Article Figures & Data
Figures
- FIG. 1.
Models for different conformational (folding) states of colicin E3 when it binds to, and inserts into, a translocation pore that utilizes OmpF. (A) T domain is fully/partially folded when colicin interacts with the TolB protein. The immunity protein may or may not be bound to colicin E3 when it interacts with TolB protein. In this case, the TolB extensor region (37 residues), extending from residue 46 to 83, has no extra length, thus causing steric limitations reflected in the requirement for a minimum length of the TolB extensor. The R domain is depicted to be folded as a coiled coil. (B) Colicin/T domain is unfolded when it interacts with TolB. In this case, there is extra length in the TolB extensor. The coiled coil of the R domain is depicted as partly unfolded. (C) Outer membrane BtuB/OmpF translocon for the import of colicin E3. Abbreviations: C, catalytic domain; R, receptor binding domain; T, translocation domain; Imm, immunity protein; OM, outer membrane; PGL, peptidoglycan layer.
- FIG. 2.
TolB binding affinity of colicin constructs measured by the area under the elution peaks formed by colicin/TolB complex separated on a Superdex 75 column, as described in Materials and Methods. The affinity is normalized to the total amount of TolB added (1.0 on the ordinate).
- FIG. 3.
Effect of the various colicin constructs on the growth of E. coli K17 indicator cells. (A) Colicins were added at a concentration of 3.2 nM. E3 Δ55-83, E3 Δ65-83, and E3 Δ43-52 did not inhibit cell growth, while E3 Δ65-73 and E3 Δ72-80 were as active as wild-type E3. (B) E3 Δ65-78 and E3 79-83/Δ65-78 had a similar activity, which was slightly less than that of wild-type E3. E3 60-64/Δ65-78 showed decreased cytotoxicity compared to E3 Δ65-78. Error bars are contained within symbols if they are not explicitly displayed. (C) Effective multiplicity of colicin and constructs that inhibited cell growth in panel B. *, The m value and fractional survival for E3 Δ65-83 have been assumed to be similar to the control in which no colicin was added on the basis of their identical growth curves and is shown here for purposes of comparison.
- FIG. 4.
E3 Δ55-83 occludes OmpF channels in planar bilayers. OmpF was added to the cis side of the membrane and the solution stirred until channels appeared. The transmembrane current was recorded upon application of transmembrane potentials, 50mV and −50mV (sign of the potential is that of cis side). (A) E3 Δ55-83 was added to the cis side, and no occlusion was seen upon application of potential. (B) When the colicin was added from the trans side, it occluded OmpF channels upon application of a cis-negative potential. The occlusion effect was negated upon changing the potential to cis positive. (C) OmpF occlusion by colicin E3 deletion constructs. 1, The presence (+) or absence (−) of occlusion denotes the ability of 0.5 to 1 μg/ml of colicin to block OmpF ion channels.
