Development of efficient modules for recombinant protein expression and periplasmic localisation in Pseudomonas bharatica CSV86T.

Escherichia coli has been widely employed as a host for heterologous protein expression. However, due to certain limitations, alternative hosts like Pseudomonas, Lactococcus and Bacillus are being explored. Pseudomonas bharatica CSV86T, a novel soil isolate, preferentially degrades wide range of aromatics over simple carbon sources like glucose and glycerol. Strain also possesses advantageous eco-physiological traits, making it an ideal host for engineering xenobiotic degradation pathways, which necessitates the development of heterologous expression systems. Based on the efficient growth, short lag-phase and rapid metabolism of naphthalene, Pnah and Psal promoters (regulated by NahR) were selected for expression. Pnah was found to be strong and leaky as compared to Psal, using 1-naphthol 2-hydroxylase (1NH, ∼66 kDa) as reporter gene in strain CSV86T. The Carbaryl hydrolase (CH, ∼72 kDa) from Pseudomonas sp. C5pp was expressed under Pnah in strain CSV86T and could successfully be translocated to the periplasm due to the presence of the Tmd + Sp sequence. The recombinant CH was purified from the periplasmic fraction and the kinetic characteristics were found to be similar to the native protein from strain C5pp. These results potentiate the suitability of P. bharatica CSV86T as a desirable host, while Pnah and the Tmd + Sp can be employed for overexpression and periplasmic localisation, respectively. Such tools find application in heterologous protein expression and metabolic engineering applications.

Two LysR Family Transcriptional Regulators, McbH and McbN, Activate the Operons Responsible for the Midstream and Downstream Pathways, Respectively, of Carbaryl Degradation in Pseudomonas sp. Strain XWY-1.

Previously, a LysR family transcriptional regulator, McbG, that activates the mcbBCDEF gene cluster involved in the upstream pathway (from carbaryl to salicylate) of carbaryl degradation in Pseudomonas sp. strain XWY-1 was identified by us (Z. Ke, Y. Zhou, W. Jiang, M. Zhang, et al., Appl Environ Microbiol 87:e02970-20, 2021, https://doi.org/10.1128/AEM.02970-20). In this study, we identified McbH and McbN, which activate the mcbIJKLM cluster (responsible for the midstream pathway, from salicylate to gentisate) and the mcbOPQ cluster (responsible for the downstream pathway, from gentisate to pyruvate and fumarate), respectively. They both belong to the LysR family of transcriptional regulators. Gene disruption and complementation study reveal that McbH is essential for transcription of the mcbIJKLM cluster in response to salicylate and McbN is indispensable for the transcription of the mcbOPQ cluster in response to gentisate. The results of electrophoretic mobility shift assay (EMSA) and DNase I footprinting showed that McbH binds to the 52-bp motif in the mcbIJKLM promoter area and McbN binds to the 58-bp motif in the mcbOPQ promoter area. The key sequence of McbH binding to the mcbIJKLM promoter is a 13-bp motif that conforms to the typical characteristics of the LysR family. However, the 12-bp motif that is different from the typical characteristics of the LysR family regulator binding site sequence is identified as the key sequence for McbN to bind to the mcbOPQ promoter. This study revealed the regulatory mechanisms for the midstream and downstream pathways of carbaryl degradation in strain XWY-1 and further our knowledge of (and the size of) the LysR transcription regulator family. IMPORTANCE The enzyme-encoding genes involved in the complete degradation pathway of carbaryl in Pseudomonas sp. strain XWY-1 include mcbABCDEF, mcbIJKLM, and mcbOPQ. Previous studies demonstrated that the mcbA gene, responsible for hydrolysis of carbaryl to 1-naphthol, is constitutively expressed and that the transcription of mcbBCDEF was regulated by McbG. However, the transcription regulation mechanisms of mcbIJKLM and mcbOPQ have not been investigated yet. In this study, we identified two LysR-type transcriptional regulators, McbH and McbN, which activate the mcbIJKLM cluster (responsible for the degradation of salicylate to gentisate) and the mcbOPQ cluster (responsible for the degradation of gentisate to pyruvate and fumarate), respectively. The 13-bp motif is critical for McbH to bind to the promoter of mcbIJKLM, and 12-bp motif different from the typical characteristics of the LysR-type transcriptional regulator (LTTR) binding sequence affects the binding of McbN to the promoter. These findings help to expand the understanding of the regulatory mechanism of microbial degradation of carbaryl.

