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Infect Immun. Dec 2002; 70(12): 6534–6540.
PMCID: PMC132963

Construction of a Novel Transposon Mutagenesis System Useful in the Isolation of Streptococcus parasanguis Mutants Defective in Fap1 Glycosylation

Abstract

Streptococcus parasanguis, a primary colonizer of the tooth surface, has long, peritrichous fimbriae. A fimbria-associated protein, Fap1, is identified as an adhesin of S. parasanguis FW213. The mature Fap1 protein is glycosylated, and the glycosylation is required for fimbria biogenesis and bacterial adhesion. Little is known about the mechanism of Fap1 glycosylation due to the lack of identifiable mutants. A novel transposon mutagenesis system was established and used to generate a mutant library. Screening of the library with a monoclonal antibody specific for a glycan epitope of Fap1 yielded six mutants with decreased expression levels of surface-associated glycosylated Fap1 protein. Southern blot analyses revealed that three of the mutants had the transposon inserted in the fap1 locus, whereas the other three mutants had insertions in other genes. Among the latter three mutants, two expressed Fap1 polypeptides on which no glycosylation was detected by glycan-specific antibodies; the other mutant expressed a partially glycosylated Fap1 polypeptide. These data suggest that three mutants were isolated with defects in genes implicated in Fap1 glycosylation.

Streptococcus parasanguis FW213 initiates dental plaque formation by adhering to the tooth surface and acting as a substrate for colonization of other species. The adherence of this organism to saliva-coated hydroxyapatite (SHA), an in vitro tooth model, is attributed to the presence of peritrichous long fimbriae on the cell surface (12, 14). Fimbrial assembly and adhesion to SHA are both mediated by a fimbria-associated protein (Fap1), the first streptococcal fimbrial structural subunit described (35). Mature Fap1 migrates in sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) gels as a 200-kDa protein. The protein has an unusual feature: 80% of it consists of dipeptide serine repeats (34). It also contains the traditional cell wall sorting signal associated with gram-positive surface proteins (28). A number of experimental observations have shown that Fap1 is a glycosylated protein (30); however, it is not clear how glycosylation of the fimbriae occurs.

Monoclonal antibodies (MAbs) F51 and D10 block S. parasanguis FW213 adhesion by binding to glycan epitopes on Fap1 (12, 30). Competition experiments have demonstrated that MAb F51 and MAb D10 are specific for different glycan epitopes in the dipeptide repeat region, whereas another antibody, MAb E42, is specific for a peptide epitope in the nonrepetitive region of Fap1 (11). These antibodies should be useful in selecting mutants that are defective in various stages of glycosylation.

A variety of nonadhesive, nonfimbriated mutants have been isolated previously (10, 14). Western blot analyses of wild-type FW213 and these mutants probed with various specific antibodies reveal two additional Fap1-related bands at approximately 360 and 470 kDa. These bands are detected at low intensities in the wild type, but they are never present in the fap1 null mutant. Some mutants, e.g., the VT508 mutant, express only the 360-kDa polypeptide, which is detected only by peptide-specific antibodies, such as MAb E42. These mutants are thought to be defective in glycosylation, since they fail to react with antibodies that are specific for glycan epitopes and never produce the mature 200-kDa species. Other mutants which do not make the mature 200-kDa species, such as the VT324 mutant, express a 470-kDa polypeptide, which is detected by both peptide-specific MAbs and only one of the glycan-specific antibodies (MAb D10). The inference is that these mutants have partially glycosylated Fap1 (29).

These immunological data suggest that some of these chemical mutants are defective in glycosylation. However, the genetic basis for the defect is not easily determined, as the locus is not “tagged” and complementation is not yet possible in S. parasanguis FW213. Thus, in this study we have developed a transposon mutagenesis system in order to generate glycosylation-defective mutants with identifiable genotypes.

