A Synergistic Consortium Involved in rac-Dichlorprop Degradation as Revealed by DNA Stable Isotope Probing and Metagenomic Analysis.

rac-Dichlorprop, a commonly used phenoxyalkanoic acid herbicide, is frequently detected in environments and poses threats to environmental safety and human health. Microbial consortia are thought to play key roles in rac-dichlorprop degradation. However, the compositions of the microbial consortia involved in rac-dichlorprop degradation remain largely unknown. In this study, DNA stable isotope probing (SIP) and metagenomic analysis were integrated to reveal the key microbial consortium responsible for rac-dichlorprop degradation in a rac-dichlorprop-degrading enrichment. OTU340 (Sphingobium sp.) and OTU348 (Sphingopyxis sp.) were significantly enriched in the rac-[13C]dichlorprop-labeled heavy DNA fractions. A rac-dichlorprop degrader, Sphingobium sp. strain L3, was isolated from the enrichment by a traditional enrichment method but with additional supplementation of the antibiotic ciprofloxacin, which was instructed by metagenomic analysis of the associations between rac-dichlorprop degraders and antibiotic resistance genes. As revealed by functional profiling of the metagenomes of the heavy DNA, the genes rdpA and sdpA, involved in the initial degradation of the (R)- and (S)-enantiomers of dichlorprop, respectively, were mostly taxonomically assigned to Sphingobium species, indicating that Sphingopyxis species might harbor novel dichlorprop-degrading genes. In addition, taxonomically diverse bacterial genera such as Dyella, Sphingomonas, Pseudomonas, and Achromobacter were presumed to synergistically cooperate with the key degraders Sphingobium/Sphingopyxis for enhanced degradation of rac-dichlorprop. IMPORTANCE Understanding of the key microbial consortium involved in the degradation of the phenoxyalkanoic acid herbicide rac-dichlorprop is pivotal for design of synergistic consortia used for enhanced bioremediation of herbicide-contaminated sites. However, the composition of the microbial consortium and the interactions between community members during the biodegradation of rac-dichlorprop are unclear. In this study, DNA-SIP and metagenomic analysis were integrated to reveal that the metabolite 2,4-dichlorophenol degraders Dyella, Sphingomonas, Pseudomonas, and Achromobacter synergistically cooperated with the key degraders Sphingobium/Sphingopyxis for enhanced degradation of rac-dichlorprop. Our study provides new insights into the synergistic degradation of rac-dichlorprop at the community level and implies the existence of novel degrading genes for rac-dichlorprop in nature.

Pigmentiphaga sp. Strain D-2 Uses a Novel Amidase To Initiate the Catabolism of the Neonicotinoid Insecticide Acetamiprid.

Acetamiprid, a chloronicotinyl neonicotinoid insecticide, is among the most commonly used insecticides worldwide, and its environmental fate has caused considerable concern. The compound 1-(6-chloropyridin-3-yl)-N-methylmethanamine (IM 1-4) has been reported to be the main intermediate during acetamiprid catabolism in microorganisms, honeybees, and spinach. However, the molecular mechanism underlying the hydrolysis of acetamiprid to IM 1-4 has not yet been elucidated. In this study, a novel amidase (AceAB) that initially hydrolyzes the C-N bond of acetamiprid to generate IM 1-4 was purified and characterized from the acetamiprid-degrading strain Pigmentiphaga sp. strain D-2. Based on peptide profiling of the purified AceAB and the draft genome sequence of strain D-2, aceA (372 bp) and aceB (2,295 bp), encoding the α and ß subunits of AceAB, respectively, were cloned and found to be necessary for acetamiprid hydrolysis in strain D-2. The characteristics of AceAB were also systematically investigated. Though AceA and AceB showed 35% to 56% identity to the α and ß subunits of the N,N-dimethylformamidase from Paracoccus aminophilus, AceAB was specific for the hydrolysis of acetamiprid and showed no activities to N,N-dimethylformamide or its structural analogs.IMPORTANCE Acetamiprid, among the top neonicotinoid insecticides used worldwide, is one of the most important commercial insecticides. Due to its extensive use, the environmental fate of acetamiprid, especially its microbial degradation, has caused considerable concern. Although the catabolic pathways of acetamiprid in microorganisms have been extensively studied, the molecular mechanisms underlying acetamiprid biodegradation (except for a nitrile hydratase) remain largely unknown, and the enzyme responsible for the biotransformation of acetamiprid into its main intermediate, IM 1-4, have not yet been elucidated. The amidase AceAB and its encoding genes, aceA and aceB, characterized in this study, were found to be necessary and specific for the initial hydrolysis of the C-N bond of acetamiprid to generate IM 1-4 in Pigmentiphaga sp. strain D-2. The finding of the novel amidase AceAB will greatly enhance our understanding of the microbial catabolism of the widely used insecticide acetamiprid at the molecular level.