Display of a novel carboxylesterase CarCby on Escherichia coli cell surface for carbaryl pesticide bioremediation.

BACKGROUND: Carbamate pesticides have been widely used in agricultural and forestry pest control. The large-scale use of carbamates has caused severe toxicity in various systems because of their toxic environmental residues. Carbaryl is a representative carbamate pesticide and hydrolase/carboxylesterase is the initial and critical enzyme for its degradation. Whole-cell biocatalysts have become a powerful tool for environmental bioremediation. Here, a whole cell biocatalyst was constructed by displaying a novel carboxylesterase/hydrolase on the surface of Escherichia coli cells for carbaryl bioremediation. RESULTS: The carCby gene, encoding a protein with carbaryl hydrolysis activity was cloned and characterized. Subsequently, CarCby was displayed on the outer membrane of E. coli BL21(DE3) cells using the N-terminus of ice nucleation protein as an anchor. The surface localization of CarCby was confirmed by SDS-PAGE and fluorescence microscopy. The optimal temperature and pH of the engineered E. coli cells were 30 °C and 7.5, respectively, using pNPC4 as a substrate. The whole cell biocatalyst exhibited better stability and maintained approximately 8-fold higher specific enzymatic activity than purified CarCby when incubated at 30 °C for 120 h. In addition, ~ 100% and 50% of the original activity was retained when incubated with the whole cell biocatalyst at 4 ℃ and 30 °C for 35 days, respectively. However, the purified CarCby lost almost 100% of its activity when incubated at 30 °C for 134 h or 37 °C for 96 h, respectively. Finally, approximately 30 mg/L of carbaryl was hydrolyzed by 200 U of the engineered E. coli cells in 12 h. CONCLUSIONS: Here, a carbaryl hydrolase-containing surface-displayed system was first constructed, and the whole cell biocatalyst displayed better stability and maintained its catalytic activity. This surface-displayed strategy provides a new solution for the cost-efficient bioremediation of carbaryl and could also have the potential to be used to treat other carbamates in environmental bioremediation.

McbG, a LysR Family Transcriptional Regulator, Activates the mcbBCDEF Gene Cluster Involved in the Upstream Pathway of Carbaryl Degradation in Pseudomonas sp. Strain XWY-1.

Although enzyme-encoding genes involved in the degradation of carbaryl have been reported in Pseudomonas sp. strain XWY-1, no regulator has been identified yet. In the mcbABCDEF cluster responsible for the upstream pathway of carbaryl degradation (from carbaryl to salicylate), the mcbA gene is constitutively expressed, while mcbBCDEF is induced by 1-naphthol, the hydrolysis product of carbaryl by McbA. In this study, we identified McbG, a transcriptional activator of the mcbBCDEF cluster. McbG is a 315-amino-acid protein with a molecular mass of 35.7 kDa. It belongs to the LysR family of transcriptional regulators and shows 28.48% identity to the pentachlorophenol (PCP) degradation transcriptional activation protein PcpR from Sphingobium chlorophenolicum ATCC 39723. Gene disruption and complementation studies reveal that mcbG is essential for transcription of the mcbBCDEF cluster in response to 1-naphthol in strain XWY-1. The results of the electrophoretic mobility shift assay (EMSA) and DNase I footprinting show that McbG binds to the 25-bp motif in the mcbBCDEF promoter area. The palindromic sequence TATCGATA within the motif is essential for McbG binding. The binding site is located between the -10 box and the transcription start site. In addition, McbG can repress its own transcription. The EMSA results show that a 25-bp motif in the mcbG promoter area plays an important role in McbG binding to the promoter of mcbG This study reveals the regulatory mechanism for the upstream pathway of carbaryl degradation in strain XWY-1. The identification of McbG increases the variety of regulatory models within the LysR family of transcriptional regulators.IMPORTANCEPseudomonas sp. strain XWY-1 is a carbaryl-degrading strain that utilizes carbaryl as the sole carbon and energy source for growth. The functional genes involved in the degradation of carbaryl have already been reported. However, the regulatory mechanism has not been investigated yet. Previous studies demonstrated that the mcbA gene, responsible for hydrolysis of carbaryl to 1-naphthol, is constitutively expressed in strain XWY-1. In this study, we identified a LysR-type transcriptional regulator, McbG, which activates the mcbBCDEF gene cluster responsible for the degradation of 1-naphthol to salicylate and represses its own transcription. The DNA binding site of McbG in the mcbBCDEF promoter area contains a palindromic sequence, which affects the binding of McbG to DNA. These findings enhance our understanding of the mechanism of microbial degradation of carbaryl.

