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. Author manuscript; available in PMC: 2011 Jan 1.

Published in final edited form as: Methods Mol Biol. 2010;634:343–354. doi: 10.1007/978-1-60761-652-8_24

Fig. 2 — Effect of dsbA mutation on the activities of AcrB Cys mutants evaluated by drug susceptibility of the host strains. (A) Side view of a gradient plate containing cholate in the lower layer. (B) An example of measurement of bacterial drug susceptibility using the gradient plate. Fully functional control, the ΔacrB strain harboring wild-type AcrB-expressing-plasmid, pSCLBH, grew well up to the region containing cholate at high concentrations, while the nonfunctional control, the host with vector only (pSPORT1), grew only for a short length. The relative activity of each mutant was calculated from the length of its growth compared to the two controls, pSCLBH (100%) and pSPORT1 (0%). (C) Activities of AcrB single- and double-Cys mutants and the effect of dsbA mutation. AcrB proteins containing double-Cys mutations on both sides of the cleft are strongly compromised in function in dsbA⁺ host strain AG100YB (gray bars, three columns on the right). In contrast in the dsbA host AG100YBD (black bars), the CL-F666C/T678C and CL-F666C/Q830C double mutants largely retain their transport activities, indicating that the inactivation of these two double-Cys mutants in the dsbA⁺ host is due to the formation of disulfide bonds. This is one of the biochemical evidences that the conformational changes, including the closure of the cleft suggested from the asymmetrical trimer structure, indeed occur in vivo and that they are required for AcrB function. (Reproduced from ref. 12.)

Fig. 2 — Effect of dsbA mutation on the activities of AcrB Cys mutants evaluated by drug susceptibility of the host strains. (A) Side view of a gradient plate containing cholate in the lower layer. (B) An example of measurement of bacterial drug susceptibility using the gradient plate. Fully functional control, the ΔacrB strain harboring wild-type AcrB-expressing-plasmid, pSCLBH, grew well up to the region containing cholate at high concentrations, while the nonfunctional control, the host with vector only (pSPORT1), grew only for a short length. The relative activity of each mutant was calculated from the length of its growth compared to the two controls, pSCLBH (100%) and pSPORT1 (0%). (C) Activities of AcrB single- and double-Cys mutants and the effect of dsbA mutation. AcrB proteins containing double-Cys mutations on both sides of the cleft are strongly compromised in function in dsbA⁺ host strain AG100YB (gray bars, three columns on the right). In contrast in the dsbA host AG100YBD (black bars), the CL-F666C/T678C and CL-F666C/Q830C double mutants largely retain their transport activities, indicating that the inactivation of these two double-Cys mutants in the dsbA⁺ host is due to the formation of disulfide bonds. This is one of the biochemical evidences that the conformational changes, including the closure of the cleft suggested from the asymmetrical trimer structure, indeed occur in vivo and that they are required for AcrB function. (Reproduced from ref. 12.)