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. 2009 Jul 6;191(17):5428–5440. doi: 10.1128/JB.00477-09

FIG. 2.

FIG. 2.

Participation of bacterial and phage lysins in phage release. (A) Phage release patterns. The culture media of R36AP, R36AP Δsvl, R36AP ΔlytA, and R36AP ΔlytA Δsvl treated with MitC were filtered (0.45 μm) at 20-min intervals after the start of lysis in the R36AP strain, and the supernatants were used directly in phage plaque assays of the indicator strain R36A. The time after MitC addition is indicated. The results are representative of three independent experiments. (B) Phage plaque assay using indicator strains differing only in the presence or absence of LytA. The supernatant of the culture medium of R36AP was collected 180 min after MitC treatment and used on indicator strains R36A and R36A ΔlytA. The results are representative of three independent experiments. (C) Indicator lawn lysis is due to phage induction. As a control, R36A indicator lawns were exposed to C+Y medium with MitC at the same concentration used for phage induction. For R36AP ΔlytA Δsvl, the supernatant collected 180 min post-MitC addition produced no phage plaques. The supernatant of an R36AP culture treated for 180 min with MitC was filtered through a 100-kDa-cutoff membrane to retain phages while eliminating small proteins. The <100-kDa filtrate showed no phage plaques, unlike the retained fraction (>100-kDa filtrate; 10−1 dilution), demonstrating that the plaques were due to phage and were not due to the action of bacterial and phage lysins, holins, or bacteriocins (mass < 100 kDa), which can cause cell lysis. (D) Comparison of the numbers of PFU per ml detected upon phage induction. The numbers of PFU per ml were determined for strains R36AP, R36AP Δsvl, and R36AP ΔlytA after 180 min of MitC treatment on indicator lawns of strains R36A and R36A ΔlytA, as indicated. Averages and 95% confidence intervals are indicated. For R36A in the indicator lawn, the comparisons were between supernatants obtained from R36APΔ lytA and R36AP (P < 10−4), R36AP Δsvl and R36AP ΔlytA (P < 10−3), and R36AP Δsvl and R36AP (P < 10−5). When the R36AP supernatant was plated on indicator lawns of strains R36A and R36A ΔlytA, P was <10−9. Magnification (A to C), ×6.3 or ×8.0.