Journal
FRONTIERS IN MICROBIOLOGY
Volume 6, Issue -, Pages -Publisher
FRONTIERS MEDIA SA
DOI: 10.3389/fmicb.2015.00004
Keywords
RND; oligomerization; membrane transporter; efflux pump; protein thermal stability
Categories
Funding
- National Science Foundation [MCB-1158036]
- National Institute of Health [1R21AI103717]
- Direct For Biological Sciences [1158036] Funding Source: National Science Foundation
- Div Of Molecular and Cellular Bioscience [1158036] Funding Source: National Science Foundation
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AcrAB-TolC and their homologs are major multidrug efflux systems in Gram-negative bacteria. The inner membrane component AcrB functions as a trimer. Replacement of Pro223 by Gly in AcrB decreases the trimer stability and drastically reduces the drug efflux activity. The goal of this study is to identify suppressor mutations that restore function to mutant AcrBp223G and explore the mechanism of function recovery. Two methods were used to introduce random mutations into the plasmid of AcrB(P223G). Mutants with elevated drug efflux activity were identified, purified, and characterized to examine their expression level, trimer stability, interaction with AcrA, and substrate binding. Nine single-site repressor mutations were identified, including T199M, D256N, A209V, G257V, M662I, Q737L, D788K, P800S, and E810K. Except for M662I, all other mutations located in the docking region of the periplasmic domain. While three mutations, T199M, A209V, and D256N, significantly increased the trimer stability, none of them restored the turner affinity to the wild type level. M662, the only site of mutation that located in the porter domain, was involved in substrate binding. Our results suggest that the function loss resulted from compromised AcrB trimerization could be restored through various mechanisms involving the compensation of trimer stability and substrate binding.
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