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Chen, Qiang

Microbiology and Molecular Genetics

2004

Ph. D.

Streptococcus parasanguis FW213, a primary colonizer of the tooth surface, has long peritrichous fimbriae. A fimbriae-associated protein, Fap1, is identified as the fimbria structural unit. Fapl is indispensable for fimbriae biogenesis, adhesion to an in vitro tooth model and biofilm formation. The mature Fapl protein is glycosylated with an apparent molecular mass of 200 kDa. Presence of glycosylated Fapl on the bacterial surface is required for adhesion. Little is known about the mechanism of Fapl glycosylation due to the lack of genetically 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 Fapl yielded six mutants with decreased expression levels of a surface-associated glycosylated Fapl protein. The insertion sites in the six transposon mutants were localized. Three of them had the transposon inserted in the fapl locus. In one mutant, transposition had occurred in an intergenic region between a cadD and a copB locus. The last two mutants had transposition occurring in a secA2 locus. Glycosylated Fap 1 was not detected either on the surface or in the whole cell extract of the two secA2 transposon mutants, suggesting secA2 is implicated in Fap1 glycosylation and/or secretion, if secA2 was the only gene affected in these two mutants.
A non-polar mutant of secA2 was generated using a novel in-frame deletion system. The relationship of Fapl glycosylation and secretion was investigated in the secA2 mutant and the wild type. In the secA2 mutant, Fapl was not secreted. Glycosylated Fap1 was present in the membrane and cytoplasm of the mutant, although in greatly reduced amounts. In FW213, glycosylated Fap1 was present in all the subcellular fractions including the cytoplasm. These data suggested that Fapl glycosylation occurs prior to its secretion. Fapl secretion and abundance were restored when the secA2 mutant was complemented by a plasmid-borne secA2. The secretion defect of the secA2 mutation appears limited to a small group of proteins such as Fapl and FimA. These data suggested that Fapl secretion rather than glycosylation was the major effect of the deletion of secA2; however, this deletion also had an impact on Fapl abundance. In order to define the region of secA2 required for secretion, two more secA2 mutants with different regions deleted were generated and tested for their ability to secrete Fap1. One mutant was completely unable to secrete Fap1 while the other maintained the ability to secrete Fap1 in a decreased amount. These data suggest that the region containing nucleotides 2032-2337 deleted in the latter mutant, is dispensable for Fap1 secretion.