Purification and Characterization of Thin Pili of IncI1 Plasmids ColIb-P9 and R64: Formation of PilV-Specific Cell Aggregates by Type IV Pili

Abstract

Thin pili of the closely related IncI1 plasmids ColIb-P9 and R64 are required only for liquid mating and belong to the type IV family of pili. They were sedimented by ultracentrifugation from culture medium in which Escherichia coli cells harboring ColIb-P9- or R64-derived plasmids had been grown, and then the pili were purified by CsCl density gradient centrifugation. In negatively stained thin pilus samples, long rods with a diameter of 6 nm, characteristic of type IV pili, were observed under an electron microscope. Gel electrophoretic analysis of purified ColIb-P9 thin pili indicated that thin pili consist of two kinds of proteins, pilin and the PilV protein. Pilin was demonstrated to be the product of the pilS gene. Pilin was first synthesized as a 22-kDa prepilin from the pilS gene and subsequently processed to a 19-kDa protein by the function of the pilU product. The N-terminal amino group of the processed protein was shown to be modified. The C-terminal segments of the pilV products vary among six or seven different types, as a result of shufflon DNA rearrangements of the pilV gene. These PilV proteins were revealed to comprise a minor component of thin pili. Formation of PilV-specific cell aggregates by ColIb-P9 and R64 thin pili was demonstrated and may play an important role in liquid mating.

Conjugal pili are encoded by self-transmissible plasmids and play an important role in the early steps of bacterial conjugation (5, 7). IncI1 plasmids, such as R64 and ColIb-P9, form two distinct types of conjugal pili, a thick rigid pilus and a thin flexible pilus (1, 2). The thick pilus of IncI1 plasmids is required for conjugation both in liquid and on a solid surface, while the thin pilus is required only for liquid mating (12, 13).

The genetic locus of the R64 pil region responsible for the formation of R64 thin pilus has recently been revealed by DNA sequencing (11). The R64 pil region is organized into a single operon consisting of 14 genes, pilI to pilV (Fig. 1A). Based on amino acid sequence homology with known proteins, the pilS and pilV products were proposed to be type IV prepilins (19, 20, 24). These proteins contain putative type IV prepilin peptidase cleavage sites (Fig. 1C). The C-terminal end of the prepeptide is a glycine residue and the fifth amino acid residue of mature pilin is glutamic acid. The N-terminal 20-amino-acid region of mature pilin is hydrophobic. The pilU product has amino acid sequence homology with a type IV prepilin peptidase. Furthermore, the pilN, pilQ, pilR, and pilT products have sequence homology with the proteins related to type IV pilus biogenesis. Thus, the R64 thin pilus was predicted to belong to the type IV family, specifically group IVB, of pili. The requirement of the pilR and pilU genes for R64 liquid mating was demonstrated by the introduction of frameshift mutations into their coding sequences. In addition to the pil genes, the traBC genes are also necessary for thin pilus formation and are proposed to function as positive regulators for the expression of the pil operon (9).

FIG. 1.

(A) Gene organization of the traA to -D and pilI to -V regions of pKK641-A′ and pCD641-A′. Restriction sites: B, BglII; P, PstI; V, PvuII; H, HindIII; Hp, HpaI; C, ClaI; and E, EcoRI. Below each map, open reading frames are represented by arrows. tra, trc, transfer; pil, formation of thin pilus; shf, shufflon; rci, shufflon-specific recombinase. The solid lines above pKK641-A′ indicate the DNA segments present in pKK691 and pKK692. The crosses on pKK691 mark the locations of the pilS2, pilT2, and pilU2 mutations. (B) Switching of six pilV genes by DNA rearrangement of the ColIb-P9 shufflon. The gene organization of plasmid A′ expressing pilVA′ is shown at the top. Black arrows represent the six 19-bp repeats. The three DNA segments are indicated above the diagram. Open reading frames are indicated by stippling. The rci-mediated site-specific recombination of plasmid A′ between repeats 2 and 1 caused the inversion of segment A, to yield plasmid A, and the conversion of pilV from pilVA′ to pilVA. Subsequent inversion of whole segments resulted in plasmid B′, converting pilV from pilVA to pilVB′. Thus, a series of independent or group inversions of the three DNA segments made six pilV genes encoding different C-terminal segments. (C) N-terminal amino acid sequences of the pilS and pilV products. PilS and PilV sequences presented here are identical between ColIb-P9 and R64. The putative cleavage sites of type IV prepilin peptidase are indicated by the arrow. The conserved glycine and glutamic acid in type IV prepilins are indicated by boldface. The N-terminal hydrophobic region is underlined.

