a The AcrA response requires TolC. Genotypes of E. coli strains inoculated on the left or proteins/pre-killed cells applied on the right are indicated below the plates. The tolC deletion reduces SR to Kan25, therefore Kan35 was used in these plates. Strain ΔtolCΔK has its KanR marker removed (ΔK; see Supplementary Table 1 and “Methods”). b A model of the AcrAB-TolC efflux pump (PDB ID: 5NG5) visualized and drawn (not to scale) using Chimera93, with TolC and AcrA components enlarged. The enlarged view for TolC shows the residues mutated in this study, with maroon and blue indicating SR+ and SR− outcomes, and similar colors in AcrA indicating the importance of the HTH region for SR+ activity. c Summary of data delineating residues in TolC, and regions in AcrA, important for SR. For TolC analysis, tolC mutants expressed from pTrC99a plasmids in a ΔtolCΔK strain were inoculated on the left (I), with purified AcrA on the right (II). For AcrA, WT cells were inoculated on the left (III), and pre-killed cells expressing the indicated C- and N-terminal truncated versions of AcrA from pTrc plasmids in a ΔacrAΔK strain (Kan marker removed) were applied on right (IV). SR response color scheme as in Fig. 1e. See Supplementary Fig. 6 for primary data. d Imaging of AcrA binding to the outer membrane of WT swarm cells. QDot705-labeled anti-FLAG antibody was used to label AcrA-FLAG, and FM-143 dye to label the outer membrane (see “Methods”). The entire field of view is shown in Supplementary Fig. 7a. Scale bar, 10 μm. See Supplementary Fig. 7d for binding of Qdot alone (control). e AcrA-TolC co-localization in representative SR+ and SR− TolC mutants identified in (c) and treated as in (d). See Supplementary Fig. 7b for brightfield images. Scale bar, 10 μm. The last panel is a control to show that AcrA is not internalized. Here, WT swarm cells were pre-incubated with AcrA and subsequently treated with trypsin followed by addition of the Qdot (see “Methods”). See Supplementary Fig. 7c for western blot analysis of these samples. Source data are provided as a Source Data file.