position 45 [A45D]), by deletion of the three adjacent alanines, and by a frameshift mutation 22 bp upstream of the predicted Ala-Glu cleavage site. With the exception of the frameshift mutation, the alanine mutations do not prevent VirB2 processing in E. coli, while in A. tumefaciens they result in VirB2 instability, since no holo- or processed protein is detectable. All of the above mutations abolish virulence. The frameshift mutation abolishes processing in both organisms. These results indicate that VirB2 is processed into a 7.2-kDa structural protein. The cleavage site in E. coli appears to differ from that predicted in A. tumefaciens. Yet, the cleavage sites are relatively close to each other since the final cleavage products are similar in size and are produced irrespective of the length of the amino-terminal portion of the holoprotein. As we observed previously, the similarity between the processing of VirB2 in A. tumefaciens and the processing of the propilin TraA of the F plasmid now extends to E. coli.     

Analysis of the <Emphasis Type="Italic">pilU</Emphasis> gene for the prepilin peptidase involved in the biogenesis of type IV pili encoded by plasmid R64

In many type IV pili, the N-terminal amino acid of the pilin subunit is N-methylated phenylalanine. A prepilin peptidase removes the leader peptide from the precursor and methylates the amino group of the newly formed phenylalanine. PilS, the precursor of the pilin encoded by plasmid R64, is processed by the prepilin peptidase PilU, but the N-terminal amino acid of the mature pilin is a non-methylated tryptophan that is otherwise modified. To study the relationship between the structure and function of PilU, 42 missense pilU mutations were constructed by PCR and site-directed mutagenesis, and the ability of these pilU mutants to complement a pilU null mutant for mating in liquid culture was analyzed. Although practically no conjugation was noted for 21 of the mutants, the remaining 21 supported varying levels of residual plasmid transfer activity. Two mutants with aspartic acid replacements in conserved motifs exhibited no PilU activity, suggesting that the product of the pilU gene is an aspartic acid peptidase, like TcpJ, the prepilin peptidare of Vibrio cholerae. No PilS processing was detected in 21 of the mutants, but the remaining 21 exhibited varying levels of residual PilS processing. A close correlation was noted between residual PilS processing activity and conjugative transfer, suggesting that the pilU gene product possesses prepilin peptidase activity, but is unable to methylate the N-terminal tryptophan. Based on the activity of pilU-phoA and pilU-lacZ fusion genes encoding different segments of PilU, a model for the membrane topology of the protein is also proposed. Furthermore, some amino acid substitutions in the pilU portion of the pilU-phoA and pilU-lacZ fusion genes were found to alter the membrane topology of the product.     

Effects of F-encoded components and F-pilin domains on the synthesis and membrane insertion of TraA'-'PhoA fusion proteins

F-pilin, the 70-amino-acid F-pilus subunit, accumulates in the cell envelope of F⁺strains in a process that requires interactions between its precursor (the traA gene product) and other host and F-encoded proteins. Here, we have used a set of (traA-phoA) genes to explore the effects of different TraA domains on the synthesis and membrane insertion of TraA-PhoA fusion proteins, particularly in relation to other F-encoded gene products. The 51-amino-acid TraA leader peptide fused directly to alkaline phosphatase was synthesized at comparable rates and incorporated rapidly and efficiently into the inner membrane in F' and F^? cells. A second fusion gene encoded the TraA leader peptide and the first 51 amino acids of F-pilin itself fused to PhoA (TraA'-'PhoA-102 polypeptide). Alkaline phosphatase activities and patterns of pulse-labelled polypeptides indicated that TraA'-'PhoA-102 was synthesized at comparable rates in F' and F^? cells, but in neither was the TraA'-'PhoA-102 polypeptide efficiently processed as a membrane protein. A third gene encoded the entire 121-amino-acid TraA polypeptide fused to PhoA (TraA-'PhoA-121 polypeptide). About 70% of the pulse-labelled TraA-'PhoA-121 polypeptide was rapidly processed in F'cells, where it accumulated in the cell envelope as active alkaline phosphatase, whereas in F- cells, >5% of the pulse-labelled polypeptide was processed. Additionally, the apparent rate of TraA-'PhoA-121 polypeptide synthesis was threefold higher in F'cells. The traQ gene alone could not substitute for F in restoring TraA-'PhoA-121 (or wild-type F-pilin) accumulation.     

