Functional genomics of the bacterial degradation of the emerging water contaminants: 1,4-dioxane and N-nitrosodimethylamine (NDMA) by Christopher Michael Sales A dissertation submitted in partial satisfaction of the requirements for the degree of Doctor of Philosophy in Engineering-Civil and Environmental Engineering in the Graduate Division of the University of California, Berkeley Committee in charge: Professor Lisa Alvarez-Cohen, Chair Professor Kara L. Nelson Professor Mary K. Firestone Spring 2012 Functional genomics of the bacterial degradation of the emerging water contaminants: 1,4-dioxane and N-nitrosodimethylamine (NDMA) Copyright 2012 By Christopher Michael Sales Abstract Functional genomics of the bacterial degradation of the emerging water contaminants: 1,4-dioxane and N-nitrosodimethylamine (NDMA) by Christopher Michael Sales Doctor of Philosophy in Engineering - Civil & Environmental Engineering University of California, Berkeley Professor Lisa Alvarez-Cohen, Chair The emerging water contaminants 1,4-dioxane and N-nitrosodimethylamine (NDMA) are toxic and classified as probable human carcinogens. Both compounds are persistent in the environment and are highly mobile in groundwater plumes due to their hydrophilic nature. The major source of 1,4-dioxane is due to its use as a stabilizer in the chlorinated solvent 1,1,1-trichloroethane. The presence of NDMA as a water contaminant is related to the release of rocket fuels and its formation in the disinfection of water and wastewater. Prior studies have demonstrated that bacteria expressing monooxygenases are capable of degrading 1,4-dioxane and NDMA. While growth on 1,4-dioxane as a sole carbon and energy source has been reported in Pseudonocardia dioxanivorans CB1190 and Pseudonocardia benzenivorans B5, it is also co-metabolically degradable by a variety of monooxygenase-expressing strains. In contrast, NDMA has only been observed to biode- grade co-metabolically after growth on some monooxygenase-inducing substrates. The fastest rates of NDMA degradation occur in Rhodococcus sp. RR1 and Mycobacterium vaccae JOB5 after growth on propane. Pathways have been proposed for 1,4-dioxane biodegradation in P. dioxanivorans and for NDMA biodegradation in propane-induced Rhodococcus sp. RR1 based only on identified intermediates. The overall goal of this dissertation is to gain a better understanding of the genes and biological pathways respon- sible for degradation of 1,4-dioxane and NDMA. Due to the lack of molecular sequence information for organisms capable of growth on 1,4-dioxane, the genome of P. dioxanivorans strain CB1190 was sequenced in this study. The genome has a total size of 7,440,794 bp and consists of four replicons: a circular chromosome, a circular plasmid pPSED01, an unclosed circular plasmid pPSED02, and a linear plasmid pPSED03. Analysis of this genome sequence revealed the presence of eight multicomponent monooxygenases: a propane monooxygenase, a phenol mono- oxygenase, a 4-hydroxyphenylacetate monooxygenase, four aromatic (toluene) mono- oxygenases, and a tetrahydrofuran (THF) monooxygenase. A total of 92 genes identified as putative dioxygenases were identified. Protein-encoding genes involved in transport systems, signal transduction systems, secretion systems, and heavy-metal and antibiotic resistance were identified. The presence of pathways for carbon and nitrogen metabolism was examined. A complete Calvin-Benson-Bassham carbon fixation pathway was found and a number of carboxylases that function in other carbon fixation pathways were identi- fied. AlthoughP. dioxanivorans has been reported to fix nitrogen, no nitrogenase genes 1 were found. The genome sequence of P. dioxanivorans was compared to other sequenced genomes of members in the family Pseudonocardiaceae, including Amycolatopsis medi- terranei S699, Amycolatopsis mediterranei U32, Pseudonocardia sp. P1, Pseudonocardia sp. P2, Saccharomonaspora azurea NA-128, Saccharomonospora paurometabolica YIM 9007, Sacharomonospora viridis DSM43017, Saccharopolyspora erythraea NRRL2338, and Saccharopolyspora spinosa NRRL18395. Genome-aided approaches were employed to identify the protein-encoding genes in- volved in the metabolic degradation of 1,4-dioxane in P. dioxanivorans. These ap- proaches included whole genome expression microarray transcriptomics, quantitative reverse transcriptase PCR (qRT-PCR), enzyme assays, and heterologous expression clones. Genes differentially expressed during growth on 1,4-dioxane, glycolate (a pre- viously identified degradation intermediate), and pyruvate (control) were analyzed to determine genes differentially expressed and involved in the metabolism of 1,4-dioxane. Based on the differentially expressed genes, the 1,4-dioxane degradation pathway was