Author ORCID Identifier

Year of Publication


Degree Name

Doctor of Philosophy (PhD)

Document Type

Doctoral Dissertation


Arts and Sciences



First Advisor

Dr. Yinan Wei


Efflux pumps and low permeability of the outer membrane of gram-negative bacteria are major culprits in drug resistance. The most studied of these multidrug efflux pumps is the AcrAB-TolC, a complex of three proteins, the inner membrane transporter AcrB, the periplasmic adaptor AcrA and outer membrane factor TolC. This pump exports a wide range of molecules such as dyes, detergents, and antibiotics out of the bacteria system into the external environment. AcrB and TolC exist as obligate trimers, but the structure of AcrA during the assembly process is not well defined. My research aims to explore the functional and structural dynamics of the AcrAB-TolC pump to target the efflux pumps for effective antibiotic development. To achieve this, it is important to understand its structure, behavior, and function within the cell. The dominant negative effect of inactive AcrA and AcrB mutants was investigated, and we proved that once formed, the complex remains bound and does not dissociate easily. We also speculated that the assembly of the AcrAB-TolC is a precisely controlled process involving delicate proof-reading mechanisms that prevent the formation of futile complex.

I explored the role of the unresolved N-and C-terminal ends (NT and CT) in the function and activity of AcrA. At the NT, we removed residues Q31QGG34 resulting in loss of activity. We found that upon replacement of the signal peptide of AcrA with that of OmpA, function of this mutant was restored. The removal of residues 26-37 resulted in a significant loss of AcrA activity even with the OmpA signal peptide. We also found that the CT unresolved residues were not important for the function of AcrA. At the C terminus, truncation of the last 20 residues from E377 to 397 had no detectable impact on activity until lysine at position 374 (K374) when the protein became fully inactive.

Disulfide trapping was also used to probe the structure and conformation of AcrA oligomers in E. coli cells, and the interaction of AcrA and AcrB. Sites were chosen based on the tip-to-tip model from the cryo-EM AcrABZ-TolC complex structure and were used as probes to examine factors that affects the hexameric structure of AcrA, that is if the presence of AcrB or TolC affects oligomer formation. We speculate that formation of inter molecular disulfide links between AcrA and AcrB, and between neighboring AcrA subunits, would indicate that the tip-to-tip model truly reflect the assembled structure of the complex in bacteria cells. This is important since most supporting evidence for the tip-to-tip model comes from cryo-EM studies using purified proteins.

Digital Object Identifier (DOI)

Funding Information

National Institute of Health grant (number 1R56AI137020, 1R21AI142063-01,NIH/NHLBI HL142640, and NIH/NIGMS GM132443) and National Science Foundation grant (number CHE-1709381)

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