Degradation of the herbicide dicamba by two sphingomonads via different O-demethylation mechanisms
Introduction
The herbicide dicamba (2-methoxy-3,6-dichlorobenzoic acid) is a chlorinated benzoic acid herbicide. The mode of action of dicamba is to affect cell wall plasticity and nucleic acid metabolism. Due to its high efficiency and low-cost, dicamba has been widely used in a variety of crops to control annual and perennial broadleaf weeds since it was first approved in 1962 (Tomlin, 2006). Recently, biotech giant Monsanto Company has successfully developed genetically modified (GM) dicamba-resistant soybean and cotton by transferring a dicamba monooxygenase gene (DMO) (Behrens et al., 2007). The GM soybean has passed the safety assessment on October 5, 2012 (http://www.inspection.gc.ca/plants/plants-with-novel-traits/approved-under-review/decision-documents/dd-2012-94/eng/1364910295912/1364910444165). Once this GM crop is planted on a large scale, the usage of dicamba would be significantly increased. Thus, the persistence and degradation mechanism of dicamba in the environment are of great interest.
Dicamba is rapidly degraded in unsterilized soil but not in sterilized soil under both aerobic and anaerobic conditions, indicating that microbial metabolism is the major environmental dicamba removal process (Krueger et al., 1989, Taraban et al., 1993, Gu et al., 2001). To date, two isolates that are able to degrade dicamba have been characterized. Aerobic strain Stenotrophomonas (formerly Pseudomonas) maltophilia DI-6, which is able to metabolized dicamba to CO2, was isolated from sludge sample obtained from a storm water retention pond at a dicamba manufacturing plant in Beaumont, TX (Krueger et al., 1989). Another well-studied microorganism is the anaerobic strain Moorella thermoacetica, which can metabolize at least 20 different methoxylated aromatics, such as dicamba, vanillate and syringate under anaerobic condition (Naidu and Ragsdale, 2001). Metabolic studies have shown that the degradation of dicamba in both strains is initiated by O-demethylation to generate 3,6-dichlorosalicylic acid (DCSA), a compound without herbicidal activity (Behrens et al., 2007, Naidu and Ragsdale, 2001). However, these two bacterial strains have different O-demethylation mechanisms: in Stenotrophomonas maltophilia DI-6, the demethylation is catalyzed by a dicamba monooxygenase (DMO) (Wang et al., 1997, Herman et al., 2005, Behrens et al., 2007), and in M. thermoacetica, a tetrahydrofolate (THF)-dependent demethylase system (Mtv) is responsible for the demethylation (Naidu and Ragsdale, 2001).
In this study, two sphingomonads Ndbn-10 and Ndnn-20, capable of degrading dicamba, were isolated from activated sludge from dicamba-manufacturing wastewater and compost sample, respectively. Enzymatic studies revealed that the O-demethylation of dicamba in the two sphingomonads is different: the O-demethylation in Sphingobium sp. Ndbn-10 is catalyzed by DMO, whereas the O-demethylation in Sphingomonas sp. Ndbn-20 is catalyzed by a novel tetrahydrofolic acid (THF)-dependent demethylase, which is different from the DMO and Mtv systems.
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Chemicals and samples
Dicamba (99.3% purity) and THF were obtained from Sigma–Aldrich (Shanghai, China). DCSA (98% purity) and cobalamin were purchased from Qingdao Chemical Reagent Co., Ltd, Qingdao, China. Luria–Bertani (LB) broth and LB agar were obtained from Difco Laboratories (Detroit, MI). The minimal salts medium (MSM) consisted of the following components: 1.3 g K2HPO4, 0.86 g KH2PO4, 0.66 g (NH4)2SO4, 0.097 g MgSO4, 0.025 g MnSO4·H2O, 0.005 g FeSO4·7H2O and 0.0013 g CaSO4·6H2O per liter water, pH 7.0.
Screen and taxonomic study of dicamba-degrading isolates
The
Strain isolation and identification
After four transfer rounds, both of enrichment cultures displayed good dicamba-degrading abilities. Enrichment with activated sludge as inoculant completely degraded the added 2.25 mM dicamba after 24 h of incubation. Four different colonies were obtained from this enrichment; however, only one colony, designated as Ndbn-10, could degrade dicamba. The enrichment inoculated with the compost sample degraded 95 percent of the 2.25 mM dicamba within 48 h. Most colonies obtained from this enrichment
Discussion
Sphingomonads, belonging to α-proteobacteria, are characterized by glycosphingolipids on their outer membrane and include four genera: Sphingomonas, Sphingobium, Novosphingobium and Sphingopyxis. Sphingomonads are widespread in various environments and are known for their abilities to degrade a wide variety of natural and xenobiotic compounds such as biphenyl, naphthalene, dibenzo-p-dioxin, dibenzothiophene, carbazole, hexachlorocyclohexane (HCH), estradiol, phenanthrene and pyrethroids (Stolz,
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These authors contributed equally to this work.