Biodegradation of synthetic pyrethroids by <Emphasis Type="Italic">Ochrobactrum tritici</Emphasis> strain pyd-1

A synthetic pyrethroid (SP)-degrading bacterium, designated pyd-1, was isolated from SPcontaminated soil. Based on its phenotypic and genotypic properties, the strain was identified as Ochrobactrum tritici. Strain pyd-1 was able to degrade a wide range of SPs, and its degradation efficiencies were dependent on the molecular structure of the SP. Interestingly, the strain degraded cis- and trans-permethrin (cypermethrin) at nearly the same rate and possessed approximately equal hydrolysis activities toward the two enantiomers of fenpropathrin. These results suggest that different isomers of SPs are degraded with equal efficiency by strain pyd-1. We studied the metabolic pathway of fenpropathrin degradation in strain pyd-1 by metabolite identification and enzymatic analysis. Fenpropathrin is degraded by hydrolysis of the carboxylester linkage to yield 2,2,3,3-tetramethylcyclopropanecarboxylic acid and 3-phenoxybenzaldehyde, which is converted to 3-phenoxybenzoic acid (PBA). PBA is further metabolized to 4-hydroxy-3-phenoxybenzoic acid (4-hydroxy-PBA). 4-Hydroxy-PBA is oxidized to protocatechuate and p-hydroquinone. Protocatechuate is further oxidized through an ortho-cleavage pathway, and p-hydroquinone is degraded via 1,2,4-benzenetriol.     

Simultaneous determination of five characteristic stilbene glycosides in root bark of Morus albus L. (Cortex Mori) using high-performance liquid chromatography

Introduction – Cortex Mori, one of the well‐known traditional Chinese herbal medicines, is derived from the root bark of Morus alba L. according to the China Pharmacopeia. Stilbene glycosides are the main components isolated from aqueous extracts of Morus alba and their content varies depending on where Cortex Mori was collected. We have established a qualitative and quantitative method based on the bioactive stilbene glycosides for control of the quality of Cortex Mori from different sources. Objective – To develop a high‐performance liquid chromatography coupled with ultraviolet absorption detection for simultaneous quantitative determination of five major characteristic stilbene glycosides in 34 samples of the root bark of Morus alba L. (Cortex Mori) from different sources. Methodology – The analysis was performed on an ODS column using methanol‐water‐acetic acid (18: 82: 0.1, v/v/v) as the mobile phase and the peaks were monitored at 320 nm. Results – All calibration curves showed good linearity (r ≥ 0.9991) within test ranges. This method showed good repeatability for the quantification of these five components in Cortex Mori with intra‐ and inter‐day standard deviations less than 2.19% and 1.45%, respectively. Conclusion – The validated method was successfully applied to quantify the five investigated components, including a pair of cis‐trans‐isomers 1 and 2 and a pair of isomers 4 and 5 in 34 samples of Cortex Mori from different sources. Copyright © 2010 John Wiley & Sons, Ltd.     

Stereoselective Degradation of alpha‐Cypermethrin and Its Enantiomers in Rat Liver Microsomes

Alpha‐cypermethrin (α‐CP), [(RS)‐a‐cyano‐3‐phenoxy benzyl (1RS)‐cis‐3‐(2, 2‐dichlorovinyl)‐2, 2‐dimethylcyclopropanecarboxylate], comprises a diastereoisomer pair of cypermethrin, which are (+)‐(1R‐cis‐αS)–CP (insecticidal) and (?)‐(1S‐cis‐αR)–CP (inactive). In this experiment, the stereoselective degradation of α‐CP was investigated in rat liver microsomes by high‐performance liquid chromatography (HPLC) with a cellulose‐tris‐ (3, 5‐dimethylphenylcarbamate)‐based chiral stationary phase. The results revealed that the degradation of (?)‐(1S‐cis‐αR)‐CP was much faster than (+)‐(1R‐cis‐αS)‐CP both in enantiomer monomers and rac‐α‐CP. As for the enzyme kinetic parameters, there were some variances between rac‐α‐CP and the enantiomer monomers. In rac‐α‐CP, the V_max and CL_int of (+)‐(1R‐cis‐αS)–CP (5105.22 ± 326.26 nM/min/mg protein and 189.64 mL/min/mg protein) were about one‐half of those of (?)‐(1S‐cis‐αR)–CP (9308.57 ± 772.24 nM/min/mg protein and 352.19 mL/min/mg protein), while the K_m of the two α‐CP enantiomers were similar. However, in the enantiomer monomers of α‐CP, the V_max and K_m of (+)‐(1R‐cis‐αS) ‐CP were 2‐fold and 5‐fold of (?)‐(1S‐cis‐αR)‐CP, respectively, which showed a significant difference with rac‐α‐CP. The CL_int of (+)‐(1R‐cis‐αS)–CP (140.97 mL/min/mg protein) was still about one‐half of (?)‐(1S‐cis‐αR)–CP (325.72 mL/min/mg protein) in enantiomer monomers. The interaction of enantiomers of α‐CP in rat liver microsomes was researched and the results showed that there were different interactions between the IC₅₀ of (?)‐ to (+)‐(1R‐cis‐αS)‐CP and (+)‐ to (?)‐(1S‐cis‐αR)‐CP(IC_50(?)/(+) / IC_50(+)/(?) = 0.61). Chirality 28:58–64, 2016. © 2015 Wiley Periodicals, Inc.     

