Date of Award

5-2022

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Department

Chemical and Biomolecular Engineering

Committee Chair/Advisor

Mark A. Blenner

Committee Member

Nicole E. Martinez

Committee Member

Jessica Larsen

Committee Member

Marc Birtwistle

Abstract

Precise detection and monitoring of nuclear fuel cycle, enrichment, and weapon development activities are critical for supporting warfighter preparation in chemical, biological, radiological, nuclear, and explosives (CBRNE) operations, clandestine activities, and nuclear compliance. A biological sensing system could serve as an alternative to traditional detection methods by using organic material naturally present in the environment to discreetly detect residual trace nuclear material. Microorganisms provide an optimal platform for an alternative sensing system; however, their response to low levels of ionizing radiation is poorly characterized. Combining the power of next-generation sequencing and transcriptomic analysis, this dissertation takes an approach to obtain a comprehensive picture of microbial response to low-dose ionizing radiation and, through characterization analysis, elucidate transcriptional signatures unique to radiation for future RNA-based sensing applications.

In this work, we aim to characterize the response of model and environmental microorganisms to in situ low-dose ionizing radiation. This dissertation is the first to describe the transcriptional response of stationary phase microorganisms, Escherichia coli, Pseudomonas putida, Saccharomyces cerevisiae, and Rhodosporidium toruloides, exposed to in situ radionuclide sources at an absorbed dose rate of 9 mGy d-1, where the biological response of microorganisms is poorly characterized. Furthermore, internal radionuclides representative of the nuclear fuel cycle, plutonium-239 (239Pu), tritium (3H), and iron-55 (55Fe), were selected to represent three major radiation types: alpha, beta, and gamma respectively.

Initially, two bacteria, E. coli and P. putida, were selected to characterize the microbial response to low-dose radiation. Cultures were subjected to internal radiation exposures for a 15-day period before preparing samples for next-generation sequencing. RNA-Seq was used to process sequencing data, and differential expression analysis was performed to identify significant changes from non-irradiated controls. Global transcriptional analysis found exposure to 239Pu initiated a moderate change in gene expression following one-day, but a negligible response after 15-days in both E. coli and P. putida. The beta emitter, 3H, had the opposite response with a minimal change after one-day of exposure in E. coli and P. putida, but the most substantial change of the radionuclides studied after 15-days. Exposure to 55Fe resulted in similar changes in gene expression in the bacteria after both one and 15-days.

Differential expression analysis revealed key similarities and differences in the gene expression of E. coli with the three radionuclide sources, 239Pu, 3H, and 55Fe, and two times, one and 15-days, studied. The transcriptional response was broad with genes encoding biosynthesis pathways of nuclear envelope components, amino acids, and siderophores, transport systems such as ABC transporters and Type II secretion proteins, and initiation of stress response and regulatory systems of temperature stress, the RpoS regulon, and oxidative stress. Differential expression analysis of the P. putida data identified significant changes in gene expression to membrane components, central metabolic processes, and motility by multiple radiation types. In addition, genes uniquely changed by a single radiation type were also observed.

The next step was to expand the comprehensive picture to a simple eukaryotic system. Again, a model yeast, S. cerevisiae, and an environmental organism, R. toruloides, were selected. Utilizing a similar analysis, the yeast systems did not produce as large of a response as their bacterial counterparts. We hypothesize the robust and adaptive nature of R. toruloides prevented any significant changes in gene expression with exposure to the internal radionuclides at this dose rate. Similarly, S. cerevisiae had a smaller response to 239Pu and 55Fe with significant gene expression changes in Ty1 transposons and metal ion processes.

This dissertation demonstrates an initial characterization of the effects of low levels of ionizing radiation on microbial species. Genes unique to a single radiation type and those commonly differentially expressed by multiple radiation types provide components for future engineering of discriminatory and general RNA-based detection of nuclear material.

Available for download on Thursday, April 13, 2023

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