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Journal of Bacteriology, February 2008, p. 1202-1208, Vol. 190, No. 4
0021-9193/08/$08.00+0     doi:10.1128/JB.01204-07
Copyright © 2008, American Society for Microbiology. All Rights Reserved.

Novel Class of Mutations of pilS Mutants, Encoding Plasmid R64 Type IV Prepilin: Interface of PilS-PilV Interactions

Eriko Shimoda, Tatsuya Muto, Takayuki Horiuchi, Nobuhisa Furuya, and Teruya Komano^*

Department of Biology, Tokyo Metropolitan University, Minamiohsawa, Hachioji, Tokyo 192-0397, Japan

Received 27 July 2007/ Accepted 21 November 2007

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The type IV pili of plasmid R64 belonging to the type IVB group are required only for liquid mating. They consist of the major and minor components PilS pilin and PilV adhesin, respectively. PilS pilin is first synthesized as a 22-kDa prepilin from the pilS gene and is then processed to a 19-kDa mature pilin by PilU prepilin peptidase. In a previous genetic analysis, we identified four classes of the pilS mutants (T. Horiuchi and T. Komano, J. Bacteriol. 180:4613-4620, 1998). The products of the class I pilS mutants were not processed by prepilin peptidase; the products of the class II mutants were not secreted; in the class III mutants type IV pili with reduced activities in liquid mating were produced; and in the class IV mutants type IV pili with normal activities were produced. Here, we describe a novel class, class V, of pilS mutants. Mutations in the pilS gene at Gly-56 or Tyr-57 produced type IV pili lacking PilV adhesin, which were inactive in liquid mating. Residues 56 and 57 of PilS pilin are suggested to function as an interface of PilS-PilV interactions.

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Type IV pili are long fibers with a diameter of 6 to 8 nm that are up to 20 µm long (for reviews, see references 16, 17, and 25). They are produced by gram-negative bacteria, such as Pseudomonas aeruginosa, Neisseria gonorrhoeae, Myxococcus xanthus, Vibrio cholerae, and Salmonella enterica serovar Typhi. Some plasmids, such as R64, ColIb-P9, R721, pO113, and pHG1, and enteropathogenic Escherichia coli enteroadherent factor also encode type IV pili (13, 23, 28). These pili participate in various bacterial processes, including adhesion to prokaryotic and eukaryotic cells and abiotic surfaces, target cell specificity, twitching motility, social gliding motility, biofilm formation, DNA uptake, and bacteriophage adsorption. Thus, they are key virulence factors for many important bacterial pathogens.

The type IV pilus is a polymer of type IV pilin, which is produced by the processing of prepilin (25) (Fig. 1A). Type IV prepilin has an N-terminal leader peptide or prepeptide, which is removed by cognate prepilin peptidase. Leader peptides usually contain two or more basic amino acids, and their C-terminal amino acid is invariantly glycine. A long hydrophobic segment is present in the N-terminal region of mature pilins. The fifth amino acid of the mature pilins is usually glutamic acid. The type IV pilins are further classified into groups IVA and IVB based on the length of the leader peptide and the amino acid sequence of the mature pilin. The leader peptides of type IVA and IVB prepilins are usually short and long, respectively. The N-terminal amino acid of type IVA pilin is N-methylated phenylalanine, while various amino acids are present at the N terminus of type IVB pilin. More than 10 proteins are required for the biogenesis of the type IV pilus, and the prepilins, prepilin leader peptidases, secretins, integral membrane proteins, and ATPases are widely conserved. The proteins required for type II protein secretion systems of gram-negative bacteria, flagellum formation in archaebacteria, and DNA uptake systems are closely related to those of type IV pilus biogenesis systems (19).