Identification and characterization of a novel bacterial carbohydrate esterase from the bacterium Pantoea ananatis Sd-1 with potential for degradation of lignocellulose and pesticides.

OBJECTIVE: Identification and characterization of a novel bacterial carbohydrate esterase (PaCes7) with application potential for lignocellulose and pesticide degradation. RESULTS: PaCes7 was identified from the lignocellulolytic bacterium, Pantoea ananatis Sd-1 as a new carbohydrate esterase. Recombinant PaCes7 heterologously expressed in Escherichia coli showed a clear preference for esters with short-chain fatty acids and exhibited maximum activity towards α-naphthol acetate at 37 °C and pH 7.5. Purified PaCes7 exhibited its catalytic activity under mesophilic conditions and retained more than 40% activity below 30 °C. It displayed a relatively wide pH stability from pH 6-11. Furthermore, the enzyme was strongly resistant to Mg2+, Pb2+, and Co2+ and activated by K+ and Ca2+. Both P. ananatis Sd-1 and PaCes7 could degrade the pesticide carbaryl. Additionally, PaCes7 was shown to work in combination with cellulase and/or xylanase in rice straw degradation. CONCLUSIONS: The data suggest that PaCes7 possesses promising biotechnological potential.

Identification of the key amino acid sites of the carbofuran hydrolase CehA from a newly isolated carbofuran-degrading strain Sphingbium sp. CFD-1.

A novel carbofuran-degrading strain CFD-1 was isolated and preliminarily identified as Sphingbium sp. This strain was able to utilize carbofuran as the sole carbon source for growth. The carbofuran hydrolase gene cehA was cloned from strain CFD-1 and expressed in Escherichia coli. CehA could hydrolyze carbamate pesticides including carbofuran and carbaryl efficiently, while it showed poor hydrolysis ability against isoprocarb, propoxur, oxamyl and aldicarb. CehA displayed maximal enzymatic activity at 40 °C and pH 7.0. The apparent Km and Kcat values of CehA for carbofuran were 133.22 ±â€¯5.70 µM and 9.48 ±â€¯0.89 s-1, respectively. The site-directed mutation experiment showed that His313, His315, His453 and His495 played important roles in the hydrolysis of carbofuran by CehA. Furthermore, the sequence of cehA is highly conserved among different carbofuran-degrading strains, and there are mobile elements around cehA, indicating that it may be transferred horizontally between different strains.

Production of a specific monoclonal antibody for 1-naphthol based on novel hapten strategy and development of an easy-to-use ELISA in urine samples.

