d 2-Methyl-6-ethylaniline (MEA) is the main microbial degradation intermediate of the chloroacetanilide herbicides acetochlor and metolachlor. Sphingobium sp. strain MEA3-1 can utilize MEA and various alkyl-substituted aniline and phenol compounds as sole carbon and energy sources for growth. We isolated the mutant strain MEA3-1Mut, which converts MEA only to 2-methyl-6-ethyl-hydroquinone (MEHQ) and 2-methyl-6-ethyl-benzoquinone (MEBQ). MEA may be oxidized by the P450 monooxygenase system to 4-hydroxy-2-methyl-6-ethylaniline (4-OH-MEA), which can be hydrolytically spontaneously deaminated to MEBQ or MEHQ. The MEA microbial metabolic pathway was reconstituted based on the substrate spectra and identification of the intermediate metabolites in both the wild-type and mutant strains. Plasmidome sequencing indicated that both strains harbored 7 plasmids with sizes ranging from 6,108 bp to 287,745 bp. Among the 7 plasmids, 6 were identical, and pMEA02= in strain MEA3-1Mut lost a 37,000-bp fragment compared to pMEA02 in strain MEA3-1. Two-dimensional electrophoresis (2-DE) and protein mass fingerprinting (PMF) showed that MEA3-1Mut lost the two-component flavin-dependent monooxygenase (TC-FDM) MeaBA, which was encoded by a gene in the lost fragment of pMEA02. MeaA shared 22% to 25% amino acid sequence identity with oxygenase components of some TC-FDMs, whereas MeaB showed no sequence identity with the reductase components of those TC-FDMs. Complementation with meaBA in MEA3-1Mut and heterologous expression in Pseudomonas putida strain KT2440 resulted in the production of an active MEHQ monooxygenase.
Chloroacetanilide herbicides are widely used throughout the world (1) for the control of most annual grasses and certain broadleaf weeds (2). The majority of the commonly used chloroacetanilide herbicides, such as alachlor, acetochlor, butachlor, and metolachlor, are N-alkoxyalkyl-N-chloroacetyl-substituted aniline derivatives (3). Long-term application of these herbicides has caused negative impacts on both the aquatic environment and agricultural ecosystems (4, 5). Acetochlor has been classified by the U.S. Environmental Protection Agency (EPA) (4) as a B-2 carcinogen and a probable human carcinogen, and several chloroacetanilide herbicides have been proven to cause tumors in rats (6). Thus, there is great concern about the behavior and fate of chloroacetamide herbicides and their degradation metabolites in the environment.Although chloroacetamide herbicides may be degraded through chemical and physical processes, microbial metabolism is the main mechanism responsible for herbicidal degradation in natural soils (7,8). A variety of bacterial strains that are able to degrade butachlor, alachlor, acetochlor, and metolachlor have been characterized (9, 10). In the degradation pathway, these herbicides are N-dealkylated to 2-chloro-N-(2,6-diethylphenyl) acetamide (CDEPA) (for alachlor and butachlor) or 2-chloro-N-(2-methyl-6-ethylphenyl) acetamide (CMEPA) (for acetochlor and metolachlor), which are then converted to 2,6-dieth...