FIG 3.

PA reassembly after HP exposure is an energy-dependent process not dependent on the synthesis of novel proteins. (A) Treatment of an LMM1010 ibpA-yfp population with indicated concentrations of CCCP after HP exposure (300 MPa, 20°C, 15 min) decreases the fraction of cells (n ≥ 115 per independent experiment) that are able to assemble their PAs. Asterisks indicate statistically significant differences from the control (i.e., no addition of CCCP) (Student's t test, α = 0.01, P = 0.003 for 20 μM and 5 μM CCCP, and P = 0.005 for 1 μM CCCP). Addition of chloramphenicol (30 μg/ml) to the growth medium of an HP-exposed (300 MPa, 20°C, 15 min) LMM1010 ibpA-yfp population does not influence the ability of cells (n ≥ 120 per independent experiment) to reassemble their PAs (Student's t test, P = 0.28). Reassembly was considered completed when a cell was able to reassemble its PAs into one or two larger PAs (located in the cell poles) within 3 h after HP exposure. The means of three independent experiments are shown, with error bars representing the standard deviation. (B and C) Representative images of two time-lapse fluorescence image sequences indicating the reversibility of CCCP-induced energy depletion. (B) PA assembly does not occur after HP exposure (300 MPa, 20°C, 15 min) in the presence of CCCP (20 μM, for 180 min). (C) PA assembly resumes normally after CCCP is washed away (after 180 min) and cells are supplied with fresh nutrients. YFP epifluorescence images (reporting PAs) in combination with cell outlines are shown at the indicated times after HP exposure. The scale bar corresponds to 1 μm.