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FIG. 1. (A) Conservation of the GY sequence in PilS in PilV-containing type IVB pilus biogenesis systems. N-terminal amino acid sequences of various prepilins of the PilV-containing type IVB group, the type IVB group without PilV, and the type IVA group are aligned. Prepilin peptidase cleavage sites are indicated by the spaces under the arrow. The conserved GY and GXY sequences in the PilV-containing type IVB group are enclosed in a box. Highly conserved amino acids in each group are indicated by bold type. N-terminal -helices determined by three-dimensional analyses are underlined. (B) Comparison of N-terminal amino acid sequences of R64 PilS pilin and PilV adhesin. Identical amino acids are indicated by bold type. Putative N-terminal -helices are underlined with dashed lines. The sequences are the sequences of (from top to bottom) S. enterica serovar Typhimurium plasmid R64 PilS (R64 PilS) (GenBank accession number GI:32470255), S. enterica serovar Typhi PilS (Sty PilS) (accession number 16763018), Yersinia pseudotuberculosis PilS (Yps PilS) (accession number 49658866), Yersinia enterocolitica PilS (Yen PilS) (accession number 123443690), Photorhabdus luminescens PilS (Plu PilS) (accession number 37525041), P. aeruginosa PilS (Pae PilS) (accession number 116052611), Pseudomonas syringae PilS (Psy PilS) (accession number 66044759), Pseudomonas fluorescens PilS (Pfl PilS) (accession number 70732018), E. coli plasmid pSERB1 PilS (pSERB1 PilS) (accession number 51038814), Ralstonia eutropha plasmid pHG1 PilS (pHG1 PilS) (accession number 38638007), E. coli plasmid R721 PilS (R721 PilS) (accession number 10955497), E. coli plasmid pO113 PilS (pO113 PilS) (accession number 15667836), Serratia entomophila plasmid pADAP PilS (pADAP PilS) (accession number 38201730), V. cholerae TcpA (Vch TcpA) (accession number 135542), enteropathogenic E. coli enteroadherent factor plasmid BfpA (EAF BfpA) (accession number 92108702), P. aeruginosa PilA (Pae PilA) (accession number 120438), N. gonorrhoeae PilE (Ngo PilE) (accession number 729529), M. xanthus PilA (Mxa PilA) (accession number 108761074), R64 PilS (R64 PilS), and R64 PilV (R64 PilV) (accession number 32470251).

The initial step of plasmid R64 conjugation in liquid media is thought to be the formation of donor-recipient cell aggregates using a type IV pilus called the thin flexible pilus (28). The R64 type IV pilus is required for conjugation only in liquid media. Type IV pili of R64 and ColIb-P9 were sedimented from culture medium in which Escherichia coli cells harboring R64 and ColIb-P9 derivatives, respectively, had been grown, and then they were purified by CsCl density gradient centrifugation (28). They were shown to consist of the major component PilS (19 kDa) and the minor component PilV (48 kDa).

The R64 type IV pilus is encoded in the R64 pil region (13). The pil region contains 14 genes, and 12 genes of these genes, pilK to pilV, are essential for formation of the pilus (29) (Fig. 2A). The R64 pilL, pilN, pilQ, pilR, pilS, pilT, pilU, and pilV genes encode lipoprotein, secretin, ATPase, integral membrane protein, major prepilin, lytic transglycosidase, prepilin peptidase, and adhesin, respectively (21). The product of the pilS gene is synthesized as a 22-kDa prepilin and then processed to a 19-kDa pilin by PilU prepilin peptidase. The structure of PilS prepilin indicates that the R64 type IV pilus belongs to the type IVB group. In previous genetic analyses, the R64 PilU protein was shown to be an aspartic acid peptidase (1). The N-terminal sequence of the PilV protein has the typical features of type IV prepilin, including a 7-amino-acid leader peptide, which may be cleaved off by PilU prepilin peptidase (Fig. 1B). The 20 N-terminal amino acids of the mature PilV protein are hydrophobic, and the fifth amino acid is glutamic acid. Thus, the pilV gene is most likely to encode a minor pilin, which functions as an adhesin. Type IVA pili and type II secretion systems are known to contain several pilinlike proteins, which are essential for their assembly and functions (2, 16, 19). The R64 PilV adhesin is distinct from the pilinlike proteins of type IVA pili and type II secretion systems, since (i) PilV is much longer than the pilinlike proteins and (ii) PilV adhesin is secreted extracellularly, while the pilinlike proteins are located in the membrane fraction.

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FIG. 2. (A) Organization of the R64 traABCD and pilIJKLMNOPQRSTUV genes in pKK641A'. The top horizontal line is a restriction map. Abbreviations: B, BglII; E, EcoRI; H, HindIII; Hp, HpaI; P, PstI; and V, PvuII. Below the map, the arrows indicate the coding sequences of various genes, including tra (transfer), pil (formation of type IV pilus), shf (shufflon), and rci (shufflon-specific recombinase). The positions of the pilS1 and pilV1 mutations are indicated by multiplication signs. (B) Five classes of R64 pilS mutations. Only the mature pilin region is indicated. Ovals and arrows indicate -helix and β-sheet regions, respectively, predicted on the basis of the three-dimensional structure of S. enterica serovar Typhi PilS (27). The positions of the hydrophobic region and the disulfide bridge are indicated. At the bottom, the locations of five classes of pilS mutations are indicated. The mutations at positions 53 to 59 were isolated and characterized in the present study. The results for the remaining mutations were obtained from a previous study (9).