1-naphthol (1-NAP) is the main metabolite of pesticide carbaryl and naphthalene, and is also a genotoxic and carcinogenic intermediate in the synthesis of organic compound, dyes, pigment and pharmaceutical industry. In this work, two novel haptens were designed and synthesized for developing a competitive indirect enzyme-linked immunosorbent assay (ciELISA) method for 1-NAP in urine samples. The assay showed a limit of detection of 2.21 ng/mL and working range from 4.02 ng/mL to 31.25 ng/mL for 1-NAP in optimized working buffer. The matrix effect of samples was eliminated via 15-fold dilution of optimized working buffer. Good average recoveries (102.4%-123.4%) with a coefficient of variation from 11.7% to 14.7% was obtained for spiked urine samples. Subsequent instrument verification test showed good correlation between the results of ciELISA and high-performance liquid chromatography. The developed ciELISA is a high-throughput tool to monitor 1-NAP in urine, which can provide technical support for the establishment of biological exposure level for the exposure to carbaryl, naphthalene and other related pollutants.

Genome Analysis of Carbaryl-Degrading Strain Pseudomonas putida XWY-1.

Carbaryl was a widely used pesticide in the agriculture industry. The toxicity against non-target organisms and the environmental pollution it caused became the focus of public concern. However, the microbial mechanism of carbaryl degradation was not fully investigated. In the study, we reported the complete genome of the carbaryl-degrading Pseudomonas putida strain XWY-1, which consists of a chromosome (5.9 Mbp) and a plasmid (0.4 Mbp). The carbaryl degradation genes are located on the plasmid. The study on the genome will facilitate to further elucidate the carbaryl degradation and advance the potential biotechnological applications of P. putida strain XWY-1.

Carbaryl biodegradation by Xylaria sp. BNL1 and its metabolic pathway.

Although ascomycetes occupy a vaster niche in soil than the well-studied basidiomycetes, they have received limited attention in studies related to bioremediation. In this study, the degradation of carbaryl by Xylaria sp. was studied in different culture conditions and its possible metabolic pathway was elucidated. In liquid culture, 99% of the added carbaryl was eliminated when cytochrome P450 (CYP450) was active, which was similar to the degradation rate of Pleurotus ostreatus, a fungus with strong bioremediation ability. Mn2+ is beneficial to the degradation of carbaryl. Compared to the 72.17% degradation rate in sterile soil, 59.0% carbaryl was eliminated in non-sterile soil, which suggested that Xylaria sp. BNL1 can resist microorganismal infection. Furthermore, the intracellular fractions containing laccase, CYP450, and carbaryl esterase efficiently degraded carbaryl. The presence of carbaryl metabolites suggested that Xylaria sp. BNL1 initiated its attack on carbaryl via carbaryl esterase to release α-naphthol, which was further degraded to 1,4-naphthoquinone and benzoic acid by CYP450 and laccase. Thus, our study highlights the potential of using Xylaria sp. for bioremediation.

Compartmentalization of the Carbaryl Degradation Pathway: Molecular Characterization of Inducible Periplasmic Carbaryl Hydrolase from Pseudomonas spp.

