Figure 4. Effects of HflK/C variants on bacterial recovery under aminoglycoside stress.

All assays were performed using E. coli BL21 ΔhflK/C cells expressing various HflK/C variants from a pPro24 plasmid under the control of a sodium-propionate-inducible promoter at 37°C in LB medium containing 50 μg/mL kanamycin, 35 μg/mL chloramphenicol, and 1 mM sodium propionate unless otherwise noted.

(A) Growth curves for cells expressing wild-type HflK/C (HflK/C^WT), crosslinked HflK/C (HflK/C^SS), and empty vector (EV).

(B) Growth curves for cells expressing HflK/C^WT, crosslinked HflK/C (HflK/C^SS), and EV under tobramycin stress.

(C) Top: pull-down of the HflK/C complex solubilized in DDM from E. coli BL21 ΔhflK/C cells with chromosomally encoded FLAG-tagged FtsH and either WT or M1 (R141A, E142A, and R185A) HflK, Bottom: western blot analysis of the whole-cell lysates shows a comparable expression of HflC between WT and M1 mutant.

(D and E) Growth curves of tobramycin-stressed cells expressing: HflK/C^WT, mutant M1 (HflK(R141A, E142A, R185A)/HflC), and EV under 1 mM sodium propionate (D) and 5 mM sodium propionate (E). Each growth assay was performed using a separate colony (n = 3 independent biological replicates), and data are presented as the mean ± 1 SD.