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. 2002 Feb 5;99(3):1286–1291. doi: 10.1073/pnas.022480199

Figure 1.

Figure 1

(A) Sequence of the pore-forming domain of colicin A (pfColA), ImmE2, and CTE2. The asterisk in the pfColA sequence is the insertion location. The bold letters in the CTE2 sequence are the mutated amino acids R544A/H575A. (B) In vivo activity of colicin E2 (R544A/H575A). Five hours after induction with mitomycin C, equal amounts of cells (C) and supernatants (Sn) of medium were subjected to SDS/PAGE and proteins were detected by immunoblotting with anti-colicin E2 Ab. The wt and mutated colicin E2 were secreted at the same level. Undiluted wt and mutated colicin E2 (1 μl of supernatant and indicated serial dilutions) were spotted onto a lawn of sensitive cells. The zone of inhibition on the lawn appears as a clear hole on the photo. Note that the mutated R544A/H575H colicin E2 is totally devoid of toxicity. (C) Expression of colicin A, ColA-CTE2, and ColA-ImmE2 proteins. Bacteria producing colicin A, ColA-CTE2, or ColA-ImmE2 were grown in LB medium at 37°C to OD600 = 0.8 and induced for 30 min with 300 ng/ml mitomycin C. Then total cell extracts producing colicin A (lane 1), ColA-ImmE2 (lanes 2 and 5), and ColA-CTE2 (lanes 3 and 4) were loaded onto SDS/PAGE and analyzed by immunoblotting with the indicated Ab. ColA-ImmE2 and ColA-CTE2 were detected with Abs directed against colicin A or ImmE2 and colicin E2, respectively. (D) Protein binding in solution shown by nondenaturing gels. The gel on the left (lanes 1–4) was run − to + at pH 8.8, and the gel on the right (lanes 5- 8) was run + to − at pH 5.0. Lanes 1–4 contain 2 μg of each ImmE2. In addition, lane 2 contains 6 μg of CTE2, lane 3 contains 20 μg of ColA-CTE2, and lane 4 contains 6 μg of colicin Ia. Notice that CTE2 and ColA-CTE2 bind to ImmE2 and prevent it from entering the gel, whereas the basic protein colicin Ia does not. Lanes 5–8 contain 1.2 μg each of CTE2. In addition, lane 6 contains 3 μg of ImmE2, lane 7 contains 5 μg of colicin A, and lane 8 contains 3 μg of ColA-ImmE2. Notice that ColA-ImmE2 and ImmE2 bind to CTE2 and shift its position in the gel, whereas wt colicin A does not.