Alvarez, Pedro J2019-05-172020-05-012019-052019-04-16May 2019He, Ya. "Isolation of Novel 1,4-Dioxane Degraders and Investigation of Responsible Catabolic Genes." (2019) Diss., Rice University. <a href="https://hdl.handle.net/1911/105926">https://hdl.handle.net/1911/105926</a>.https://hdl.handle.net/1911/1059261,4-Dioxane (dioxane) is a widespread groundwater contaminant with potential carcinogenicity. Several strains capable of degrading dioxane have been isolated. Nearly all these strains were isolated from wastewater treatment plants or dioxane-impacted sites. However, before this research, the potential for encountering dioxane degraders in uncontaminated environments, which is important to assess the distribution and potential ubiquity of dioxane degraders and the associated natural attenuation potential, had not been considered in the literature. Also, the genetic basis for dioxane degradation was only partially understood, limited performance assessment of bioremediation and natural attenuation. This dissertation contributes to advancing our basic understanding of both the occurrence of dioxane degraders and the genetic basis of dioxane degradation. Two bacterial consortia were enriched from uncontaminated soil by virtue of their ability to grow on dioxane as a sole carbon and energy source. Their specific dioxane degradation rates at 30℃, pH=7 (i.e., 5.7 to 7.1 g-dioxane/g-protein/day) were comparable to those of two dioxane-metabolizing archetypes: Pseudonocardia dioxanivorans CB1190 and Mycobacterium dioxanotrophicus PH-06. Based on 16S rRNA sequencing, Mycobacterium was the dominant genus. Acetylene inhibition tests suggested that dioxane degradation was mediated by monooxygenase enzymes. Although qPCR assays indicated the responsible gene was not the only previously known gene cluster (thmADBC). DNA sequence analyses also showed 3- to 7-fold enrichment of group-5 and group-6 soluble diiron monooxygenase (SDIMO) genes relative to the original soil samples. Both consortia degraded dioxane at low initial concentrations (300 µg/L) below detectable levels (5 µg/L) in bioaugmented microcosms prepared with impacted groundwater. A novel group-6 propane monooxygenase gene cluster (prmABCD) was identified in Mycobacterium dioxanotrophicus PH-06, which is a bacterium with superior dioxane degradation kinetics (Ks = 78 ± 10 mg/L, Y = 0.16 g protein / g dioxane) compared with Pseudonocardia dioxanivorans CB1190 (Ks = 145 ± 17 mg/L, Y = 0.11 g protein / g dioxane). Whole genome sequencing of PH-06 revealed the existence of a single SDIMO gene cluster. RNA sequencing and reverse transcription quantitative PCR (RT-qPCR) subsequently confirmed that all four components of this gene cluster were upregulated when PH-06 was grown on dioxane compared with growth on acetate or glucose as negative controls. A primer/probe set targeting the large hydroxylase subunit (the active site for hydroxylation) of this newly identified gene cluster (prmABCD) was designed. qPCR assays showed that this primer/probe set exhibited high selectivity (no false positives) and high sensitivity (detection limit = 3000 – 4000 gene copies / mL culture). Finally, to better monitor the existence and expression of dioxane degrading genes, we proposed a strategy to create a reporter strain by inserting an engineered gene cassette into the dioxane degrading bacteria. This gene cassette would contain the promoter of the dioxane degrading gene cluster and a reporter gene. Ideally, when the dioxane degrading gene cluster is induced by dioxane, the reporter gene would be induced and expressed accordingly. The expressed signal would be used to easily monitor the activities of dioxane degraders. Despite many attempts to build this reporter strain, including the use of different shuttle vectors, different reporter genes, and different electroporation protocols, we were not able to build this reporter strain. Nevertheless, preliminary results and recommendations are presented to facilitate future accomplishment of this task. Overall, the isolation of both consortia shows that dioxane degrading bacteria (and the associated natural attenuation potential) exist even in some uncontaminated soils and may be enriched to broaden bioaugmentation options for sites experiencing insufficient dioxane catabolic capacity. The characterization of a group-6 SDIMO associated with dioxane biodegradation suggests that dioxane degrading genes may be more diverse than previously appreciated. The newly designed primer /probe set may be useful to help assess the presence of dioxane degraders at contaminated sites and minimize false negatives. The trial and error of construction of dioxane degrading reporter strain would provide valuable advice for further work or related genetic engineering work.application/pdfengCopyright 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.1,4-dioxanebiodegradationconsortiamonooxygenasebiomarkerSDIMOsMycobacterium dioxanotrophicus PH-06Pseudonocardia dioxanivorans CB1190prmABCDreporter strainThesis2019-05-17