FIG 6.

lytF-expressing cells are abundant near the bottom of the biofilm even in a thin biofilm formed by ΔgtfB cells. (A) P_lytF reporter and ΔgtfB P_lytF reporter strains were grown in an aerobic atmosphere containing 5% CO₂ at 37°C in BHIs with sCSP for 6 h in a 6-well plate. Sonication was performed to suspend aggregated cells. lytF-expressing cells were quantified by flow cytometry. These data indicate the mean ± standard deviations of the results from three independent experiments. (B) Images showing side views of the biofilms for strains grown in an aerobic atmosphere containing 5% CO₂ at 37°C in BHIs with sCSP for 6 h in a glass-bottom dish. Nonadherent cells were removed by washing twice with PBS. The biofilm cells were stained with 5 μM SYTO 59 and observed with inverted CLSM. Z-stacks were acquired at 0.5 μm intervals. SYTO 59 (all cells) and mScarlet-I (lytF-expressing cells) are shown in green and magenta, respectively. The scale bars indicate 10 μm. Representative data from independent experiments are presented.