FIGURE 5.

Cell killing by hybrid colicin/klebicin derivatives, effect of point mutations in the LBS on toxicity, interaction with LepB in vitro and processing. A, comparison of the cytotoxicity of in vitro synthesized (Zubay-S30 system), wild-type colicin D (ColD), klebicin C (KlebC), hybrid colicin D/klebicin D (ColD-KlebD), and colicin D/klebicin C (ColD-KlebC) by growth inhibition test is shown. Undiluted aliquots were spotted, as indicated by black points, onto a lawn of AD202 wild-type or A38 LepB(N274K) mutant E. coli strain. Dark halos indicate a clear zone of growth inhibition (toxicity), and no clearing reveals the resistance to wild-type or hybrid colicins. B, nine point mutations to alanine, aspartate, or valine introduced into or close to the LepB-binding site (blue-boxed peptide sequence located in the colicin D central domain) are indicated. The killing activity of these purified mutated colicins D was quantified by spotting aliquots of undiluted colicin (numbered 0) and serial 10-fold dilutions up to 10⁻⁵ (numbered 1–5) directly onto a lawn of AD202 strain and analyzed as in A. Turbid zones indicate marginal toxicity. C, wild-type or mutated FS colicins (∼50 ng) were separated by 15% SDS-PAGE and detected by Amido Black staining (upper panel). Their interaction with purified wild-type LepB was detected in vitro by Far Western blotting (lower panel) with anti-LepB antiserum. The efficiency of interaction was quantified and expressed as a percentage of value measured with wild-type colicin D (wt, 100%). D, in vivo detection of the colicin D processed form (ColD PF, 12.4 kDa) from the S100 cytoplasmic fraction of an OmpT-deficient colicin D-sensitive strain treated by purified wild-type or mutated colicins D. Total S100 extracts were separated by 15% SDS-PAGE, and the ColD PF was detected by Western blotting. Mutations provoking a weak or an almost full defect on toxicity on the interaction with LepB and on the processing are indicated in green and red, respectively.