Site‐specific profiles of biochemical properties in the larval digestive tract of Japanese rhinoceros beetle,Trypoxylus dichotomus (Coleoptera: Scarabaeidae)

The larvae of Japanese rhinoceros beetle, Trypoxylus dichotomus, feed on dead plant material in forest soils that are derived from fallen leaves broken down by basidiomycete fungi. Our previous work provided an understanding of the degradation of polysaccharides in dead plant material by T. dichotomus larvae and reported the complexity of the physicochemical and biochemical environment of the larval gut. Here, we examined ten divisions of the digestive tract of T. dichotomus larvae for physicochemical and biochemical conditions to elucidate site‐specifically functional properties along the tract. The distribution of potassium ions, pH, and acetic acid differed markedly along the length of the digestive tract with the potassium ion concentration profile closely reflecting that of pH along the length of the digestive tract. Distinct physicochemical environments were maintained in the digestive tract along with site‐specific polysaccharide degradation. Based on these findings, we suggest that there are metabolic relationships between the activities of the enzymes involved in polysaccharide degradation, the presence of intermediate metabolites and location along the digestive tract. Furthermore, we revealed that the anterior region of the gut plays an important role in the degradation of polysaccharides in the digestive tract of T. dichotomus larvae.     

Mapping DNA damage‐dependent genetic interactions in yeast via party mating and barcode fusion genetics

Condition‐dependent genetic interactions can reveal functional relationships between genes that are not evident under standard culture conditions. State‐of‐the‐art yeast genetic interaction mapping, which relies on robotic manipulation of arrays of double‐mutant strains, does not scale readily to multi‐condition studies. Here, we describe barcode fusion genetics to map genetic interactions (BFG‐GI), by which double‐mutant strains generated via en masse “party” mating can also be monitored en masse for growth to detect genetic interactions. By using site‐specific recombination to fuse two DNA barcodes, each representing a specific gene deletion, BFG‐GI enables multiplexed quantitative tracking of double mutants via next‐generation sequencing. We applied BFG‐GI to a matrix of DNA repair genes under nine different conditions, including methyl methanesulfonate (MMS), 4‐nitroquinoline 1‐oxide (4NQO), bleomycin, zeocin, and three other DNA‐damaging environments. BFG‐GI recapitulated known genetic interactions and yielded new condition‐dependent genetic interactions. We validated and further explored a subnetwork of condition‐dependent genetic interactions involving MAG1, SLX4, and genes encoding the Shu complex, and inferred that loss of the Shu complex leads to an increase in the activation of the checkpoint protein kinase Rad53.     

Influence of lactic acid bacteria on stereoselective degradation of theta‐cypermethrin

