FIG 5.

CirA is the outer membrane receptor for Mcc1229. (A) Spontaneous mutants of PA2 were resistant to inhibition by E. coli 0.1229ΔB17::cat. Colonies were isolated from within the zones of clearing and subjected to whole-genome sequencing to identify the source of Mcc1229 resistance. Multiple independent mutants (PA2.1, PA2.2, and PA2.3) had mutations in CirA. A ΔcirA::kan mutant of PA2 is resistant to Mcc1229, and when cirA mutants are grown in the spent supernatants of E. coli 0.1229ΔB17::cat, they are insensitive to Stx amplification. Sensitivity is restored by complementation with pBR322::cirA (cirA⁺) but not with the pBR322 empty vector (“E”). Asterisks mark significant differences between a given supernatant sample and the PA2 wild type (two-way ANOVA and Dunnett’s multiple-comparison test). (B) Colicin indicator strains confirmed that CirA was the Mcc1229 receptor. NCTC 50154 (WT) and NCTC 50157 (cirA) were tested in agar overlay assays. The resulting zones of inhibition (ZOI) were significantly different (P < 0.0001 by Student’s t test).