Linking Low-Level Stable Isotope Fractionation to Expression of the Cytochrome P450 Monooxygenase-Encoding
            <i>ethB</i>
            Gene for Elucidation of Methyl
            <i>tert</i>
            -Butyl Ether Biodegradation in Aerated Treatment Pond Systems

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This enables efficient degradation of diethyl ether, diisopropyl ether, MTBE, ETBE, TAME, and tert-amyl ethyl ether (TAEE) without any lag phase in strain L108. However, ethers with larger residues, n-hexyl methyl ether, tetrahydrofuran, and alkyl aryl ethers, were not attacked by the Eth system at significant rates in resting-cell experiments, indicating that the residue in the ether molecule which is not hydroxylated also contributes to the determination of substrate specificity.

mentioning

confidence: 94%

Constitutive Expression of the Cytochrome P450 EthABCD Monooxygenase System Enables Degradation of Synthetic Dialkyl Ethers in Aquincola tertiaricarbonis L108

Schuster

Purswani

Breuer

et al. 2013

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“…Consequently, biomarkers are becoming increasingly important in bioremediation and monitoring applications for environmental contaminants, with the development of several assays that target specific genes involved in compound degradation (8,12,13). For example, quantification of the cytochrome P450 monooxygenase-encoding gene, ethB, was used as an indicator for the microbial transformation of methyl tert-butyl ether (MTBE) in samples with low levels of contamination (14). Alcohol dehydrogenase (ADH) and aldehyde dehydrogenase (ALDH) genes associated with BMMs also play an important role in the biotransformation of recalcitrant compounds.…”

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confidence: 99%

Identification of Biomarker Genes To Predict Biodegradation of 1,4-Dioxane

Gedalanga

Pornwongthong

Mora³

et al. 2014

Bacterial multicomponent monooxygenase gene targets in Pseudonocardia dioxanivorans CB1190 were evaluated for their use as biomarkers to identify the potential for 1,4-dioxane biodegradation in pure cultures and environmental samples. Our studies using laboratory pure cultures and industrial activated sludge samples suggest that the presence of genes associated with dioxane monooxygenase, propane monooxygenase, alcohol dehydrogenase, and aldehyde dehydrogenase are promising indicators of 1,4-dioxane biotransformation; however, gene abundance was insufficient to predict actual biodegradation. A time course gene expression analysis of dioxane and propane monooxygenases in Pseudonocardia dioxanivorans CB1190 and mixed communities in wastewater samples revealed important associations with the rates of 1,4-dioxane removal. In addition, transcripts of alcohol dehydrogenase and aldehyde dehydrogenase genes were upregulated during biodegradation, although only the aldehyde dehydrogenase was significantly correlated with 1,4-dioxane concentrations. Expression of the propane monooxygenase demonstrated a time-dependent relationship with 1,4-dioxane biodegradation in P. dioxanivorans CB1190, with increased expression occurring after over 50% of the 1,4-dioxane had been removed. While the fraction of P. dioxanivorans CB1190-like bacteria among the total bacterial population significantly increased with decrease in 1,4-dioxane concentrations in wastewater treatment samples undergoing active biodegradation, the abundance and expression of monooxygenase-based biomarkers were better predictors of 1,4-dioxane degradation than taxonomic 16S rRNA genes. This study illustrates that specific bacterial monooxygenase and dehydrogenase gene targets together can serve as effective biomarkers for 1,4-dioxane biodegradation in the environment.

“…T3-1. This N-deethoxymethylase was encoded by the ethABCD gene cluster involved in methyl tertbutyl ether (MTBE) and ETBE degradation (16)(17)(18)(19)(20)(21)(22)(23)(24). The functions of EthABCD in bacteria were genetically verified by gene complementation in mutant strains.…”

Section: Discussionmentioning

confidence: 99%

“…Many strains harboring the ethRABCD gene cluster have been reported, and its function was also studied by genetic methods (16)(17)(18)(19)(20)(21)(22)(23)(24). The ethABCD cluster encoded a ferredoxin reductase (EthA), a cytochrome P450 (EthB), a ferredoxin (EthC), and a protein with an unknown function (EthD) (17).…”

Section: Discussionmentioning

confidence: 99%

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Involvement of the Cytochrome P450 System EthBAD in the N -Deethoxymethylation of Acetochlor by Rhodococcus sp. Strain T3-1

Wang

Zhou

et al. 2015

c Acetochlor [2-chloro-N-(ethoxymethyl)-N-(2-ethyl-6-methylphenyl)-acetamide] is a widely applied herbicide with potential carcinogenic properties. N-Deethoxymethylation is the key step in acetochlor biodegradation. N-Deethoxymethylase is a multicomponent enzyme that catalyzes the conversion of acetochlor to 2=-methyl-6=-ethyl-2-chloroacetanilide (CMEPA). Fast detection of CMEPA by a two-enzyme (N-deethoxymethylase-amide hydrolase) system was established in this research. Based on the fast detection method, a three-component enzyme was purified from Rhodococcus sp. strain T3-1 using ammonium sulfate precipitation and hydrophobic interaction chromatography. The molecular masses of the components of the purified enzyme were estimated to be 45, 43, and 11 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Based on the results of peptide mass fingerprint analysis, acetochlor N-deethoxymethylase was identified as a cytochrome P450 system, composed of a cytochrome P450 oxygenase (43-kDa component; EthB), a ferredoxin (45 kDa; EthA), and a reductase (11 kDa; EthD), that is involved in the degradation of methyl tert-butyl ether. The gene cluster ethABCD was cloned by PCR amplification and expressed in Escherichia coli BL21(DE3). Resting cells of a recombinant E. coli strain showed deethoxymethylation activity against acetochlor. Subcloning of ethABCD showed that ethABD expressed in E. coli BL21(DE3) has the activity of acetochlor N-deethoxymethylase and is capable of converting acetochlor to CMEPA. Chloroacetanilide herbicides are a class of important herbicides used worldwide. The chloroacetanilide herbicide acetochlor [2-chloro-N-(ethoxymethyl)-N-(2-ethyl-6-methylphenyl)-acetamide] is a selective preemergent herbicide that has been used to effectively control broadleaf weeds and annual grasses in corn fields for almost 40 years (1). The excessive and frequent application of acetochlor may result in high levels of acetochlor residues, which have been detected in ground and surface waters and negatively impact both the environment and agricultural ecosystems (2). The U.S. Environmental Protection Agency (EPA) has classified acetochlor as a B-2 carcinogen and a probable human carcinogen (3). Studies of different soil types treated with acetochlor have demonstrated that 2=-methyl-6=-ethyl-2-chloroacetanilide (CMEPA), a compound derived from acetochlor N-deethoxymethylation, is a major product of the microbial degradation process (1, 3-5).We previously isolated an efficient acetochlor N-deethoxymethylation strain, T3-1, from a microbial consortium that could mineralize acetochlor completely. The strain was identified as a Rhodococcus sp. (1). The biochemical pathway of acetochlor degradation by the three bacteria in the consortium was proposed to be as follows: acetochlor to CMEPA by Rhodococcus sp. strain T3-1, CMEPA to 2-methyl-6-ethyl aniline (MEA) by Delftia sp. strain T3-6, and MEA by Sphingobium sp. strain MEA3-1, based on the identified degradation intermediates (1). In this study, a three-...