Thesis (Ph. D.)--University of Wisconsin--Madison, 1975. / Typescript. Vita. eContent provider-neutral record in process. Description based on print version record. Bibliographical references.

Thesis (Ph. D.)-- University of Washington, 2005. / Vita. Includes bibliographical references (leaves 145-163).

Thesis (Ph. D.)--University of Washington, 2000. / Vita. Includes bibliographical references (leaves 128-136).

Antibiotics are one of the most important advances in medical science, but today, antibiotic-resistant bacteria threaten this legacy. We risk losing our ability to treat acute infections, perform invasive surgeries, and exploit immunosuppressive therapies like transplantation and cancer chemotherapy. The antibiotics we use today have ancient roots and have been produced by microbial denizens of the soil for millions of years before we adopted them in the 20th century. This history has modern consequences, as strategies to resist these compounds have evolved in concert for millions of years. The result is a vast reservoir of antimicrobial resistance that exists in environmental bacteria, which have the potential to be mobilized into human pathogens and cripple our antibiotic arsenal. Here, I set out to deepen our understanding of the environmental resistome, focusing on the rifamycin antibiotics. These compounds inhibit bacterial RNA polymerase and are frontline agents for treating tuberculosis. Environmental bacteria from the phylum Actinobacteria induce the production of resistance enzymes in response to these compounds. Although mechanistic questions remain, we demonstrate that this induction stems from the inhibition of RNA polymerase by rifamycins. The induction process is known to require a specific DNA motif; here, I identify additional sequences as part of this motif and use this information to map inducible rifamycin resistance across the entire phylum. The most common rifamycin-inducible gene was an uncharacterized family of proteins annotated as DNA helicases. I investigated these proteins and discovered that they bind to RNA polymerase and displace rifamycin antibiotics, a novel mechanism of rifamycin resistance. Lastly, we repurposed this inducible system to develop an assay to screen for novel RNA polymerase inhibitors. From this screen, we identified a rifamycin immune to a common environmental resistance enzyme and a new family of rifamycin antibiotics. / Thesis / Doctor of Philosophy (PhD) / Our antibiotic arsenal consists primarily of metabolites produced by soil microbes, which humanity repurposed into life-saving medicines in the 20th century. As a direct result of the natural origin of antibiotics, resistant bacteria exist in these same environments, independent of human use. Individual genetic determinants from this reservoir can emerge in pathogenic bacteria without warning and render antibiotics ineffective. The aim of this work was to understand how environmental bacteria resist the rifamycin class of antibiotics. Firstly, I investigated the ability of some bacteria to sense the presence of rifamycins, and in response produce proteins to protect themselves. I discovered that this process requires specific DNA sequences nearby resistance genes. Using this DNA sequence as a guide I cataloged resistance genes in thousands of bacterial genomes and discovered a new mechanism of rifamycin resistance. Lastly, I exploited this rifamycin sensing system to discover new antibiotics from soil microbes.

The rifamycins are a class of antibiotics which were once used almost exclusively to treat tuberculosis, but are currently receiving renewed interest. Resistance to rifamycins is most commonly attributed to mutations in the drug target, RNA polymerase. Yet environmental isolates are also able to enzymatically inactivate rifamycins in a number of ways. Recently, rifamycin resistance determinants from the environment were found to be closely associated with a so called rifamycin associated element (RAE). The region containing the RAE from an environmental strain was shown to induce gene expression in the presence of rifamycins, hinting at an inducible system for rifamycin resistance. In this work, we examine the RAE from a model organism for Streptomyces genetics, Streptomyces venezuelae. We confirm that the promoter region containing the RAE upstream of a rifamycin monooxygenase rox is inducible by rifamycins. The strains of S. venezuelae generated in this work can be used in future genetic studies on the RAE. As well, the rifamycin monooxygenase Rox was purified for the first time and characterized biochemically. The structure of Rox was obtained with and without the substrate rifampin. Steady state kinetics for the enzyme were determined with a number of substrates, and its ability to confer resistance to rifamycins was examined. Monooxygenated rifamycin SV compound was purified and structurally characterized by NMR analysis. We proposed an aromatic hydroxylase type mechanism for Rox, in which the enzyme hydroxylates the aromatic core of the rifamycin scaffold and causes a non-enzymatic C-N bond cleavage of the macrolactam ring. This is a new mechanism of rifamycin resistance, and sheds some light on the decomposition of rifamycins mediated by monooxygenation, which is still poorly understood. / Thesis / Master of Science (MSc) / Antibiotic resistance represents a major threat to global health. Infections that were once readily treatable are no longer so due to the rise in multidrug resistant bacteria. As our arsenal of effective antibiotics is depleted, new drugs are being discovered less and less frequently. This has caused the scientific community to get creative in coming up with treatments: trying combinations of antibiotics, using antibiotics which were once considered too toxic, and repurposing antibiotics for different bacteria. Rifamycins are a class of antibiotics most commonly used in the treatment of tuberculosis. However, they are becoming more widely used as a result of antibiotic resistance. There are a number of different ways bacteria can become resistant to the harmful effects of rifamycins: by modifying the target so the drug can no longer bind to it, actively pumping the drug out of the cell, or by changing the drug in some way so it is no longer effective. Bacteria in the environment use antibiotics as a form of chemical warfare to gain an advantage over their neighbours; therefore, they have had millions of years to evolve very effective methods of antibiotic resistance. By surveying what kinds of antibiotic resistance are in the environment, we can predict what we might see one day in a medical setting. In this thesis, I have studied a protein that bacteria make to inactivate rifamycins. The rifamycin monooxygenase Rox adds an oxygen to the rifamycin scaffold; this causes spontaneous cleavage of the rifamycin backbone and changes the conformation of the drug so it can no longer bind to its target. I have also investigated the regulation of this and other genes in the bacterial strain Streptomyces venezuelae. By understanding how this process works, we can potentially design inhibitors to stop this from happening, should this method of resistance ever become clinically prevalent.

Thesis (Ph. D.)--University of Washington, 1999. / Vita. Includes bibliographical references (leaves 298-336).