Objectives: To determine the role of a putative glycosyltransferase GalT1 in Fap1 glycosylation and biofilm formation.
Methods: The galT1 mutant was constructed using allelic replacement mutagenesis strategy. Production of Fap1 was examined by Western blotting analysis using Fap1-specific antibodies. A recombinant fap1 construct with genes coding for glycosyltransferases Gtf1-2-3 was co-expressed with a gene coding for GalT1 in a glycosylation system in E. coli to determine Fap1 glycosylation in vivo. A recombinant Fap1 modified by Gtf1-2-3 was used as a Fap1 substrate in an in vitro glycosylation assay with active sugar donor 3H UPD-Glc. Bacteria grown on abiotic surface was assessed for biofilm formation. GST pull-down assays were employed to determine the GalT2 self-interaction.
Results: In vitro glycosylation assays showed that GalT1 catalyzed the direct transfer of glucosyl-residues to the Glc-GlcNAc-modified Fap1, suggesting that GalT1 is a glucosyltransferase. The N-terminal domain of unknown function is critical for the glycosyltransferase activity, while the C-terminal domain is responsible for self-association of GalT1 as determined by GST pull-down assays. In vivo studies demonstrated GalT1 is responsible for the third step of Fap1 glycosylation. The galT1 mutant had a defect in biofilm formation. Conclusions: Our results suggested GalT1 is a glucosyltransferase that catalyzes the subsequent glycosylation step of Fap1 modified by Gtf1-2-3, and is required for the biofilm formation of S. parasanguinis. Supported by NIH/NIDCR DE01795.