De-esterification is an important degradation or detoxification mechanism of sulfonylurea herbicide in microbes and plants. However, the biochemical and molecular mechanisms of sulfonylurea herbicide de-esterification are still unknown. In this study, a novel esterase gene, sulE, responsible for sulfonylurea herbicide de-esterification, was cloned from Hansschlegelia zhihuaiae S113. The gene contained an open reading frame of 1,194 bp, and a putative signal peptide at the N terminal was identified with a predicted cleavage site between Ala37 and Glu38, resulting in a 361-residue mature protein. SulE minus the signal peptide was synthesized in Escherichia coli BL21 and purified to homogeneity. SulE catalyzed the de-esterification of a variety of sulfonylurea herbicides that gave rise to the corresponding herbicidally inactive parent acid and exhibited the highest catalytic efficiency toward thifensulfuron-methyl. SulE was a dimer without the requirement of a cofactor. The activity of the enzyme was completely inhibited by Ag ؉ , Cd 2؉ , Zn 2؉ , methamidophos, and sodium dodecyl sulfate. A sulE-disrupted mutant strain, ⌬sulE, was constructed by insertion mutation. ⌬sulE lost the de-esterification ability and was more sensitive to the herbicides than the wild type of strain S113, suggesting that sulE played a vital role in the sulfonylurea herbicide resistance of the strain. The transfer of sulE into Saccharomyces cerevisiae BY4741 conferred on it the ability to de-esterify sulfonylurea herbicides and increased its resistance to the herbicides. This study has provided an excellent candidate for the mechanistic study of sulfonylurea herbicide metabolism and detoxification through de-esterification, construction of sulfonylurea herbicide-resistant transgenic crops, and bioremediation of sulfonylurea herbicide-contaminated environments. S ulfonylurea herbicides are an important class of herbicides used worldwide for controlling weeds in all major agronomic crops. The herbicides inhibit acetohydroxy acid synthase (AHAS), a key enzyme in the biosynthesis pathway of branched-chain amino acids valine, leucine, and isoleucine in bacteria, fungi, and plants (3,4,8,13). The use of sulfonylurea herbicides has developed rapidly because of their high efficacies at low dosages and multicrop selectivities. The sulfonylurea products are now the second most common kind of herbicides after the glyphosates, and more than 30 products have been commercialized.Most sulfonylurea herbicides are weak acids, vulnerable to acid hydrolysis under acidic conditions. However, in neutral to alkaline soils, some of the herbicides, such as metsulfuron-methyl, chlorsulfuron, and ethametsulfuron-methyl, are degraded at a very slow rate and persist from several months to more than 1 year (15,20,24,30). The residues of herbicides in the soil seriously damage subsequent rotation of sulfonylurea-sensitive crops, like legumes and oilseeds, which can result in serious agricultural loss. Thus, great concern and interest have been raised regarding the enviro...

Cyhalofop-butyl (CyB) is a widely used aryloxyphenoxy propanoate (AOPP) herbicide for control of grasses in rice fields. Five CyB-degrading strains were isolated from rice field soil and identified as Agromyces sp., Stenotrophomonas sp., Aquamicrobium sp., Microbacterium sp., and Pseudomonas azotoformans; the results revealed high biodiversity of CyB-degrading bacteria in rice soil. One strain, P. azotoformans QDZ-1, degraded 84.5% of 100 mg L(-1) CyB in 5 days of incubation in a flask and utilized CyB as carbon source for growth. Strain QDZ-1 could also degrade a wide range of other AOPP herbicides. An esterase gene, chbH, which hydrolyzes CyB to cyhalofop acid (CyA), was cloned from strain QDZ-1 and functionally expressed. A chbH-disrupted mutant dchbH was constructed by insertion mutation. Mutant dchbH could not degrade and utilize CyB, suggesting that chbH was the only esterase gene responsible for CyB degradation in strain QDZ-1. ChbH hydrolyzed all AOPP herbicides tested as well as permethrin. The catalytic efficiency of ChbH toward different AOPP herbicides followed the order quizalofop-P-ethyl ≈ fenoxaprop-P-ethyl > CyB ≈ fluazifop-P-butyl > diclofop-methyl ≈ haloxyfop-P-methyl; the results indicated that the chain length of the alcohol moiety strongly affected the biodegradability of the AOPP herbicides, whereas the substitutions in the aromatic ring had only a slight influence.

