Fig. 1: Degradation of apoNDM-1 in the periplasm of live bacterial cells.
From: Periplasmic protein quality control at atomic level in live cells
a Schematic representation of the protocol used for preparing bacterial samples overexpressing NDM-1 and the proteases Prc and DegP. Induction with arabinose (ARA) or IPTG was done at 37 °C and 16 °C, respectively. Incubation with spectinomycin (Spect.) was done at 20 °C. b Comparison of NDM-1 overexpression levels from the in-cell NMR samples with P. aeruginosa and K. pneumoniae clinical strains (N = 1). The dilution factor used (1/160) was determined by semi-quantitative western blot analysis and comparisons with the clinical strain Enterobacter cloacae 17464 (Supplementary Fig. 1b, c). Normalization was done considering the total number of cells estimated by OD600. c Membrane localization of overexpressed NDM-1 determined by Western blot analysis. Cyto., Perip., and Total memb. corresponds to cytosolic, periplasmic and total membrane fractions, respectively (N = 3). d Representative immunofluorescence of NDM-1 (magenta) and Prc and DegP (green) in E. coli cells (N = 3). e Mean fluorescence intensity of NDM-1 in E. coli cells. Panel shows a representative analysis of three independent repetitions (N = 3). No NDM/DPA = 0.0011 ± 0.0001, NDM/No DPA = 2.8600 ± 0.2494, NDM/DPA = 1.6590 ± 0.1227 (mean ± SEM, n = 10 per treatment). Significant difference between conditions, ****p < 0.0001, Tukey, one-way ANOVA. f Western blot analysis of periplasmic NDM-1 degradation by Prc and DegP upon metal deprivation (N = 3).