The purpose of this study was to investigate the influence of four kinds of Lactic acid bacteria (LAB) on stereoselective degradation of theta‐cypermethrin (CYP), including Lactobacillus plantarum, Lactobacillus casei, Lactobacillus delbrueckii, and Streptococcus thermophilus. An effective analytical method for (±)‐theta‐CYP in medium was developed by high‐performance liquid chromatography with cellulose tris‐(3,5‐dimethylphenylcarbamate) chiral stationary phase. theta‐Cypermethrin was spiked to LAB medium with different inoculation rates and sampled at 0, 2, 8, 24, 36, 48, 72, 120, 168, and 240 hours. The results showed that LAB influenced the half‐lives and enantiomer fractions of theta‐CYP enantiomers, which lead a closer degradation rate between the 2 stereoisomers, and no obvious difference was found among 4 LABs. Besides, the stereoselective degradation of theta‐CYP was closely related to pH. The lower the pH (pH of 3, 5, 7, and 9), the lower the enantiomer fraction (from 4.88 to 6.69). At pH of 3, 7, and 9, significant differences of half‐lives between enantiomers were observed. (?)‐theta‐Cypermethrin decreased faster than (+)‐theta‐CYP under pH of 3, while opposite results were indicated under pH of 7 and 9. Moreover, the acidic condition contributed to the higher chiral configuration stability of (±)‐theta‐CYP. (+)‐Enantiomer was influenced by pH in a greater degree than (?)‐enantiomer.     

The use of quantitative imaging to investigate regulators of membrane trafficking in Arabidopsis stomatal closure

Expansion of gene families facilitates robustness and evolvability of biological processes but impedes functional genetic dissection of signalling pathways. To address this, quantitative analysis of single cell responses can help characterize the redundancy within gene families. We developed high‐throughput quantitative imaging of stomatal closure, a response of plant guard cells, and performed a reverse genetic screen in a group of Arabidopsis mutants to five stimuli. Focussing on the intersection between guard cell signalling and the endomembrane system, we identified eight clusters based on the mutant stomatal responses. Mutants generally affected in stomatal closure were mostly in genes encoding SNARE and SCAMP membrane regulators. By contrast, mutants in RAB5 GTPase genes played specific roles in stomatal closure to microbial but not drought stress. Together with timed quantitative imaging of endosomes revealing sequential patterns in FLS2 trafficking, our imaging pipeline can resolve non‐redundant functions of the RAB5 GTPase gene family. Finally, we provide a valuable image‐based tool to dissect guard cell responses and outline a genetic framework of stomatal closure.      

Seasonal changes of resistance to fenvalerate and cypermethrin in the larval parasitoid Cotesia plutellae （Hymenoptera： Braconidae）

The seasonal changes of insecticide resistance and stability in hymenopteran Cotesia plutellae, collected from Jianxin, Fuzhou-City, and Shangjie, Minhou-County, Fujian, China, were assessed by using a dry residual film method. The resistance to two insecticides in the field populations of C. plutellae was not stable under insecticide-free conditions in the insectarium. Compared with susceptible F11 progeny of C. plutellae in the insectarium, the resistance ratios （RR） in F0 parents were 18.4 for fenvalerate and 11.4 for cypermethrin based on LC50 at 9 hours, and 32.8 for fenvalerate and 28.5 for cypermethrin based on LC50 at 24 hours when the parasitoids were left in contact with the insecticides for 1 hour and mortalities were recorded at 9 and 24 hours, respectively. However, the RR in a field population of C. plutellae were 9.2 for fenvalerate and 12.7 for cypermethrin, if the parasitoids were left in contact with the insecticides for 24 hours. The resistances to the two pyrethroids in other field populations collected from Jianxin and Shangjie from November 2000 and July 2004 were also determined. Significant seasonal variations of resistance to the two insecticides in the field populations of C. plutellae were found. The RR were 3.0-18.4 for fenvalerate and 4.8-20.6 for cypermethrin in Jianxin populations from November 2000 to April 2002 based on LC50 at 9 h, and 2.3-13.6 for fenvalerate and 3.6-16.0 for cypermethrin in Shangjie populations from May 2002 to July 2004 based on LC50 at 24 hours. The resistance levels were high in spring and autumn and decreased sharply in summer. In addition, significant recovery from the knocked-down caused by the insecticides was found in the F0 and field populations of C. plutellae which were resistant to fenvalerate and cypermethrin if the parasitoids were left in contact with the pyrethroids for 1 hour. However, no recovery was found in susceptible F11 progeny.     

