Antibiotic resistance in gram-negative bacteria is often mediated by efflux pumps such as AcrAB-TolC. TolC, the outer membrane channel of this system, is a key therapeutic target, and we have previously shown that fragments of the bacteriocin colicin E1 can bind TolC and inhibit its efflux function (Budiardjo et al., eLife 2022). However, the mechanism by which full-length colicin E1 translocates into cells remains unresolved. Based on current understanding of interactions between antimicrobial peptides and bacterial cells, two models have been proposed. Clarifying which pathway colicin E1 follows requires resolving how colicin engages the vitamin B12 transporter BtuB and TolC during translocation. To distinguish between the total thread model and the pillar model, we tracked single molecules of Cy3-labeled colicin E1 in living E. coli cells. By optimizing labeling density, incubation time, and concentration, we achieved sparse puncta suitable for single-molecule tracking. We analyzed the positioning and diffusion of colicin E1 in wild-type (WT) and αTolC strains. We detected mobile colicin E1 molecules inside the WT cells, but the colicin E1 remained surface-bound in αTolC mutants, indicating that TolC is essential for cell entry. Microplate assays confirmed this requirement: colicin E1 efficiently kills WT cells but showed no killing activity in αTolC strains. Together, these results demonstrate that TolC is required for both translocation and cytotoxic activity of colicin E1, and ongoing experiments are measuring the role of BtuB. This study provides mechanistic insight into bacteriocin entry and informs strategies to exploit outer membrane proteins for overcoming antibiotic resistance.
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