OBJECTIVE: Oral streptococci are primary colonisers of the tooth surface and are abundant in dental plaque biofilms. Bacteria growing in these relatively dense, surface-associated communities are phenotypically quite distinct from their planktonic counterparts. The purpose of the present study was to develop a method to investigate biofilm-specific surface protein expression by Streptococcus sanguis to help provide a better understanding of the critical events in plaque development. DESIGN: Biofilm cells were grown on the surface of glass beads in a biofilm device fed with mucin-containing artificial saliva. Planktonic cells were grown in continuous culture at approximately the same growth rate. Surface hydrophobicity of biofilm and planktonic cells was determined by hexadecane partitioning, and expression of streptococcal fibronectin adhesin CshA was determined in ELISA using specific antiserum. Antisera raised to glutaraldehyde-fixed whole biofilm or planktonic grown cells were used to screen an expression library of S. sanguis genomic DNA, and isolated clones were sequenced. RESULTS: Phenotypic analysis of biofilm and planktonic cells confirmed that mode of growth affected surface properties of S. sanguis. Thus, hydrophobicity and CshA expression was significantly elevated in biofilm cells. Library screening with biofilm antiserum yielded 32 recombinant clones representing 21 different S. sanguis proteins involved in adhesion and colonisation, carbohydrate utilisation or bacterial metabolism. In differential analysis of four selected Escherichia coli clones, biofilm antiserum reacted five times stronger than planktonic antiserum with cell-free extracts of clones encoding homologues of CshA and Cna collagen adhesin of Staphylococcus aureus, suggesting that these surface proteins are up-regulated in biofilm cells. In contrast, both antisera reacted equally strongly with cell-free extracts of the remaining two clones (encoding dihydrofolate synthase and an unknown protein). CONCLUSIONS: The method described represents a useful means for determining bacterial protein expression in biofilms based on a combination of molecular and immunological techniques. Surface expression of putative fibronectin and collagen adhesins was up-regulated in biofilm cells.
OBJECTIVE: Oral streptococci are primary colonisers of the tooth surface and are abundant in dental plaque biofilms. Bacteria growing in these relatively dense, surface-associated communities are phenotypically quite distinct from their planktonic counterparts. The purpose of the present study was to develop a method to investigate biofilm-specific surface protein expression by Streptococcus sanguis to help provide a better understanding of the critical events in plaque development. DESIGN: Biofilm cells were grown on the surface of glass beads in a biofilm device fed with mucin-containing artificial saliva. Planktonic cells were grown in continuous culture at approximately the same growth rate. Surface hydrophobicity of biofilm and planktonic cells was determined by hexadecane partitioning, and expression of streptococcal fibronectin adhesin CshA was determined in ELISA using specific antiserum. Antisera raised to glutaraldehyde-fixed whole biofilm or planktonic grown cells were used to screen an expression library of S. sanguis genomic DNA, and isolated clones were sequenced. RESULTS: Phenotypic analysis of biofilm and planktonic cells confirmed that mode of growth affected surface properties of S. sanguis. Thus, hydrophobicity and CshA expression was significantly elevated in biofilm cells. Library screening with biofilm antiserum yielded 32 recombinant clones representing 21 different S. sanguis proteins involved in adhesion and colonisation, carbohydrate utilisation or bacterial metabolism. In differential analysis of four selected Escherichia coli clones, biofilm antiserum reacted five times stronger than planktonic antiserum with cell-free extracts of clones encoding homologues of CshA and Cna collagen adhesin of Staphylococcus aureus, suggesting that these surface proteins are up-regulated in biofilm cells. In contrast, both antisera reacted equally strongly with cell-free extracts of the remaining two clones (encoding dihydrofolate synthase and an unknown protein). CONCLUSIONS: The method described represents a useful means for determining bacterial protein expression in biofilms based on a combination of molecular and immunological techniques. Surface expression of putative fibronectin and collagen adhesins was up-regulated in biofilm cells.