Metabolic profiles and phylogenetic diversity of microbial communities from chlorinated pesticides contaminated sites of different geographical habitats of India

Aims: To study the microbial communities in three sites contaminated with chlorinated pesticides and evaluation of dehydrodechlorinase (linA) gene variants involved in gamma‐hexachlorocyclohexane (γ‐HCH, lindane) degradation. Methods and Results: Using a culture‐independent method, 16S rRNA genes were amplified from microbial communities occurring in contaminated soils. From 375 clone libraries analysed, 55 different restriction fragment length polymorphism phylotypes were obtained. Dehydrodechlorinase (linA) gene, which initiates the γ‐HCH degradation, was directly amplified by PCR from the DNA extracted from soils. Deduced amino acid sequences of eight variant genotypes of linA showed few amino acid changes. All the variants of linA had mutations of F151L and S154T, and one of the genotype carried 12 amino acid changes when compared to a linA of Sphingomonas sp. reported from the same soil. Conclusions: The microbial communities displayed complex and diverse groups similar to bacteria involved in biodegradation. The presence of biodegradative genes like linA indicates the presence of communities with capacity to biodegrade the persistent pesticide HCH. Significance and Impact of the Study: This study provides insights to evaluate the presence of catabolic genes and assessing the bioremediation potential of the industrial soils contaminated by chlorinated pesticides.     

Biodegradation of commercial linear alkyl benzenes byNocardia amarae

Laboratory degradation studies of two indigeneously produced linear alkyl benzenes byNocardia amarae MB-11 isolated from soil showed an overall degradation of linear alkyl benzenes isomers to the extent of 57–70%. Degradation of 2-phenyl isomers of linear alkyl benzenes was complete and faster than that of other phenyl position (C3–C7) isomers which were degraded to the extent of 40–72% only. Length of alkyl side chains (C₁₀–C₁₄) had little or no impact on the degradation pattern. Major metabolities detected were 2-, 3-and 4-phenyl butyric acids, phenyl acetic acid and cis, cis-muconic acid. Minor metabolites weretrans-cinnamic acid, 4-phenyl 3-butenoic acid and 3-phenyl pentanoic acid along with two unidentified hydroxy acids. On the basis of the formation pattern of these metabolities, three catabolic pathways of linear alkyl benzenes isomers inNocardia amarae MB-11 were postulated. All the phenyl position (C2–C7) isomers of C₁₀, C₁₂, and C₁₄ linear alkyl benzenes along with 3-phenyl and 5-phenyl isomers of C₁₁ and C₁₃ linear alkyl benzenes were degraded viacis,cis-muconic acid pathway. Other phenyl position isomers of C₁₁ and C₁₃ linear alkyl benzenes with phenyl substitution at even number carbon atoms were principally degraded via phenyl acetic acid pathway whiletrans-cinnamic acid formation provided a minor pathway     

Stereolability of Chiral Ruthenium Catalysts With Frozen NHC Ligand Conformations Investigated by Dynamic‐HPLC

The stereolability of chiral Hoveyda–Grubbs II type ruthenium complexes bearing N‐heterocyclic carbene (NHC) ligands with Syn‐phenyl groups on the backbone and Syn‐ or Anti‐oriented o‐tolyl N‐substituents was studied by resorting to dynamic high‐performance liquid chromatography (D‐HPLC). A complete chromatographic picture of the involved stereoisomers (four for Anti‐ and two for Syn‐complexes) was achieved at very low temperatures (?53°C and ?40°C respectively), at which the NHC‐Ru bond rotations were frozen out. Inspection of the chromatographic profiles recorded at higher temperatures revealed the presence of plateau zones between the couples of either Syn or Anti stereoisomers, attesting to the active interconversion between the eluted species. Such dynamic chromatograms were successfully simulated through procedures based on both theoretical plate and classical stochastic models. The good superimposition achieved between experimental and simulated chromatographic profiles allowed determination of the related isomerization energy barriers (ΔG_isom^#), all derived by rotation around the NHC‐Ru bond. The obtained diastereomerization barriers between the Anti isomers were found in very good agreement with those previously measured by experimental nuclear magnetic resonance (NMR) and assessed through Density Functional Theory (DFT) calculations. With the same approach, for the first time we also determined the enantiomerization barrier of the Syn isomer. Focused changes to the structure of complex Syn, studied by a molecular modeling approach, were found suitable to strongly reduce the stereolability arising from rotation around the NHC‐Ru bond. Chirality 27:685–692, 2015. © 2015 Wiley Periodicals, Inc.     

