TABLE 1.
Phenotypic analysis of TolQ cysteine mutations
TPS13 (tolQR) cells expressing both the wild-type (WT) tolR from the pUCR plasmid and the tolQ derivatives from the pOK-QHA plasmid were tested for their susceptibility to the Tol-dependent colicins A and E2 and Tol-independent colicin D (Ton-dependent) and their susceptibility to DOC. Only the TolQ mutation is indicated, WT and tolQ corresponding to the TPS13 pUCR pOK-QHA and TPS13 pUCR pOK12 strains, respectively. Colicin sensitivities were tested on bacterial lawns by the spot dilution assay. The number indicated represents the maximal 10-fold dilution of the colicin stock (∼1 mg/ml) still able to kill the strain (R, resistant; 4, sensitive to the 104 dilution). Underlined numbers correspond to turbid halo. Deoxycholate resistance was tested by spotting 10-fold dilutions of the strain on 1% DOC LB plates. The number indicated represents the maximal dilution able to form colonies (R, resistant; S, sensitive; numbers, 10n dilution). A summary is indicated in the Phenotype column using the color code shown in Figure 2A: B (blue, WT phenotype, colicin-sensitive and DOC-resistant), R (red; tol phenotype, colicin-resistant and DOC-sensitive), and C (cyan; discriminative phenotype, colicin- and DOC-sensitive). The ability of the TolQ cysteine variants to form homodimer (see supplemental Fig. S3) is indicated by + in the “complex formation” column. OM, outer membrane.
| Mutation | Colicin activities |
OM stability (DOC) | Complex formation | Phenotype | ||
|---|---|---|---|---|---|---|
| A | E2 | D | ||||
| WT | 4 | 4 | 4 | R | − | B |
| tolQ | R | R | 4 | S | − | R |
| TMH1 | ||||||
| L19C | 4 | 4 | 4 | R | − | B |
| I20C | 4 | 4 | 4 | R | − | B |
| M21C | 4 | 4 | 4 | R | − | B |
| L22C | 4 | 4 | 4 | R | − | B |
| I23C | 4 | 4 | 4 | 3 | − | B |
| L24C | 4 | 4 | 4 | 3 | − | B |
| I25C | 4 | 4 | 4 | 3 | − | B |
| G26C | 4 | 4 | 4 | R | − | B |
| F27C | 4 | 4 | 4 | R | − | B |
| S28C | 4 | 4 | 4 | 1 | − | C |
| I29C | 4 | 4 | 4 | 3 | − | B |
| A30C | 4 | 4 | 4 | 3 | − | B |
| S31C | 3 | 3 | 4 | 1 | − | C |
| W32C | 4 | 4 | 4 | R | − | B |
| A33C | 4 | 4 | 4 | 3 | − | B |
| I34C | 4 | 4 | 4 | 3 | − | B |
| I35C | 4 | 4 | 4 | 1 | − | C |
| I36C | 4 | 4 | 4 | 3 | − | B |
| Q37C | 4 | 4 | 4 | 3 | − | B |
| TMH2 | ||||||
| P138C | 4 | 4 | 4 | 1 | − | C |
| Y139C | 4 | 4 | 4 | S | − | C |
| I140C | 4 | 4 | 4 | R | − | B |
| G141C | R | R | 4 | S | − | R |
| L142C | 4 | 4 | 4 | 1 | − | C |
| F143C | 4 | 4 | 4 | 3 | − | B |
| G144C | R | R | 4 | S | − | R |
| T145C | 4 | 4 | 4 | R | − | B |
| V146C | 3 | 4 | 4 | 1 | − | C |
| W147C | 3 | 4 | 4 | R | − | B |
| G148C | 2 | 2 | 4 | S | − | C |
| I149C | 4 | 4 | 4 | 3 | − | B |
| M150C | 4 | 4 | 4 | 1 | − | C |
| H151C | 4 | 4 | 4 | R | − | B |
| A152C | 4 | 4 | 4 | 3 | − | B |
| F153C | 4 | 4 | 4 | 2 | − | C |
| I154C | 4 | 4 | 4 | R | + | B |
| A155C | 3 | 4 | 4 | 3 | − | B |
| L156C | 3 | 4 | 4 | 3 | − | B |
| G157C | 4 | 4 | 4 | 3 | + | B |
| TMH3 | ||||||
| P169C | 3 | 4 | 4 | 1 | − | C |
| G170C | 4 | 4 | 4 | R | − | B |
| I171C | 2 | 3 | 4 | 3 | − | B |
| A172C | 4 | 4 | 4 | R | − | B |
| E173C | R | R | 4 | 1 | − | R |
| A174C | 4 | 4 | 4 | 1 | − | C |
| L175C | 3 | 4 | 4 | S | − | C |
| I176C | 4 | 4 | 4 | R | − | B |
| A177C | 3 | 3 | 4 | S | − | C |
| T178C | 4 | 4 | 4 | 1 | − | C |
| A179C | 4 | 4 | 4 | R | − | B |
| I180C | 4 | 4 | 4 | R | − | B |
| G181C | 4 | 4 | 4 | 1 | − | C |
| L182C | R | R | 4 | S | − | R |
| F183C | 4 | 4 | 4 | 3 | − | B |
| A184C | 4 | 4 | 4 | R | − | B |
| A185C | 4 | 4 | 4 | R | − | B |
| I186C | 3 | 3 | 4 | 1 | − | C |
| P187C | 4 | 4 | 4 | S | − | C |