Colicin-Mediated Transport of DNA through the Iron Transporter FepA

Abstract

Colicins are protein antibiotics deployed by Escherichia coli to eliminate competing strains. Colicins frequently exploit outer membrane (OM) nutrient transporters to penetrate the selectively permeable bacterial cell envelope. Here, by applying live-cell fluorescence imaging, we were able to monitor the entry of the pore-forming toxin colicin B (ColB) into E. coli and localize it within the periplasm. We further demonstrate that single-stranded DNA coupled to ColB can also be transported to the periplasm, emphasizing that the import routes of colicins can be exploited to carry large cargo molecules into bacteria. Moreover, we characterize the molecular mechanism of ColB association with its OM receptor FepA by applying a combination of photoactivated cross-linking, mass spectrometry, and structural modeling. We demonstrate that complex formation is coincident with large-scale conformational changes in the colicin. Thereafter, active transport of ColB through FepA involves the colicin taking the place of the N-terminal half of the plug domain that normally occludes this iron transporter. IMPORTANCE Decades of excessive use of readily available antibiotics has generated a global problem of antibiotic resistance and, hence, an urgent need for novel antibiotic solutions. Bacteriocins are protein-based antibiotics produced by bacteria to eliminate closely related competing bacterial strains. Bacteriocin toxins have evolved to bypass the complex cell envelope in order to kill bacterial cells. Here, we uncover the cellular penetration mechanism of a well-known but poorly understood bacteriocin called colicin B that is active against Escherichia coli. Moreover, we demonstrate that the colicin B-import pathway can be exploited to deliver conjugated DNA cargo into bacterial cells. Our work leads to a better understanding of the way bacteriocins, as potential alternative antibiotics, execute their mode of action as well as highlighting how they might even be exploited in the genomic manipulation of Gram-negative bacteria.

Keywords: DNA delivery; Rosetta flexible backbone; antibiotic resistance; bacteriocins; conformational changes; membrane transport.

FIG 1

ssDNA follows the ColB-RT translocation path into E. coli cells. (A) Translocation of ColB-RT-Alexa 488 (BRT) or ColB-RT ΔTonB box–Alexa 488 (ΔTonB box) constructs into E. coli MG1655 cells (WT) or E. coli BW25113 ΔFepA (FepA knockout [KO]) cells grown in minimal medium to mid-log growth phase (OD₆₀₀, ∼0.35). OM translocation was defined as fluorescent signal resistant to trypsin treatment (Tryp). Cytoplasmic localization was defined as fluorescent signal remaining after spheroplasting the cells, which results in the removal of the OM and the periplasmic peptidoglycan layer (Sphe). The averaged fluorescence intensities were calculated from at least 120 cells (30 cells × 4 biological repeats), and standard error bars of each treatment are shown. Representative cellular images are below each treatment. Scale bar, 1 μm. (B) Translocation of ColB-RT-DNA-fused constructs were ColB-RT-A₁₅ Alexa 488 (15A), ColB-RT A₅C₁₀ Alexa 488 (10C 5A), and ColB-RT A₅C₁₀ Alexa 488 + G₁₀ (A₅C₁₀ + G₁₀).

FIG 2

Structural insights on the ColB-FepA complex by pBPA cross-linking and Rosetta-based structural modeling. (A) Initial encounter complex (EC) modeled with moderate to little backbone flexibility (under 5 Å root mean square deviations [RMSD]). ColB (blue) and FepA (gray) form this encounter complex with in vitro cross-links, FepA-K639 and ColB-D202 (teal), and FepA-P642 and ColB-R205 (purple), which lie in proximity in the model. The last in vitro cross-link pair, FepA-S652 and ColB-Q55 (cyan), and the two in vivo cross-links, FepA-T58 and ColB-M19 (olive) and FepA A214 and ColB-G81 (orange), are not satisfied in this structure. (B) Mapped in vitro cross-linking sites on the ColB and FepA PDB structures (1RH1 and 1FEP, respectively). Cropped relevant cross-link gels. Self-cross-linking control to the right of each lane (full in vitro cross-linking image is in Fig. S3A). (C) Fully assembled spontaneously formed stable complex (SC) modeled with the Rosetta FlopyTail algorithm (48) simulating the partially unstructured ColB 1–55 as a floppy tail. (D) Rosetta interface score (y axis) versus interface RMSD (x axis) for output structures identified by local docking (ReplicaDock2) of ColB to FepA. RMSD is measured relative to the lowest-scoring global docking structure. There is a deep minimum resulting from the arrangement of the flexible N-linker for the FloppyTail models. Measurements corresponding to panel A are in navy blue, measurements corresponding to panel C are in yellow.

FIG 3

Partially unstructured ColB-RT 55-residue N-terminal end occupies the gap generated by the active unfolding of the FepA N-terminal half plug domain. (A) A bottom-to-top view of the hypothesized translocation pathway (stage 3) created with Rosetta by pulling the FepA N terminus into the cell. Step 1, SC complex is formed and the force-labile half-plug domain (light pink) begins to unfold. Step 2, the force-labile half-plug is partially unfolded, which allows the ColB N-terminal loop (blue) to occupy the void created by the absence of the plug domain. Step 3, the unfolding of the FepA half-plug domain creates a channel for the ColB N-terminal loop to enter. (B) The ability of ColB-81X GFP to cross-link in vivo as a function of both ColB and FepA TonB boxes. GFP fluorescence (right) and Coomassie blue stain (left) are shown. Cross-linked band is circled. (C) The top-scoring model portraying the translocation state (step 3). In this final-stage model, all the cross-link constraints are satisfied and the model is energetically favorable over other states (see Fig. S7 for energy calculations).