Figure 3
From: Type 1 secretion necessitates a tight interplay between all domains of the ABC transporter
Quantification of chHlyB expression and pro-HlyA secretion. SDS-PAGE (a) and immunoblot analysis (b) of pro-HlyA from supernatants of E. coli cells expressing the indicated chHlyB and HlyD in two replicates; the used antibody targeted the secretion signal of pro-HlyA. Immunoblot (c) of whole E. coli cells show the amount of expressed chHlyB in two replicates; the used antibody targeted the NBD of chHlyB. The samples were diluted to match the same OD600. The contrast of the SDS-PAGE in (a) was adjusted to improve visibility of secreted pro-HlyA in the case of HlyB-EEK. Schematic representation of the chHlyB variants are depicted above the respective chimera. Blue domains originate from HlyB (E. coli) and red domains from RtxB (K. kingae). The quantification revealed reduced secretion efficiencies for both HlyB-EKE (dark grey) and HlyB-EEK (light grey) by a factor of ~ 3 when compared to HlyB-EEE (black). Uncropped Western blots and SDS-PAGE gels are shown in Supplementary Fig. S6. (d) The left panel shows the calculations using signals from Western blots for quantifications of pro-HlyA, while the right panel shows quantifications using Coomassie stained gels (CBB). Shown are the mean values with standard deviations as error bars (n ≥ 16, biological replicates). Statistical analysis was performed using a one-way ANOVA test (****p < 0.0001, ns: p = 0.5138 for EKE vs. EEK (Western Blot) and p = 0.9995 for EKE vs. EEK (CBB)). M: Protein marker, the approximated molecular weight of the marker proteins is given on the left; x h: time after induction, when the samples were taken.
