Table 1. Spot titer assay results.
TonB system activity of strains expressing variants of ExbD and TonB was evaluated using spot titer assays. Cultures of the mutants expressed to near-chromosomal levels (verified through Western blot—not shown) were plated on T-plates and spotted with fivefold serial dilutions of colicins and tenfold serial dilutions of bacteriophage ϕ80. Values were recorded as the reciprocal of the highest dilution at which clearing of the bacterial lawn was evident after 18 hours of incubation at 37°C. “T” indicates tolerance (no sensitivity).
| Strain | Phenotype | Sensitivitya | |||
|---|---|---|---|---|---|
| Colicin B | Colicin Ia | Colicin M | ϕ80 | ||
| W3110 | WT | 8,8,8 | 7,7,7 | 6,6,6 | 8,8,8 |
| RA1045 | ExbD-,TolQR- | T,T,T | T,T,T | T,T,T | T,T,T |
| KP1509 | ExbD- TonB- | T,T,T | T,T,T | T,T,T | T,T,T |
| KP1344/pKP381 | TonB(H20A) | T,T,T | T,T,T | T,T,T | T,T,T |
| KP1344/pKP1054 | TonB(H20D) | T,T,T | T,T,T | T,T,T | T,T,T |
| RA1045/pKP1055 | ExbD(D25H) | T,T,T | T,T,T | T,T,T | T,T,T |
| KP1509/pKP1054/pKP1055 | ExbD(D25H)/TonB(H20D) | T,T,T | T,T,T | T,T,T | T,T,T |
| RA1045/pKP999 | ExbD | 8,8,7 | 7,7,7 | 6,6,6 | 8,8,8 |
| RA1045/pKP1064 | ExbD(D25N) | T,T,T | T,T,T | T,T,T | T,T,T |
| RA1045/pKP1191 | ExbD(D25A) | T,T,T | T,T,T | T,T,T | T,T,T |
Scored as the highest fivefold dilution of a standard colicin preparation, or tenfold dilution of bacteriophage ϕ80, that provided an evident zone of clearing on a cell lawn. “T” indicates tolerance (i.e. no clearing of the lawn) to undiluted colicin or phage. The values of three platings are presented for each strain/plasmid and colicin or phage pairing.