A variety of transposon mutagenesis systems have been developed for use in the streptococci. All of these systems work in some, but not in all, streptococcal strains. A suicide vector, pMGC57, has been previously exploited for transposon mutagenesis in Streptococcus pyogenes (21). It contains IS256 (4), an insertion sequence of the class I composite-type transposon Tn4001 (20). IS256 transposes with a high degree of randomness in FW213 (unpublished data), but its usefulness is limited because of its low frequency of transformation and transposition. Another transposon system that utilizes a streptococcal temperature-sensitive replicon (23) and transposon Tn917 (32) has been developed (17) for poorly transformable streptococci. Unfortunately, transposition of Tn917 is not random in FW213 (unpublished data). Therefore, we developed a transposon mutagenesis system that overcame the problems associated with other systems. Successful utilization of this system allowed us to isolate three mutants with defective glycosylation of Fap1, as well as three fap1 insertion mutants.

MATERIALS AND METHODS

Bacterial strains and growth conditions.

Bacterial strains and plasmids used in this study are summarized in Table Table1.1. HB101 (rpsL20 recA13) was chosen for molecular cloning in Escherichia coli because erm is not toxic to this strain (J. A. Gutierrez, personal communication). E. coli was cultured in Luria-Bertani medium at 37°C or, when harboring temperature-sensitive plasmids, at 30°C. S. parasanguis preserved in 10% dimethyl sulfoxide was streaked onto blood agar or Todd-Hewitt (TH) plates (Becton Dickinson, Cockeysville, Md.) and incubated aerobically under 5% CO2. Broth cultures were prepared by inoculation of single colonies from plates into TH broth and grown statically under 5% CO2. All strains were cultivated at 37°C except where noted otherwise. When appropriate, antibiotics were added to the medium at the following concentrations: erythromycin, 500 μg/ml for E. coli and 10 μg/ml for S. parasanguis; kanamycin, 50 μg/ml for E. coli and 125 μg/ml for S. parasanguis; chloramphenicol, 25 μg/ml for E. coli.

TABLE 1.
Bacterial strains and plasmids used in this work

PCR.

The transposon IS256.erm was amplified by PCR with the following primers derived from the nucleotide sequence of IS256 (GenBank accession number M18086): EIR (5′-GCT GAA TTC AGT CAA GTC CAG ACT CC-3′) and PIR (5′-TCG CTG CAG GAT AAA GTC CGT ATA ATT G-3′). This introduced an EcoRI and a PstI site (underlined) immediately adjacent to the inverted repeats of IS256. The PCR was set up using 2.5 U of Taq DNA polymerase, 200 ng of pVT1515 DNA, 2 mM each deoxynucleoside triphosphate, 1.0 μM primers, and 2 mM MgCl2 in 100-μl volumes. A single cycle consisting of denaturation at 94°C for 1 min, annealing at 52°C for 1 min, and extension at 72°C for 3 min was repeated for 35 cycles in a Techne (Princeton, N.J.) Genius thermocycler.

Construction of the transposon plasmid.

Routine DNA manipulations were performed as described previously (26). A 1.8-kb HindIII-ClaI fragment containing the erm gene of pVA838 was blunt ended using T4 DNA polymerase and ligated to EcoRV-digested pMGC57. The ligation mixture was electroporated into E. coli HB101 by using a Gene Pulser apparatus (Bio-Rad, Richmond, Calif.). VT1515 was obtained by selecting a single transformant on erythromycin-chloramphenicol agar. IS256.erm was amplified from pVT1515 by PCR using primers EIR and PIR. The 3.1-kb product was gel purified and ligated into a 3.4-kb EcoRI-PstI digest of pTV1-OK conferring repA-ts and kan. The ligation mixture was transformed as described above. VT1528 was generated by selection of a single colony on erythromycin-kanamycin agar. The plasmid constructed, pVT1528, was confirmed by PCR, restriction analysis, and sequencing and was then transformed into FW213 by electroporation (13).

Transposon mutagenesis of S. parasanguis.

