Characterization of two novel hydrolases from Sphingopyxis sp. DBS4 for enantioselective degradation of chiral herbicide diclofop-methyl.

Diclofop-methyl, an aryloxyphenoxypropionate (AOPP) herbicide, is a chiral compound with two enantiomers. Microbial detoxification and degradation of various enantiomers is garnering immense research attention. However, enantioselective catabolism of diclofop-methyl has been rarely explored, especially at the molecular level. This study cloned two novel hydrolase genes (dcmA and dcmH) in Sphingopyxis sp. DBS4, and characterized them for diclofop-methyl degradation. DcmA, a member of the amidase superfamily, exhibits 26.1-45.9% identity with functional amidases. Conversely, DcmH corresponded to the DUF3089 domain-containing protein family (a family with unknown function), sharing no significant similarity with other biochemically characterized proteins. DcmA exhibited a broad spectrum of substrates, with preferential hydrolyzation of (R)-(+)-diclofop-methyl, (R)-(+)-quizalofop-ethyl, and (R)-(+)-haloxyfop-methyl. DcmH also preferred (R)-(+)-quizalofop-ethyl and (R)-(+)-haloxyfop-methyl degradation while displaying no apparent enantioselective activity towards diclofop-methyl. Using site-directed mutagenesis and molecular docking, it was determined that Ser175 was the fundamental residue influencing DcmA's activity against the two enantiomers of diclofop-methyl. For the degradation of AOPP herbicides, DcmA is an enantioselective amidase that has never been reported in research. This study provided novel hydrolyzing enzyme resources for the remediation of diclofop-methyl in the environment and deepened the understanding of enantioselective degradation of chiral AOPP herbicides mediated by microbes.

Experimental and computational approaches to characterize a novel amidase that initiates the biodegradation of the herbicide propanil in Bosea sp. P5.

The herbicide propanil and its major metabolite 3,4-dichloroaniline (3,4-DCA) are difficult to biodegrade and pose great health and environmental risks. However, studies on the sole or synergistic mineralization of propanil by pure cultured strains are limited. A two-strain consortium (Comamonas sp. SWP-3 and Alicycliphilus sp. PH-34), obtained from a swep-mineralizing enrichment culture that can synergistically mineralize propanil, has been previously reported. Here, another propanil degradation strain, Bosea sp. P5, was successfully isolated from the same enrichment culture. A novel amidase, PsaA, responsible for initial propanil degradation, was identified from strain P5. PsaA shared low sequence identity (24.0-39.7 %) with other biochemically characterized amidases. PsaA exhibited optimal activity at 30 °C and pH 7.5 and had kcat and Km values of 5.7 s-1 and 125 µM, respectively. PsaA could convert the herbicide propanil to 3,4-DCA but exhibited no activity toward other herbicide structural analogs. This catalytic specificity was explained by using propanil and swep as substrates and then analyzed by molecular docking, molecular dynamics simulation and thermodynamic calculations, which revealed that Tyr138 is the key residue that affects the substrate spectrum of PsaA. This is the first propanil amidase with a narrow substrate spectrum identified, providing new insights into the catalytic mechanism of amidase in propanil hydrolysis.

Effects of elevated CO2 and warming on the root-associated microbiota in an agricultural ecosystem.

Plant root-associated microbial communities profoundly affect plant nutrition and productivity. Although elevated atmospheric CO2 and warming affect above- and belowground plant processes, it remains unclear how root-associated microbial communities respond to elevated CO2 and warming. In this study, an open-air field experiment was conducted to assay the interactive effects of elevated CO2 (500 ppm) and warming (+2°C) on the root-associated microbiota and soil enzyme activities in a rice-wheat rotation ecosystem. The results revealed that elevated CO2 significantly increased rhizosphere soil organic carbon (SOC) and total nitrogen contents. In addition, glucosidase, ß-xylosidase, and phosphatase activities significantly increased. The richness and Shannon diversity indices were significantly higher in rhizosphere soil than in root endosphere. Elevated CO2 and warming significantly impacted the rhizosphere soil microbiota and altered their composition by changing the relative abundance of some specific groups. Soil pH, SOC, and available potassium content significantly altered the dominant bacterial phyla in the rhizosphere. SOC affected root endophytic bacterial phyla. Bacterial and fungal genera were significantly correlated with soil variables in the rhizosphere than in the root endosphere. These results indicate that microbial communities in the rhizosphere are more sensitive to elevated CO2 and warming than those in the root endosphere.

Agriterribacter soli sp. nov., isolated from herbicide-contaminated soil.

A Gram-stain-negative, non-spore-forming and rod-shaped bacterium, designated strain NS-102T, was isolated from herbicide-contaminated soil sampled in Nanjing, PR China, and its taxonomic status was investigated by a polyphasic approach. Cell growth of strain NS-102T occurred at 16-42 °C (optimum, 30 °C), at pH 5.0-8.0 (optimum, pH 6.0) and in the presence of 0-3.5 % (w/v) NaCl (optimum, without addition of NaCl). The 16S rRNA gene sequence of strain NS-102T shows high similarity to that of Agriterribacter humi YJ03T (96.9 % similarity), followed by Terrimonas terrae T16R-129T (93.8 %) and Terrimonas pekingensis QHT (93.6 %). Average nucleotide identity, average amino acid identity and digital DNA-DNA hybridization values between the draft genomes of strain NS-102T and A. humi YJ03T were 72.5, 69.4 and 18.6%, respectively. The only respiratory quinone was MK-7, and phosphatidylethanolamine and unidentified lipids were the major polar lipids. The major cellular fatty acids of strain NS-102T contained high amounts of iso-C15 : 0 (24.6 %), iso-C17 : 03-OH (24.1 %), iso-C15 : 0 G (16.6 %) and summed feature 3 (C16 : 1 ω6c and/or C16 : 1 ω7c) (15.6 %). The G+C content of the total DNA was determined to be 40.0 mol%. The morphological, physiological, chemotaxonomic and phylogenetic analyses clearly distinguished this strain from its closest phylogenetic neighbours. Thus, strain NS-102T represents a novel species of the genus Agriterribacter, for which the name Agriterribacter soli sp. nov. is proposed. The type strain is NS-102T (=CCTCC AB 2017249T=KCTC 62322T).