Sequences encoding seven PilV adhesins with different C-terminal segments can be created by shufflon multiple DNA inversion (7, 14). The seven PilV C-terminal segments determine the recipient specificity of R64 liquid mating through recognition of lipopolysaccharides (LPSs) of recipient cells (10). The recipient E. coli K-12 strain was recognized by the PilVA', PilVC, and PilVC' adhesins. E. coli B was recognized only by PilVA' adhesin. E. coli R1, S. enterica serovar Typhimurium LT2, and Shigella flexneri were recognized by the PilVA or PilVA', PilVA' or PilVB', and PilVA' or PilVD' adhesin, respectively. Liquid mating experiments using various waa mutants showed that the specific receptor structures for the PilVA, PilVB', PilVC, and PilVC' adhesins are GlcNAc(β1-3)Glc, GlcNAc(1-2)Glc, GlcNAc(β1-7)Hep, and Glc(1-2)Glc or Glc(1-2)Gal, respectively, of LPS from E. coli R1, E. coli K-12, or S. enterica serovar Typhimurium LT2 (10, 11). The binding of PilV adhesins or their C-terminal segments to LPSs has been demonstrated using filter overlay assays and surface plasmon resonance analyses (11).

We previously isolated 32 missense or deletion mutants with mutations in the R64 pilS gene, including 9 mutants with mutations in the leader peptide region and 23 mutants with mutations in the mature protein region (9) (Fig. 2B). Four classes of pilS mutations were identified based on their phenotypes for type IV pilus formation and liquid mating. The products of the class I mutants were not processed by prepilin peptidase; the extracellular secretion of the products of the class II mutants was inhibited; in the class III mutants type IV pili with reduced activity in liquid mating were produced; and in the class IV mutants type IV pili with normal mating activity were produced. Point mutations in the class I mutants were distributed throughout the prepilin sequence, indicating that the processing of the pilS product requires the entire prepilin sequence. In the class III and IV mutants, extracellular type IV pili containing both PilS pilin and PilV adhesins were produced, while in the class I and II mutants, no extracellular type IV pili were produced.

In the present work, a novel class of pilS mutants was isolated and characterized. E. coli cells harboring R64 derivatives with pilS mutations at Gly-56 or Tyr-57 produced type IV pili lacking PilV adhesins, which were inactive in liquid mating. Residues 56 and 57 of PilS are suggested to function as an interface of PilS-PilV interactions.

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Bacterial strains, plasmids, phages, and media. E. coli DH5 [supE44 lacU169 hsdR17 recA1 endA1 gyrA96 thi-1 relA1 80dlacZM15), NF83 [recA56 ara (lac-proAB) rpsL 80dlacZM15], BW25113 (lacI^q rrnB_T14 lacZWJ16 hsdR514 araBAD_AH33 rhaBAD_LD78), and TN102 Nal^r were used (5, 9, 22).

The plasmid vector pUC119 was also used (22). pKK641A', pKK641A' pilS1, pKK641A' pilV1, pKK661, pKK692 pilS⁺, and pKD46 have been described previously (5, 15, 29).

The IncI1-specific phages I and PR64FS were used (9).

Luria-Bertani medium was prepared as described previously (22). The solid medium contained 1.5% agar. Antibiotics were added to the liquid and solid media at the following concentrations: ampicillin, 100 µg/ml; chloramphenicol, 25 µg/ml; kanamycin, 50 µg/ml; and nalidixic acid, 20 µg/ml.

Construction of pilS mutants. Recombinant DNA techniques were performed as described previously (22). Random mutagenesis of the pilS gene was performed by PCR using appropriate primers and pKK692 DNA as the template. To increase the mutation frequency, 0.5 nM MnCl₂ was added to the PCR mixture. In addition, pilS mutants were constructed by site-directed mutagenesis with synthetic oligonucleotides (9). pKK641A' pilS1 pilV1 was constructed as previously described (5).

