Journal: Cell reports

Article Title: Structural plasticity of the membrane-bound protein degradation assembly supports bacterial adaptation to stress

doi: 10.1016/j.celrep.2026.117231

Figure Lengend Snippet: All assays were performed using E. coli BL21 Δ hflK/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.

Article Snippet: For the DDM-solubilized FtsH·HflK/C complex extracted from the tobramycin treated cells, 21,895 movies were collected with EPU using aberration-free image shift (AFIS) and hole-clustering method on a Titan Krios G3i with an acceleration voltage of 300 kV and magnification of 130,000×, detected in super-resolution mode on a Gatan K3 detector for an effective pixel size of 0.654 Å (binned by 2).

Techniques: Expressing, Plasmid Preparation, Control, Western Blot, Mutagenesis, Growth Assay