Proteomic analysis of Burkholderia zhejiangensis CEIB S4-3 during the methyl parathion degradation process.

Methyl parathion is an organophosphorus pesticide widely employed worldwide to control pests in agricultural and domestic environments. However, due to its intensive use, high toxicity, and environmental persistence, methyl parathion is recognized as an important ecosystem and human health threat, causing severe environmental pollution events and numerous human poisoning and deaths each year. Therefore, identifying and characterizing microorganisms capable of fully degrading methyl parathion and its degradation metabolites is a crucial environmental task for the bioremediation of pesticide-polluted sites. Burkholderia zhejiangensis CEIB S4-3 is a bacterial strain isolated from agricultural soils capable of immediately hydrolyzing methyl parathion at a concentration of 50 mg/L and degrading the 100% of the released p-nitrophenol in a 12-hour lapse when cultured in minimal salt medium. In this study, a comparative proteomic analysis was conducted in the presence and absence of methyl parathion to evaluate the biological mechanisms implicated in the methyl parathion biodegradation and resistance by the strain B. zhejiangensis CEIB S4-3. In each treatment, the changes in the protein expression patterns were evaluated at three sampling times, zero, three, and nine hours through the use of two-dimensional polyacrylamide gel electrophoresis (2D-PAGE), and the differentially expressed proteins were identified by mass spectrometry (MALDI-TOF). The proteomic analysis allowed the identification of 72 proteins with differential expression, 35 proteins in the absence of the pesticide, and 37 proteins in the experimental condition in the presence of methyl parathion. The identified proteins are involved in different metabolic processes such as the carbohydrate and amino acids metabolism, carbon metabolism and energy production, fatty acids ß-oxidation, and the aromatic compounds catabolism, including enzymes of the both p-nitrophenol degradation pathways (Hydroquinone dioxygenase and Hydroxyquinol 1,2 dioxygenase), as well as the overexpression of proteins implicated in cellular damage defense mechanisms such as the response and protection of the oxidative stress, reactive oxygen species defense, detoxification of xenobiotics, and DNA repair processes. According to these data, B. zhejiangensis CEIB S4-3 overexpress different proteins related to aromatic compounds catabolism and with the p-nitrophenol  degradation pathways, the higher expression levels observed in the two subunits of the enzyme Hydroquinone dioxygenase, suggest a preferential use of the Hydroquinone metabolic pathway in the p-nitrophenol degradation process. Moreover the overexpression of several proteins implicated in the oxidative stress response, xenobiotics detoxification, and DNA damage repair reveals the mechanisms employed by B. zhejiangensis CEIB S4-3 to counteract the adverse effects caused by the methyl parathion and p-nitrophenol exposure.

Transcriptomic analysis of Burkholderia cenocepacia CEIB S5-2 during methyl parathion degradation.

Methyl parathion (MP) is a highly toxic organophosphorus pesticide associated with water, soil, and air pollution events. The identification and characterization of microorganisms capable of biodegrading pollutants are an important environmental task for bioremediation of pesticide impacted sites. The strain Burkholderia cenocepacia CEIB S5-2 is a bacterium capable of efficiently hydrolyzing MP and biodegrade p-nitrophenol (PNP), the main MP hydrolysis product. Due to the high PNP toxicity over microbial living forms, the reports on bacterial PNP biodegradation are scarce. According to the genomic data, the MP- and PNP-degrading ability observed in B. cenocepacia CEIB S5-2 is related to the presence of the methyl parathion-degrading gene (mpd) and the gene cluster pnpABA'E1E2FDC, which include the genes implicated in the PNP degradation. In this work, the transcriptomic analysis of the strain in the presence of MP revealed the differential expression of 257 genes, including all genes implicated in the PNP degradation, as well as a set of genes related to the sensing of environmental changes, the response to stress, and the degradation of aromatic compounds, such as translational regulators, membrane transporters, efflux pumps, and oxidative stress response genes. These findings suggest that these genes play an important role in the defense against toxic effects derived from the MP and PNP exposure. Therefore, B. cenocepacia CEIB S5-2 has a great potential for application in pesticide bioremediation approaches due to its biodegradation capabilities and the differential expression of genes for resistance to MP and PNP.

Omics Approaches to Pesticide Biodegradation.