Conjugal transfer and phage sensitivity. The frequency of conjugation in a liquid medium was determined as described previously (9, 15). Log-phase cultures of E. coli NF83 donor cells harboring pKK641A' pilS1, pKK661, and pKK692 with or without pilS mutations were mixed with overnight cultures of E. coli TN102 recipient cells. The mixtures were incubated for 90 min at 37°C and plated at various dilutions onto selective media. The transfer frequency was the ratio of the number of transconjugants to the number of donor cells (expressed as a percentage).

The sensitivity of E. coli cells harboring pKK641A' pilS1, pKK661, and pKK692 with or without pilS mutations to phages I and PR64FS was determined as described previously (9).

Crude type IV pilus fraction. A crude type IV pilus fraction was prepared as described previously (9, 28). E. coli cells harboring pKK641A' pilS1, pKK661, and pKK692 with or without pilS mutations were grown until they reached the late log phase. Each culture was centrifuged three times at 9,200 x g for 10 min to remove the bacterial cells. The supernatant was centrifuged at 140,000 x g for 1 h to obtain a crude type IV pilus fraction.

Western blot analysis. Proteins of total cells or crude type IV pilus fractions were separated by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE) and transferred to a nitrocellulose membrane. The membrane was blocked, reacted with anti-pilin or anti-PilV rabbit antiserum, and then reacted with biotin-labeled anti-rabbit immunoglobulin G goat antibody. The pilin or adhesin was detected using an ABC-POD kit (Wako, Osaka, Japan).

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Isolation of a novel class of mutants with mutations in the pilS gene. We previously identified four classes of pilS mutations (9). Here, a novel class of pilS mutants, pilS(Y57C), was identified among R64 pilS mutants obtained by PCR random mutagenesis. E. coli cells harboring the pilS(Y57C) derivative of plasmid R64 produced type IV pili lacking PilV adhesin which were inactive in liquid mating, as described below. To investigate the effects of mutations around residue Tyr-57 of the PilS pilin on type IV pilus biogenesis of R64, 16 additional mutants with mutations in residues 53 to 59 were constructed by site-directed mutagenesis (Table 1).

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TABLE 1. Characteristics of the products of various mutant pilS genes

Activity of the mutant pilS genes in R64 transfer. The abilities of mutant pilS genes to complement the pilS1 mutation, in which a frameshift mutation was induced by insertion of a 22-bp fragment at the sixth codon, were determined by liquid mating. For this purpose, the pKK641-pKK661 system was used to avoid the effects of shufflon multiple inversions (15). E. coli donor cells harboring pKK641A' pilS1, pKK661, and pUC119 exhibited practically no transfer of pKK661 (Table 1). In contrast, when donor cells harbored pKK692 pilS⁺ instead of pUC119, the transfer frequency of pKK661 was 1.6%, indicating that there was complementation of the pilS1 mutation by the wild-type pilS gene. pKK692 derivatives carrying mutant pilS genes were introduced into E. coli donor cells harboring pKK641A' pilS1 and pKK661. Donor cells carrying the pilS mutations at positions 53, 54, 55, 58, and 59 exhibited transfer frequencies similar to that of the wild type (Table 1). These results indicated that pilS(G53D), pilS(S54R), pilS(D55A), pilS(T58G), pilS(T58I), and pilS(F59A) were class IV pilS mutations. Cells carrying mutations at Gly-56, including pilS(G56A), pilS(G56E), pilS(G56P), pilS(G56R), and pilS(G56), exhibited no transfer or a very low transfer frequency. Cells having most of the pilS mutations at Tyr-57, pilS(Y57A), pilS(Y57C), pilS(Y57L), and pilS(Y57W), exhibited no transfer or a very low transfer frequency. The pilS(Y57F) mutation did not affect the transfer frequency, suggesting that replacement of Tyr-57 by phenylalanine is tolerated due to the structural similarity of the amino acids. The pilS(Y57) mutation exhibited residual transfer. In this case, Phe-59 may function as a substitute for Tyr-57.

To examine the transdominance of the mutant pilS gene over the wild-type pilS gene, pKK692 derivatives carrying mutant pilS genes were introduced into E. coli donor cells harboring pKK641A' and pKK661. Among the pilS mutants with mutations at Gly-56 and Tyr-57, the mutants with a very low transfer frequency exhibited slight dominant-negative effects on the wild-type allele (Table 1). The products of these pilS mutants might competitively inhibit the function of the products of the wild-type allele. The dominant-negative effects of these pilS mutants were slightly weaker than those of the class I and class II pilS mutants observed in a previous study (9).