Pseudomonas sp. strains C5pp and C7 degrade carbaryl as the sole carbon source. Carbaryl hydrolase (CH) catalyzes the hydrolysis of carbaryl to 1-naphthol and methylamine. Bioinformatic analysis of mcbA, encoding CH, in C5pp predicted it to have a transmembrane domain (Tmd) and a signal peptide (Sp). In these isolates, the activity of CH was found to be 4- to 6-fold higher in the periplasm than in the cytoplasm. The recombinant CH (rCH) showed 4-fold-higher activity in the periplasm of Escherichia coli The deletion of Tmd showed activity in the cytoplasmic fraction, while deletion of both Tmd and Sp (Tmd+Sp) resulted in expression of the inactive protein. Confocal microscopic analysis of E. coli expressing a (Tmd+Sp)-green fluorescent protein (GFP) fusion protein revealed the localization of GFP into the periplasm. Altogether, these results indicate that Tmd probably helps in anchoring of polypeptide to the inner membrane, while Sp assists folding and release of CH in the periplasm. The N-terminal sequence of the mature periplasmic CH confirms the absence of the Tmd+Sp region and confirms the signal peptidase cleavage site as Ala-Leu-Ala. CH purified from strains C5pp, C7, and rCHΔ(Tmd)a were found to be monomeric with molecular mass of ∼68 to 76 kDa and to catalyze hydrolysis of the ester bond with an apparent Km and Vmax in the range of 98 to 111 µM and 69 to 73 µmol · min-1 · mg-1, respectively. The presence of low-affinity CH in the periplasm and 1-naphthol-metabolizing enzymes in the cytoplasm of Pseudomonas spp. suggests the compartmentalization of the metabolic pathway as a strategy for efficient degradation of carbaryl at higher concentrations without cellular toxicity of 1-naphthol.IMPORTANCE Proteins in the periplasmic space of bacteria play an important role in various cellular processes, such as solute transport, nutrient binding, antibiotic resistance, substrate hydrolysis, and detoxification of xenobiotics. Carbaryl is one of the most widely used carbamate pesticides. Carbaryl hydrolase (CH), the first enzyme of the degradation pathway which converts carbaryl to 1-naphthol, was found to be localized in the periplasm of Pseudomonas spp. Predicted transmembrane domain and signal peptide sequences of Pseudomonas were found to be functional in Escherichia coli and to translocate CH and GFP into the periplasm. The localization of low-affinity CH into the periplasm indicates controlled formation of toxic and recalcitrant 1-naphthol, thus minimizing its accumulation and interaction with various cellular components and thereby reducing the cellular toxicity. This study highlights the significance of compartmentalization of metabolic pathway enzymes for efficient removal of toxic compounds.

Carbaryl as a Carbon and Nitrogen Source: an Inducible Methylamine Metabolic Pathway at the Biochemical and Molecular Levels in Pseudomonas sp. Strain C5pp.

Carbaryl is the most widely used carbamate family pesticide, and its persistent nature causes it to pollute both soil and water ecosystems. Microbes maintain the Earth's biogeochemical cycles by metabolizing various compounds present in the matter, including xenobiotics, as a sole source of carbon, nitrogen, and energy. Soil isolate Pseudomonas sp. strain C5pp metabolizes carbaryl efficiently as the carbon source. Periplasmic carbaryl hydrolase catalyzes the conversion of carbaryl to 1-naphthol and methylamine. 1-Naphthol was further used as a carbon source via gentisate, whereas the metabolic fate of methylamine is not known. Here, we demonstrate that strain C5pp showed efficient growth on carbaryl when supplied as a carbon and nitrogen source, suggesting that the methylamine generated was used as the nitrogen source. Genes involved in the methylamine metabolism were annotated and characterized at the biochemical and molecular level. Transcriptional and enzyme activity studies corroborate that the γ-glutamylmethylamide/N-methylglutamate (GMA/NMG) pathway is involved in the metabolism of carbaryl and methylamine as a nitrogen source. Compared to carbaryl, methylamine was found to be an effective inducer for the metabolic and transporter genes. Strain C5pp also harbored genes involved in sarcosine metabolism that were cotranscribed and induced by sarcosine. The presence of inducible pathways for metabolism of carbaryl as a nitrogen and carbon source helps in complete and efficient mineralization of carbaryl by strain C5pp, thereby maintaining the biogeochemical cycles.IMPORTANCE The degradation of xenobiotics plays a significant role in the environment to maintain ecological systems as well as to prevent the imbalance of biogeochemical cycles via carbon-nitrogen cycling. Carbaryl is the most widely used pesticide from the carbamate family. Pseudomonas sp. strain C5pp, capable of utilizing carbaryl as a carbon and nitrogen source for its growth, subsequently helps in complete remediation of carbaryl. Thus, it maintains the ecosystem by balancing the biogeochemical cycles. The metabolic versatility and genetic diversity of strain C5pp for the transformation of contaminants like carbaryl and 1-naphthol into less harmful products make it a suitable candidate from the perspective of bioremediation.

Insights into metabolism and sodium chloride adaptability of carbaryl degrading halotolerant Pseudomonas sp. strain C7.

