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ABSTRACTAntibiotic resistance is generally selected within a window of concentrations high enough to inhibit wild-type growth but low enough for new resistant mutants to emerge. We studied de novoevolution of resistance to ciprofloxacin in an Escherichia coliknockout library. Five null mutations had little or no effect on intrinsic antibiotic susceptibility but increased the upper antibiotic dosage to which initially sensitive populations could adapt. These mutations affect mismatch repair, translation fidelity, and iron homeostasis.