The C-terminal segments of the ColIb-P9 and R64 pilV gene products convert as a result of the DNA rearrangements of the shufflon (10, 15, 16) (Fig. 1B). The ColIb-P9 shufflon consists of three DNA segments, designated A, B, and C, which are flanked and separated by six 19-bp repeat sequences in either direction, while the R64 shufflon is comprised of four segments, A, B, C, and D, with seven 19-bp repeat sequences. The site-specific recombination mediated by the rci product occurs between any two inverted repeats, resulting in the inversion of three or four DNA segments independently or in groups. Consequently, the shufflon may act as a biological switch to select one of six or seven pilV genes, resulting in a constant N-terminal region and a variable C-terminal region. The shufflon determines recipient specificity in liquid mating by switching the six or seven C-terminal segments encoded by the pilV gene (13, 14).

In this study, thin pilus was purified from Escherichia coli cells harboring ColIb-P9- and R64-derived plasmids. The pilV gene product was revealed to be a component of thin pilus. Pilin, the major component of thin pilus, was demonstrated to be the product of the pilS gene and was characterized in detail.

MATERIALS AND METHODS

Bacterial strains.

E. coli K-12 strains JM83 Δ(lac-proAB) rpsL thi ara φ80 dlacZΔM15 and JM109 recA1 Δ(lac-proAB) endA1 gyrA96 thi hsdR17 supE44 relA1/F′ traD36 proAB lacI^qZΔM15 were used (27). E. coli C strain C-1 was also used (14).

Plasmid vectors pUC118 and pUC119 (25) were used for cloning and sequencing. pUEX03 (3) was used to construct a lacZ fusion gene. pKK641 has been described previously (12).

Media.

Luria-Bertani (LB) broth was prepared as described previously (22). Solid media contained 1.5% agar. Antibiotics were added to liquid or solid media, when necessary, at the following concentrations: ampicillin, 100 μg/ml; chloramphenicol, 25 μg/ml; kanamycin, 50 μg/ml; and tetracycline, 12.5 μg/ml.

Construction of plasmids.

Recombinant plasmids were constructed according to the method of Sambrook et al. (22). pCD641, which contained a 21.2-kb SmaI-HpaI fragment of ColIb-P9drd-1 and a DNA fragment for kanamycin resistance from Tn5, was constructed by the in vivo recombination method (12) (Fig. 1A). Since DNA rearrangement of the shufflon did not occur due to the absence of rci activity, the pilV gene in pCD641 was fixed. The first clone (pCD641-A′) obtained was shown by restriction enzyme analysis to carry the fixed pilVA′ gene. To create six pCD641 series plasmids carrying the fixed pilVA, pilVA′, pilVB, pilVB′, pilVC, and pilVC′ genes, pCD641-A′ was introduced into E. coli cells which harbored pKK024 containing the R64 rci gene. To isolate each pCD641 series plasmid, plasmid DNA isolated from cells harboring both pCD641 and pKK024 was again introduced into E. coli cells with kanamycin selection. The pilV C-terminus-encoding segments of various plasmids were determined by restriction enzyme analysis.

To sequence the ColIb-P9 pilS gene, a 0.9-kb HincII-BglII fragment of pCD641 was cloned into pUC119 to generate pCD692. The DNA sequence of the pilS gene was determined by the dideoxy chain termination method (23).