Pseudomonas sp. strain C7 isolated from sediment of Thane creek near Mumbai, India, showed the ability to grow on glucose and carbaryl in the presence of 7.5 and 3.5% of NaCl, respectively. It also showed good growth in the absence of NaCl indicating the strain to be halotolerant. Increasing salt concentration impacted the growth on carbaryl; however, the specific activity of various enzymes involved in the metabolism remained unaffected. Among various enzymes, 1-naphthol 2-hydroxylase was found to be sensitive to chloride as compared to carbaryl hydrolase and gentisate 1,2-dioxygenase. The intracellular concentration of Cl- ions remained constant (6-8 mM) for cells grown on carbaryl either in the presence or absence of NaCl. Thus the ability to adapt to the increasing concentration of NaCl is probably by employing chloride efflux pump and/or increase in the concentration of osmolytes as mechanism for halotolerance. The halotolerant nature of the strain will be beneficial to remediate carbaryl from saline agriculture fields, ecosystems and wastewaters.

Expanded insecticide catabolic activity gained by a single nucleotide substitution in a bacterial carbamate hydrolase gene.

Carbofuran-mineralizing strain Novosphingobium sp. KN65.2 produces the CfdJ enzyme that converts the N-methylcarbamate insecticide to carbofuran phenol. Purified CfdJ shows a remarkably low KM towards carbofuran. Together with the carbaryl hydrolase CehA of Rhizobium sp. strain AC100, CfdJ represents a new protein family with several uncharacterized bacterial members outside the proteobacteria. Although both enzymes differ by only four amino acids, CehA does not recognize carbofuran as a substrate whereas CfdJ also hydrolyzes carbaryl. None of the CfdJ amino acids that differ from CehA were shown to be silent regarding carbofuran hydrolytic activity but one particular amino acid substitution, i.e., L152 to F152, proved crucial. CfdJ is more efficient in degrading methylcarbamate pesticides with an aromatic side chain whereas CehA is more efficient in degrading the oxime carbamate nematicide oxamyl. The presence of common flanking sequences suggest that the cfdJ gene is located on a remnant of the mobile genetic element Tnceh carrying cehA. Our results suggest that these enzymes can be acquired through horizontal gene transfer and can evolve to degrade new carbamate substrates by limited amino acid substitutions. We demonstrate that a carbaryl hydrolase can gain the additional capacity to degrade carbofuran by a single nucleotide transversion.

Isolation and characterization of fenobucarb-degrading bacteria from rice paddy soils.

Forty-five fenobucarb-degrading bacteria were isolated from rice paddy soils, and their genetic and phenotypic characteristics were investigated. The isolates were able to utilize fenobucarb as a sole source of carbon and energy. Analysis of the 16S rRNA gene sequence indicated that all the isolates were related to members of the genera Sphingobium and Novosphingobium. Among 45 isolates, 21 different chromosomal DNA fingerprinting patterns were obtained. All these strains exhibited similar growth and degradation patterns on fenobucarb. 2-sec-butylphenol was identified as an intermediate during fenobucarb degradation by HPLC analysis. All of the isolates were able to degrade another carbamate insecticide, carbaryl, and 2-sec-butylphenol, but not other fenobucarb related compounds such as aldicarb and fenoxycarb. Representative strains of the different repetitive extragenic palindromic sequence PCR fingerprint types had one to six plasmids. The plasmid-cured strains lost their degradation abilities, suggesting that fenobucarb degradative genes were on their plasmid DNAs in these strains. When analyzed with PCR amplification using the primers targeting for the previously reported carbamate hydrolase genes, most of the isolates did not exhibit any positive signals for different genes involved in carbamate degradation such as mcd, cahA and cehA genes. This is the first report that microorganisms involved in the degradation of fenobucarb have been isolated and the intermediate of fenobucarb biodegradation was identified.

Metabolic regulation and chromosomal localization of carbaryl degradation pathway in Pseudomonas sp. strains C4, C5 and C6.