E. coli cells producing R64 type IV pili are sensitive to phages I and PR64FS (9). E. coli cells producing type IV pili with pilS mutations at positions 53, 54, 55, 58, and 59, which exhibited transfer frequencies similar to that of the wild type, were sensitive to phages I and PR64FS (Table 1). In contrast, E. coli cells producing type IV pili with pilS mutations at Gly-56 and Tyr-57, which exhibited very low transfer frequencies, were resistant to these phages.

Production, processing, and assembly of pilus proteins in the pilS mutants. The R64 type IV pilus consists of the major PilS pilin and the PilV adhesin (28). The intracellular production of the PilS and PilV proteins in E. coli cells harboring pKK641A', pKK661, and pKK692 with or without pilS mutations was assessed by Western blot analysis using anti-pilin and anti-PilV antisera, respectively. PilS prepilin was produced in all strains except the vector control (Fig. 3A). Approximately one-third of the PilS prepilin was processed into pilin, indicating that all the mutant PilS prepilin can be normally processed. Since the pilS gene was complemented by the multicopy vector pUC119 in the present experiments, not all PilS prepilin was processed. PilV protein was produced intracellularly in all strains, including the vector control (Fig. 3B).

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FIG. 3. Production, processing, and assembly of PilS and PilV proteins in pilS mutants. Total proteins of E. coli cells harboring pKK641A' pilS1, pKK661, and pKK692 with or without pilS mutations were separated by SDS-PAGE and subjected to Western blot analysis using (A) anti-pilin or (B) anti-PilV antisera. The crude type IV pilus fractions prepared from the proteins were separated by SDS-PAGE and subjected to Western blot analysis using (C) anti-pilin or (D) anti-PilV antisera. Lane 1, pKK641A' pilS1, pKK661, and pUC119; lane 2, pKK641A' pilS1, pKK661, and pKK692 pilS+; lanes 3 to 19, pKK641A' pilS1, pKK661, and pKK692 with the indicated pilS mutations. The positions of prepilin (22 kDa), pilin (19 kDa), and PilV adhesin (45 kDa) are indicated on the right.

The formation of extracellular type IV pilus in these mutants was assessed by analysis of the PilS and PilV proteins in crude type IV pilus fractions. The crude type IV pilus fractions were prepared from culture media in which E. coli cells harboring pKK641A', pKK661, and pKK692 with or without pilS mutations had been grown. In all strains except the vector control, type IV pili containing PilS pilin were produced (Fig. 3C). The crude type IV pilus fractions of the pilS mutants with mutations at positions 53, 54, 55, 58, and 59 contained levels of PilV adhesin similar to that of the wild type (Fig. 3D). In contrast, the crude type IV pilus fractions of the pilS(G56A), pilS(G56E), pilS(G56P), pilS(G56R), pilS(G56), pilS(Y57A), pilS(Y57C), pilS(Y57L), and pilS(Y57W) mutants did not contain PilV adhesin. The crude type IV pilus fraction of the pilS(Y57F) mutant contained the wild-type level of PilV adhesin, which might have been related to its wild-type pilS activity in liquid mating (Table 1). The crude type IV pilus fraction of the pilS(Y57) mutant contained a reduced level of PilV adhesin, which might have been related to its reduced pilS activity. One possibility is that a small amount of type IV pili was produced in the pilS(Y57) mutant, since the amount of PilS pilin was also reduced in this mutant (Fig. 3C).

The production of PilS and PilV proteins in the crude type IV pilus fractions of the pilS mutants is summarized in Table 1. The results indicate that the failure of liquid mating with the pilS mutants with mutations at positions 56 and 57 was due to the fact that these mutants produced type IV pili lacking PilV adhesin. We tentatively designated this type of pilS mutants class V pilS mutants, since they have not been previously reported.

Indispensability of the pilV gene for extracellular type IV pilus formation in class V pilS mutants. The pilV gene was previously shown to be essential for the type IV pilus biogenesis of R64 (29). Two alternative interpretations are possible for the phenotype of the class V pilS mutants: (i) in the class V pilS mutants type IV pili without PilV adhesin are produced; or (ii) in the class V pilS mutants type IV pili with PilV adhesin are produced first, and then PilV adhesin is detached from the pili due to weak PilS-PilV interactions.

