Figure 4.
The Dependence between Persister Formation Frequency and TolC Expression Level
(A) TolC expression level in total cells of TC tag-TolC strain measured by Tetracysteine-based protein detection (FlAsH imaging, upper panel); TolC expression level in persister cells that survived antibiotic treatment (lower panel).
(B) Time-lapse microscopy showing that persister cells express a high level of TolC (from Movie S3). The first and fifth images are the merged bright field and fluorescent images (separate bright field and fluorescent images are shown in Figure S3D). The different media added during the experiment are indicated below (killing medium: 90% [v/v] M9 + 10% [v/v] LB + 150 μg/ml carbenicillin + 0.15% [w/v] methylcellulose; growth medium: 90% [v/v] M9 + 10% [v/v] LB + 5% [w/v] methylcellulose). The persister cell (arrow), shows a significantly higher expression of TolC, and regrows after removal of antibiotic (from Movie S3). (Scale bar, 3 μm; t = time in min).
(C) Experimental procedure for sorting cells by expression level of persistence related genes.
(D) Distribution of fluorescence intensity indicating TolC expression levels of 76RP strain. Stationary-phase cells were sorted into three groups: group A containing the total cells (100.0%), group B including the majority of total cells except for those with highest fluorescence intensity (97.8%), and group C containing a sub-population with highest fluorescence intensity (1.0%).
(E) Bar plot representing cell survival rate (percentage log scale) after 4 hr carbenicillin treatment of three groups sorted by flow cytometer, revealing that the survival rate significantly increases in group C. The bars indicate mean of at least three independent experiments; error bar indicates SD.