Roles of Two Glutathione-Dependent 3,6-Dichlorogentisate Dehalogenases in Rhizorhabdus dicambivorans Ndbn-20 in the Catabolism of the Herbicide Dicamba.

The herbicide dicamba is initially demethylated to 3,6-dichlorosalicylate (3,6-DCSA) in Rhizorhabdus dicambivorans Ndbn-20 and is subsequently 5-hydroxylated to 3,6-dichlorogentisate (3,6-DCGA). In the present study, two glutathione-dependent 3,6-DCGA dehalogenases, DsmH1 and DsmH2, were identified in strain Ndbn-20. DsmH2 shared a low identity (only 31%) with the tetrachlorohydroquinone (TCHQ) dehalogenase PcpC from Sphingobium chlorophenolicum ATCC 39723, while DsmH1 shared a high identity (79%) with PcpC. In the phylogenetic tree of related glutathione S-transferases (GSTs), DsmH1 and DsmH2, together with PcpC and the 2,5-dichlorohydroquinone dehalogenase LinD, formed a separate clade. DsmH1 and DsmH2 were synthesized in Escherichia coli BL21 and purified as His-tagged enzymes. Both enzymes required glutathione (GSH) as a cofactor and could 6-dechlorinate 3,6-DCGA to 3-chlorogentisate in vitro DsmH2 had a significantly higher catalytic efficiency toward 3,6-DCGA than DsmH1. Transcription and disruption analysis revealed that DsmH2 but not DsmH1 was responsible for the 6-dechlorination of 3,6-DCGA in strain Ndbn-20 in vivo Furthermore, we propose a novel eta class of GSTs to accommodate the four bacterial dehalogenases PcpC, LinD, DsmH1, and DsmH2.IMPORTANCE Dicamba is an important herbicide, and its use and leakage into the environment have dramatically increased since the large-scale planting of genetically modified (GM) dicamba-resistant crops in 2015. However, the complete catabolic pathway of dicamba has remained unknown, which limits ecotoxicological studies of this herbicide. Our previous study revealed that 3,6-DCGA was an intermediate of dicamba degradation in strain Ndbn-20. In this study, we identified two glutathione-dependent 3,6-DCGA dehalogenases, DsmH1 and DsmH2, and demonstrated that DsmH2 is physiologically responsible for the 6-dechlorination of 3,6-DCGA in strain Ndbn-20. GSTs play an important role in the detoxification and degradation of a variety of endogenous and exogenous toxic compounds. On the basis of their sequence identities, phylogenetic status, and functions, the four bacterial GSH-dependent dehalogenases (PcpC, LinD, DsmH1, and DsmH2) were reclassified as a new eta class of GSTs. This study helps us to elucidate the microbial catabolism of dicamba and enhances our understanding of the diversity and functions of GSTs.

A Tetrahydrofolate-Dependent Methyltransferase Catalyzing the Demethylation of Dicamba in Sphingomonas sp. Strain Ndbn-20.

UNLABELLED: 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. IMPORTANCE: Dicamba 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.

Rhizorhabdus dicambivorans sp. nov., a dicamba-degrading bacterium isolated from compost.

Strain Ndbn-20T, a Gram-staining-negative, non-spore-forming bacterium, was isolated from compost of plant litter. The strain was able to degrade dicamba. Phylogenetic analysis based on 16S rRNA gene sequences indicated that Ndbn-20Trepresented a member of the family Sphingomonadaceae of the Alphaproteobacteria and showed high sequence similarities to Rhizorhabdusargentea SP1T (98.8 %), Sphingomonaswittichii RW1T (97.9 %), Sphingomonasstarnbergensis 382T (97.7 %) and Sphingomonashistidinilytica UM2T (97.7 %). However, the strain showed low DNA sequence relatedness with R. argentea SP1T (45.6±1.9 %), S. wittichii RW1T (33.5±2.3 %), S.histidinilytica UM2T (39.4±3.6 %) and S. starnbergensis382T (42.1±4.1 %). Ndbn-20T possessed Q-10 as the predominant ubiquinone, spermidine as the major polyamine, and summed feature 8 (comprising C18 : 1ω7c/C18 : 1ω6c), summed feature 3 (comprising C16 : 1ω7c/C16 : 1ω6c), C17 : 1ω6c, C16 : 0 and C14 : 02-OH as the major fatty acids (>5 % of the total). The profile of polar lipids consisted of diphosphatidylglycerol, phosphatidylethanolamine, phosphatidylcholine, glycolipid, sphingoglycolipid, phosphatidyldimethylethanolamine and phosphatidylglycerol. The DNA G+C content was 65.4 mol%. Based on a polyphasic taxonomic analysis, strain Ndbn-20T is proposed to represent a novel species of the genus Rhizorhabdus, with the proposed name of Rhizorhabdus dicambivorans sp. nov. The type strain is Ndbn-20T (=CCTCC AB 2016143=KACC 18661).

Growth inhibition of Escherichia coli and methicillin-resistant Staphylococcus aureus by targeting cellular methionine aminopeptidase.

Methionine aminopeptidase (MetAP) catalyzes the N-terminal methionine excision from the majority of newly synthesized proteins, which is an essential cotranslational process required for cell survival. As such, MetAP has become an appealing target for the development of antimicrobial therapeutics with novel mechanisms of action. By screening a library of small organic molecules, we previously discovered a class of compounds that selectively inhibit the Fe(II)-form of MetAP. Herein, we demonstrate that some of these compounds and their newly synthesized derivatives halt the growth of Escherichia coli and Staphylococcus aureus cells with significant potency. The most potent compound inhibited methicillin-resistant S. aureus (MRSA) growth with an IC(50) value of 1 µM and MIC of 0.7 µg/ml. Two cell-based assays were used to verify that MetAP is the intracellular target in E. coli cells. These findings can serve as foundation for the development of novel therapeutics against an ever increasing threat by drug resistant staphylococcal infections.

Rationally tuned micropores within enantiopure metal-organic frameworks for highly selective separation of acetylene and ethylene.

Separation of acetylene and ethylene is an important industrial process because both compounds are essential reagents for a range of chemical products and materials. Current separation approaches include the partial hydrogenation of acetylene into ethylene over a supported Pd catalyst, and the extraction of cracked olefins using an organic solvent; both routes are costly and energy consuming. Adsorption technologies may allow separation, but microporous materials exhibiting highly selective adsorption of C(2)H(2)/C(2)H(4) have not been realized to date. Here, we report the development of tunable microporous enantiopure mixed-metal-organic framework (M'MOF) materials for highly selective separation of C(2)H(2) and C(2)H(4). The high selectivities achieved suggest the potential application of microporous M'MOFs for practical adsorption-based separation of C(2)H(2)/C(2)H(4).