FIG 6.

Mutations in CRP confer resistance to gentamicin and are pleiotropic for resistance to other antibiotics. (A) Verification of mutations enriched in the regulator library for resistance to gentamicin at a 30-μg/ml concentration. (B) Position of mutated residues (blue) on the CRP structure (gray) and their proximity to DNA (red). (C) Evaluation of the extent of synergistic pleiotropy for the mutation in crp conferring resistance to gentamicin for resistance to thiamphenicol at a 100-μg/ml concentration. (D) Evaluation of the extent of synergistic pleiotropy for the mutation in crp conferring resistance to gentamicin for resistance to azithromycin at a 120-μg/ml concentration. The blue lines indicate variants, and the black lines indicate wild-type E. coli. All strains used to verify resistance and pleiotropy were cultured in LB medium (10-g/liter tryptone, 5-g/liter yeast extract, 10-g/liter NaCl) with the corresponding antibiotic at 37°C and 220 rpm in 250-ml conical flasks with a 50-ml final volume of the medium.