The Sorting Mechanism of the Surface Proteins in Streptococcus gordoniiV288

Objectives: The srtAgene product, sortase, is required for efficient cell wall anchoring of the surface proteins of Gram-positive bacteria. We sought to test the hypothesis that disruption of the srtAgene changes the array of surface proteins of Streptococcus gordoniiV288, decreasing in vitro biofilm formation ability, and adhesion to saliva-coated hydroxylapatite. Methods: A srtAmutant was constructed by insertion duplication using the pCR2.1:B210 plasmid (Siga Research Laboratories, Oregon). Three different protein fractions were isolated from both S. gordoniiV288 and the srtAmutant: cell wall, cytoplasmic, and released proteins in the bacterial growth media. Proteins expressed in these fractions were compared on SDS-PAGE gels. Results: Results show unambiguous differences between strains V288 and the srtAmutant in the protein patterns of the supernatant phases, slight differences in the cell wall proteins, and no differences in the cytoplasmic fractions. In vitro assay of the biofilm-forming ability of the mutant bacteria cultured on plastic showed a 21% reduction compared with the wild-type. The adhesion ability of the mutant was also reduced when compared to the wild-type. Conclusion: These results suggest that the srtAmutation manifests in altered cell surface anchoring of proteins, modifying adhesion and in vitro biofilm formation. Supported by NIH Grant R01 DE08590.