Delivery of IS256.erm into the host chromosome was achieved by a temperature shift, as described previously (17), with some modifications. Stationary culture (48 h) of VT1529, grown at the permissive temperature (30°C) in TH broth containing erythromycin and kanamycin, was subcultured by 1/5,000 dilution into fresh TH broth. Following incubation at 44°C for 24 to 48 h, the subculture was plated onto TH agar containing erythromycin. The putative transposon insertion mutants (Ermr Kans) were selected by plating all the Ermr colonies onto TH agar containing kanamycin. Ermr Kans clones were then preserved in dimethyl sulfoxide at −70°C for future screening.

Southern blot analysis.

Genomic DNA of S. parasanguis was prepared by using the Puregene DNA isolation kit (Gentra Systems Inc., Minneapolis, Minn.). Southern blot analysis was carried out using the ECL direct nucleic acid labeling and detection system according to the manufacturer's protocol (Amersham plc, Little Chalfont, United Kingdom). To compare the restriction pattern of fap1 in FW213 with those in the transposon mutants, an internal fragment of fap1 (GenBank accession number AF100426; nucleotides 610 to 1380) was used as a probe.

Mutant screening by colony hybridization.

S. parasanguis mutants in the Ermr Kans transposon library were tested for reactivity with MAb F51. Ermr Kans colonies on TH-erythromycin agar plates were transferred to nitrocellulose membranes (Schleicher & Schuell Inc., Keene, N.H.). After colony lifts were performed, the nitrocellulose membranes were blocked with 5% nonfat dry milk dissolved in Tris-buffered saline (TBS) for 1 h. Membranes were probed with 300 ng of MAb F51/ml in TBS with 0.1% Tween 20 (TBST) for 1 h and then incubated with peroxidase-conjugated goat anti-mouse immunoglobulins for 1 h. Antibody conjugates were detected using the ECL Western blotting reagents (Amersham Pharmacia Biotech Inc., Piscataway, N.J.). Membranes were washed three times with TBST prior to the addition of each solution.

SDS-PAGE and Western blot analysis.

S. parasanguis cells grown to exponential phase were harvested by centrifugation. For each sample, the same number of cells, enumerated by counting CFU, was resuspended in sample buffer (0.0625 M Tris [pH 6.8], 2% SDS, 10% glycerol, 0.01% bromophenol blue) and boiled for 10 min before electrophoresis. Commercially available precast 4-to-12% gradient gels were utilized for SDS-PAGE according to the manufacturer's protocol (BioWhittaker Molecular Applications, Rockland, Maine). Separated proteins were transferred from gels onto nitrocellulose membranes in a Bio-Rad Mini Trans-blot apparatus. Membranes were probed with antibodies (3 μg of MAb F51/ml or 4 μg of MAb E42/ml) and detected as described above.

BactELISA.

The presence of Fap1 epitopes on the cell surface was examined by using a whole-bacterial-cell enzyme-linked immunosorbent assay (BactELISA) as described previously (9) with some modifications. S. parasanguis cells in 50 mM sodium carbonate, pH 9.6, were dried onto 96-well microtiter plates. Wells were blocked with 1% bovine serum albumin in TBS for 1 h. MAb F51 or MAb E42 was diluted in TBST to 300 or 134 ng/ml, respectively, and added to wells for 1 h. After incubation for 1 h with a peroxidase-conjugated goat anti-mouse immunoglobulin, hydrogen peroxide and o-phenylenediamine were added for color development. Enzyme activity was quantified by measurement of the absorbance at 490 nm using a Microplate Autoreader EL311 (Biotek Instruments, Winooski, Vt.). Wells were washed three times with TBST prior to the addition of each solution.

Electron microscopy.

S. parasanguis cells grown to mid-exponential phase were harvested and washed three times in TBS. A drop of cell suspension was added to a copper grid coated with Formvar. Dried grids were subjected to platinum-carbon shadow casting and examined by scanning electron microscopy.

RESULTS

Construction of a transposon delivery system.