Sphingomonas sp. strain Ndbn-20 degrades and utilizes the herbicide dicamba as its sole carbon and energy source. In the present study, a tetrahydrofolate (THF)-dependent dicamba methyltransferase gene, dmt, was cloned from the strain, and three other genes, metF, dhc, and purU, which are involved in THF metabolism, were found to be located downstream of dmt. A transcriptional study revealed that the four genes constituted one transcriptional unit that was constitutively transcribed. Lysates of cells grown with glucose or dicamba exhibited almost the same activities, which further suggested that the dmt gene is constitutively expressed in the strain. Dmt shared 46% and 45% identities with the methyltransferases DesA and LigM from Sphingomonas paucimobilis SYK-6, respectively. The purified Dmt catalyzed the transfer of methyl from dicamba to THF to form the herbicidally inactive metabolite 3,6-dichlorosalicylic acid (DCSA) and 5-methyl-THF. The activity of Dmt was inhibited by 5-methyl-THF but not by DCSA. The introduction of a codon-optimized dmt gene into Arabidopsis thaliana enhanced resistance against dicamba. In conclusion, this study identified a THF-dependent dicamba methyltransferase, Dmt, with potential applications for the genetic engineering of dicamba-resistant crops. IMPORTANCEDicamba is a very important herbicide that is widely used to control more than 200 types of broadleaf weeds and is a suitable target herbicide for the engineering of herbicide-resistant transgenic crops. A study of the mechanism of dicamba metabolism by soil microorganisms will benefit studies of its dissipation, transformation, and migration in the environment. This study identified a THF-dependent methyltransferase, Dmt, capable of catalyzing dicamba demethylation in Sphingomonas sp. Ndbn-20, and a preliminary study of its enzymatic characteristics was performed. Introduction of a codon-optimized dmt gene into Arabidopsis thaliana enhanced resistance against dicamba, suggesting that the dmt gene has potential applications for the genetic engineering of herbicide-resistant crops.

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Dicamba, a broad-spectrum and highly efficient herbicide, is an excellent target herbicide for the engineering of herbicide-resistant crops. In this study, a new tetrahydrofolate (THF)-dependent dicamba methyltransferase gene, dmt50, was cloned from the dicamba-degrading strain Rhizorhabdus dicambivorans Ndbn-20. Dmt50 catalyzed the methyl transfer from dicamba to THF, generating the herbicidally inactive product 3,6-dichlorosalicylic acid (3,6-DCSA) and 5-methyl-THF. A dmt50 transgenic Arabidopsis thaliana clearly showed dicamba resistance (560 g/ha, the normal field application rate). However, Dmt50 demethylation activity was inhibited by the product 5-methyl-THF. Mthfr66, encoded by the 5,10-methylene-THF reductase gene mthf r66 could relieve the inhibition by removing 5-methyl-THF in vitro. Compared with expression of dmt50 alone, simultaneous expression of dmt50 and mthfr66 further improved the dicamba resistance (1120 g/ha) of transgenic A. thaliana. This study provides new genes for dicamba detoxification and a strategy for the engineering of dicamba-resistant crops.

The synergistic relationships between plants and their rhizospheric microbes can be used to develop a combinational bioremediation method, overcoming the constraints of individual phytoremediation or bioaugmentation method. Here, we provide a combinational transgenic plant-microbial remediation system for a more efficient removal of phenylurea herbicides (PHs) from contaminated-sites. The transgenic synthesizing the bacterial-demethylase PdmAB in the chloroplast was developed. The constructed transgenic exhibited significant tolerance to isoproturon (IPU), a typical PH, and it took up the IPU through roots and transported to leaves, where the majority of the IPU was demethylated to 3-(4-isopropylphenyl)-1-methylurea (MDIPU). The produced intermediate was released outside of roots and further metabolized by the combinationally inoculated MDIPU-mineralizing bacterium sp. strain 1017-1 in the rhizosphere, resulting in an enhanced and complete removal of IPU from soil. Mutual benefits were built for both transgenic and strain 1017-1. The transgenic offered strain 1017-1 a suitable accommodation and in return, the latter protected the plant from the phytotoxicity of MDIPU. The biomass of the transgenic and the residence of the inoculated degrading microbes in the combinational treatment increased significantly compared to that in their respective individual transgenic plant treatment or bioaugmentation treatment. The influence of the structure of bacterial community by combinational treatment was between that of the two individual treatments. Overall, the combination of two approaches, phytoremediation by transgenic plants and bioaugmentation with intermediate-mineralizing microbes in the rhizosphere, represents an innovative strategy for the enhanced and complete remediation of pollutant-contaminated sites. Phytoremediation of organic pollutant-contaminated sites using transgenic plants expressing bacterial enzyme has been well described. The major constraint of transgenic plants transferred with a single catabolic gene is that they can also accumulate/release intermediates, still causing phytotoxicity or additional environmental problems. On the other hand, bioaugmentation with degrading strains also has its drawbacks including instability of the inoculated strains and low bioavailability of pollutants. In this study, the synergistic relationship between transgenic expressing the bacterial-demethylase PdmAB in the chloroplast and the inoculated intermediate-mineralizing bacterium sp. strain 1017-1 in the rhizosphere is used to develop an intriguing bioremediation method. The combinational transgenic plant-microbe remediation system shows a more efficient and complete removal of phenylurea herbicides from contaminated-sites, and can overcome the constraints of individual phytoremediation or bioaugmentation methods.

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