Rhodococci are common soil heterotrophs that possess diverse functional enzymatic activities with economic and ecological significance. In this study, the correlation between gene expression and biological removal of the water contaminant N-nitrosodimethylamine (NDMA) is explored. NDMA is a hydrophilic, potent carcinogen that has gained recent notoriety due to its environmental persistence and emergence as a widespread micropollutant in the subsurface environment. In this study, we demonstrate that Rhodococcus sp. strain RHA1 can constitutively degrade NDMA and that activity toward this compound is enhanced by approximately 500-fold after growth on propane. Transcriptomic analysis of RHA1 and reverse transcriptase quantitative PCR assays demonstrate that growth on propane elicits the upregulation of gene clusters associated with (i) the oxidation of propane and (ii) the oxidation of substituted benzenes. Deletion mutagenesis of prmA, the gene encoding the large hydroxylase component of propane monooxygenase, abolished both growth on propane and removal of NDMA. These results demonstrate that propane monooxygenase is responsible for NDMA degradation by RHA1 and explain the enhanced cometabolic degradation of NDMA in the presence of propane.Recently recognized as a drinking water contaminant (19), N-nitrosodimethylamine (NDMA) is now closely monitored by municipal water providers to minimize human exposure (3,6,20). Concern has developed due to NDMA's potent mutagenicity and carcinogenicity (11) coupled with increasing awareness of its presence as a groundwater contaminant associated with liquid rocket propellants, certain industrial processes, and chlorine-based water reuse projects (19,20,23). The combination of high subsurface mobility coupled with poor attenuation by volatilization, sorption, and abiotic and biological processes (19) has resulted in groundwater plumes that contain measurable quantities of NDMA following decades and miles of subsurface propagation (31). Despite its recalcitrance in groundwater, it has recently been shown that NDMA can be attenuated in wastewater treatment systems (23) and soils (2, 5, 32), presumably through the involvement of microorganisms. This dichotomy between persistence and potential biodegradability necessitates a more detailed understanding of the biochemical mechanisms that contribute to NDMA degradation.Microorganisms grown on substrates such as propane, methane, and toluene have been shown to rapidly oxidize NDMA in the laboratory (7,25). In these cases, evidence from inhibition and induction experiments along with observations of requisite oxygen consumption suggests that propane monooxygenases (PrMO), soluble methane monooxygenases (sMMO), and toluene monooxygenases (TMO) are most likely involved in these transformations. In addition, experiments with Escherichia coli clones expressing TMO inserts confirmed the role of toluene 4-monooxygenase (T4MO) in NDMA oxidation, while cupric selection for soluble rather than particulate MMO confirmed the role of sMMO (25). T...

ABSTRACTThe groundwater contaminant 1,4-dioxane (dioxane) is transformed by several monooxygenase-expressing microorganisms, but only a few of these, includingPseudonocardia dioxanivoransstrain CB1190, can metabolize the compound as a sole carbon and energy source. However, nothing is yet known about the genetic basis of dioxane metabolism. In this study, we used a microarray to study differential expression of genes in strain CB1190 grown on dioxane, glycolate (a previously identified intermediate of dioxane degradation), or pyruvate. Of eight multicomponent monooxygenase gene clusters carried by the strain CB1190 genome, only the monooxygenase gene cluster located on plasmid pPSED02 was upregulated with dioxane relative to pyruvate. Plasmid-borne genes for putative aldehyde dehydrogenases, an aldehyde reductase, and an alcohol oxidoreductase were also induced during growth with dioxane. With both dioxane and glycolate, a chromosomal gene cluster encoding a putative glycolate oxidase was upregulated, as were chromosomal genes related to glyoxylate metabolism through the glyoxylate carboligase pathway. Glyoxylate carboligase activity in cell extracts from cells pregrown with dioxane and inRhodococcus jostiistrain RHA1 cells expressing the putative strain CB1190 glyoxylate carboligase gene further demonstrated the role of glyoxylate metabolism in the degradation of dioxane. Finally, we used13C-labeled dioxane amino acid isotopomer analysis to provide additional evidence that metabolites of dioxane enter central metabolism as three-carbon compounds, likely as phosphoglycerate. The routing of dioxane metabolites via the glyoxylate carboligase pathway helps to explain how dioxane is metabolized as a sole carbon and energy source for strain CB1190.

The bacterium Pseudonocardia dioxanivorans CB1190 grows on the cyclic ethers 1,4-dioxane (dioxane) and tetrahydrofuran (THF) as sole carbon and energy sources. Prior transcriptional studies indicated that an annotated THF monooxygenase (THF MO) gene cluster, thmADBC, located on a plasmid in CB1190 is upregulated during growth on dioxane. In this work, transcriptional analysis demonstrates that upregulation of thmADBC occurs during growth on the dioxane metabolite ␤-hydroxyethoxyacetic acid (HEAA) and on THF. Comparison of the transcriptomes of CB1190 grown on THF and succinate (an intermediate of THF degradation) permitted the identification of other genes involved in THF metabolism. Dioxane and THF oxidation activity of the THF MO was verified in Rhodococcus jostii RHA1 cells heterologously expressing the CB1190 thmADBC gene cluster. Interestingly, these thmADBC expression clones accumulated HEAA as a dead-end product of dioxane transformation, indicating that despite its genes being transcriptionally upregulated during growth on HEAA, the THF MO enzyme is not responsible for degradation of HEAA in CB1190. Similar activities were also observed in RHA1 cells heterologously expressing the thmADBC gene cluster from Pseudonocardia tetrahydrofuranoxydans K1.

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