Microbial ecology of chlorinated solvent biodegradation

This study focused on the microbial ecology of tetrachloroethene (PCE) degradation to trichloroethene, cis‐1,2‐dichloroethene and vinyl chloride to evaluate the relationship between the microbial community and the potential accumulation or degradation of these toxic metabolites. Multiple soil microcosms supplied with different organic substrates were artificially contaminated with PCE. A thymidine analogue, bromodeoxyuridine (BrdU), was added to the microcosms and incorporated into the DNA of actively replicating cells. We compared the total and active bacterial communities during the 50‐day incubations by using phylogenic microarrays and 454 pyrosequencing to identify microorganisms and functional genes associated with PCE degradation to ethene. By use of this integrative approach, both the key community members and the ecological functions concomitant with complete PCE degradation could be determined, including the presence and activity of microbial community members responsible for producing hydrogen and acetate, which are critical for Dehalococcoides‐mediated PCE degradation. In addition, by correlation of chemical data and phylogenic microarray data, we identified several bacteria that could potentially oxidize hydrogen. These results demonstrate that PCE degradation is dependent on some microbial community members for production of appropriate metabolites, while other members of the community compete for hydrogen in soil at low redox potentials.     

生物冶金过程中的关键微生物及其碳循环代谢途径研究

【目的】深入研究极端酸性环境中微生物的碳循环过程。【方法】应用16S r RNA高通量测序和功能基因芯片技术对德兴铜矿中浸矿堆(LH)和积液池(LS)两个子系统中的微生物群落结构组成和功能基因组成进行分析;并运用PICRUSt功能基因预测的方法对群落功能进行预测。【结果】功能基因芯片和功能预测分析都表明碳循环基因在子系统间存在显著差异(P0.05),且碳固定相关的卡尔文循环、还原性三羧酸循环等基因以及碳降解相关的己聚糖和纤维素等基因在LS系统中都要明显高于LH系统。碳循环功能基因在子系统之间的差异与环境条件相关,其中TON、Ca、ES、Fe3+和P作用显著。【结论】在极端酸性环境中,环境条件的差异会对微生物群落碳循环功能基因产生筛选作用,参与碳循环的微生物的种类和相对丰度都发生变化,最终改变了群落碳循环模式。     

Molecular diversity of PBAN family peptides from fire ants

The PBAN/Pyrokinin peptide family is a major neuropeptide family characterized with a common FXPRLamide in the C‐termini. These peptides are ubiquitously distributed in the Insecta and are involved in many essential endocrinal functions, e.g., pheromone production. Previous work demonstrated the localization of PBAN in the fire ant central nervous system, and identified a new family of PBAN from the red imported fire ant, Solenopsis invicta. In this study, we identified five more PBAN/Pyrokinin genes from S. geminata, S. richteri, S. pergandii, S. carolinensis, and a hybrid of S. invicta and S. richteri. The gene sequences were used to determine the phylogenetic relationships of these species and hybrid, which compared well to the morphologically defined fire ant subgroup complexes. The putative PBAN and other peptides were determined from the amino acid sequences of the PBAN/pyrokinin genes. We summarized all known insect PBAN family neuropeptides, and for the first time constructed a phylogenetic tree based on the full amino acid sequences translated from representative PBAN cDNAs. The PBAN/pyrokinin gene is well conserved in Insecta and probably extends into the Arthropod phylum; however, translated pre‐propeptides may vary and functional diversity may be retained, lost, or modified during the evolutionary process. Published 2010 Wiley Periodicals, Inc.     

A Simple Method for Resolution of Endo‐/Exo‐Monoesters of Trans‐Norborn‐5‐Ene‐2,3‐Dicarboxylic Acids Into Their Enantiomers

Separation of optical isomers obtainable from trans‐norborn‐5‐ene‐2,3‐dicarboxylic acid methyl and tert‐butyl monoesters was performed by crystallization of the respective salts prepared with (R)‐ and (S)‐1‐phenylethylamine. Starting from racemic endo‐monomethyl ester of trans‐norborn‐5‐ene‐2,3‐dicarboxylic acid, prepared by partial hydrolysis of the cyclopentadiene‐dimethyl fumarate adduct, the corresponding (2R,3R)‐endo‐monoester was isolated in 97% enantiomeric excess (ee) yield after seven repeated crystallizations from tetrachloromethane. Starting from exo‐mono‐tert‐butyl ester of the same acid, prepared by alcoholysis of the cyclopentadiene‐maleic anhydride adduct followed by isomerization, (2R,3R)‐exo‐monoester was isolated in >98% ee yield after four repeated crystallizations from ethanol. Crystallization of the acids from the mother liquor containing (S)‐1‐phenylethylamine yielded products with inverse stereochemical configuration. Chirality 27:151–155, 2015. © 2014 Wiley Periodicals, Inc.