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.

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 primers EIR and PIR. The amplicon was ligated with an EcoRI-PstI digest of pTV1-OK consisting of repA-ts and kan to generate pVT1528. (B) Detailed diagram of IS256.erm on pVT1528. The transposon encodes a transposase gene (tnp) and an erythromycin resistance gene (erm). Inverted repeats (IR) are flanked by restriction sites introduced by PCR.

FIG. 2.

Southern blot analysis of S. parasanguis transposon mutants. Southern blot analysis was carried out with EcoRI chromosomal digests of 11 randomly chosen Erm^r Kan^s transposon mutants with IS256.erm as the probe. Molecular size markers are given on the right.

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 markers are given on the left.

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 detected as described in Materials and Methods. Molecular sizes are given on the left.

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, standard deviations.

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.