Figure 4

Testing MCrg* with inhibitors of translation. Cell-based assay: MCrg* cells were cultured in Erlenmeyer flasks at 25°C in M9 medium for 7 hours in the absence and presence of antibiotics, as indicated. Samples were taken every hour. (A) OD₆₀₀ values were determined and (B) mAzami and mCherry fluorescence emission were detected and ratios were calculated. Fluorescence ratios of MCrg* were normalized to 1. Exemplary growth curves are given and fluorescence ratios are means from three independent experiments; error bars show standard deviation. Analyses of isolated ribosomal particles: Sucrose density gradient (10-25%) centrifugation profiles from (C) control cells with no antibiotic (none), (D) chloramphenicol (Cam), (E) erythromycin (Ery), (F) kanamycin (Kan) and (G) neomycin (Neo) treated cells. Sucrose gradient fractions from (C) to (G) were analyzed for fluorescence by a microplate reader. A₂₅₄ profiles and fluorescence bar charts were superimposed for (H) control cells with no antibiotic (none), (J) chloramphenicol (Cam), (K) erythromycin (Ery), (L) kanamycin (Kan) and (M) neomycin (Neo) treated cells. Cells in presence and absence of antibiotics were cultured in LB medium at 25°C for 3 hours before subsequent polysome analysis. Left shifted peaks of the large subunit are indicated by horizontal arrows, abnormal portions of the small subunit by vertical arrows. Red bars: normalized mCherry fluorescence; Green bars: normalized mAzami fluorescence. Fluorescence was normalized to the first polysome peak (“disome”) where subunits are present in 1:1 ratio. Experiments were done in duplicates, representative profiles are shown.