A pKK641A' pilS1 pilV1 double mutant was constructed. Mutant PilS prepilin was produced and processed in E. coli cells harboring pKK641A' pilS1 pilV1, pKK661, and pKK692 pilS(Y57A) or pilS(Y57C) (Fig. 4A). E. coli cells harboring pKK641A' pilS1, pKK661, and pKK692 pilS(Y57A) or pilS(Y57C) produced extracellular pili (Fig. 4B, lanes 1 and 2), although they lacked PilV adhesin. In contrast, E. coli cells harboring pKK641A' pilS1 pilV1, pKK661, and pKK692 pilS(Y57A) or pilS(Y57C) did not produce any extracellular pili (Fig. 4B, lanes 3 and 4), indicating that the pilV gene is essential for the production of extracellular type IV pili in the class V pilS mutants. These results strongly suggest that the second possibility described above is true.

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FIG. 4. Indispensability of the pilV gene for the extracellular production of the type IV pilus in the class V pilS mutants. (A) Total proteins and (B) crude type IV pilus fractions of E. coli cells harboring pKK641A' pilS1 or pKK641A' pilS1 pilV1, pKK661, and pKK692 pilS(Y57A) or pilS(Y57C) were separated by SDS-PAGE and subjected to Western blot analysis using anti-pilin antiserum. Lane 1, pKK641A' pilS1, pKK661, and pKK692 pilS(Y57A); lane 2, pKK641A' pilS1, pKK661, and pKK692 pilS(Y57C); 3, pKK641A' pilS1 pilV1, pKK661, and pKK692 pilS(Y57A); lane 4, pKK641A' pilS1 pilV1, pKK661, and pKK692 pilS(Y57C). The positions of prepilin (22 kDa) and pilin (19 kDa) are indicated on the right.

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In the present work, a novel class, class V, of R64 pilS mutants was identified. E. coli cells harboring pKK641 with pilS mutations at Gly-56 or Tyr-57 produced type IV pili lacking PilV adhesin, which were inactive in liquid mating. Amino acid residues 53 to 59 were examined, and only pilS mutants with mutations at residues 56 and 57 exhibited the class V phenotype (Fig. 2B). In addition, none of 23 mutants with mutations in the PilS mature region isolated previously exhibited the class V phenotype. Therefore, it is likely that the class V pilS mutations are restricted to amino acid residues 56 and 57. This type of pilS mutants is of special interest, since it has not been reported previously for type IV pili and type II secretion systems.

Recently, many PilV-containing type IVB pilus biogenesis systems have been reported (3, 8, 23, 30). These systems exhibit gene organizations similar to that of R64. The glycine-tyrosine (GY) sequences are conserved in PilS pilin in the corresponding region in many PilV-containing type IVB pili (Fig. 1A). In some cases, GXY sequences are found in the corresponding region. In contrast, the GY sequence was not found in most of the pilin at the corresponding region in type IVA and type IVB pili, in which PilV adhesins have not been found. These results suggest the importance of the GY or GXY sequence for PilS function in PilV-containing type IVB pili.

The pilS(Y57A) pilV or pilS(Y57C) pilV double mutants did not produce any extracellular type IV pili, whereas the pilS(Y57A) or pilS(Y57C) mutants produced extracellular type IV pili lacking PilV adhesin. The requirement for PilV for extracellular production of type IV pili in the class V pilS mutants suggests that there is a process in which the class V pilS mutants produce type IV pili with PilV adhesins and then PilV adhesins are detached from the pili due to weak PilS-PilV interactions.

