Degree

Doctor of Philosophy (PhD)

Department

Biological Sciences

Document Type

Dissertation

Abstract

Multiple Antibiotic Resistance Regulators (MarRs) are DNA binding proteins involved in bacterial survival and pathogenesis. My PhD dissertation focuses on a MarR homolog, OstR, which regulates genes encoding major multidrug transporters, a resistance-nodulation-division (RND) and an E. coli multidrug resistance B (EmrB) efflux pump homolog in Burkholderia thailandensis. The ostR gene is divergently oriented from genes encoding a hypothetical protein (Hyp) and an RND transporter and adjacent to a gene encoding an EmrB transporter. OstR binds to the promoters of each gene (Kd = 64.9 ± 8.0 nM, Kd = 126.1 ± 10.9 nM and Kd = 6.7 ± 0.8, respectively), and expression of rnd and hyp is ~2-fold upregulated in the ΔostR strain, whereas emrB expression is increased ~25-fold. DNA binding is differentially altered on oxidation of OstR, depending on the nature of the oxidant, as reflected in altered levels of gene expression in vivo. OstR is a dimeric protein harboring six cysteine residues. Under conditions of oxidative stress, OstR forms inter- and intra-molecular disulfide bonds. Notably, the three cysteine residues (Cys-3, -4 and -169) contribute differentially to the structural stability of OstR. This dissertation also elucidates mechanisms used to extrude common antibiotics. Inactivation of emrB leads to increased sensitivity to trimethoprim. Inactivation of ostR leads to resistance to tetracycline and nalidixic acid, perhaps due to increased emrB expression. Unexpectedly, inactivation of emrB also led to increased resistance to tetracycline; induction of amrB (encoding RND efflux system AmrAB-OprA) in the ΔemrB strain suggests that this resistance is due to failure to export tetracycline through EmrB. I also report a transcription factor (Hyp) belonging to a novel protein family, which regulates expression of both rnd and emrB. Expression of emrB and rnd is increased ~14-fold and ~2-fold in the Δhyp strain, with relative changes similar to that of the ΔostR strain. My study also identifies gentamicin-mediated downregulation of oxyR. OxyR was previously shown to activate hyp expression; consistent with this observation, expression of both hyp and ostR is repressed by gentamicin. Thus, this study illustrates a web of mechanisms involved in controlling expression of genes encoding multidrug efflux pumps.

Date

3-14-2019

Committee Chair

Grove, Anne

Available for download on Wednesday, March 11, 2026

Share

COinS