Characterization of traX, the F plasmid locus required for acetylation of F-pilin subunits.

Acetylation of F-pilin subunits has previously been shown to depend upon expression of the F plasmid transfer operon gene traX. To assess the requirement for pilin acetylation in conjugative transfer of F, we constructed traX::kan insertion mutations and crossed them onto the transmissible F derivative pOX38. Under standard conditions, the function of traX seemed to be dispensable. Although pilin synthesized by mutant plasmids pOX38-traX482 and pOX38-traX483 was not acetylated, F-pilus production and F-pilus-specific phage infection appeared to be normal and transfer occurred at wild-type frequency. Analysis of labeled products showed that TraX+ plasmids expressed two approximately 24- (TraX1) and 22-kDa (TraX2) polypeptides that localized in the cytoplasmic membranes of cells. No product that was similar in size to the product predicted from the traX open reading frame (27.5 kDa) was detected. Therefore, we used site-directed mutagenesis, stop codon linker insertions, and phoA fusion analysis to investigate traX expression. Both TraX1 and TraX2 appeared to be encoded by the traX open reading frame. Insertion of a stop codon linker into the traX C-terminal coding region led to synthesis of two correspondingly truncated products, and fusions to phoA indicated that only the traX reading frame was translated. Expression was also very dependent on the traX M1 start codon; when this was altered, no protein products were observed. However, pilin acetylation activity was still detectable, indicating that some other in-frame start codon(s) can also be used. All sequences that are essential for activity are contained between traX codons 29 and 225. Sequence analysis indicated that traX mRNA is capable of forming a variety of base-paired structures. We suggest that traX expression is translationally controlled and that F-pilin acetylation activity may be regulated by physiological conditions in cells.     

Requirements for substrate recognition by bacterial leader peptidase.

Many secreted and membrane proteins have amino-terminal leader peptides which are essential for their insertion across the membrane bilayer. These precursor proteins, whether from prokaryotic or eukaryotic sources, can be processed to their mature forms in vitro by bacterial leader peptidase. While different leader peptides have shared features, they do not share a unique sequence at the cleavage site. To examine the requirements for substrate recognition by leader peptidase, we have truncated M13 procoat, a membrane protein precursor, from both the amino- and carboxy-terminal ends with specific proteases or chemical cleavage agents. The fragments isolated from these reactions were assayed as substrates for leader peptidase. A 16 amino acid residue peptide which spans the leader peptidase cleavage site is accurately cleaved. Neither the basic amino-terminal region nor most of the hydrophobic central region of the leader peptide are essential for accurate cleavage.     

Cysteine residues in the transmembrane regions of M13 procoat protein suggest that oligomeric coat proteins assemble onto phage progeny

The M13 phage assembles in the inner membrane of Escherichia coli. During maturation, about 2,700 copies of the major coat protein move from the membrane onto a single-stranded phage DNA molecule that extrudes out of the cell. The major coat protein is synthesized as a precursor, termed procoat protein, and inserts into the membrane via a Sec-independent pathway. It is processed by a leader peptidase from its leader (signal) peptide before it is assembled onto the phage DNA. The transmembrane regions of the procoat protein play an important role in all these processes. Using cysteine mutants with mutations in the transmembrane regions of the procoat and coat proteins, we investigated which of the residues are involved in multimer formation, interaction with the leader peptidase, and formation of M13 progeny particles. We found that most single cysteine residues do not interfere with the membrane insertion, processing, and assembly of the phage. Treatment of the cells with copper phenanthroline showed that the cysteine residues were readily engaged in dimer and multimer formation. This suggests that the coat proteins assemble into multimers before they proceed onto the nascent phage particles. In addition, we found that when a cysteine is located in the leader peptide at the -6 position, processing of the mutant procoat protein and of other exported proteins is affected. This inhibition of the leader peptidase results in death of the cell and shows that there are distinct amino acid residues in the M13 procoat protein involved at specific steps of the phage assembly process.     