revised and annotated with the enzymes known to catalyze specific transformation steps. Up-regulation of genes were confirmed and quantified by qRT-PCR. Transcriptional analyses, isotopic tracer analyses with 1,4-[U-13C]-dioxane, and glyoxylate carboligase enzymatic activity assays confirmed the role of glyoxylate as a central intermediate in the degradation of 1,4-dioxane. Specifically, transcriptional analyses indicated that the THF monooxygenase, encoded by thmADBC, is up-regulated during growth on 1,4-dioxane and THF. Furthermore, Rhodococcus jostii RHA1 clones heterologously expressing the P. dioxanivorans genes thmADBC demonstrated 1,4-dioxane and THF degradation activity. A surprising result with the THF monooxygenase expressing clones was the accumulation of the intermediate β-hydroxyethoxyacetic acid (HEAA). This result, combined with the non-inhibitory effect of acetylene on HEAA degradation by 1,4-dioxane grown P. dioxa- nivorans indicates that a monooxygenase is not involved in the transformation of HEAA as previously hypothesized. Transcriptomic microarray analysis of THF- and succinate- grown cells led to the first proposed THF metabolic degradation pathway forP. dioxaniv- orans. A novel finding of this transcriptomic microarray analysis was the identification of an alcohol dehydrogenase up-regulated during growth on THF that could catalyze the conversion of 2-hydroxytetrahydrofuran to γ-butyrolactone. The genome sequence of Rhodococcus jostii RHA1 was utilized to determine the pro- pane-induced monooxygenase responsible for NDMA degradation. The degradation of NDMA was characterized in R. jostii RHA1 grown on propane and on non-inducing substrates (LB medium, soy broth, and pyruvate). Propane enhanced the removal rate of NDMA by nearly two orders of magnitude compared to constitutive degradation during growth on non-inducing substrate. Transcriptomic microarray and qRT-PCR analyses demonstrated that propane elicits the up-regulation of a propane monooxygenase gene cluster. Genetic knockouts of the prmA gene encoding the large hydroxylase component of the propane monooxygenase were unable to grow on propane and degrade NDMA. These results indicate that the propane monooxygenase is responsible for NDMA degra- dation by R. jostii and explain the enhanced co-metabolic degradation of NDMA in the presence of propane. With the newly gained knowledge of the role of propane monooxy- genase in NDMA degradation, oligonucleotide degenerate primers targeting prmA were designed and were identify and quantify the presence of propane monooxygenase genes 2 in Rhodoccocus sp. RR1 and M. vaccae JOB5. A homolog to prmA was found in Rhodoc- cocus sp. RR1 but not in M. vaccae JOB5. The prmA gene in Rhodococcus sp. RR1 was up-regulated during growth on propane relative to pyruvate. Characterization of the kinet- ics of propane-enhanced NDMA degradation showed that Rhodococcus sp. RR1 and M. vaccae possess similar maximum transformation rates (44 ± 5 and 28 ± 3 mg NDMA(mg -1 -1 protein) h , respectively). However, a comparison of half saturation constants (Ks,n) and NDMA degradation in the presence of propane revealed pronounced differences be- -1 tween the strains. The Ks,n for Rhodococcus sp. RR1 was 36 ± 10 mg NDMA L while the propane concentration needed to inhibit NDMA rates by 50% (Kinh) occurred at 7,700 mg propane L-1 (R2 = 0.9669). In contrast, M. vaccae had a markedly lower affinity for -1 NDMA verses propane with a calculated Ks,n of 2,200 ± 1,000 mg NDMA L and Kinh of 120 mg propane L-1 (R2 = 0.9895). Differences between the propane monooxygenases in Rhodococcus sp. RR1 and the unidentified enzyme(s) inM. vaccae may explain the disparities in NDMA degradation and inhibition kinetics between these strains. 3 To Hilary i ii Table of Contents Abstract………………………………………………………………………………….....1 Dedication………………………………………………………………………………….i Table of Contents…………………………………………………………………………iii List of Figures…………………………………………………………………………...viii List of Table………………………………………………………………………...…..…x Acknowledgements…………………………………………………………………...….xii Chapter 1: Introduction and Objectives...........................................................................1 1.1 Introduction……………………………………………………………………………2 1.2 Research goal and specific objecties.......……………………………………………...3 1.3 Dissertation overview……………………………………………………………........4 Chapter 2: Background and Literature Review..............................................................5 2.1 Introduction…………………………………………………………………...............6 2.2 1,4-dioxane and NDMA as emerging contaminants…………………………..............7 2.2.1 Drinking water regulations and health effects of 1,4-dioxane…………................7 2.2.2 Uses of 1,4-dioxane………………………………………………….....................9 2.2.3 Occurrence