An ideal transposon delivery system for FW213 would comprise a conditional origin of replication, a transposon that would randomly insert into the chromosome, and an antibiotic resistance marker that was functional in both E. coli and the streptococci. A chimeric plasmid, pVT1528, that made use of the replication-conditional origin of pTV1-OK, transposon IS256 from pMGC57, and an erythromycin resistance (erm) cassette from pVA838, was constructed in E. coli (Fig. (Fig.1A).1A). A HindIII-ClaI fragment containing erm was isolated from pVA838 and blunt-ended with T4 DNA polymerase. The fragment was inserted into the EcoRV site of pMGC57 to generate pVT1515. An EcoRI-tagged primer (EIR) and a PstI-tagged primer (PIR) were designed and used to PCR amplify the new transposon IS256.erm (Fig. (Fig.1B)1B) from pVT1515. After restriction with EcoRI and PstI, the amplicon was ligated with an EcoRI-PstI digest of pTV1-OK which contained a repA-ts (temperature-sensitive origin of replication for both E. coli and streptococci) and a kan (kanamycin resistance gene). The composition of the resultant plasmid, pVT1528, was confirmed by PCR, extensive digestion, and sequencing. FW213 was transformed with pVT1528 by electroporation. A single transformant, VT1529, was isolated and screened by growth on a solid medium containing kanamycin at 30°C (permissive temperature). It failed to grow when the temperature was raised to 44°C (nonpermissive temperature), demonstrating that the repA-ts was operable in FW213.

FIG. 1.
(A) Construction of pVT1528. Plasmid pVA838 was digested with HindIII and ClaI, treated with T4 DNA polymerase, and inserted into the EcoRV site of pMGC57 to generate pVT1515. A fragment containing IS256 and erm was PCR amplified from pVT1515 by using ...

Transposon mutagenesis of S. parasanguis.

VT1529 was grown at the permissive temperature (30°C) to stationary phase. It was then subcultured at the restrictive temperature (44°C) and grown to exponential phase to promote transposition. The resultant cultures were plated onto TH agar with erythromycin. The Ermr clones were patched onto TH agar with kanamycin. More than 3,500 out of approximately 10,000 clones were Ermr Kans, suggesting that transposition had occurred. These clones were isolated and frozen down as the transposon mutant library. The remaining Ermr Kanr clones resulted primarily from replicon fusion between the chromosome and the plasmid or failure of the origin of replication to be temperature sensitive.

Southern blotting was performed with chromosomal DNA from 11 randomly chosen Ermr Kans mutants, which was probed with IS256.erm (Fig. (Fig.2).2). All of the clones had random insertions, and 80% of them were single insertions. These clones were frozen, then cultured without antibiotic selection and analyzed by the same Southern blot analysis. The locations of the transposons in all mutants remained the same (data not shown), suggesting that the transposon was stably maintained at its original integration site.

FIG. 2.
Southern blot analysis of S. parasanguis transposon mutants. Southern blot analysis was carried out with EcoRI chromosomal digests of 11 randomly chosen Ermr Kans transposon mutants with IS256.erm as the probe. Molecular size markers are given on the ...

Screening the transposon library for putative glycosylation-defective mutants.

We tested the usefulness of the transposon library by screening mutants that exhibited defects in Fap1 glycosylation. MAb F51, which binds specifically to the glycan moiety of the Fap1 protein, was chosen to screen the transposon library for glycosylation mutants. Colony lifts of Ermr Kans mutants were probed with MAb F51. Six (VT1531, VT1532, VT1533, VT1534, VT1535, and VT1537) of the 2,300 transposon mutants screened to date displayed decreased reactivity compared to FW213.

Southern blot analyses of these six mutants probed with erm indicated that a single random insertion of the transposon had taken place in each mutant (Fig. (Fig.3A).3A). Blots were stripped and probed with an internal fragment of fap1 (Fig. (Fig.3B)3B) to determine if any of the insertions were in fap1. Mutants VT1531, VT1532, and VT1533 had a hybridization pattern identical to that of wild-type FW213 after the chromosomal DNA digestion with HindIII and PstI, indicating that the fap1 locus in these three mutants was not affected. The remaining three mutants displayed smaller fap1 bands than wild-type FW213, which is consistent with an insertion into the fap1 locus, as IS256.erm has an internal HindIII site (Fig. (Fig.1B1B).

