Supporting Information

Supporting Information

TABLE S1. MIC (*) value of D39 WT, D39DclpL, and D39ClpL overexpressing strain (RT172) on THY blood agar plate (A) and THY broth (B).
D39 WT / D39DclpL / RT172
A / B / A / B / A / B
0.040 / 0.035 / 0.010 / 0.014 / 0.160 / 0.200
·  (*) Penicillin concentration was calculated as µg/ml.
·  The table shows the average data from three independent experiments. The MIC value obtained was exactly same in all 3 repeated times. Therefore, the standard deviation was essentially 0.

Figure S1

Figure S1. Optical densities of DOC, penicillin, and Triton X-100- treated cultures. A, B, and C. D39ClpL overexpressing strain was resistant to lysis triggered by DOC (A), Triton X-100 (B), and penicillin (C). D39 WT, D39DclpL, and D39ClpL overexpressing strain were cultured in THY broth followed by addition of DOC (A), Triton X-100 (B), or penicillin (C) to the culture at final concentrations of 0.1%, 0.05%, or 0.1 μg/ml, respectively. The optical density of the culture at 550 nm was determined every 20 sec (A) or 1 h (B, C) after treatment. D. D39DclpL and D39DclpL containing the empty pMV158 vector were incubated in THY broth until mid-log phase (OD 550 = 0.3). Penicillin was added into the culture at a final concentration of 0.1 µg/ml. The number of viable bacteria was counted at 1.5-h intervals for 6 h. E. Heat shock-induced ClpL induction increased resistance of D39 WT to penicillin-triggered lysis. D39 WT and D39DclpL were cultured in THY broth at 30°C until OD 550 nm was 0.2, then heat shocked at 42°C for 30 min. After heat shock, penicillin was added to the culture at a final concentration of 0.2 μg/ml. The optical density at 550 nm was measured every 1 h after addition of penicillin. Pen: Penicillin.

Figure S2

Figure S2. Heat shock does not change generation time of both D39WT and D39DclpL. D39 WT and its isogenic DclpL were inoculated in THY broth at the same OD 550 nm = 0.05, cultured at 30°C until mid-exponential phase (OD 550 = 0.3), and then heat shocked at 42°C (black arrows) for 30 min. The culture temperature was then shifted to 37°C (gray arrows). The optical density at 550 nm was determined every one hour.

Figure S3

Figure S3. Capsule production of D39 WT, D39DclpL, and D39ClpL overexpressing strain. Bacteria were cultured in THY broth until mid-log phase (OD 550 = 0.3). Bacterial pellet was washed twice in sterile PBS, and fixed in 5% of formalin for 15 min followed by washing twice with PBS again. Bacterial capsule was probed with anti-capsular type 2 antibody followed by FITC-labelled anti-rabbit IgG. The pictures were taken by phase contrast (left panel) and fluorescent (right panel) microscope (Olympus BX51TRF Fluorescence Research Microscope).

Figure S4

Figure S4. Effect of penicillin on gene expressions. D39 WT was cultured in THY broth until mid-log phase (OD550 = 0.3). Penicillin was added into the culture at the final concentration of 0 – 1 μg/ml, and incubated at 37°C for 1 h. Bacterial RNA was isolated by the hot phenol method from cultures grown with and without added penicillin. The mRNA level was analyzed by RT-PCR (A). Band density was analyzed by Photoshop (B, C). The figure shows standard deviation from three independent experiments. Statistical differences were analyzed by ANOVA. * P=0.04 compared to non-treated group; ** P=0.0025 compared to non-treated group.

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