Squatter colonies and mutations in the EnvZ/OmpR system. (A) Squatter colonies of NCTC86 and ATCC 4157 isolates in the growth inhibition zone of the benzylpenicillin E test. Three colonies of each isolate were sequenced (yellow and red circles, respectively). (B) Nonsynonymous mutations in OmpR, EnvZ, OmpF, and OmpC found in the squatter colonies compared to NCTC86 and ATCC 4157. (C) Schematic representation of the EnvZ/OmpR system showing the mutations indicated by a star (yellow and red for the NCTC86 and ATCC 4157 isolates, respectively). OM, outer membrane; IM, inner membrane. (D) Benzylpenicillin E tests of NCTC86_S1, NCTC86_S3, NCTC86_S1/pompR10, and NCTC86_S3/pompR10 (complemented isolates were tested on plates containing kanamycin). The absence of inhibition zone corresponds to a MIC > 32 mg/liter. Note that the sensitive phenotype with some squatter colonies is restored by pompR10 complementation only in the NCTC86_S1 isolate.