To construct R64 pilSTU⁺ plasmid pKK691 and pilS⁺ plasmid pKK692, 3.2-kb BclI-EcoRV and 0.9-kb HincII-BglII fragments from pKK641-A′ were inserted into the BamHI-HincII sites of pUC118 and pUC119, respectively, in the orientation in which the pilSTU and pilS genes were expressed by the lac promoter of the vector (Fig. 1A). The BglII site of pKK691 was treated with DNA polymerase I Klenow fragment to create the pilT2 mutation. Into the Eco47III and HpaI sites of pKK691 an EcoRI linker, 5′-GGAATTCC-3′, was inserted to create the pilS2 and pilU2 mutations, respectively.

To construct a lacZ-pilVA′ fusion gene, a 1,111-bp NdeI fragment of pKK010-079 was treated with Klenow fragment and then inserted into the SmaI site of pUEX03 in the proper orientation. The resulting plasmid, pKKV1, carried a lacZ-pilVA′ gene, which consisted of the R64 pilVA′ gene (encoding a 326-amino-acid C-terminal segment) fused to the E. coli lacZ gene.

Preparation of thin pili.

All the preparation steps were performed at 4°C. First, E. coli cells harboring pCD641 or pKK641 series plasmids were grown overnight with shaking at 37°C. The culture was centrifuged twice at 9,200 × g for 10 min to remove the bacterial cells. The supernatant was again centrifuged at 140,000 × g for 1 h. The pellet was used as a crude thin pilus fraction. Thin pili were further purified by CsCl density gradient centrifugation as described previously (4). Three aliquots of CsCl solution with different densities were layered in a Hitachi 13 PET tube as follows: 3.5 ml of dense CsCl solution (0.56 g/ml), 3.5 ml of CsCl solution (0.44 g/ml) containing the crude thin pilus fraction, and 3.5 ml of less dense CsCl solution (0.27 g/ml) on the top. Centrifugation was performed in a Hitachi RPS40T rotor at 218,000 × g for 20 h.

Electron microscopy.

Electron microscopic observation of thin pilus samples negatively stained with 4% uranyl acetate on collodion mesh was performed with a JEM-1010 electron microscope (JEOL).

Purification of pilin.

The pilin protein was purified from the crude thin pilus fraction by gel filtration chromatography with the Tosoh high-performance liquid chromatography system. A TSKgel G3000SW column (Tosoh) was used with 80 mM Tris-HCl (pH 7.0) containing 0.1% sodium dodecyl sulfate (SDS).

Purification of tryptic peptides of pilin.

Digestion of pilin with trypsin was performed in 80 mM Tris-HCl (pH 7.0) containing 2 M urea for 12 h at 37°C. Peptides derived from tryptic digestion of pilin were purified by reverse-phase chromatography with a TSK gel ODS80T column (Tosoh). Elution of peptide was performed by a linear gradient (from 20 to 80%) of acetonitrile in 0.05% trifluoroacetic acid.

The N-terminal amino acid sequence of pilin or its tryptic peptides was determined by Edman degradation in a model 477A/120A protein sequencer (Applied Biosystems Inc.).

Molecular weight determination by mass spectrometry.

The molecular weight of mature pilin was determined by matrix-assisted laser desorption/time-of-flight mass spectrometry (MALDI/TOFMS) on a Finnigan-Mat Vision-2000 mass spectrometer. One microliter of the purified protein, dissolved in 5 mM N-cyclohexyl-3-aminopropanesulfonic acid–HCl buffer (pH 10.5) containing 0.1% n-octylglucoside and 0.1 mM dithiothreitol at a concentration of 0.1 μg/μl, was placed on a target probe, mixed with an equal volume of 2,5-dihydroxybenzoic acid (10 mg/ml in 10% acetonitrile–0.1% trifluoroacetic acid), and subjected to mass spectrometry after the mixture was dried at room temperature.

Preparation of antiserum.

