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Infection and Immunity, December 2002, p. 6534-6540, Vol. 70, No. 12
0019-9567/02/$04.00+0     DOI: 10.1128/IAI.70.12.6534-6540.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.

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

Qiang Chen,¹ Hui Wu,² and Paula M. Fives-Taylor^1*

Department of Microbiology and Molecular Genetics,¹ Department of Medicine, University of Vermont, Burlington, Vermont 05405²

Received 25 March 2002/ Returned for modification 29 June 2002/ Accepted 3 September 2002

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.

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* Corresponding author. Mailing address: Department of Microbiology and Molecular Genetics, College of Medicine and College of Agriculture and Life Sciences, 116 Stafford Hall, 95 Carrigan Dr., University of Vermont, Burlington, VT 05405. Phone: (802) 656-1121. Fax: (802) 656-8749. E-mail: pfivesta{at}zoo.uvm.edu.

Editor: V. J. DiRita

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Infection and Immunity, December 2002, p. 6534-6540, Vol. 70, No. 12
0019-9567/02/$04.00+0     DOI: 10.1128/IAI.70.12.6534-6540.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.

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