FIG. 2.

Determination of pyocin activity in biofilms of strain PAO1. ΔprtN, the prtN mutant. (A) Cell density of 2-day biofilms. White and shaded columns represent biofilms grown under aerobic and anaerobic conditions, respectively. (B) Results obtained using the pyocin overlay assay. Ten microliters of neat supernatant and 10-fold dilutions of supernatant were spotted onto TSA and covered with a soft agar overlay containing an indicator strain (DWW1). A, aerobic biofilm supernatant; An, anaerobic biofilm supernatant. Similar results were obtained when proteinase K (100 μg/ml), which is active against S-type pyocins (6), was incorporated into the overlay agar. Although this observation does not eliminate the possibility that S-type pyocins are active against strain DWW1, it is clear from this result that bactericidal activity can be attributed to R- or F-type pyocins. Results obtained using the planktonic growth inhibition assay, showing the effect of biofilm supernatant (aerobic [C] or anaerobic [D]) on growth of indicator strain DWW1. Filled diamonds, control (no added supernatant); open squares, 10-μl PAO1 biofilm supernatant added; open triangle, 10-μl ΔprtN mutant biofilm supernatant added.