FIGURE 3.

ES24 impairs Hbp secretion and requires reduction by NfsA/B to impose this effect. (a) Schematic overview of the domain organization of the autotranporter Hbp. (b) E. coli TOP10F′ bacteria were grown in a round bottom tube and exposed to 0.5× MIC ES24 and NFT or DMSO as control. Hbp and GFP were expressed from pEH3 using IPTG for induction. After 10 and 30 min of exposure whole cell lysates were analyzed by SDS‐PAGE and Western blotting using antibodies against GFP and Hbp β‐barrel. The spent medium was TCA precipitated and analyzed by Western blotting using antibodies against the Hbp passenger domain. (c) E. coli NER502 ∆nfsA/B and its wild‐type parental strain were grown as described above and exposed to 0.5× MIC ES24 and NFT or DMSO as control. After 30 min of Hbp expression from pEH3, using IPTG for induction, whole cell lysates were analyzed by SDS‐PAGE and Western blotting using antibodies against the Hbp β‐barrel domain. The spent medium was TCA precipitated and analyzed by Western blotting using antibodies against the Hbp passenger domain. Data are representative of three independent experiments. Samples were corrected for growth (OD₆₀₀) in order to load equal amounts [Colour figure can be viewed at wileyonlinelibrary.com]