Article Figures & Data
Figures
- FIG. 1.
Cellular distribution of ColV activity expressed from pRR-ColV plasmid in TnMK. Periplasmic and cytoplasmic fractions were prepared from TnMK grown in the presence of arabinose. (A) Bactericidal activities were assessed by loading 5 μl of the fractions on the top of soft agar, which was prepared by mixing 1 ml of overnight test strain cultures of MC4100A (WT), TnMK (sdaC), SC44 (tolC), and WB591 (tonB) with 1 ml of 1% agar solution and poured on the surface of LB plates. Inhibition halos were recorded after overnight incubation of plates at 37°C. (B) The quality of the periplasmic and cytoplasmic fractions was analyzed by immunoblotting with anti-RBP (periplasmic marker) and anti-GroEL (cytoplasmic marker).
- FIG. 2.
The ColV-resistant phenotype of the TnMK mutant is the direct consequence of the sdaC mutation. Portions (5 μl) of either undiluted or 10-fold-serial-diluted ColV solution prepared from the cytoplasmic fraction of the tatC mutant expressing RR-ColV were loaded on the soft agar containing MC4100A, TnMK/pUC19, TnMK/pUSCB, or TnMK/pUSC prepared as described in Fig. 1.
- FIG. 3.
Sensitivity of the sdaC mutant to different colicins. Portions (10 μl) either undiluted (lane 1) or 10-fold diluted (lane 2) of ColA (A), ColE3 (B), or ColIa (C) were loaded on cellular lawns of the wild-type strain MC4100A, the sdaC mutant TnMK, or the tolQR or tonB mutant. The growth inhibition effect was analyzed after overnight incubation at 37°C.
- FIG. 4.
Sensitivity of TnMK to endogenously produced or exogenously added EtpM-ColV fusion. (A) CFU of TnMK/pEtpM167-ColV grown on an LB-AMP plate supplemented with glucose (Glc +) or arabinose (Ara +) without (pcvi −) or with (pcvi +) coexpression of ColV immunity protein. (B) Crude extracts containing ColV (lanes 1 and 3) or ColV and EtpM-ColV (lanes 2 and 4) were resolved on 0.1% sodium dodecyl sulfate-20% polyacrylamide gels. The gels were then incubated for 30 min in a 50% methanol-10% acetic acid solution, washed twice with 250 ml of sterile water, and placed onto LB plates. Soft agar containing the wild-type MC4100A strain (lanes 1 and 2) or the sdaC mutant (lanes 3 and 4) was poured on the top, and the petri dishes were incubated overnight at 37°C. The migration positions of ColV and EtpM-ColV are indicated.
