Article Figures & Data
Figures
- FIG 1
Fitness of transposon insertional mutants in the operon mexAB-oprM (Psyr_4007-9) both in a WT background and in cells in which mexAB-oprM has been disrupted. “KB” and “DMSO” represent the media and media plus solvent control, respectively. Fitness was calculated as log2 change in relative insertion strain barcode abundance for a given gene.
- FIG 2
Fitness of transposon insertional mutants in the operon mexEF-oprN (Psyr_2967-9) both in a WT background and in cells in which mexAB-oprM has been disrupted. “KB” and “DMSO” represent the media and media plus solvent control, respectively. Fitness was calculated as log2 change in relative insertion strain barcode abundance for a given gene.
- FIG 3
Fitness of transposon insertional mutants in the operon muxABC-opmB (Psyr_2482-5) both in a WT background and in cells in which mexAB-oprM has been disrupted. “KB” and “DMSO” represent the media and media plus solvent control, respectively. Fitness was calculated as log2 change in relative insertion strain barcode abundance for a given gene.
- FIG 4
Fitness of transposon insertional mutants in the operon Psyr_0344-6 both in a WT background and in cells in which mexAB-oprM has been disrupted. “KB” and “DMSO” represent the media and media plus solvent control, respectively. Fitness was calculated as log2 change in relative insertion strain barcode abundance for a given gene.
- FIG 5
Fitness of transposon insertional mutants in the major facilitator superfamily transporter gene Psyr_0228 both in a WT background and in cells in which mexAB-oprM has been disrupted. “KB” and “DMSO” represent the media and media plus solvent control, respectively. Fitness was calculated as the log2 change in relative insertion strain barcode abundance for a given gene.
- FIG 6
Fitness of transposon insertional mutants in the SMR gene Psyr_0541 both in a WT background and in cells in which mexAB-oprM has been disrupted. “KB” and “DMSO” represent the media and media plus solvent control, respectively. Fitness was calculated as the log2 change in relative insertion strain barcode abundance for a given gene.
- FIG 7
Likely substrates of P. syringae B728a multidrug resistance RND transporters, MFS transporter Psyr_0228, and SMR pump Psyr_0541. Blue cells indicate the compound is an apparent substrate of that transporter, with insertional mutants of a given gene having decreased fitness (fitness value less than −1) in the WT and/or the ΔmexB genetic backgrounds. Red cells indicate increased fitness of insertional mutants in this operon.
Tables
- TABLE 1
RND operons homologous to mexAB-oprM that likely contribute to multidrug resistancea
↵a The number of experimental conditions where a significant contribution to fitness was seen (fitness value less than −1) is shown for mutants in a WT or ΔmexB background. A total of 23 treatments were examined, including 2 KB controls, 2 DMSO controls, and 16 unique compounds (3 at two concentrations). NA, not applicable.
- TABLE 2
Antimicrobial susceptibility of P. syringae B728a wild-type, derivative mutant, and complementation strains
FIG S1
Insertion coverage of transposon mutant libraries. Data represent frequencies of mapped transposon insertions across the chromosome of P. syringae B728a WT (left) and P. syringae B728a ΔmexB (right). Download FIG S1, PDF file, 0.1 MB.
This is a work of the U.S. Government and is not subject to copyright protection in the United States. Foreign copyrights may apply.
TABLE S1
Characteristics of the barcoded mariner transposon libraries in B728a WT and ΔmexB backgrounds. We defined genic strains as those that contain an insertion within the central 10% to 90% of a gene; only these insertions were used to calculate fitness. Total number of B728a genes, 5,216. Download Table S1, PDF file, 0.04 MB.
This is a work of the U.S. Government and is not subject to copyright protection in the United States. Foreign copyrights may apply.
FIG S2
Zone of growth inhibition assays to test antibiotic sensitivity of B728a ΔmexB complementation variants. Mean values are displayed as column heights. For each compound tested, means marked with the same letter do not differ at the P value of 0.01 (Tukey’s honestly significant difference [HSD] test). The lower limit of measurement is 6 mm (dotted line). Download FIG S2, PDF file, 0.1 MB.
This is a work of the U.S. Government and is not subject to copyright protection in the United States. Foreign copyrights may apply.
TABLE S2
Antimicrobial compounds used in growth assays of the barcoded mariner transposon libraries. For compounds with known (and different) MIC values for the WT and ΔmexB genotypes, 0.25 MIC values were used. Otherwise, the same concentration was used for both libraries. DMSO solvent controls were used at concentrations representative of the highest concentration used. Download Table S2, PDF file, 0.04 MB.
This is a work of the U.S. Government and is not subject to copyright protection in the United States. Foreign copyrights may apply.
TABLE S3
Shared amino acid identity between P. syringae B728a Psyr_2482-5 and P. aeruginosa PAO1 MuxABC-OpmB. Amino acid sequence homology was calculated using NCBI blastp. “Positives” includes amino acids having similar chemical properties. Download Table S3, PDF file, 0.04 MB.
This is a work of the U.S. Government and is not subject to copyright protection in the United States. Foreign copyrights may apply.
FIG S3
Fitness of transposon insertional mutants in the operon Psyr_2282-Psyr_2283 both in a WT background and in cells in which mexAB-oprM has been disrupted. KB and DMSO, media and media plus solvent control, respectively. Fitness was calculated as the log2 change in relative insertion strain barcode abundance for a given gene. Download FIG S3, PDF file, 0.1 MB.
This is a work of the U.S. Government and is not subject to copyright protection in the United States. Foreign copyrights may apply.
FIG S4
Fitness of transposon insertional mutants in the operons Psyr_0917-Psyr_0918 (left) and Psyr_1754-Psyr_1759 (right) both in a WT background and in cells in which mexAB-oprM has been disrupted. KB and DMSO, media and media plus solvent control, respectively. Fitness was calculated as the log2 change in relative insertion strain barcode abundance for a given gene. Download FIG S4, PDF file, 0.1 MB.
This is a work of the U.S. Government and is not subject to copyright protection in the United States. Foreign copyrights may apply.
FIG S5
Zone of growth inhibition assays to test antibiotic sensitivity of B728a efflux pump deletion mutants (A) ΔmexB ΔmexF, (B) ΔmexB ΔmuxB, and (C) ΔmexB ΔmexK. Mean values are displayed as column heights. For each compound tested, means marked with the same letter do not differ at the P value of 0.01 (Tukey’s HSD test). The lower limit of measurement is 6 mm (dotted line). Download FIG S5, PDF file, 0.1 MB.
This is a work of the U.S. Government and is not subject to copyright protection in the United States. Foreign copyrights may apply.
FIG S6
Relationship between the change in MIC and fitness value of mutants when exposed to a given compound. MIC fold change was calculated as the change in log2 dilution factor relative to the B728a WT strain for single mutants or relative to the ΔmexB mutant for double-deletion mutants. Fitness was calculated as the log2 change in relative insertion strain barcode abundance for a given gene. All values were measured in kilobases. Download FIG S6, PDF file, 0.1 MB.
This is a work of the U.S. Government and is not subject to copyright protection in the United States. Foreign copyrights may apply.
TABLE S4
Strains, plasmids, and oligonucleotides used in this study. Download Table S4, PDF file, 0.1 MB.
This is a work of the U.S. Government and is not subject to copyright protection in the United States. Foreign copyrights may apply.
