FIG 6.

RpoS-dependent GFP expression of gadX::gfp promoter fusions in the stationary phase at 15°C and 37°C by fluorescence microscopy (A) and Western blotting using anti-GFP antibody (B). E. coli strains containing the reporter fusion were cultured in LB broth supplemented with kanamycin (50 μg/ml) with agitation. Samples were taken at stationary phase (17 h at 37°C and 36 h at 15°C), fixed with ethanol-methanol (1:1) solution, and resuspended in PBS, and 2 μl was placed on a slide and imaged with a Leica DMI3000 B microscope. RpoS activity indicated by fluorescence was higher at 37°C than at 15°C (A), and this correlated with GFP detection by immunoblotting (B). COB585 carrying the reporter fusion had the lowest RpoS activity among strains with intact RpoS. Fluorescence was not detected in the BW25113ΔrpoS strain either by microscopy or immunoblotting. Fluorescent images presented are representatives of 2 independent experiments with >3 field captures in each experiment. Western blot image is representative of 3 independent experiments.