Identification and characterization of monooxygenase enzymes involved in 1,4-dioxane degradation in Pseudonocardia sp. strain ENV478, Mycobacterium sp. strain ENV421, and Nocardia sp. strain ENV425

The first part of this dissertation deals with the identification and analysis of oxygenases possibly involved in a biodegradation of a possible human carcinogen, 1, 4-dioxane.;The cometabolic oxidations of 1, 4-dioxane in three gram positive bacteria were analyzed. In Mycobacterium sp. ENV421 and Nocardia sp. ENV425, 1, 4-dioxane is oxidized during growth on propane. Three putative propane oxidizing enzymes (alkane monooxygenase, soluble diiron monooxygenase, and cytochrome P450) were identified in both strains. While in strain ENV425 only soluble diiron monooxygenase was expressed after growth on propane, in strain ENV421 all three genes were expressed. Although 1,4-dioxane oxidation activity could not be detected in heterologous host gene expression studies, aliphatic oxidation activity was observed with cytochrome P450 CYP153 from strain ENV421.;In strain Pseudonocardia sp. ENV478, 1, 4-dioxane oxidation activity was induced in the presence of tetrahydrofuran (THF). A THF-inducible soluble diiron monooxygenase was identified in strain ENV478. The oxygenase gene was transcribed in a complex operonic fashion yielding multiple transcripts with varying lengths. A gene knockdown experiment using antisense technology was employed to show that this gene is essential in THF and 1,4-dioxane degradation.;The second part of this dissertation dealt with the isolation and characterization of a novel solid alkane-utilizing beta-Proteobacterium, Aquabacterium njensis sp. nov.;A novel liquid and solid alkane-degrading bacterium was isolated from hydrocarbon-contaminated soil in New Jersey, USA. The morphological, genomic, and metabolic properties of this organism were examined, including, observation with scanning electron microscopy, DNA-DNA hybridization, and nutritional screening of mainly aliphatic compounds as the sole source of carbon for growth. The preliminary screen of known alkane oxidation enzymes identified two alkane monooxygenase (AlkB) genes in the HMGZ-01 strain.;Sequence analysis of 16S rRNA showed that it has the highest similarity to the members of the genus Aquabacterium. The ability to hydrolyze urea and gelatin, NaCl tolerance, substrate utilization pattern and anaerobic growth distinguish the isolated strain from other members of this genus. We proposed the new species, Aquabacterium njensis, to be created with a single member and a type strain (HMGZ-01T ).