Pedobacter helvus sp. nov., isolated from farmland soil.

A strictly aerobic, Gram-stain-negative, rod-shaped, yellow, non-spore-forming bacterial strain, designated P-25T, was isolated from soil collected in Yantai, Shandong Province, PR China. The temperature, pH and NaCl concentration ranges for the growth of strain P-25T were 10-37 °C (optimum, 28-30 °C), pH 6.0-9.0 (optimum, pH 7.5-8.0) and 0-4 % (w/v) (optimum, 1 % w/v), respectively. Phylogenetic analysis based on 16S rRNA gene sequences showed that strain P-25T was most closely related to Pedobacter xixiisoli S27T (98.1 % 16S rRNA gene sequence similarity), followed by Pedobacter chitinilyticus CM134L-2T (97.2 %) and Pedobacter ureilyticus THG-T11T (97.1 %). The genomic DNA G+C content of strain P-25T based on its draft genome sequence was 38.1 %. MK-7 was the major respiratory quinone, and iso-C15 : 0, C16 : 1ω7c and/or C16 : 1ω6c (summed feature 3) and iso-C17 : 0 3-OH were the major fatty acids. The major polar lipids were phosphatidylethanolamine, one unidentified aminophospholipid, one unidentified phospholipid, two unidentified lipids, five unidentified aminolipids and two unidentified glycolipids. Average nucleotide identity values for the draft genomes between strain P-25T and strains S27T, CM134L-2T and THG-T11T were 81.8, 77.6 and 81.2 %, respectively, and the digital DNA-DNA hybridization (dDDH) values were 30.0, 19.2 and 27.6 %, respectively. Based on their phylogenetic and phenotypic characteristics, chemotaxonomic data, and dDDH results, strain P-25T is considered to represent a novel species of the genus Pedobacter, for which the name Pedobacter helvus sp. nov. is proposed; the type strain is strain P-25T (KCTC 62821T=CCTCC AB 2018185T).

Steroidobacter soli sp. nov., isolated from farmland soil.

A Gram-stain-negative bacterial strain, designated JW-3T, was isolated from a soil sample collected from farmland in Yantai, Shandong Province, PR China. Cells of strain JW-3T are motile rods and strictly aerobic, showing catalase- and oxidase-positive reactions. Strain JW-3T could grow at 16-37 °C (optimum, 30 °C), at pH 6.0-9.0 (pH 7.0) and in the presence of 0-1 % (w/v) NaCl (0.5 %, in Luria-Bertani broth). The major fatty acids were summed feature 3 (C16 : 1 ω7c and/or C16 : 1 ω6c; 35.5 %), iso-C16 : 0 (16.7 %) and C12 : 0 (10.8 %). The major respiratory quinone was ubiquinone-8 (Q8). The polar lipids of strain JW-3T consisted of diphosphatidylglycerol, phosphatidylethanolamine, phosphatidylglycerol, four unidentified phospholipids, two unidentified lipids, two unidentified glycolipids and a partial unidentified aminophospholipid. Strain JW-3T was most closely related to Steroidobacter agariperforans KA5-BT with 97.67 % 16S rRNA gene sequence similarity. Results of phylogenetic analyses, based on 16S rRNA gene sequencing, showed that strain JW-3T forms a distinct phylogenic lineage within the genus Steroidobacter of the family Sinobacteraceae. The DNA G+C content of strain JW-3T was 62.57 mol%, based on its draft genome sequence. Average nucleotide identity values and digital DNA-DNA hybridization values for draft genomes, between strain JW-3T and strain KA5-BT, were 84.54 and 30.80 %, respectively. Based on its phenotypic, chemotaxonomic and molecular features, and DNA-DNA hybridization results, strain JW-3T represents a novel species of the genus Steroidobacter, for which the name Steroidobactersoli sp. nov. is proposed. The type strain is JW-3T (=CCTCC AB 2018184T=KCTC 62820T).

Spatial and temporal changes of social vulnerability of cities to natural hazards in Zhejiang province, China.

