Fig. 1

OMP-bound ColE9 reduces the lateral mobility of TolA in the IM. a Left-hand panel, central components of the Tol-Pal complex (TolQ, TolR and TolA in the IM, TolB in the periplasm and the lipoprotein Pal in the OM) and the OMPs BtuB and OmpF. ColE9 exploits all these proteins during its import with the exception of Pal. TolA was tagged with GFP at its N-terminus while TolQ was fused to mCherry at its C-terminus (not shown). Also shown is the location of a histidine residue (H22) in the transmembrane helix of TolA that is critical for coupling to the PMF via TolQ and TolR³⁶. Right-hand panel, binding of fluorescently labelled nuclease bacteriocin ColE9 to BtuB/OmpF recruits TolB, which in turn binds TolA in the IM⁴³ and establishes a protein bridge across the cell envelope. The colicin contains an internal disulphide bond that blocks import. Figure adapted from Rassam, P., Copeland, N.A., Birkholz, O., Tóth, C., Chavent, M., Duncan, A.L., Cross, S.J., Housden, N.G., Kaminska, R., Seger, U., Quinn, D.M., Garrod, T.J., Sansom, M.S.P., Piehler, J., Baumann, C.G. & Kleanthous, C. Supramolecular assemblies underpin turnover of outer-membrane proteins in bacteria. Nature 523, 333–336 (2015). b Confocal FRAP data for E. coli JW0729/pNP4, pRP5 cells expressing GFP-TolA (not induced) and BtuB (induced), respectively, in the absence (−) and in the presence (+) of ColE9 (300 nM) and the corresponding recovery curves (averages from 30 cells). Asterisk, p < 0.01 from a Student’s t test. c Single-molecule TIRFM tracking data of GFP-TolA in the IM before and after ColE9 forms its transenvelope complex showing how OMP-bound colicin restricts the lateral mobility of TolA. Measurements of the TolA diffusion coefficient from mean square displacement plots of single-molecule trajectories are shown alongside, where n = 30. Those of TolA in the presence of ColE9 are typical of OMPs such as BtuB¹⁶. Asterisk, p < 0.001 from a Mann–Whitney U-test. Scale bars, 1 μm