FIG. 4.

Effect of phage holin activity on S. pneumoniae cell membrane permeabilization. Cultures of SVMC28, SVMC28 Δsvl ΔlytA, and SVMC28 Δsvl were treated with MitC and tested for membrane permeabilization at various times by flow cytometry analysis using a mixture of Syto 9 and PI staining (Live/Dead BacLight bacterial viability kit; Invitrogen, Carlsbad, CA). Experimentally defined gates R2 and R3 were used to differentiate between damaged and undamaged cell populations and were designed over gate R1, which included the total stained population (Fig. 3). The left column shows a shift in the Syto 9/PI staining pattern through time after phage induction of strain SVMC28 Δsvl ΔlytA, which lacks both the phage endolysin and the S. pneumoniae autolysin, LytA. In the presence of lytic enzymes (middle and right columns), a different scenario was observed, with only a few damaged cells detected. The data are from a representative experiment of a minimum of three independent experiments.