Fig. 5
From: Gram-negative bacteria resist antimicrobial agents by a DzrR-mediated envelope stress response
The regulatory linkage between DzrR and desABC in Dickeya, Ralstonia, and Burkholderia strains. A Genetic organization of dzrR and desABC in Dickeya, Ralstonia, and Burkholderia strains. The numbers indicate the sequence identity (similarity) percentage compared to dzrR and desABC, respectively. B The dzrR box was revealed by SeqLogo analysis. C EMSA of DzrR25416 binding to the promoter fragment of desAB homologs from B. cepacia ATCC 25416 (PdesB25416). Free probes, i.e., biotin-labeled DNA fragment of PdesB25416 (222 bp) and biotin-labeled control DNA (60 bp), and PdesB25416 probe with DzrR25416 (Bound probe) are indicated by arrows. D DNase I footprinting assay performed between DzrR25416 and PdesB25416. E Expression of RND-8H111 efflux operon in the presence of chlorpromazine or chlorhexidine. Cell cultures of H111 (pPRND-8-Gfp) and ∆dzrRH111(pPRND-8-Gfp) at an optical density at 600 nm (OD600) about 0.5 were treated with chlorpromazine or chlorhexidine, respectively, at 37 °C for 4 h. After incubation, the average fluorescence intensity of 50,000 bacterial cells was measured by a CytoFLEX flow cytometer (Beckman Coulter, Brea, CA, USA) for determining the relative fluorescence of bacterial strains under different treatments. The relative fluorescence was expressed as the fluorescence of bacterial cells treated with chlorpromazine or chlorhexidine normalized to the fluorescence of bacterial cells treated with the same amount of solvent (DMSO). Chlorpromazine at a final concentration of 10 μg/ml and chlorhexidine at a final concentration of 2 μg/ml were used in this assay. The relative fluorescence is presented as mean ± SE, n = 3. Statistical analysis was performed using a two-tailed unpaired Student’s t test versus the solvent control. **P < 0.01. F DzrRH111 mediated chlorhexidine tolerance in B. cenocepacia H111. Cell cultures at an OD600 of 1.0 (5 × 108 CFU/ml) were serially diluted in twofold dilutions. Two microliters of each dilution were spotted on LB agar plates supplemented with chlorhexidine (20 μg/ml) and incubated at 37 °C before photography. The image is a representative of three repeats