Insights into the molecular network underlying phytotoxicity and phytoaccumulation of ciprofloxacin.

Ciprofloxacin (CIP) is frequently detected in agricultural soils and can be accumulated by crops, causing phytotoxicities and food safety concerns. However, the molecular basis of its phytotoxicity and phytoaccumulation is hardly known. Here, we analyzed physiological and molecular responses of choysum (Brassica parachinensis) to CIP stress by comparing low CIP accumulation variety (LAV) and high accumulation variety (HAV). Results showed that the LAV suffered more severe inhibition of growth and photosynthesis than the HAV, exhibiting a lower tolerance to CIP toxicity. Integrated transcriptome and proteome analyses suggested that more differentially expressed genes/proteins (DEGs/DEPs) involved in basic metabolic processes were downregulated to a larger extent in the LAV, explaining its lower CIP tolerance at molecular level. By contrast, more DEGs/DEPs involved in defense responses were upregulated to a larger extent in the HAV, showing the molecular basis of its stronger CIP tolerance. Further, a CIP phytotoxicity-responsive molecular network was constructed for the two varieties to better understand the molecular mechanisms underlying the variety-specific CIP tolerance and accumulation. The results present the first comprehensive molecular profile of plant response to CIP stress for molecular-assisted breeding to improve CIP tolerance and minimize CIP accumulation in crops.

Substrate-enzyme interactions and catalytic mechanism in a novel family VI esterase with dibutyl phthalate-hydrolyzing activity.

Microbial degradation has been confirmed as effective and environmentally friendly approach to remediate phthalates from the environment, and hydrolase is an effective element for contaminant degradation. In the present study, a novel dibutyl phthalate (DBP)-hydrolyzing carboxylesterase (named PS06828) from Pseudomonas sp. PS1 was heterogeneously expressed in E. coli, which was identified as a new member of the lipolytic family VI. Purified PS06828 could efficiently degrade DBP with a wide range of temperature (25-37 °C) and pH (6.5-9.0). Multi-spectroscopy methods combined with molecular docking were employed to study the interaction of PS06828 with DBP. Fluorescence and UV-visible absorption spectra revealed the simultaneous presence of static and dynamic component in the fluorescence quenching of PS06828 by DBP. Synchronous fluorescence and circular dichroism spectra showed inconspicuous alteration in micro-environmental polarity around amino acid residues but obvious increasing of α-helix and reducing of ß-sheet and random coil in protein conformation. Based on the information on exact binding sites of DBP on PS06828 provided by molecular docking, the catalytic mechanism mediated by key residues (Ser113, Asp166, and His197) was proposed and subsequently confirmed by site-directed mutagenesis. The results can strengthen our mechanistic understanding of family VI esterase involved in hydrolysis of phthalic acid esters, and provide a solid foundation for further enzymatic modification.

Mechanistic insight into esterase-catalyzed hydrolysis of phthalate esters (PAEs) based on integrated multi-spectroscopic analyses and docking simulation.

A novel PAE-hydrolyzing esterase (named Hyd) gene was screened from the genomic library of Rhodococcus sp. 2G and was successfully expressed in heterologous E. coli, which was defined as a new family of esterolytic enzymes. The purified Hyd could efficiently degrade various PAEs, displaying high activity and stability with a broad range of pH (4-10) and temperature (20-60 °C). Interaction mechanism of Hyd with dibutyl phthalate (DBP) was investigated by integrated multi-spectroscopic and docking simulation methods. Fluorescence and UV-vis spectra revealed that DBP could quench the fluorescence of Hyd through a static quenching mechanism. The results from synchronous fluorescence and CD spectra confirmed that the DBP binding to Hyd triggered conformational and micro-environmental changes of Hyd, which were characterized by increased stretching extent and random coil, and decreased α-helix and ß-sheet. Molecular docking study showed that DBP could be bound to the cavity of Hyd with hydrogen bonding and hydrophobic interaction. A novel and distinctive catalytic mechanism was proposed: two key residues Thr190 and Ser191 might catalyze the hydrolysis of DBP, instead of the conserved catalytic triad (Ser-His-Asp) reported elsewhere, which was confirmed by site-directed mutagenesis.

Novel phosphate-solubilising bacteria isolated from sewage sludge and the mechanism of phosphate solubilisation.

