polyGlcNAc-containing exopolysaccharides are necessary for <i>E</i>. <i>faecalis</i> semisolid media penetration.

(A and B) Immunofluorescence analysis of cells stained with either Texas red-conjugated WGA or mAb F598, which specifically binds to β-1,6-linked GlcNAc polysaccharides. To visualize antibody binding to GlcNAc-containing polymers, cells were reacted with a secondary anti-human IgG conjugated to Alexa Fluor-488 (green fluorescence). DAPI was used to stain bacterial DNA (blue fluorescence). (A) Staphylococcus aureus MN8 (positive control) and E. faecalis MMH594 non-penetrating cells from MOLP-grown colonies were treated with or without 300 μg/mL Dispersin B (DspB), an enzyme that degrades β-1,6 bonds. (B) Cells of E. faecalis WT and transposon mutants grown on MOLP for 6 days with (+) or without (-) 10 mM GlcNAc were tested for binding to mAb F598 as described above. (C) Colony hybridization blot (top panel) of 24 hours-grown-colonies of WT and glnA::TnM and rpiA::TnM alone or harboring the pAT28 vector containing their corresponding WT allele (p-glnA and p-rpiA). The relative intensity obtained upon incubation with mAb F598 was calculated for each colony using Image J (lower panel); (mean±SE; n = 8; ****PA and B): 20 μm and (C): 1000 μm.