To prepare anti-PilVA′ antiserum, E. coli JM83 cells harboring pKKV1 were heat induced. The LacZ-PilVA′ fusion protein was recovered from the pellet of the cell extract and purified by gel filtration chromatography. Final purification was performed by SDS-polyacrylamide gel electrophoresis (SDS-PAGE). The gel slices containing the LacZ-PilVA′ protein were used to immunize a rabbit.

To prepare antipilin antiserum, ColIb-P9 pilin was finally purified by SDS-PAGE. The gel slices containing pilin were used to immunize a rabbit.

Western blot analysis.

Thin pilus preparations or lysates of E. coli cells harboring various plasmids were analyzed by SDS-PAGE. Proteins were transferred to a polyvinylidene difluoride membrane with a model AE-6675 semidry-transfer apparatus (Atto Corp.) and detected with anti-PilVA′ or antipilin antiserum by using anti-rabbit immunoglobulin G conjugated to horseradish peroxidase with chromogenic substrates.

Nucleotide sequence accession number.

The nucleotide sequence of the ColIb-P9 pilS gene will appear in the DDBJ, EMBL, and GenBank nucleotide sequence databases under the accession no. AB007463.

RESULTS

Purification of ColIb-P9 thin pili.

E. coli cells harboring pKK641-A′, which contained the traA to -D and pilI to -V genes as well as the rep region of R64drd-11 (Fig. 1A), were previously shown to form thin pili and to be sensitive to phages Iα and PR64FS (12). We found that phages Iα and PR64FS formed clear plaques on E. coli cells harboring ColIb-P9drd-1, while they formed only very turbid plaques on cells harboring R64drd-11 (data not shown). Hence, we cloned a ColIb-P9drd-1 segment corresponding to pKK641-A′ to obtain pCD641-A′ (Fig. 1A). pCD641-A′ contains the pilVA′ gene, which encodes a product with the C-terminal segment fixed in the A′ configuration due to the lack of rci activity. The organization of ColIb-P9 pil genes responsible for thin pilus formation appears to be similar to that of the R64 pil genes as judged by restriction mapping and partial sequencing (Fig. 1A). Major differences are as follows. (i) The ColIb-P9 traA product is 95 amino acid residues in length, while the R64 traA product has a length of 58 amino acid residues. (ii) ColIb-P9 carries the trcD gene instead of the R64 traD gene. The ColIb-P9 trcD and R64 traD genes are not related. (iii) The product of the ColIb-P9 pilJ gene is 98 amino acid residues in length, while the R64 pilJ product is 150 amino acid residues in length. (iv) The ColIb-P9 shufflon contains three invertible DNA segments, while the R64 shufflon consists of four DNA segments (10) (Fig. 1B).

ColIb-P9 thin pili were purified from culture medium in which E. coli cells harboring pCD641-A′ were grown overnight with shaking. First, E. coli cells were removed from the culture medium by two cycles of low-speed centrifugation. Then, thin pili were collected from the culture medium by ultracentrifugation (crude thin pilus fraction). To purify thin pili, CsCl density gradient centrifugation was performed. After centrifugation, the thin pili formed a band at a density of 1.40 g/cm³ (data not shown). Peak fractions of the CsCl density gradient were used as purified thin pili. About 500 μg of purified thin pili was obtained from 1 liter of culture medium. From E. coli cells harboring pKK641-A′, R64 thin pili were purified by a similar method. However, the yield of thin pili from cells harboring pKK641-A′ was much lower than that from cells harboring pCD641-A′.

The morphology of a negatively stained sample of purified ColIb-P9 thin pili was observed under an electron microscope (Fig. 2). ColIb-P9 thin pili appeared to be long rods with a diameter of 6 nm. Thin pili tended to aggregate laterally. Their morphology was similar to those of the other type IV pili (19, 24).

FIG. 2.

Electron micrograph of purified ColIb-P9 thin pili. Samples were stained with 4% uranyl acetate. Bar, 50 nm.

Protein contents of thin pili.