Understanding how social dynamics interact with natural hazards is one of the main challenges at global and local scales in the world for studying social vulnerability to natural hazards. In this study, we explore the spatial and temporal changes of social vulnerability of cities in Zhejiang province to natural hazards in China for the last decade. Based on the Zhejiang province's census data and the demographics and socioeconomic data during the period from 2009 to 2018, we have characterized social vulnerability through the Social Vulnerability Index (SoVI) for 11 cities throughout the province during 2009-2018 and examined spatial changes in social vulnerability using equal interval method. The results indicated that although the comprehensive vulnerability of Zhejiang province shows a declining trend at a county level, the social vulnerability of different city at the provincial level has obvious differences.

Remediation of isoproturon-contaminated soil by Sphingobium sp. strain YBL2: Bioaugmentation, detoxification and community structure.

The widely used phenylurea herbicide isoproturon (IPU) and its residues can inhibit the growth of subsequently planted crops. However, reports on bioremediation of IPU-contaminated soil are scarce. In this study, Sphingobium sp. strain YBL2-gfp (a derivative of the IPU-degrading Sphingobium sp. strain YBL2 isolated by our lab) was constructed to bioremediate IPU-contaminated soil. In pot experiments, strain YBL2-gfp colonized the roots of wheat and eliminated IPU residues in the soil within 21 d, effectively alleviating its toxicity and restoring wheat growth. IPU treatment reduced the richness and diversity of soil bacteria, while inoculation YBL2-gfp mainly affected richness with less impact on diversity. The high concentrations of IPU and inoculation of YBL2-gfp alone reduced the soil microbial community connections, while bioaugmentation treatment enhanced the soil microbial community connections. Additionally, strain YBL2-gfp stimulated the metabolic capacity of the indigenous microbes, promoting the degradation of IPU and reducing the negative impact of high concentrations of IPU on microbial community. Taken together, this study offers relatively comprehensive insights into the practical application of bioaugmentation, demonstrating that strain YBL2 has the potential to remediate IPU-contaminated soils.

Metagenomic insights into the diversity of 2,4-dichlorophenol degraders and the cooperation patterns in a bacterial consortium.

2,4-Dichlorophenol, which is largely employed in herbicides and industrial production, is frequently detected in ecosystems and poses risks to human health and environmental safety. Microbial communities are thought to perform better than individual strains in the complete degradation of organic contaminants. However, the synergistic degradation mechanisms of the microbial consortia involved in 2,4-dichlorophenol degradation are still not widely understood. In this study, a bacterial consortium named DCP-2 that is capable of degrading 2,4-dichlorophenol was obtained. Metagenomic analysis, cultivation-dependent functional verification, and co-occurrence network analysis were combined to reveal the primary 2,4-dichlorophenol degraders and the cooperation patterns in the consortium DCP-2. Metagenomic analysis showed that Pseudomonas, Achromobacter, and Pigmentiphaga were the primary degraders for the complete degradation of 2,4-dichlorophenol. Thirty-nine phylogenetically diverse bacterial genera, such as Brucella, Acinetobacter, Aeromonas, Allochromatium and Bosea, were identified as keystone taxa for 2,4-dichlorophenol degradation by keystone taxa analysis of the co-occurrence networks. In addition, a stable synthetic consortium of isolates from DCP-2 was constructed, consisting of Pseudomonas sp. DD-13 and Brucella sp. FZ-1; this synthetic consortium showed superior degradation capability for 2,4-dichlorophenol in both mineral salt medium and wastewater compared with monoculture. The findings provide valuable insights into the practical bioremediation of 2,4-dichlorophenol-contaminated sites.

Modeling microbial communities from atrazine contaminated soils promotes the development of biostimulation solutions.

Microbial communities play a vital role in biogeochemical cycles, allowing the biodegradation of a wide range of pollutants. The composition of the community and the interactions between its members affect degradation rate and determine the identity of the final products. Here, we demonstrate the application of sequencing technologies and metabolic modeling approaches towards enhancing biodegradation of atrazine-a herbicide causing environmental pollution. Treatment of agriculture soil with atrazine is shown to induce significant changes in community structure and functional performances. Genome-scale metabolic models were constructed for Arthrobacter, the atrazine degrader, and four other non-atrazine degrading species whose relative abundance in soil was changed following exposure to the herbicide. By modeling community function we show that consortia including the direct degrader and non-degrader differentially abundant species perform better than Arthrobacter alone. Simulations predict that growth/degradation enhancement is derived by metabolic exchanges between community members. Based on simulations we designed endogenous consortia optimized for enhanced degradation whose performances were validated in vitro and biostimulation strategies that were tested in pot experiments. Overall, our analysis demonstrates that understanding community function in its wider context, beyond the single direct degrader perspective, promotes the design of biostimulation strategies.