FIG. 3.
Southern blot analysis of FW213 and S. parasanguis transposon mutants. HindIII and PstI digests of genomic DNA from wild-type FW213 and six transposon mutants (VT numbers shown) were probed with erm (A) or an internal fragment of fap1 (B). Molecular size ...

Expression of Fap1 in nonfimbriated mutants.

Mutants isolated by MAb F51 were assayed for defects in Fap1 expression and/or in Fap1 glycosylation. A peptide-specific antibody, MAb E42, was utilized to examine Fap1 expression in Western blot analyses (Fig. (Fig.4).4). VT324 and VT508, two previously characterized mutants, were used as controls. FW213 exhibited a prominent 200-kDa protein, representing the mature form of Fap1 (35), as well as the 470-kDa polypeptide. The three transposon mutants with insertions outside fap1 (VT1531, VT1532, and VT1533) displayed phenotypes similar to those of the control mutants. VT1531 and VT1533 appeared to overexpress the 360-kDa Fap1 polypeptide, a smaller amount of which was present in VT508. VT1532 yielded a weak band similar in size to the high-molecular-weight Fap1 band in VT324. Two of the three transposon mutants (VT1534 and VT1537) that had insertions in fap1 produced polypeptides of approximately 180 kDa. This suggested that the transposon had inserted downstream in fap1 and that the mutants had produced a truncated protein. VT1535, the other fap1 insertion mutant, yielded a small amount of the 470-kDa polypeptide. No bands were associated with VT1393, the fap1 null mutant (35), demonstrating that all of the bands observed are Fap1 specific. Low-molecular-weight bands (<68 kDa) were observed in FW213 and in some of the mutants. The nature of these bands is unknown, but they are presumed to be degradation products of Fap1 (29, 35). Additional evidence will be required to explain the origins of these bands in FW213 and the mutants.

FIG. 4.
Western blot analysis of S. parasanguis probed with MAb E42. Whole-cell extracts from wild-type FW213 and mutants (VT numbers shown) were subjected to SDS-PAGE and transferred onto a nitrocellulose membrane. The antibody reactivity of the membrane was ...

The same blot was probed with MAb F51, the MAb specific for a glycan epitope which is present only in the fully glycosylated form of Fap1 (29, 30). A 200-kDa band in FW213 and a smaller band at about 180 kDa in VT1534 and VT1537 were detected (data not shown), indicating that only these three strains produced fully glycosylated Fap1. None of the high-molecular-weight (>200 kDa) or low-molecular-weight (<68 kDa) bands were detected in these strains, because these forms of Fap1 do not react with MAb F51 and are presumed not to be fully glycosylated.

Cell surface Fap1 expression and morphology of fimbriae.

A BactELISA was employed to investigate Fap1 expression on the surfaces of FW213 and nonfimbriated mutant cells (Fig. (Fig.5).5). FW213 manifested strong reactivity with both MAb F51 and MAb E42. VT1531 and VT1533, mutants which were shown to express the 360-kDa Fap1 polypeptide, retained some of the reactivity with MAb E42 but failed to react with MAb F51. VT1532 did not react well with any of the antibodies, suggesting that very little Fap1, either glycosylated or unglycosylated, was transported to the cell surface. Two of the fap1 transposon insertion mutants, VT1534 and VT1537, had reduced reactivity with both MAbs compared to that of FW213. VT1535, the other fap1 insertion mutant, did not express any form of Fap1 on its surface. As expected, neither antibody reacted with VT1393, the fap1 null mutant.

FIG. 5.
BactELISA of S. parasanguis. Immobilized whole cells from wild-type FW213 and mutants (VT numbers shown) were probed with glycan-specific MAb F51 (shaded bars) and peptide-specific MAb E42 (solid bars). Assays were performed in triplicate. Error bars, ...