A great amount of insoluble phosphate in agricultural soils is not available for crops. Three strains of bacteria (Bacillus megaterium YLYP1, Pseudomonas prosekii YLYP6 and Pseudomonas sp. YLYP29) isolated from activated sludge and soil could efficiently solubilise tricalcium phosphate. In particular, the novel strain P. prosekii YLYP6 produced 716 mg L-1 of available phosphate within 6 days under the optimal culture conditions [20 °C, pH 7.9, inoculum size of 0.5% (v:v)] determined by response surface methodology. P. prosekii YLYP6 demonstrated efficient phosphate solubilisation in response to broad variations in pH (5-9) and temperature (15-35 °C). The phosphate solubilisation curves of the strains fit well with a first-order kinetic model (R2 > 0.939), with a half-life of 1.51-5.94 d for 5.0 g L-1 calcium phosphate. Continuous culture experiments combined with scanning electron microscopic observations and gas chromatography-mass spectrometry analysis revealed that 2,3-dimethylfumaric acid, gluconic and N-butyl-tert-butylamine that were produced by P. prosekii YLYP6 were responsible for phosphate solubilisation by supplying H+ ions and organic anions. Efficient phosphate solubilisation in actual soil by P. prosekii YLYP6 demonstrated the strong application potential to reduce the use of chemical P fertilisers and the resulting agricultural nonpoint pollution.

Differences in Root Physiological and Proteomic Responses to Dibutyl Phthalate Exposure between Low- and High-DBP-Accumulation Cultivars of Brassica parachinensis.

Di- n-butyl phthalate (DBP), as an endocrine-disrupting chemical that tends to be accumulated in crops, poses great risks to human health through the food chain. To identify the molecular mechanism underlying differences in their DBP accumulation, the root physiological and proteomic responses to DBP stress of two Brassica parachinensis cultivars, a high-DBP accumulator (Huaguan) and a low-DBP accumulator (Lvbao), were investigated. Root damage of greater severity and significantly greater ( p < 0.05) decreases in root protein content and root activity were detected in Lvbao than in Huaguan, suggesting that Lvbao had lower tolerance to DBP. In total, 52 DBP-responsive proteins were identified by two-dimensional electrophoresis and MALDI-TOF mass spectrometry. More proteins involved in basic metabolic processes, such as protein synthesis and energy metabolism, were downregulated in Lvbao, possibly explaining its lower tolerance and root damage. Several proteins involved in starch metabolism, cell-wall biosynthesis and modification, and stress response were activated in Huaguan, suggesting greater tolerance to DBP. Overall, differences in root proteome between the two cultivars might be responsible for the genotype-dependent DBP tolerance and accumulation in B. parachinensis.

Global Picture of Protein Regulation in Response to Dibutyl Phthalate (DBP) Stress of Two Brassica parachinensis Cultivars Differing in DBP Accumulation.

iTRAQ analysis was used to map the proteomes of two Brassica parachinensis cultivars that differed in dibutyl phthalate (DBP) accumulation. A total of 5699 proteins were identified to obtain 152 differentially regulated proteins, of which 64 and 48 were specific to a high- and a low-DBP-accumulation cultivar, respectively. Genotype-specific biological processes were involved in coping with DBP stress, accounting for the variation in DBP tolerance and accumulation. Formation of high DBP accumulation in B. parachinensis might attribute to the more effective regulation of protein expression in physiology and metabolism, including (a) enhanced cell wall biosynthesis and modification, (b) better maintenance of photosynthesis and energy balance, (c) greatly improved total capacity for antioxidation and detoxification, and (d) enhanced cellular transport and signal transduction. Our novel findings contribute to a global picture of DBP-induced alterations of protein profiles in crops and provide valuable information for the development of molecular-assisted breeds of low-accumulation cultivars.

Intraspecific variability of ciprofloxacin accumulation, tolerance, and metabolism in Chinese flowering cabbage (Brassica parachinensis).

To investigate the mechanism of genotype differences in ciprofloxacin (CIP) accumulation, this study was designed to compare the tolerance and metabolic responses to CIP exposure between low (Cutai) and high (Sijiu) CIP-accumulation cultivars of Brassica parachinensis. Decreases in biomass and chlorophyll content were significantly greater (p < 0.05) and toxicities were more severe within cell ultrastructures of Cutai compared to Sijiu. A sequential growth test also revealed that Sijiu was more tolerant to CIP stress compared to Cutai. Meanwhile, significantly higher (p < 0.05) root parameters and higher areas of the stele and xylem may be responsible for the increased uptake and transport of CIP in Sijiu. Ultra performance liquid chromatography-electrospray ionization tandem mass spectrometry (UPLC-ESI-MS/MS) analysis revealed that CIP was metabolized to three major metabolites by the hydroxylation and breakdown of the piperazinyl substituent in the CIP molecule. The enhanced metabolic transformation of CIP in Sijiu indicated a more efficient capacity to detoxify, which in turn favored an increased accumulation of CIP in this cultivar. Thus, the present study demonstrated that the stronger tolerance and metabolism of Sijiu to CIP were responsible for its high CIP accumulation, suggesting an evolutionary mechanism for adaptation to environmental stress.