Response of soil bacterial communities to lead and zinc pollution revealed by Illumina MiSeq sequencing investigation.

Soil provides a critical environment for microbial community development. However, microorganisms may be sensitive to substances such as heavy metals (HMs), which are common soil contaminants. This study investigated bacterial communities using 16S ribosomal RNA (rRNA) gene fragment sequencing in geographic regions with and without HM pollution to elucidate the effects of soil properties and HMs on bacterial communities. No obvious changes in the richness or diversity of bacterial communities were observed between samples from mining and control areas. Significant differences in bacterial richness and diversity were detected between samples from different geographic regions, indicating that the basic soil characteristics were the most important factors affecting bacterial communities other than HMs. However, the abundances of several phyla and genera differed significantly between mining and control samples, suggesting that Zn and Pb pollution may impact the soil bacterial community composition. Moreover, regression analyses showed that the relative abundances of these phyla and genera were correlated significantly with the soil-available Zn and Pb contents. Redundancy analysis indicated that the soil K, ammoniacal nitrogen (NH4+-N), total Cu, and available Zn and Cu contents were the most important factors. Our results not only suggested that the soil bacteria were sensitive to HM stresses but also indicated that other soil properties may affect soil microorganisms to a greater extent.

Anti-inflammatory effects and pharmacokinetics study of geniposide on rats with adjuvant arthritis.

The aim of this study was to explore the anti-inflammatory effects of Geniposide (GE), an iridoid glycoside compound extracted from Gardenia jasminoides Ellis (GJ) fruit in adjuvant-induced arthritis (AA) rats and its pharmacokinetic (PK) basis. AA was induced by injecting with Freund's complete adjuvant (FCA). Male SD rats were subjected to treatment with GE (30, 60 and 120mg/kg) from day 17 to 24 after immunization. Fibroblast-like synoviocyte (FLS) proliferation was assessed by MTT. Interleukin (IL)-1, IL-6, TNF-α and IL-10 were determined using double-sandwich enzyme-linked immunosorbent assay (ELISA). Expression of p38 mitogen-activated protein kinases (p38MAPKs) related proteins in FLS was detected by Western blotting. PK profiles were simultaneously detected by ultra-performance liquid chromatography-electrospray ionization-tandem mass spectrometry (UPLC-ESI-MS/MS) in AA rat plasma after oral administration of GE on day 17 after immunization. As a result, GE promoted the recovery of arthritis and inhibited the colonic inflammation damage in AA rats by decreasing the expression level of TNF-α, IL-1 and IL-6, increasing the production of IL-10 and inhibiting the expression of phospho-p38 (p-p38) related proteins in FLS. PK parameters (AUC, Cmax and t1/2) tended to be associated with dosage-related decreasing of efficacy index.

Determination of geniposide in adjuvant arthritis rat plasma by ultra-high performance liquid chromatography tandem mass spectrometry method and its application to oral bioavailability and plasma protein binding ability studies.

A specific, sensitive and high throughput ultra-high performance liquid chromatography-electrospray ionization tandem mass spectrometric method (UHPLC-ESI-MS/MS) was established and validated to assay geniposide (GE), a promising anti-inflammatory drug, in adjuvant arthritis rat plasma: application to pharmacokinetic and oral bioavailability studies and plasma protein binding ability. Plasma samples were processed by de-proteinised with ice-cold methanol and separated on an ACQUITY UPLC™ HSS C18 column (100 mm × 2.1mm i.d., 1.8 µm particle size) at a gradient flow rate of 0.2 mL/min using acetonitrile-0.1% formic acid in water as mobile phase, and the total run time was 9 min. Mass detection was performed in selected reaction monitoring (SRM) mode with negative electro-spray ionization includes the addition of paeoniflorin (Pae) as an internal standard (IS). The mass transition ion-pair was followed as m/z 387.4 → 122.4 for GE and m/z 479.4 → 449.0 for IS. The calibration curves were linear over the concentration range of 2-50,000 ng/mL with lower limit of quantification of 2 ng/mL. The intra-day and inter-day precisions (RSD, %) of the assay were less than 8.4%, and the accuracy was within ± 6.4% in terms of relative error (RE). Extraction recovery, matrix effect and stability were satisfactory in adjuvant arthritis rat plasma. The UHPLC-ESI-MS/MS method was successfully applied to a pharmacokinetic study of GE after oral administration of depurated GE at 33, 66, 132 mg/kg and intravenous injection at 33, 66, 132 mg/kg in adjuvant arthritis (AA) rats. In addition, it was found that GE has rapid absorption and elimination, low absolute bioavailability, high plasma protein binding ability in AA rats after oral administration within the tested dosage range. It suggested that GE showed slow distribution into the intra- and extracellular space, and the binding rate was not proportionally dependent on plasma concentration of GE when the concentration of GE was below 5.0 µg/mL.