Proteins of the purified ColIb-P9 thin pilus preparation were separated by SDS-PAGE and stained with Coomassie brilliant blue (Fig. 3). Two protein bands, a faint band at 45 kDa and a dense band at 19 kDa, were detected in the purified thin pilus preparation (Fig. 3, lane 3). These two proteins were present in the crude thin pilus preparation from E. coli cells harboring pCD641-A′ (lane 1) but absent in the preparation from E. coli cells without the plasmid (lane 2).

FIG. 3.

Protein analysis of ColIb-P9 thin pili. Proteins were separated by SDS-PAGE (17.5% polyacrylamide) and stained with Coomassie brilliant blue (lanes 1 to 3) or analyzed by Western blotting with anti-R64 PilVA′ antiserum (lanes 4 to 6). Lanes 1 and 4, crude thin pilus fraction from E. coli cells harboring pCD641-A′; lanes 2 and 5, crude thin pilus fraction from cells without plasmid; lanes 3 and 6, purified thin pilus fraction. The locations of molecular mass markers (in kilodaltons) are indicated on the left.

Since the 45-kDa protein reacted with anti-PilVA′ antiserum (Fig. 3, lanes 4 and 6), it appears to be the pilVA′ product, indicating that the purified thin pili contain the pilVA′ product as a minor component.

Characterization of pilin.

The results of SDS-PAGE of the purified ColIb-P9 thin pilus preparation indicated that the 19-kDa protein is pilin, a major component of thin pili (Fig. 3). To characterize ColIb-P9 pilin, the pilin protein was purified from the ColIb-P9 crude thin pilus preparation by gel filtration chromatography. Edman degradation analysis of the N-terminal sequence of the purified pilin indicated that the N-terminal amino group of ColIb-P9 pilin was blocked.

The purified ColIb-P9 pilin was digested with trypsin. Tryptic peptides were purified by reverse-phase chromatography and sequenced. The N-terminal amino acid sequences of two tryptic peptides were determined to be NH₂-Met-Thr-Gly-Ala-Leu-Ile-Gln-Met-Gly-Val and NH₂-Leu-Ile-Phe-Thr-Ile-Asn-Gly. These sequences corresponded to the amino acid sequence of ColIb-P9 PilS protein predicted from the DNA sequence (data not shown). The DNA sequence of the ColIb-P9 pilS gene was shown to be identical with that of the R64 pilS gene (11) except for a single nucleotide replacement, which results in the replacement of serine in the deduced PilS product of ColIb-P9 with asparagine in the R64 product at the 169th amino acid.

The molecular mass (19 kDa) of mature pilin, estimated by SDS-PAGE, was significantly lower than that (21,238 Da) predicted from the DNA sequence of the pilS gene. Hence, the molecular mass of mature ColIb-P9 pilin was accurately determined by mass spectroscopy. The result of MALDI/TOFMS indicates that the molecular weight of the purified pilin is 18,947 (Fig. 4). The significance of this value is discussed below.

FIG. 4.

MALDI/TOFMS spectrum of mature ColIb-P9 pilin. The molecular weight of pilin was estimated with horse heart myoglobin (16,951) and human serum albumin (66,402) as external standards.

Processing of the R64 pilS product.

Antipilin antiserum was raised by injecting purified ColIb-P9 pilin into a rabbit. The raised antiserum reacted with ColIb-P9 pilin as well as with R64 thin pilus pilin, which was prepared from E. coli cells harboring pKK641-A′ (Fig. 5).

FIG. 5.

Processing of the R64 pilS product. Whole proteins of E. coli cells harboring pKK691, pKK691 pilS2, pKK691 pilT2, pKK691 pilU2, and pKK692 were separated by SDS-PAGE and subjected to Western blot analysis with antipilin antiserum. Lanes 1 to 5, E. coli cells harboring pKK691, pKK691 pilS2, pKK691 pilT2, pKK691 pilU2, and pKK692, respectively; lane 6, crude thin pilus fraction from cells harboring pKK641-A′; lane 7, cells without plasmid. Numbers on the left are the sizes (in kilodaltons) of marker proteins.

