Figure 7.

VUF15259 interferes with targeting or insertion of β‐barrel type OMPs. (A) E. coli TOP10F’ cells, carrying the pEH3 vector expressing σ^E factor and TOP10F’ cells, carrying the empty pEH3 vector, were treated with VUF15259 or with 1% DMSO. Cells were grown in 96‐well plates for 3 h and the σ^E stress was measured using the PrpoE‐mNG reporter construct. The fold mNG fluorescence is depicted compared to the DMSO‐treated empty vector cells, with the error bars representing the standard deviation of triplicate samples. Bacteria in the 96‐well plate were collected and separated from medium by centrifugation. Cell envelopes were isolated using ultracentrifugation and analyzed by (B) SDS‐PAGE and (C) Western blotting analysis using antibodies against BamA and LepB. For the empty vector, control cells also an antibody staining against the whole Bam‐complex was performed analyzed under (D) denaturing conditions by SDS‐PAGE, and under (E) native conditions by Blue Native PAGE. (F) TOP10F’ cells, expressing phoE from the pEH3 plasmid, were grown in 96‐well plates and treated with VUF15259 for 3 h. Cell envelopes were isolated and analyzed by a semi‐native PAGE and Western blotting. To examine heat‐modifiability, samples were either incubated at room temperate (RT) or at 95°C for 10 min. The inner membrane protein SecG was used as loading control.