Identification and distribution of four metabolites of geniposide in rats with adjuvant arthritis.

Geniposide (GE), also called Jasminoidin, is the major active ingredient of Gardenia jasminoides Ellis (GJ) fruit, which has long been used in traditional Chinese medicine (TCM). Growing evidences suggested that GE has a great potentiality for treating rheumatoid arthritis (RA). However, GE is rapidly metabolized, and we know little about its availability or metabolites in tissues. To elucidate the distribution of GE and its metabolites in tissues, three groups of adjuvant arthritis (AA) rats were given GE (33, 66 and 120 mg/kg) from days 18 to 24, and the biotransformation of GE in plasma, liver, spleen, synovium, urine and mesenteric lymph node (MLN) of rats was investigated by a novel approach named Information-Dependent Acquisition (IDA)-Mediated LC-MS/MS method. As a result, GE and its four major metabolites were detected as follows: GE, G1, G2 in plasma; GE, G2 in MLNs; only GE in liver and synovium; GE, G2, G3 and G4 in spleen; and GE, G1, G2 and G4 in urine. In total four metabolites (G1-G4) involved in the in vivo metabolism processes were identified. The results of this work have demonstrated the IDA-Mediated LC-MS/MS could screen rapidly and reliably the characterization of metabolites from iridoid compounds.

Effects and mechanisms of Geniposide on rats with adjuvant arthritis.

Geniposide (GE), an iridoid glycoside compound, is the major active ingredient of Gardenia jasminoides Ellis (GJ) fruit which has anti-inflammatory and other important therapeutic activities. The aim of this study was to investigate the effects of GE on adjuvant arthritis (AA) rats and its possible mechanisms. AA was induced by injecting with Freund's complete adjuvant (FCA). Male SD rats were subjected to treatment with GE at 30, 60 and 120mg/kg from days 18 to 24 after immunization. Lymphocyte proliferation was assessed by MTT. Interleukin (IL)-6, IL-17, IL-4 and transforming growth factor-beta 1 (TGF-ß1) were determined by ELISA. c-Jun N-terminal kinase (JNK) and phospho-JNK (p-JNK) were detected by Western blot. GE (60, 120mg/kg) significantly relieved the secondary hind paw swelling and arthritis index, along with decreased Th17-cells cytokines and increased Treg-cell cytokines in mesenteric lymph node lymphocytes (MLNL) and peripheral blood lymphocytes (PBL) of AA rats. In addition, GE decreased the expression of p-JNK in MLNL and PBL of AA rats. In vivo study, it was also observed that GE attenuated histopathologic changes of MLN in AA rats. Collectively, GE might exert its anti-inflammatory and immunoregulatory effects through inducing Th17 cell immune tolerance and enhancing Treg cell-mediated activities by down-regulating the expression of p-JNK. The mechanisms of GE on JNK signaling in MLNL and PBL may play critical roles in the pathogenesis of rheumatoid arthritis.

Comparative pharmacokinetics study after oral administration of geniposide in normal rats and adjuvant-induced arthritis rats by UPLC-MS/MS.

A simple and rapid ultra-performance liquid chromatography-electrospray ionization tandem mass spectrometry (UPLC-ESI-MS/MS) method for quantitative analysis of geniposide (GE) in rat plasma was developed, validated and applied to determine the level of GE in rat plasma after oral administration of GE in adjuvant-induced arthritis (AA) and normal rats. The investigation showed that there were significant differences in the groups between the normal rat and AA rat in pharmacokinetics parameters, such as the area under the time versus drug concentration curve (AUC(0-∞)) (3.77 ± 0.68 versus 2.27 ± 0.42, p < 0.05), the apparent volume of distribution (V) (140.41 ± 2.07 versus 136.51 ± 1.03, p < 0.05), the mean residence time (MRT) (3.98 ± 0.90 versus 3.80 ± 0.50, p < 0.05) and the clearance from the total body (CL) (16.10 ± 2.87 versus 26.44 ± 4.94, p < 0.05). The results indicated that AA could alter the pharmacokinetics of the drug and these experimental findings could be useful for the further study of the clinical applications of GE.