Pseudomonas sp. strains C4, C5 and C6 degrade carbaryl (1-naphthyl N-methylcarbamate) via 1-naphthol, 1,2-dihydroxynaphthalene, salicylate and gentisate. Carbon source-dependent metabolic studies suggest that enzymes responsible for carbaryl degradation are probably organized into 'upper' (carbaryl to salicylate), 'middle' (salicylate to gentisate) and 'lower' (gentisate to TCA cycle) pathway. Carbaryl and 1-naphthol were found to induce all carbaryl pathway enzymes, while salicylate and gentisate induce middle and lower pathway enzymes. The strains were found to harbor plasmid(s), and carbaryl degradation property was found to be stable. Genes encoding enzymes of the degradative pathway such as 1-naphthol 2-hydroxylase, salicylaldehyde dehydrogenase, salicylate 5-hydroxylase and gentisate 1,2-dioxygenase were amplified from chromosomal DNA of these strains. The gene-specific PCR products were sequenced from strain C6, and phylogenetic tree was constructed. Southern hybridization and PCR analysis using gel eluted DNA as template supported the presence of pathway genes onto the chromosome and not on the plasmid(s).

Carbaryl and 1-naphthol tissue levels and related cholinesterase inhibition in male Brown Norway rats from preweaning to senescence.

Studies incorporating both toxicokinetic and dynamic factors provide insight into chemical sensitivity differences across the life span. Tissue (brain, plasma, liver) levels of the N-methyl carbamate carbaryl, and its metabolite 1-naphthol, were determined and related to brain and RBC cholinesterase (ChE) inhibition in the same animals. Dose-response (3, 7.5, 15, or 22.5 mg/kg, 40-45 min postdosing) and time course (3 or 15 mg/kg at 30, 60, 120, or 240 min postdosing) of acute effects of carbaryl (oral gavage) in preweanling (postnatal day [PND] 18) and adult male Brown Norway rats from adolescence to senescence (1, 4, 12, 24 mo) were compared. At all ages there were dose-related increases in carbaryl and 1-naphthol in the dose-response study, and the time-course study showed highest carbaryl levels at 30 min postdosing. There were, however, age-related differences in that the 1- and 4-mo rats showed the lowest levels of carbaryl and 1-naphthol, and PND18 and 24-mo rats had similar, higher levels. The fastest clearance (shortest half-lives) was observed in 1- and 4-mo rats. Carbaryl levels were generally higher than 1-naphthol in brain and plasma, but in liver, 1-naphthol levels were similar to or greater than carbaryl. Brain ChE inhibition closely tracked brain carbaryl concentrations regardless of the time after dosing, but there was more variability in the relationship between RBC ChE and plasma carbaryl levels. Within-subject analyses suggested somewhat more brain ChE inhibition at lower carbaryl levels only in the PND18 rats. These findings may reflect maturation followed by decline in kinetic factors over the life span.

Metabolomic and proteomic insights into carbaryl catabolism by Burkholderia sp. C3 and degradation of ten N-methylcarbamates.

Burkholderia sp. C3, an efficient polycyclic aromatic hydrocarbon degrader, can utilize nine of the ten N-methylcarbamate insecticides including carbaryl as a sole source of carbon. Rapid hydrolysis of carbaryl in C3 is followed by slow catabolism of the resulting 1-naphthol. This study focused on metabolomes and proteomes in C3 cells utilizing carbaryl in comparison to those using glucose or nutrient broth. Sixty of the 867 detected proteins were involved in primary metabolism, adaptive sensing and regulation, transport, stress response, and detoxification. Among the 41 proteins expressed in response to carbaryl were formate dehydrogenase, aldehyde-alcohol dehydrogenase and ethanolamine utilization protein involved in one carbon metabolism. Acetate kinase and phasin were 2 of the 19 proteins that were not detected in carbaryl-supported C3 cells, but detected in glucose-supported C3 cells. Down-production of phasin and polyhydroxyalkanoates in carbaryl-supported C3 cells suggests insufficient carbon sources and lower levels of primary metabolites to maintain an ordinary level of metabolism. Differential metabolomes (~196 identified polar metabolites) showed up-production of metabolites in pentose phosphate pathways and metabolisms of cysteine, cystine and some other amino acids, disaccharides and nicotinate, in contract to down-production of most of the other amino acids and hexoses. The proteomic and metabolomic analyses showed that carbaryl-supported C3 cells experienced strong toxic effects, oxidative stresses, DNA/RNA damages and carbon nutrient deficiency.

