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theses

A novel strain of Burkholderia fungorum was isolated from the Department of Energy’s Rifle Integrated Field-Scale Subsurface Research Challenge Site in Rifle Colorado which can utilize uranium as a terminal electron acceptor under anaerobic conditions. The Rifle strain demonstrated growth with uranyl acetate as the terminal electron acceptor (1-10 μM) in a dose-dependent manner concomitant with decreases in soluble uranium. Each 1 μM increase of uranium initiated an increase of ~ 0.5 x 106 + 1.3 x 105 cells/ml, concurrent with a shift in uranium solubility from 90% soluble to 70-97% insoluble at the end of the time course. Cultures exposed to 5-10 μM uranium did not grow, but exhibited significantly decreased levels of soluble uranium at time point zero (78-18%), indicating either an additional cellular precipitation/detoxification mechanism in response to increased uranium concentrations or a media solubility threshold. To identify the capability of Burkholderia fungorum strain Rifle to precipitate elevated levels uranium, cultures were grown aerobically in the absence and presence of uranium (0-200 μM). Cultures grown in the presence of uranium exhibited variable lag phases, indicating a toxic response to uranium. However, the fraction of uranium which remained within the soluble fraction exhibited no difference over the course of growth, excluding bio-precipitation as mechanism of toxicity resistance under aerobic conditions. To identify possible genes of interest in regards to the anaerobic reduction of uranium or the overall toxicity resistance mechanism, the genome of Burkholderia fungorum strain Rifle was sequenced. The draft genome was assembled and annotated for prospective gene functions. A subset of seven genes sharing homology with genes of known uranium reducing species was earmarked for redox reaction capabilities. Additionally, genes associated with heavy metal toxic response were tested for up-regulation by transcription analysis using cultures shifted from uranium (-) to uranium (+) conditions. Reverse Transcriptase-PCR analysis demonstrated no variation in transcriptional level from day 0 of uranium transition to day 3 compared to the expression levels of the maleate isomerase indicating that while all select genes chosen were constitutively expressed. The nucleotide sequencing reads for these strains were also submitted for functional annotation of sequencing reads via mifaser to generate an enzymatic enrichment profile for these strains. This profile revealed that the Rifle strain exhibited enrichment for c-class cytochrome and P-type ATPase functions. An in silico proteome was also generated to identify genes of functionally similar phenotype, including a number which belong to a different genus, using Fusion phenotypic classification. Comparisons for proteins of similar function identified 42 proteins shared across genus, most with clearly defined functions but including 4 hypothetical proteins of interest. However, no gene was identified which was up-regulated by exposure to uranium. Further study of the remaining hypothetical proteins is warranted to identifying the genes involved with uranium respiration/detoxification in B. fungorum Rifle.

ETD_8497

Ph.D.

Includes bibliographical references

by Nicole Michele Koribanics

doi:10.7282/T3CZ3B9G

ETD doctoral

The author owns the copyright to this work.