Cleavage, methylation, and localization of the Pseudomonas aeruginosa export proteins XcpT, -U, -V, and -W.

Four components of the apparatus of extracellular protein secretion of Pseudomonas aeruginosa, Xcpt, -U, -V, and -W (XcpT-W), are synthesized as precursors with short N-terminal leader peptides that share sequence similarity with the pilin subunit of this organism. A specialized leader peptidase/methylase, product of the pilD gene, has been shown to cleave the leader peptide from prepilin and to methylate the N-terminal phenylalanine of the mature pilin. Antibodies were prepared against XcpT-W and used to purify each of these proteins. Sequence analysis of XcpT-W has shown that these proteins, like mature pilin, contain N-methylphenylalanine as the N-terminal amino acid. Analysis of cellular fractions from wild-type and pilD mutant strains of P. aeruginosa showed that the precursor forms of XcpT-W are located predominantly in the bacterial inner membrane, and their localization is not altered after PilD-mediated removal of the leader sequence. These studies demonstrate that the biogenesis of the apparatus of extracellular protein secretion and that of type IV pili share a requirement for PilD. This bifunctional enzyme, acting in the inner membrane, cleaves the leader peptides from precursors of pilins and XcpT-W and subsequently methylates the amino group of the N-terminal phenylalanine of each of its substrates.     

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.     

The gene encoding the prepilin peptidase involved in biosynthesis of pilus colonization factor antigen III (CFA/III) of human enterotoxigenic Escherichia coli.

The assembly of pilus colonization factor antigen III (CFA/III) of human enterotoxigenic Escherichia coli requires the processing of CFA/III major pilin (CofA) by a peptidase, likely another type IV pilus formation system. Western blot analysis of CofA reveals that CofA is produced initially as a 26.5-kDa preform pilin (prepilin) and then processed to 20.5-kDa mature pilin by a prepilin peptidase. This processing is essential for exportation of the CofA from the cytoplasm to the periplasm. In this experiment, the structural gene, cofP, encoding CFA/III prepilin peptidase which cleavages at the Gly-30-Met-31 junction of CofA was identified, and the nucleotide sequence of the gene was determined. CofP consists of 819 bp encoding a 273-amino acid protein with a relative molecular mass of 30,533 Da. CofP is predicted to be localized in the inner membrane based on its hydropathy index. The amino acid sequence of CofP shows a high degree of homology with other prepilin peptidases which play a role in the assembly of type IV pili in several gram-negative bacteria.     

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.     

The reaction specificities of the thylakoidal processing peptidase and Escherichia coli leader peptidase are identical.

Proteins which are transported across the bacterial plasma membrane, endoplasmic reticulum and thylakoid membrane are usually synthesized as larger precursors containing amino-terminal targeting signals. Removal of the signals is carried out by specific, membrane-bound processing peptidases. In this report we show that the reaction specificities of these three peptidases are essentially identical. Precursors of two higher plant thylakoid lumen proteins are efficiently processed by purified Escherichia coli leader peptidase. Processing of one precursor, that of the 23 kd photosystem II protein, by both the thylakoidal and E. coli enzymes generates the correct mature amino terminus. Similarly, leader (signal) peptides of both eukaryotic and prokaryotic origin are cleaved by partially purified thylakoidal processing peptidase. No evidence of incorrect processing was obtained. Both leader peptidase and thylakoidal peptidase are inhibited by a synthetic leader peptide.