Electron microscopy was performed on the six transposon mutants and on FW213, which served as a positive control (Fig. (Fig.6).6). Long peritrichous fimbriae on the cell surface were visualized for FW213. Electron microscopy revealed that fimbriae were absent or greatly reduced in all the mutants. These data support the BactELISA data, which revealed that these transposon mutants expressed fewer of the mature Fap1 proteins on the cell surface than FW213 did.

FIG. 6.
Electron micrographs of fimbriae on surfaces of S. parasanguis cells. FW213 (A) and mutant (B) cells were shadowed with platinum-carbon and examined by electron microscopy. Only one mutant (VT1532) is shown in panel B. Bars, 0.25 μm.

DISCUSSION

A number of transposons were considered as candidates for transposon mutagenesis in FW213; however, all had limitations. Tn4001 or its insertion sequence IS256 has been successfully applied in a wide variety of gram-positive bacteria, such as Mycoplasma (8), Spiroplasma (15), Staphylococcus (24), and Streptococcus (19, 21) spp. IS256 has a small size (1.3 kb), a broad host range, and a simple structure that needs only the inverted repeats and transposase for transposition. A key feature of IS256 is its randomness of insertion in streptococcal chromosomes (21). However, introduction of IS256 from a suicide vector entails both transformation and transposition, which often are low-frequency events. This makes it extremely difficult to use for FW213, a poorly transformable strain (13).

In order to increase the efficiency of the transposition, we moved the IS256 into a conditional replicative plasmid. The temperature-sensitive plasmid pTV1-OK is particularly efficient for transformation-independent mutagenesis in several poorly transformable streptococcal species (16, 17). Unfortunately, hot spots exist in Tn917 transposition in some species (18, 36), including S. parasanguis FW213 (unpublished data).

In this study, we constructed the novel transposon plasmid pVT1528 on the backbone of pTV1-OK. Advantages of mutagenesis with pVT1528 in our strain are as follows: (i) the small size of IS256.erm compared with those of Tn4001 (4.5 kb) and Tn917 (5.2 kb); (ii) the relative ease of mutant generation, as only one transformant (VT1529) is needed to produce thousands of transposon mutants after a temperature shift; (iii) no hot spots for insertion; (iv) a high frequency of single insertion, which is not the case with other Tn4001 or IS256 derivatives (19, 21); (v) the ability of repA-ts, originally from pWVO1, to work in many species of gram-positive and gram-negative bacteria (23).

We demonstrated the usefulness of our transposon mutagenesis system in S. parasanguis by isolating nonfimbriated mutants with diverse characteristics. Southern analyses of the six transposon mutants suggested that the insertions were randomly distributed in the genome. Southern and Western blot analyses, as well as BactELISA, suggested that VT1531 and VT1533 have identical phenotypes and that VT1534 and VT1537 are identical. The transposon IS256.erm in VT1531, VT1533, and VT1532 was inserted into at least two different non-fap1 sites, whereas the transposon in VT1534, VT1537, and VT1535 was in at least two different sites within the coding region of fap1.

These distinct locations of transposition generate distinct phenotypes among the six mutants. Although the genotypes of VT1531 and VT1533 have not been identified yet, this pair of mutants has phenotypic features in common with VT508, a spontaneous mutant studied previously (29). These mutants (i) are deficient in formation of mature Fap1 and long fimbriae, (ii) express a MAb E42-specific 360-kDa Fap1 polypeptide which is depicted as a fap1 gene product prior to glycosylation, (iii) have an unglycosylated form of Fap1 associated with the cell surface, and (iv) do not have a mutation in the fap1 locus. This is consistent with previous findings which led to the hypothesis that some nonfimbriated mutants, represented by VT508, lack glycosylation capability, even though the identity of the responsible genes is unknown. Given the convenience of defining the mutation in the tractable transposon mutants VT1531 and VT1533, we are now able to shed more light on the Fap1 glycosylation pathway.