The three-dimensional structures of type IV pilins from N. gonorrhoeae, P. aeruginosa, V. cholerae, S. enterica serovar Typhi, and enteropathogenic E. coli have been determined by X-ray crystallography and nuclear magnetic resonance spectroscopy (4, 6, 12, 18, 20, 27). The five type IV pilins exhibit globally similar β-roll structures, while each type IV pilin has a distinct difference. The monomer structure of the five type IV pilins consists of an β-roll fold with an 85-Å N-terminal -helical spine. The gross monomer structure resembles a ladle, with the hydrophobic N-terminal half of the -helical spine forming the handle. From the monomer structures, fiber structures consisting of right-handed and left-handed five-turn helices were proposed for N. gonorrhoeae and P. aeruginosa type IVA pili, respectively (6, 12, 18). Fiber structures consisting of left-handed, three-stranded, and six-turn helices were also proposed for type IVB pili of V. cholerae, S. enterica serovar Typhi, and enteropathogenic E. coli (4, 20, 27). In all fibers, the pilin N-terminal -helices gather in the center of the fiber to form a core of coiled -helices surrounded by β-sheets (Fig. 5). The type IV pilus of R64 is closely related to that of S. enterica serovar Typhi. The amino acid sequence of the PilS pilin of R64 is 50% identical to that of the PilS pilin of S. enterica serovar Typhi for the entire molecule. The three-dimensional structure model of R64 PilS pilin was constructed using that of S. enterica serovar Typhi PilS piln as a template by Swiss Model, an automated protein homology-modeling server (http://swissmodel.expasy.org) (24) (Fig. 2B). R64 PilS pilin was predicted to fold and assemble into a type IV pilus in a manner similar to that of S. enterica serovar Typhi (27). Tyrosine residue 57 of PilS is located in the 14-amino-acid loop region between -helix-1 and -helix-2 and extends to the outside from the pilin molecule, pointing to the pilus tip when it is assembled.

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FIG. 5. Hypothetical model of type IV pilus of plasmid R64. The diagram is a cross section of a six-turn helix of the type IV pilus. The PilV adhesin is located at the tip of the PilS fiber. The putative N-terminal -helices of the PilS and PilV proteins are indicated by rods. They are thought to form mixed coiled -helices. The Tyr-57 residue of PilS pilin indicated by a hexagon may interact with an unidentified residue of the PilV adhesin.

We propose a hypothetical model of the R64 type IV pilus, where an oligomer of PilV adhesins is connected to the tip of the PilS fiber (Fig. 5). Localization of the PilV adhesin at the tip of the type IV pilus has been demonstrated for S. enterica serovar Typhi type IV pili by immunogold electron microscopy (26). When R64 type IV pili containing PilVB' or PilVC adhesin were analyzed by SDS-PAGE after incubation in nonreducing SDS sample buffer at 37°C for 5 min, oligomer bands of PilVB' or PilVC adhesin were found in addition to monomer bands (11). LPS from E. coli K-12 bound to the oligomer but not the monomer band of PilVC adhesin. These results suggest that PilV adhesin is assembled into an oligomer within the type IV pilus. An oligomer of PilV adhesin may be stably attached to the tip of the type IV pilus (Fig. 5). Computer analysis (http://www.predictprotein.org) predicted that the 50 N-terminal amino acids of the mature PilV adhesin form a hydrophobic -helix like that seen in PilS pilin (Fig. 1B). PilV N-terminal -helices may be embedded at the center of the tip of the type IV pilus. Long N-terminal -helices of PilV and PilS pilins can form a core of coiled -helices. The similarity of the PilV N-terminal amino acid sequence to the PilS N-terminal amino acid sequence (Fig. 1B) is likely to play an important role in their interaction at the tip of R64 type IV pili. The phenol ring of PilS Tyr-57 is expected to interact with an unidentified residue of the PilV adhesin to form stable PilS-PilV interactions. Extension of the phenol ring of Tyr-57 from the outside of the pilin molecule (27) may be advantageous for PilS-PilV interactions. For this purpose, the benzene ring of phenylalanine can be used instead of the phenol ring of tyrosine. The location of Tyr-57 within a long loop region may help induce its specific interaction with the counterpart of PilV. Gly-56 may be required to provide a physical space to accept the interacting residue of PilV. These results suggest that Gly-56 and Tyr-57 of the R64 PilS pilin function as an interface of PilS-PilV interactions.

 
This work was supported in part by a grant from the Ministry of Education, Culture, Sports, Science and Technology of Japan.

 
* Corresponding author. Mailing address: Department of Biology, Tokyo Metropolitan University, Minamiohsawa, Hachioji, Tokyo 192-0397, Japan. Phone: 81-42-677-2754. Fax: 81-42-677-2559. E-mail: komano-teruya{at}c.metro-u.ac.jp

Published ahead of print on 7 December 2007.

Present address: Neo-Morgan Laboratory Incorporated, Biotechnology Research Center, 907 Nogawa, Miyamae-ku, Kawasaki, Kanagawa 216-0001, Japan.

Top ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 

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Journal of Bacteriology, February 2008, p. 1202-1208, Vol. 190, No. 4
0021-9193/08/$08.00+0     doi:10.1128/JB.01204-07
Copyright © 2008, American Society for Microbiology. All Rights Reserved.

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