Journal: bioRxiv

Article Title: Proton-coupled alternating access in a versatile Spns drug efflux pump from Mycobacterium smegmatis

doi: 10.64898/2026.05.09.724020

Figure Lengend Snippet: ( a ) Expression of cysteine-less (CL) Ms Spns in cell-growth assays at different IPTG concentrations (0–2.5 μM), visualized by SDS–PAGE and InVision His-tag staining. Purified CL Ms Spns is shown as a standard. 1 μM IPTG was used for protein expression in growth assays. ( b ) Expression of CL Ms Spns enhances cell survival at elevated capreomycin concentrations relative to the vector control. Measurements were performed in triplicate after 8 h at 37 °C. A 600nm values were normalized to the 0 μ g/mL capreomycin control, and standard deviations are shown. The optimal capreomycin concentration was 37.5 μg/mL. ( c ) Cells expressing CL Ms Spns show improved growth relative to the vector control at all time points in the presence of 37.5 μg/mL capreomycin. A 600nm values were normalized to the 0 μg/mL control; data points represent mean ± s.d. from triplicate measurements, as in b. ( d ) Growth of Ms Spns mutants at 37.5 μg/mL capreomycin, normalized to the 0 μg/mL control. Bars show mean ± S.E.M. for at least three replicates, with replicate numbers indicated in parentheses. For n > 3, data were collected from multiple biological replicates. One-way ANOVA showed significant differences among mutants at the 0.05 level F (40, 234) = 28.81, p < 0.00001, whereas Tukey’s multiple-comparison test indicated that the double-cysteine mutants did not differ significantly from WT or CL Ms Spns, showing that cysteine substitutions generally do not impair capreomycin resistance.

Article Snippet: Cultures were incubated while being shaken at 37 °C until they reached an absorbance at 600 nm (Abs 600nm ) of ∼ 0.8, at which time Ms Spns expression was induced by the addition of 1 mM IPTG (Gold Biotechnology).

Techniques: Expressing, Structural Proteomics, SDS Page, Staining, Purification, Plasmid Preparation, Control, Concentration Assay, Comparison