To investigate the processing of the pilS product into mature pilin, we constructed plasmids pKK691 and pKK692 carrying the R64 pilSTU and pilS genes, respectively (Fig. 1A). In addition, frameshift mutations were introduced into the pilS, pilT, and pilU genes of pKK691 by modifying various restriction sites to generate pilS2, pilT2, and pilU2 mutations, respectively. Formation of the pilS product in E. coli cells harboring various plasmids was analyzed by SDS-PAGE followed by Western blot analysis with antipilin antiserum (Fig. 5). E. coli cells harboring pKK691 produced 22- and 19-kDa proteins which reacted with antipilin antiserum (Fig. 5, lane 1), while those harboring pKK692 produced only the 22-kDa protein (lane 5). Cells harboring pKK691 pilS2 produced neither the 22- nor the 19-kDa protein (lane 2), confirming that both of these proteins are the products of the pilS gene. Cells carrying pKK691 pilU2 produced only the 22-kDa protein (lane 4). A crude thin pilus preparation contained some degradation products detected by Western blotting (lane 6). These results suggest the following model for processing of the pilS product: a 22-kDa precursor protein is first synthesized from the pilS gene and then processed into a 19-kDa protein by the function of the pilU product.

Selection of the six different ColIb-P9 PilV proteins by shufflon DNA rearrangement.

We have postulated that the shufflon is a biological switch that selects one of several pilV genes encoding different C-terminal segments (10, 15). The constant region of the ColIb-P9 PilV proteins consists of 361 amino acid residues, while the number of amino acid residues in the six PilV variable regions fluctuates between 69 and 113. To test this hypothesis, we constructed six pCD641 series plasmids carrying fixed pilVA, pilVA′, pilVB, pilVB′, pilVC, and pilVC′ genes (Fig. 1A and B) by introducing pCD641-A′ into E. coli cells harboring pKK024, which carries the R64 rci gene (16). The Rci protein produced from pKK024 promoted DNA rearrangement of shufflon in pCD641-A′, and pCD641 series plasmids with six pilV genes were generated. From E. coli cells harboring the six pCD641 series plasmids crude thin pilus fractions were prepared. The PilV proteins in the crude thin pilus fractions from E. coli cells with various pCD641 series plasmids were subjected to Western blot analysis with anti-PilVA′ antiserum (Fig. 6). The apparent molecular masses of the PilVA, PilVA′, PilVB, PilVB′, PilVC, and PilVC′ proteins correlated well with the calculated values of the putative mature proteins (48.7, 44.9, 46.8, 46.1, 46.5, and 45.3 kDa, respectively). These results indicate that the six pilV genes, selected by shufflon DNA rearrangement, encode the corresponding proteins with different molecular masses, which may then be processed and assembled into thin pili together with the pilS product.

FIG. 6.

Detection of the six different PilV proteins in the thin pilus fractions of E. coli cells harboring six pCD641 series plasmids. Crude thin pilus fractions were separated by SDS-PAGE (12.5% polyacrylamide) and subjected to Western blot analysis with anti-PilVA′ antiserum. Lanes 1 to 6, thin pilus fraction from E. coli cells harboring pCD641-A, pCD641-A′, pCD641-B, pCD641-B′, pCD641-C, and pCD641-C′, respectively; lane 7, control from cells without plasmid. Numbers on the left are the sizes (in kilodaltons) of marker proteins.

Formation of specific cell aggregates by ColIb-P9 thin pili.

