Abstract
Colicin D import into Escherichia coli requires an interaction via its TonB box with the energy transducer TonB. Colicin D cytotoxicity is inhibited by specific tonB mutations, but it is restored by suppressor mutations in the TonB box. Here we report that there is a second site of interaction between TonB and colicin D, which is dependent upon a 45-amino acid region, within the uncharacterized central domain of colicin D. In addition, the 8th amino acids of colicin D (a glycine) and colicin B (a valine), adjacent to their TonB boxes, are also required for TonB recognition, suggesting that high affinity complex formation involves multiple interactions between these colicins and TonB. The central domain also contributes to the formation of the immunity complex, as well as being essential for uptake and thus killing. Colicin D is normally secreted in association with the immunity protein, and this complex involves the following two interactions: a major interaction with the C-terminal tRNase domain and a second interaction involving the central domain of colicin D and, most probably, the alpha4 helix of ImmD, which is on the opposite side of ImmD compared with the major interface. In contrast, formation of the immunity complex with the processed cytotoxic domain, the form expected to be found in the cytoplasm after colicin D uptake, requires only the major interaction. Klebicin D has, like colicin D, a ribonuclease activity toward tRNAArg and a central domain, which can form a complex with ImmD but which does not function in TonB-mediated transport.
Highlights
FEBRUARY 22, 2008 VOLUME 283 NUMBER 8 obtaining hybrid colicins by recombination suggest that colicin evolution is in part based on the exchange and mixing of similar domains from heterologous colicins or bacteriophages (2, 4 – 6)
The crystal structure of the heterodimer complex formed between the colicin D cytotoxic domain and its cognate immunity protein showed that ImmD mimics the tRNA substrate backbone, thereby protecting colicin-producer cells against colicin molecules [8]
The synthesis of certain colicin hybrids in E. coli was found to produce aggregates in the cytoplasm, or in some cases the colicin derivatives were shown to be more susceptible to degradation when their release into the extracellular medium was impaired [40]
Summary
Bacterial Strains, Growth, and Recombinant DNA Manipulations—E. coli strains D10 [37] and XAC were used as wild-type strains. Construction of Colicins D Molecules Mutated in the Central Domain—Single or double point mutations were introduced into the central domain of colicin D by two-step PCR amplification as described previously [8], and they were expressed in vitro (Zubay-S30). Killing Activity Test—The cytotoxicity of wild-type, hybrid, or internally deleted colicins D, as well as colicin D molecules carrying point mutations in the central domain, synthesized in vitro (Zubay-S30), was quantified in vivo by growth inhibition (halo) assay. The in vitro (ZubayS30) synthesized [35S]Met-labeled, wild-type or mutated colicin D-ImmD complexes (3 g) were incubated in a concentrated crude cell extract of E. coli XAC wild-type strain enriched with added purified LepB (0.025 g) for 1 h at 37 °C, as described previously by de Zamaroczy et al [39]. Full-sized and cleaved colicin D forms were precipitated with acetone and separated by (8%) SDS-PAGE, and the labeled proteins were detected and quantified by phosphorimagery (GE Healthcare)
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