Oxidation of the cyclic ethers 1,4-dioxane and tetrahydrofuran by a monooxygenase in two Pseudonocardia species

Abstract

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 β-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.

Fig 1

THF and 1,4-dioxane degradation pathways. Proposed reaction pathways for THF and dioxane metabolism in CB1190, annotated with enzymes based on transcriptomic results in this study. The THF pathway is adapted from the pathways proposed by Bernhardt and Diekmann (1), Skinner et al. (7), and Thiemer et al. (22). The 1,4-dioxane pathway is adapted from Grostern et al. (17). The gene locus tags for the enzymes in the THF pathway are Psed_6976 to Psed_6979 for the THF monooxygenase thmADBC, Psed_0131 for alcohol dehydrogenase, Psed_6971 for hydroxyacid-oxoacid transhydrogenase, Psed_6970 for 4-hydroxybutyrate dehydrogenase, Psed_6975 for succinate semialdehyde dehydrogenase (sad), and Psed_6981 for aldehyde dehydrogenase (aldH). The locus tags are the same for the dioxane pathway, except for those for the secondary alcohol dehydrogenase (Psed_0131, Psed_2070, Psed_4156, or Psed_6971) and the aldehyde dehydrogenase (Psed_6971, Psed_6975, or Psed_6981). TCA, tricarboxylic acid cycle.

Fig 2

Functional activity of heterologous CB1190 THF MO expression clones. (A) THF removal by RHA1 containing plasmid pTip-CB1190-thfmo (■), the empty vector pTip-QC2 (control) (□), and abiotic samples (○). (B) Removal of dioxane and accumulation of HEAA by plasmid pTip-CB1190-thfmo clones, (■, dioxane; ▲, HEAA), empty vector pTip-QC2 clones (□, dioxane; △, HEAA), and abiotic samples (○, dioxane; ×, HEAA). Error bars indicate standard deviations. All conditions were run in triplicate.

Fig 3

HEAA and dioxane degradation by CB1190. (A) Disappearance of HEAA during growth of CB1190 on HEAA as the sole carbon and energy source. Effect of acetylene exposure on HEAA (B) and dioxane (C) degradation by CB1190. All experiments with acetylene-exposed cells (■), non-acetylene-exposed cells (○), and abiotic controls (△) were performed in triplicate. Error bars indicate standard deviations.