1,4-Dioxane Biodegradation by Mycobacterium dioxanotrophicus PH-06 Is Associated with a Group-6 Soluble Di-Iron Monooxygenase

1,4-Dioxane (dioxane) is a groundwater contaminant of emerging concern for which bioremediation may be a promising strategy. Several bacterial strains can metabolize dioxane or degrade it cometabolically. However, the molecular basis of dioxane biodegradation is only partially understood, and the gene coding for dioxane/tetrahydrofuran (THF) monooxygenase in Pseudonocardia dioxanivorans CB1190 is the only well-characterized catabolic gene. Here, we identify a novel group-6 propane monooxygenase gene cluster (prmABCD) in Mycobacterium dioxanotrophicus PH-06, which is a bacterium with superior dioxane degradation kinetics compared with CB1190. Whole genome sequencing of PH-06 revealed the existence of a single soluble di-iron monooxygenase (SDIMO). RNA sequencing and reverse transcription quantitative PCR (RT-qPCR) subsequently confirmed that all four components of this gene cluster are upregulated when PH-06 is grown on dioxane compared with growth on acetate or glucose as negative controls. This first characterization of a group-6 SDIMO associated with dioxane biodegradation suggests that dioxane-degrading genes may be more diverse than previously appreciated. A primer/probe set designed to target the large hydroxylase subunit of this gene cluster exhibited high selectivity (no false positives) and high sensitivity (detection limit = 3000-4000 gene copies/mL culture), which may be useful to help assess the presence of dioxane degraders at contaminated sites and minimize false negatives.