LAUSR

The bacterial exopolysaccharide poly-β-1,6-N-acetylglucosamine is a major extracellular matrix component in biofilms of both Gram-positive and Gram-negative organisms. We have leveraged the specificity of the biofilm-dispersing glycoside hydrolase Dispersin B (DspB) to generate a probe (Dispersin B PNAG probe, DiPP) for monitoring PNAG production and localization during biofilm formation. Mutation of the active site of Dispersin B gave DiPP which was an effective probe despite its low affinity for PNAG oligosaccharides, observed in fluorescence quenching and mass spectrometry binding experiments. Imaging of PNAG-dependent and -independent biofilms stained with a fluorescent-protein fusion of DiPP (GFP-DiPP) demonstrated the specificity of the probe for the structure of PNAG on both single-cell and biofilm levels. Through quantitative bacterial cell binding assays and confocal microscopy analysis using GFP-DiPP, discrete areas of local high concentrations of PNAG were detected on the surface of early log phase cells. These distinct areas were seen to grow, slough from cells and accumulate in interbacterial regions over the course of several cell divisions, showing the development of a PNAG-dependent biofilm. A potential helical distribution of staining was also noted, suggesting some degree of organization of PNAG production at the cell surface prior to cell aggregation. Together these experiments shed light on the early stages of PNAG dependent biofilm formation and demonstrate the value of a low-affinity-high-specificity probe for monitoring the production of bacterial exopolysaccharides.