Characterization of acetylcholinesterase inhibition and energy allocation in Daphnia magna exposed to carbaryl.

The inhibition of acetylcholinesterase (AChE) activity and energy allocation in the freshwater organism Daphnia magna exposed to carbaryl and potential recovery from the effects was examined. The binding of carbaryl-AChE was characterized through in vitro assays. To evaluate the recovery from inhibition and the alteration in energy budget, in vivo exposure and recovery regime tests were conducted. In comparison to diazoxon, the active metabolite of the insecticide diazinon, the stability of enzyme-carbaryl complex was fifteen times lower and the reactivity toward the active site was two times lower, resulting in approximately 30 times lower overall inhibition rate than for diazoxon. The in vitro reactivation rate constant of the inhibited enzyme and the in vivo recovery rate constant of AChE activity were 1.9 h⁻¹ and 0.12 h⁻¹ for carbaryl, respectively, which are much higher than the corresponding rate constants for diazoxon. The lower AChE inhibition and greater reactivation/recovery rates are in accordance with the lower toxicity of carbaryl compared to diazinon. Carbaryl exposure also altered the profile of the energy reserve: the decrease in lipid and glycogen and the increase in protein content resulted in the reduction of the total energy budget by about 45 mJ/g(ww). This corresponds to 26 percent of the available energy, which might allocate for external stressors. The mechanistic model of AChE inhibition is helpful to get an insight into (eco-)toxicological effects of AChE inhibitors on freshwater crustaceans under environmentally realistic conditions.

Co-inducible Catabolism of 2-Naphthol Initiated by Hydroxylase CehC1C2 in Rhizobium sp. X9 Removed Its Ecotoxicity.

2-Naphthol, which originates from various industrial activities, is widely disseminated through the discharge of industrial wastewater and is, thus, harmful to the water ecosystem, agricultural production, and human health. In this study, the carbaryl degrading strain Rhizobium sp. X9 was proven to be able to degrade 2-naphthol and reduce its toxicity to rice (Oryza sativa) and Chlorella ellipsoidea. Two-component hydroxylase CehC1C2 is responsible for the initial step of degradation and generates 1,2-dihydroxynaphthalene, which is further degraded by the ceh cluster. The transcription of gene cluster cehC1C2 could be induced when both 2-naphthol and glucose were added. A bioinformatic analysis revealed that two transcriptional regulators, the inhibitor CehR2 and the activator CehR3, could be involved in this process. Our study elucidated the molecular mechanism of microbial degradation of 2-naphthol and provided an effective strategy for the in situ remediation of 2-naphthol contamination in the environment.

The Potential Binding Interaction and Hydrolytic Mechanism of Carbaryl with the Novel Esterase PchA in Pseudomonas sp. PS21.

Microbial bioremediation is a very potent and eco-friendly approach to alleviate pesticide pollution in agricultural ecosystems, and hydrolase is an effective element for contaminant degradation. In the present study, a novel Mn2+-dependent esterase, PchA, that efficiently hydrolyzes carbamate pesticides with aromatic structures was identified from Pseudomonas sp. PS21. The hydrolytic activity was confirmed to be related closely to the core catalytic domain, which consists of six residues. The crucial residues indirectly stabilized the position of carbaryl via chelating Mn2+ according to the binding model clarified by molecular simulations, and the additional hydrophobic interactions between carbaryl with several hydrophobic residues also stabilized the binding conformation. The residue Glu398, by serving as the general base, might activate a water molecule and facilitate PchA catalysis. This work offers valuable insights into the binding interaction and hydrolytic mechanism of carbaryl with the hydrolase PchA and will be crucial to designing strategies leading to the protein variants that are capable of degrading related contaminants.