DOI: 10.5061/dryad.bnzs7h4qz
title: Drug susceptibility of <em>Burkholderia pseudomallei</em> multidrug transporters assessed by an agar-based assay

description: The Gram-negative pathogen Burkholderia pseudomallei possesses multiple
 resistance-nodulation-division superfamily transporters that contribute to
 multidrug resistance, including BpeB and BpeF. Structural studies of BpeB
 and BpeF have identified a hydrophilic patch in their substrate-binding
 pocket. To investigate the relationship between this hydrophilic patch and
 substrate specificity, mutant analyses were performed using an Escherichia
 coli recombinant expression system. Drug susceptibility tests of BpeB and
 BpeF mutants showed up to a 64-fold increase in susceptibility compared
 with the wild type. Growth curve analyses revealed that BpeB mutants
 exhibited increased resistance to aminoglycosides, which are not
 transported by the wild type. These findings suggest that the hydrophilic
 patches in the substrate-binding pockets of BpeB and BpeF are involved in
 the substrate specificity.
description: # Drug susceptibility of *Burkholderia pseudomallei* multidrug
 transporters assessed by an agar-based assay Dataset DOI:
 [10.5061/dryad.bnzs7h4qz](https://doi.org/10.5061/dryad.bnzs7h4qz) ##
 Dataset overview This dataset contains photographic images of agar plates
 obtained from drug susceptibility assays using *Escherichia coli* strains
 heterologously expressing multidrug transporters derived from
 *Burkholderia pseudomallei*. The images document bacterial growth in the
 presence of various drugs and compounds and were used to evaluate the
 contribution of the expressed transporters to drug susceptibility
 phenotypes. #### Experimental context Drug susceptibility agar plate
 assays were performed to assess the effects of *Burkholderia*-derived
 multidrug transporters on bacterial drug susceptibility. Recombinant *E.
 coli* strains expressing individual transporters were plated on agar media
 containing different drugs or compounds, and bacterial growth was
 documented by photography. These assays were performed using both
 wild-type transporters and multiple mutant variants to assess their
 effects on substrate specificity. The dataset consists of image files
 documenting bacterial growth across drug concentration series. In
 addition, a table summarizing the minimum inhibitory concentration (MIC)
 values determined from these assays is provided as a separate file
 (MIC.xlsx). #### Description of Data Files #### File: AmrB.zip
 **Description:** Image files from the drug susceptibility agar plate
 assays for AmrB #### File: BpeB.zip **Description:** Image files from the
 drug susceptibility agar plate assays for BpeB #### File: BpeF.zip
 **Description:** Image files from the drug susceptibility agar plate
 assays for BpeF #### File: MIC.xlsx **Description:** Table summarizing the
 MIC values determined from the agar plate assays #### Dataset organization
 The dataset is organized hierarchically by protein and drug. The top-level
 folders correspond to three multidrug transporters, AmrB, BpeB, and BpeF.
 Within each protein folder, subfolders are organized by drug. A total of
 14 drugs and compounds were evaluated, and each drug-specific subfolder
 contains photographs of agar plates obtained from assays performed with
 that drug. #### File description Each image file is a photograph of an
 agar plate showing bacterial growth in the presence of a specific drug or
 compound. Within each drug-specific subfolder, the images correspond to
 agar plates containing different concentrations of the indicated drug.
 Drug susceptibility assays were performed using a two-fold serial dilution
 series. The numbers following the drug abbreviations written on the plates
 indicate the drug concentration. A value of “1” corresponds to a
 concentration of 100 µg/mL, and each subsequent increase in number
 represents a two-fold decrease in concentration (e.g., “2” indicates 50
 µg/mL, and “3” indicates 25 µg/mL). For DDM and UDM, the concentrations
 are ten-fold higher than those of other drugs; in these cases, “1”
 corresponds to 1000 µg/mL. No image processing was applied to the files.
 #### Plate layout In each agar plate image, the top two rows contain
 strains expressing the following transporters, from left to right: 1.
 pBAD24 (empty vector) 2. BpeB (wild type) 3. BpeB_D407N+D408N
 (transport-inactive mutant, negative control) 4. BpeB_S132A 5.
 BpeB_S132A+S133A 6. BpeB_S132A+S133A+S134D 7. BpeB_T176V The middle two
 rows contain strains expressing the following transporters, from left to
 right: 1. pBAD24 (empty vector) 2. BpeF (wild type) 3. BpeF_D410N+D411N
 (transport-inactive mutant, negative control) 4. BpeF_S134A 5.
 BpeF_S134A+S135A 6. BpeF_S134A+S135A+P136D 7. BpeF_Q178V 8.
 BpeF_S134A+S135A+Q178V The bottom two rows contain strains expressing the
 following transporters, from left to right: 1.
  BpeB_S132A+S133A+S134D+T176E 2.  BpeB_S132A+S133A+S134D+S135N+F136A+L137Q
 3.  BpeB_S167R 4.  pBAD24 (empty vector) 5.  AmrB (wild type) 6.
  AmrB_D406N+D407N (transport-inactive mutant, negative control) #### MIC
 data The file MIC.xlsx contains a table summarizing the MIC values
 determined from the agar plate assays. MIC values were determined based on
 the presence or absence of colony growth observed in the corresponding
 agar plate images. MIC values reported as “&gt; 4000” indicate that no
 growth inhibition was observed at the highest concentration evaluated.
 #### Drug abbreviations The abbreviations used for drugs and compounds in
 folder names are as follows: *  ACR, acriflavine * CHL, chloramphenicol *
 CLI, clindamycin * DDM, n-dodecyl-β-D-maltoside * EB, ethidium bromide *
 ERY, erythromycin * GEN, gentamicin * KAN, kanamycin * LVX, levofloxacin *
 NOR, norfloxacin * TET, tetracycline * TMP, trimethoprim * TOB, tobramycin
 * UDM, n-undecyl-β-D-maltoside #### Related publication This dataset
 supports the findings reported in the following publication: **Title:**
 Substrate specificity of Burkholderia pseudomallei multidrug transporters
 is influenced by the hydrophilic patch in the substrate-binding pocket
 **Journal:** FEBS Letters (2025)  **DOI:**
 [https://doi.org/10.1002/1873-3468.70248](https://doi.org/10.1002/1873-3468.70248) The MIC values provided in MIC.xlsx correspond to those reported in the related publication. A summary of the strains used and the basic experimental procedures is provided above. For additional methodological details including strain construction, expression conditions, and full assay protocols, please refer to the related publication. #### Contact information For questions regarding this dataset, please contact: **Corresponding author:** Ui Okada **Affiliation:** Institute of Science Tokyo **E-mail:** [uiokada@life.isct.ac.jp](mailto:uiokada@life.isct.ac.jp)