Spontaneous mutations have been isolated in Escherichia coli that result in the constitutive expression of an emrKY promoter. These mutations were found to be single-nucleotide substitutions within the linker region of the sensor protein EvgS, which is part of a two-component regulatory system along with EvgA. In the linker mutants (evgSI and evgS4), emrKY expression became constitutive and MIC against sodium deoxycholate was 20 mg/ml, eight-fold higher than in the wild type. Furthermore, the start site of transcription from the promoter of emrKY was identified; EvgA was shown to bind at the -52 to -84 region by the footprinting experiment.