Colonies of E. coli K-12 cells harboring pCD641-A′, -C, and -C′ were found to be deeply rough, while colonies of E. coli K-12 cells harboring pCD641-A, -B, and -B′ were rather smooth (Fig. 7). In contrast, colonies of E. coli C cells harboring pCD641-A and -A′ were deeply rough, while those harboring the other plasmids were rather smooth (Fig. 7). In liquid culture, E. coli K-12 cells harboring pCD641-A′, -C, and -C′ and E. coli C cells with pCD641-A and -A′ tended to precipitate rapidly and to attach to the surface of culture glassware (data not shown). Phase microscopic observation indicated that E. coli K-12 cells harboring pCD641-A′, -C, and -C′ and E. coli C cells with pCD641-A and -A′ formed large cell aggregates in liquid culture, while E. coli K-12 and C cells with the other plasmids did not (Fig. 7).

FIG. 7.

Colony and cell morphology of E. coli K-12 and C strains harboring pCD641 series plasmids expressing one of the pilVA, pilVA′, pilVB, pilVB′, pilVC, and pilVC′ genes. pCD641 series plasmids carrying one of these genes were introduced into E. coli strains K-12 and C. Cells were grown on LB agar or in LB media. The cells were photographed under a Nikon microscope. Bars: colonies, 1 cm; cells, 10 μm.

In the case of the R64-derived pKK641 series plasmids, E. coli K-12 cells harboring pKK641-A′, -C, and -C′ and E. coli C cells harboring pKK641-A and -A′ formed rough colonies on agar media and formed large aggregates in liquid media, while cells harboring the other pKK641 series plasmids did not (data not shown). However, aggregates of E. coli K-12 cells harboring pKK641-A′, -C, and -C′ were smaller than those of K-12 cells with pCD641-A′, -C, and -C′.

During liquid mating, E. coli K-12 recipient cells were recognized by the PilVA′, PilVC, and PilVC′ proteins, and E. coli C recipient cells were recognized by the PilVA and PilVA′ proteins (13, 14). Therefore, formation of specific cell aggregates by thin pili is likely to play a crucial role(s) in the initiation of liquid mating.

DISCUSSION

It has been known for quite some time that IncI and related plasmids produce thin flexible pili (1, 2, 7). A recent sequencing study of the R64 pil region predicted that the R64 thin pilus belongs to the type IV pilus family (11). Type IV pili are long flexible rods, with diameters of 6 to 7 nm, protruding from the bacterial cell surface. They are produced by gram-negative bacteria, such as Pseudomonas aeruginosa, Neisseria gonorrhoeae, Moraxella bovis, Myxococcus xanthus, Vibrio cholerae, and enteropathogenic and enterotoxigenic E. coli (19, 20, 24). The pili play important roles in the attachment of bacterial pathogens to membranes of eukaryotic host cells. ColIb-P9 and R64 thin pili were purified for the first time in the present work. The purified thin pili appear as long rods with a diameter of 6 nm under an electron microscope, which concurs well with the characteristics of type IV pili.

ColIb-P9 and R64 thin pili were found to be composed of the products of the pilS and pilV genes. The results are quite consistent with our observations that the pilS and pilV products have features of type IV prepilins (Fig. 1C). They have putative type IV prepilin peptidase cleavage sites. PilS processing was mediated by the pilU gene product: the pilS product was first synthesized as a 22-kDa protein and then processed into a 19-kDa protein by the function of the pilU product. The pilU product exhibits amino acid sequence homology with other type IV prepilin peptidases (11).

In light of research on other type IV prepilins (20, 24), the cleavage site of the PilS protein was predicted to be located between the glycine and tryptophan residues encoded by the 23rd and 24th codons, respectively (Fig. 1C). The size of mature pilin was estimated to be 18,947 Da by mass spectrometry, while the size of the processed PilS protein was calculated to be 18,688 Da. ColIb-P9 pilin could not be stained by periodic acid-Schiff reagent, suggesting that it is not a glycoprotein (data not shown). Based on the absence of data from Edman degradation analysis, the difference in molecular size (approximately 259 Da) is suggested to be due to the N-terminal modification of mature pilin. The value indicates that modification of the N-terminal group of mature pilin is not methylation, although the N-terminal amino acid of many type IV pilins is N-methylphenylalanine (24). Therefore, it is likely that the N-terminal group of the 19-kDa processed PilS protein is modified subsequent to processing. The nature of modification of the N-terminal group of mature ColIb-P9 pilin is presently unknown.