Interestingly, VT1532, a mutant in another region of the genome, behaved like some other nonfimbriated mutants. These mutants, represented by VT324, which were generated by chemical mutagenesis, express a 470-kDa Fap1 polypeptide that is reactive with peptide-specific antibodies and one glycan-specific antibody, MAb D10. They are proposed to be the mutants that can undergo some degree of glycosylation, so that MAb D10-recognized glycans are added while MAb F51-recognized glycans are not (29). Like VT324, VT1532 expressed a 470-kDa Fap1 polypeptide, which did not react with MAb F51. Surface expression of Fap1 and biogenesis of fimbriae were both abolished. The amenability of the transposon mutant will enable us to recover the affected gene in VT1532 which is suspected to be indispensable for full glycosylation.

The Fap1 expression patterns varied among four of the fap1 insertion mutants, VT1393, VT1535, VT1534, and VT1537. VT1393 is an allelic replacement mutant with an aphA-3 gene introduced into the nonrepetitive region close to the 5′ end of fap1 (35), which terminates Fap1 expression. This explains why we have not detected any antibody reactivity by immunological analyses either in this or in previous studies (30, 35). Transposition sites of VT1535, VT1534, and VT1537 were in the extensive repetitive region proximal to the 3′ end of Fap1 based on our Southern blot analyses. The 180-kDa Fap1 polypeptide detected by both peptide- and glycan-specific MAbs in VT1534 and VT1537 suggested that truncated Fap1 was expressed at a reduced level and was glycosylated in these mutants. Interestingly, VT1535 produced a small amount of the 470-kDa Fap1 polypeptide reacting with peptide-specific MAb E42, but not with MAb F51, which is specific for the mature Fap1 only. The fact that this polypeptide reacted with another glycan-specific antibody, MAb D10 (data not shown), suggested that Fap1 was partially glycosylated. Why VT1535 did not achieve full glycosylation of Fap1 needs further investigation.

Collectively, we have gained four types of transposition mutants with various regions of the genomes mutagenized. The mutations all influence the glycosylation of Fap1 and/or formation of fimbriae. In addition to Fap1, prokaryotic glycoproteins have been described recently for both Archaea and Bacteria. It is now well established that prokaryotic glycosylation affects protection from proteolysis, conformational stability, immune evasion, cell shape maintenance, adhesion, and recognition (25, 27). Fap1 is not the only surface-associated prokaryotic glycoprotein that has multiple variants derived from a single gene product through posttranslational modifications, including glycosylation. Some other examples are AgC in Streptococcus salivarius strain K+ (33), SLP in Clostridium difficile (5), and AIDA in E. coli strain 2787 (2). The scenario most similar to Fap1 is that of three proteases encoded by one rgpA gene in Porphyrmonas gingivalis W50. One enzyme, HRgpA, has essentially little to no carbohydrate. The other two, RgpA and mt-RgpA, are glycosylated forms with different amounts of glycan addition. The half-life of HRgpA is much shorter than those of RgpA and mt-RgpA (7). Little information has been published on the genes relevant to prokaryotic glycosylation pathways, except for a few gram-negative bacteria (1, 2, 31).

In conclusion, through the use of a novel transposon mutagenesis system, we isolated four types of transposon mutants, differing from each other in their insertion sites and phenotypes. Fap1 glycosylation was suspected to be blocked at certain steps in VT1531, VT1533, and VT1532, despite the fact that there were some glycan epitopes on the Fap1 proteins of VT1531 and VT1533. VT1534, VT1537, and VT1535 might express various lengths of truncated Fap1 proteins that lack the C-terminal cell wall sorting signal. Experiments to identify genes involved in Fap1 glycosylation by using the transposon mutants are currently under way. The information will give us a better understanding of Fap1 glycosylation, fimbrial biogenesis, and bacterial adhesion.

Acknowledgments

We thank Diane Hutchins Meyer for helpful comments and review of the manuscript.

This work was supported by Public Health Service grant R37-DE11000 from the National Institutes of Health.

Notes

Editor: V. J. DiRita

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