Experimental evolution of TetX2: Correlating changes in fitness to their structural and functional origins

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dc.contributor.advisorShamoo, Yousif
dc.creatorWalkiewicz, Katarzyna
dc.date.accessioned2013-03-08T00:39:47Z
dc.date.available2013-03-08T00:39:47Z
dc.date.issued2012
dc.description.abstractThe study of protein evolution and adaptation resides at the junction between the disciplines of biological chemistry and evolutionary biology. We chose the B. thetaiotaomicron tetracycline resistant enzyme TetX2, as our model system to study the biophysical basis for adaptation to antibiotics; a phenomenon that continuously poses global health challenges. In the work presented here, experimental evolution and biophysical characterization were used to identify and link the physicochemical properties of TetX2 and its adaptive mutants to increased resistance to minocycline. Bacteroides thetaiotaomicron TetX2 was previously identified as a novel oxidoreductase with broad activity against tetracyclines. Experimental evolution of E. coli expressing a chromosomal copy of tet(X2) was used to identify an adaptive mutation (TetX2 T280A ) that confers higher resistance to minocycline and tigecycline. In addition to TetX2 T280A , a family of variants of TetX2 with single amino acid changes in TetX2 sequence that conferred equal or higher resistance towards MCN was identified by error-prone mutagenesis. Changes in fitness of E. coli carrying a single chromosomal copy of either wild-type or one of the mutant alleles were assessed by growth rate assays over a range of minocycline concentrations. Despite similar in vivo performances of TetX2 T280A and two other variants (TetX2 N371I and TetX2 N371T ), TetX2 T280A was the only successful mutant in the adaption experiment suggesting that mutational supply may play an important role in evolutionary dynamics of populations undergoing adaptation. The most surprising result is that the differences in growth rates among TetX2 variants arise from small changes in in vitro catalytic activity and in vivo protein expression. The steady-state kinetic studies with minocycline and NADPH suggest a binary mechanism for antibiotic inactivation by TetX2 which is supported by the structural characteristics of the enzyme. The atomic structures of the best adaptive mutant TetX2 T280A in complex with minocycline and tigecycline reveal the details of substrate recognition and show that the site of the mutation is ∼18 Å away from the active site suggesting an indirect mechanism for improved catalysis. Taken together, our data show that very small changes in the in vitro biochemical properties and expression levels can have surprisingly large fitness effects and are important during adaption. In addition, a promising preliminary mathematical model suggests that based on kinetic activity and in vivo expression levels the success of bacteria undergoing adaptation to antibiotics can be predicted.
dc.format.extent141 p.en_US
dc.format.mimetypeapplication/pdf
dc.identifier.callnoTHESIS BIOCHEM. 2012 WALKIEWICZ
dc.identifier.citationWalkiewicz, Katarzyna. "Experimental evolution of TetX2: Correlating changes in fitness to their structural and functional origins." (2012) Diss., Rice University. <a href="https://hdl.handle.net/1911/70481">https://hdl.handle.net/1911/70481</a>.
dc.identifier.digitalWalkiewiczKen_US
dc.identifier.urihttps://hdl.handle.net/1911/70481
dc.language.isoeng
dc.rightsCopyright is held by the author, unless otherwise indicated. Permission to reuse, publish, or reproduce the work beyond the bounds of fair use or other exemptions to copyright law must be obtained from the copyright holder.
dc.subjectPure sciences
dc.subjectTetX2
dc.subjectProtein evolution
dc.subjectAntibiotic resistance
dc.subjectAdaptation
dc.subjectBiochemistry
dc.titleExperimental evolution of TetX2: Correlating changes in fitness to their structural and functional origins
dc.typeThesis
dc.type.materialText
thesis.degree.departmentBiochemistry and Cell Biology
thesis.degree.disciplineNatural Sciences
thesis.degree.grantorRice University
thesis.degree.levelDoctoral
thesis.degree.nameDoctor of Philosophy
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