In liquid culture, E. coli K-12 cells harboring pCD641-A′, -C, and -C′ and E. coli C cells harboring pCD641-A and -A′ formed large cell aggregates, while cells harboring the other plasmids did not. The differences in the ability to form cell aggregates are closely related to their transfer frequency in liquid media. In liquid matings, transfer frequencies from E. coli K-12 donor cells harboring pCD641-A′, -C, and -C′ together with pKK661 to E. coli K-12 recipient cells were 10 to 30%, while those from donor cells with pCD641-A, -B, and -B′ were less than 0.1% (data not shown). When the recipient cells were E. coli C, pCD641-A and -A′ exhibited high transfer frequencies (50 and 30%, respectively) while pCD641-B, -B′, -C, and -C′ exhibited low frequencies (less than 0.3%). These results with ColIb-P9-derived plasmids concurred with our previous results with R64-derived pKK641 series plasmids (13, 14). In summary, the transfer frequencies of ColIb-P9 and R64 liquid matings were high among the combinations of bacterial strains and PilV proteins, with which large cell aggregates were formed.

The cell aggregates produced by ColIb-P9 thin pili do not appear to be stable. By extensive vortexing of liquid cultures with cell aggregates, they were broken into small fragments of cell aggregates (data not shown). Furthermore, by using a standard plating technique, similar viable cell numbers were observed from the same densities of E. coli cells in liquid cultures (measured as optical density units) with and without cell aggregates.

The requirement of the pilV genes for R64 liquid mating was previously demonstrated by constructing a ΔpilV derivative of pKK641-A (12). Recent experiments suggest that the pilV genes are required for the biogenesis of R64 thin pili (28).

The densitometric analysis whose results are shown in Fig. 3 indicated that the molar ratio of the PilVA′ protein to the PilS protein in ColIb-P9 thin pili is approximately 1:100. Fiber diffraction on P. aeruginosa pili indicated that type IV pili have five subunits per turn of helix, with a 4.0- to 4.1-nm pitch (6, 26). Hence, it is unlikely that the PilVA′ protein is equally distributed along the pilus fiber. Since the PilV proteins may function as adhesins, it is most likely that the proteins are located at the tip of the ColIb-P9 thin pilus. The location of adhesins at the tip of pili has been reported for N. gonorrhoeae type IV pilus and pap pilus (8, 17, 18, 21). However, no significant amino acid sequence similarity between ColIb-P9 or R64 PilV proteins and PilC1 or PilC2 adhesins of N. gonorrhoeae was found. PilV proteins have type IV prepilin peptidase cleavage sequences at their N termini (Fig. 1C), while PilC1 or PilC2 proteins do not.

A two-step mechanism of ColIb-P9 and R64 liquid mating can be proposed. The first step is the formation of donor and recipient cell aggregates mediated by ColIb-P9 and R64 thin pili as shown in Fig. 7. In this step, ColIb-P9 and R64 thin pili of the donor cells may attach to the surfaces of the recipient cells. Furthermore, the presence of six or seven PilV proteins in thin pili determines attachment or nonattachment to the recipient cells. The second step is a process of conjugation including stable mating-pair formation, DNA transfer, and DNA replication. Thick pili may be involved in this step. In surface mating, the first step can be skipped, since donor and recipient cell aggregation may be formed by physical contact of donor and recipient cells on the medium surface. The attachment by type IV pili of donor cells to recipient cells is reminiscent of attachment of bacterial pathogens to host cells by their type IV pili.

Analysis of putative PilV receptors at the surfaces of the recipient cells might further elucidate recipient determination by PilV proteins in ColIb-P9 or R64 liquid matings. Our preliminary results with rfa mutants of E. coli K-12 suggest that lipopolysaccharide of E. coli cells may function as a PilV receptor (8a).