Figure 1: B. subtilis strain 3610 lyses due to oxygen depletion.
(A) B. subtilis cultures lyse when not shaking. The biofilm-forming wild-type strain 3610 (WT) was grown in a test tube and then incubated at room temperature without shaking for 10 h. Phase-contrast images were acquired at 0 and 10 h. Scale bar: 5 μm; arrowhead points to cell debris.
(B) Oxygen is depleted during exponential growth and remains low throughout subsequent oxygen depletion while cultures are sealed. Cells were cultured with oxygen-sensitive nanoparticles (STAR Methods). OD600 (top) and the relative oxygen levels (bottom, oxygen level at t0growth set to 1) of the cultures were measured. Lines represent the mean and shading represents one standard deviation (SD), n=3.
(C) LytC is necessary for lysis. Left: Despite differences in OD, the ΔlytC 3610 mutant has similar viability to wild type (p=0.07 at 24 h and 0.67 at 48 h, Student’s t-test). Cultures were grown to OD600~1 and then oxygen was depleted at t=0 h and OD600 was monitored. Lines represent the mean and shading represents 1 SD, n=3. Right: ΔlytC cells have phase gray “ghosts” at 24 h post-oxygen depletion. Scale bar: 5 μm; arrowhead points to phase-gray, lysed cell.
(D) Culture lysis is strongly correlated with initial cell density when initial OD600>0.45. Representative lysis curves of 6 cultures that varied in initial OD600 (see Figure S1 for independent replicates). Inset: maximum lysis rate (absolute value of the minimum slope of ln(OD600)) vs. initial OD600. A linear regression analysis was performed on data with initial OD600>0.45.