Pesticides are xenobiotic molecules necessary to control pests in agriculture, home, and industry. However, water and soil can become contaminated as a consequence of their extensive use. Therefore, because of its eco-friendly characteristics and efficiency, bioremediation of contaminated sites is a powerful tool with advantages over other kinds of treatments. For an efficient pesticides bioremediation, it is necessary to take into account different aspects related to the microbial metabolism and physiology. In this respect, OMICs studies such as genomics, transcriptomics, proteomics, and metabolomics are essential to generate relevant information about the genes and proteins involved in pesticide degradation, the metabolites generated by microbial pesticide degradation, and the cellular strategies to contend against stress caused by pesticide exposition. Pesticides as organochlorines and organophosphorus are the more commonly studied using OMIC approaches. To date, many genomes of microorganisms capable of degrading pesticides have been published, mainly bacterial strains from Burkholderia, Pseudomonas, and Rhodococcus genera. Following the genomic reports, transcriptomic studies, using microarrays and more recently next-generation sequencing technology RNA-Seq, in pesticide microbial degradation are the most numerous. Proteomics, metabolomics, as well as studies that combine different OMIC are gained interest. This review aims to describe a brief overview of pesticide biodegradation mechanisms; new tools to study microorganisms in natural environments; basic concepts of the OMICs approaches; as well as advances in methodologies associated with the analysis of that tools. Additionally, the most recent reports on genomics, transcriptomics, proteomics, and metabolomics during the degradation of pesticides are also analyzed.

Transcriptional analysis reveals the metabolic state of Burkholderia zhejiangensis CEIB S4-3 during methyl parathion degradation.

Burkholderia zhejiangensis CEIB S4-3 has the ability to degrade methyl parathion (MP) and its main hydrolysis byproduct p-nitrophenol (PNP). According to genomic data, several genes related with metabolism of MP and PNP were identified in this strain. However, the metabolic state of the strain during the MP degradation has not been evaluated. In the present study, we analyzed gene expression changes during MP hydrolysis and PNP degradation through a transcriptomic approach. The transcriptional analysis revealed differential changes in the expression of genes involved in important cellular processes, such as energy production and conversion, transcription, amino acid transport and metabolism, translation, ribosomal structure and biogenesis, among others. Transcriptomic data also exhibited the overexpression of both PNP-catabolic gene clusters (pnpABA'E1E2FDC and pnpE1E2FDC) present in the strain. We found and validated by quantitative reverse transcription polymerase chain reaction the expression of the methyl parathion degrading gene, as well as the genes responsible for PNP degradation contained in two clusters. This proves the MP degradation pathway by the strain tested in this work. The exposure to PNP activates, in the first instance, the expression of the transcriptional regulators multiple antibiotic resistance regulator and Isocitrate Lyase Regulator (IclR), which are important in the regulation of genes from aromatic compound catabolism, as well as the expression of genes that encode transporters, permeases, efflux pumps, and porins related to the resistance to multidrugs and other xenobiotics. In the presence of the pesticide, 997 differentially expressed genes grouped in 104 metabolic pathways were observed. This report is the first to describe the transcriptomic analysis of a strain of B. zhejiangensis during the biodegradation of PNP.

Partial characterization of a biosurfactant extracted from Pseudomonas sp. B0406 that enhances the solubility of pesticides.

Biodegradation of some organochlorine and organophosphate pesticides is difficult because of their low solubility in water and, therefore, their low bioavailability. To overcome the hydrophobicity problem and the limited pesticide availability, biosurfactants play a major role. In this study, we evaluated the effect of an extract from Pseudomonas sp. B0406 strain with surfactant properties, on the solubility of two pesticides: endosulfan (ED) and methyl parathion (MP). Such a process was performed in order to increase the aqueous solubility of both pesticides, to increase its availability to microorganisms and to promote their biodegradation. The extract from Pseudomonas sp. B0406 showed a critical micellar concentration of 1.4 g/L and the surface tension at that point was 40.4 mN/m. The preliminary chemical and physical partial characterization of the extract with surfactant properties indicated that it is an anionic glycolipid, which increases the solubility of both pesticides of 0.41 at 0.92 mg/L for ED and of 34.58 at 48.10 mg/L for MP. The results of this study suggest the effectiveness of this extract in improving the solubility of both pesticides ED and MP in water and, therefore, of its potential use to enhance the degradation of these pesticides.

Characterization of methyl parathion degradation by a Burkholderia zhejiangensis strain, CEIB S4-3, isolated from agricultural soils.

