Full metadata
Title
Genetics of functional AcrAB-TolC tripartite complex assembly
Description
Intrinsic antibiotic resistance is of growing concern in modern medical treatment. The primary action of multidrug resistant strains is through over-expression of active transporters which recognize a broad range of antibiotics. In Escherichia coli, the TolC-AcrAB complex has become a model system to understand antibiotic efflux. While the structures of these three proteins (and many of their homologs) are known, the exact mechanisms of interaction are still poorly understood. By mutational analysis of the TolC turn 1 residues, a drug hypersensitive mutant has been identified which is defective in functional interactions with AcrA and AcrB. Antibiotic resistant revertants carry alterations in both TolC and AcrA act by stabilizing functional complex assembly and opening of the TolC aperture, as monitored by stability of a labile TolC mutant and sensitivity to vancomycin, respectively. Alterations in the AcrB periplasmic hairpin loops lead to a similar antibiotic hypersensitivity phenotype and destabilized complex assembly. Likewise, alterations in TolC which constitutively open the aperture suppress this antibiotic sensitivity. Suppressor alterations in AcrA and AcrB partially restore antibiotic resistance by mediating stability of the complex. The AcrA suppressor alterations isolated in these studies map to the three crystallized domains and it is concluded they alter the AcrA conformation such that it is permanently fixed in an active state, which wild type only transiently goes through when activated by AcrB. Through this genetic evidence, a direct interaction between TolC and AcrB which is stabilized by AcrA has been proposed. In addition to stabilizing the interactions between TolC and AcrB, AcrA is also responsible for triggering opening of the TolC aperture by mediating energy flow from AcrB to TolC. By permanently altering the conformation of AcrA, suppressor mutants allow defective TolC or AcrB mutants to regain functional interactions lost by the initial mutations. The data provide the genetic proof for direct interaction between AcrB and that AcrA mediated opening of TolC requires AcrB as a scaffold.
Date Created
2012
Contributors
- Weeks, Jon William (Author)
- Misra, Rajeev (Thesis advisor)
- Stout, Valerie (Committee member)
- Shi, Yixin (Committee member)
- Clark-Curtiss, Josephine (Committee member)
- Arizona State University (Publisher)
Topical Subject
Resource Type
Extent
xi, 190 p. : ill. (some col.)
Language
eng
Copyright Statement
In Copyright
Primary Member of
Peer-reviewed
No
Open Access
No
Handle
https://hdl.handle.net/2286/R.I.15167
Statement of Responsibility
by Jon William Weeks
Description Source
Retrieved on August 1, 2013
Level of coding
full
Note
thesis
Partial requirement for: Ph.D., Arizona State University, 2012
bibliography
Includes bibliographical references (p. 182-189)
Field of study: Microbiology
System Created
- 2012-08-24 06:31:27
System Modified
- 2021-08-30 01:45:19
- 3 years ago
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