Figure 3.

QnrB increases DNA replication stress. A. CIP induces replication stress (oriC/ter ratio; left panel) and qnrB mRNA expression (right panel) in a concentration‐dependent manner. The K. pneumoniae KP48 strain harboring a plasmid expressing QnrB was treated with or without CIP (0, 32, 128 or 192 mg/l) for 90 min. The oriC/ter ratio and qnrB mRNA level were measured using real‐time quantitative PCR. Data are shown as the mean ± SD of three replicates. A significant difference in the oriC/ter ratio was observed between the strain with CIP and the strain without CIP (two‐sided Student’s t‐test, ^** p < 0.01, ^*** p < 0.001). The data shown are representative of three independent experiments. B. TRIM induces replication stress. The K. pneumoniae KP48 and KP49strains were treated with or without CIP (128 mg/l), TRIM (0.7 mg/l) or tetracycline (Tet; 1 mg/l) for approximately 90 min at 37°C. The oriC/ter ratio was determined by real‐time quantitative PCR. Data are shown as the mean ± SD of three replicates. A significant difference in mRNA was observed between mRNA of the strain expressing QnrB and the strain lacking QnrB (two‐sided Student’s t‐test. ^*** p < 0.001). The data shown are representative of three independent experiments. TRIM, trimethoprim; Tet, tetracycline. C. Overexpression of QnrB induces DNA replication stress in E. coli. Induction of E. coli BW25113 cells harboring pBAD‐qnrB or pBAD was performed at an OD₆₀₀ of 0.3 by adding arabinose (0.5% w/v; red column). An untreated culture served as the negative control (blue column). The cells were harvested 40 min after treatment. Replication stress (oriC/ter ratio) was measured by real‐time quantitative PCR. Data are shown as the mean ± SD of three replicates. A significant difference in the oriC/ter ratio was observed between the strain expressing QnrB and the strain lacking QnrB (two‐sided Student’s t‐test. ^**** p < 0.0001). The data shown are representative of five independent experiments. [Colour figure can be viewed at wileyonlinelibrary.com]