Through the use of an enrichment technique, we isolated from the agricultural soils of Morelos in central México a strain of Burkholderia zhejiangensis identified as CEIB S4-3, it's could use the pesticide methyl parathion (MP) as the only source of carbon and degrade completely p-nitrophenol (PNP). For more efficient MP and PNP degradation by the CEIB S4-3 strain, the absence of an extra carbon source, a large inoculum and an MP concentration up to 50 mg/l are required. Sequence and annotation analysis of the draft genome, showed presence of mpd functional gene, which was expressed and its activity on the MP was confirmed. Additionally, the genes coding for enzymes in the benzoquinone pathway (conducted by Gram-negative bacteria) and the benzenotriol pathway (conducted by Gram-positive bacteria) were found, which was corroborated by identification of intermediary metabolites by HPLC. Thus, we propose that B. zhejiangensis CEIB S4-3 uses both degradation pathways.

Photoelectrocatalytic inactivation of fecal coliform bacteria in urban wastewater using nanoparticulated films of TiO2 and TiO2/Ag.

Photocatalysis has shown the ability to inactivate a wide range of harmful microorganisms with traditional use of chlorination. Photocatalysis combined with applied bias potential (photoelectrocatalysis) increases the efficiency of photocatalysis and decreases the charge recombination. This work examines the inactivation of fecal coliform bacteria present in real urban wastewater by photoelectrocatalysis using nanoparticulated films of TiO2 and TiO2/Ag (4%w/w) under UV light irradiation. The catalysts were prepared with different thicknesses by the sol-gel method and calcined at 400°C and 600°C. The urban wastewater samples were collected from the sedimentation tank effluent of the university sewage treatment facility. The rate of bacteria inactivation increases with increasing the applied potential and film thicknesses; also, the presence of silver on the catalyst surface annealed at 400°C shows better inactivation than that at 600°C. Finally, a structural cell damage of Escherichia coli (DH5α), inoculated in water, is observed during the photoelectrocatalytic process.

Penicillium sp. as an organism that degrades endosulfan and reduces its genotoxic effects.

Endosulfan is an organochloride and persistent pesticide that has caused concern because of its impact in the environment and its toxicity to and bioaccumulation in living organisms. In this study, we isolated an endosulfan-degrading fungus from the activated sludge from an industrial wastewater treatment plant. Through repetitive enrichment and successive subculture in media containing endosulfan as the sole carbon source, a fungus designated CHE 23 was isolated. Based on a phylogenetic analysis, strain CHE 23 was assigned to the genus Penicillium sp. In a mineral salt medium with 50 mg/l endosulfan as the sole source carbon, CHE 23 removed the added endosulfan in a period of six days. To verify the decrease in endosulfan toxicity due to the activity of the fungus, we performed genotoxicity tests trough the single cell gel electrophoresis assay or comet assay, with Eisenia fetida as the bioindicator species. This organism was exposed to the supernatants of the culture of the fungus and endosulfan. Our results indicated that the genotoxicity of endosulfan was completely reduced due the activity of this fungus. These results suggest that the Penicillium sp. CHE 23 strain can be used to degrade endosulfan residues and/or for water and soil bioremediation processes without causing toxicity problems, which are probably due to the generation of no-toxic metabolites during biodegradation.

Greenhouse gas emissions in the state of Morelos, Mexico: a first approximation for establishing mitigation strategies.

In this study, the authors report the first greenhouse gas emission inventory of Morelos, a state in central Mexico, in which the emissions of carbon dioxide (CO2), methane (CH4), and nitrous oxide (N2O) have been identified using the Intergovernmental Panel on Climate Change (IPCC) methodology. Greenhouse gas (GHG) emissions were estimated as CO2 equivalents (CO2 eq) for the years 2005, 2007, and 2009, with 2005 being treated as the base year. The percentage contributions from each category to the CO2 eq emissions in the base year were as follows: 38% from energy, 30% from industrial processes, 23% from waste, 5% from agriculture, and 4% from land use/land use change and forestry (LULUCF). As observed in other state inventories in Mexico, road transportation is the main source of CO2 emissions, wastewater handling and solid waste disposal are the main sources of CH4 emissions, and agricultural soils are the source of the most significant N2O emissions. The information reported in this inventory identifies the main emission sources. Based on these results, the government can propose public policies specifically designed for the state of Morelos to establish GHG mitigation strategies in the near future.