Project description:Pseudomonas putida S12 is exceptionally tolerant to various organic solvents. To obtain further insight in this bacterium’s primary defence mechanisms towards these potentially harmful substances, we studied its genome wide transcriptional response to sudden addition of toluene. Global gene expression profiles were monitored for 30 minutes after toluene addition. During toluene exposure, high oxygen-affinity cytochrome c oxidase is specifically expressed to provide for an adequate proton gradient supporting solvent efflux mechanisms. Concomitantly, the glyoxylate bypass route was up-regulated, to repair an apparent toluene stress-induced redox imbalance. A knock-out mutant of trgI, a recently identified toluene-repressed gene, was investigated in order to identify TrgI function. Remarkably, upon addition of toluene the number of differentially expressed genes initially was much lower in the trgI-mutant than in the wild-type strain. This suggested that after deletion of trgI cells were better prepared for sudden organic solvent stress. Before, as well as after, addition of toluene many genes of highly diverse functions were differentially expressed in trgI-mutant cells as compared to wild-type cells. This led to the hypothesis that TrgI may not only be involved in the modulation of solvent-elicited responses but in addition may affect basal expression levels of large groups of genes.
Project description:Pseudomonas putida S12 is exceptionally tolerant to various organic solvents. To obtain further insight in this bacteriumM-bM-^@M-^Ys primary defence mechanisms towards these potentially harmful substances, we studied its genome wide transcriptional response to sudden addition of toluene. Global gene expression profiles were monitored for 30 minutes after toluene addition. During toluene exposure, high oxygen-affinity cytochrome c oxidase is specifically expressed to provide for an adequate proton gradient supporting solvent efflux mechanisms. Concomitantly, the glyoxylate bypass route was up-regulated, to repair an apparent toluene stress-induced redox imbalance. A knock-out mutant of trgI, a recently identified toluene-repressed gene, was investigated in order to identify TrgI function. Remarkably, upon addition of toluene the number of differentially expressed genes initially was much lower in the trgI-mutant than in the wild-type strain. This suggested that after deletion of trgI cells were better prepared for sudden organic solvent stress. Before, as well as after, addition of toluene many genes of highly diverse functions were differentially expressed in trgI-mutant cells as compared to wild-type cells. This led to the hypothesis that TrgI may not only be involved in the modulation of solvent-elicited responses but in addition may affect basal expression levels of large groups of genes. Differential gene expression after a sudden addition of 5 mM toluene was analysed in early exponential phase cultures (optical density at 600 nm of 0.5-0.6) of P. putida strains S12 (wild-type) and S12M-NM-^TtrgI. Samples were drawn immediately before (t=0) and at set intervals (1, 2, 5, 10 and 30 minutes) after toluene exposure. Duplicat samples were drawn. This resulted in 12 samples per strain, 24 in total.
Project description:Bioavailability of electron acceptors is probably the most limiting factor in the restoration of anoxic, contaminated environments. The oxidation of contaminants such as aromatic hydrocarbons, particularly in aquifers, often depends on the reduction of ferric iron or sulphate. We have previously detected a highly active fringe zone beneath a toluene plume at a tar-oil contaminated aquifer in Germany, where a specialized community of contaminant degraders co-dominated by Desulfobulbaceae and Geobacteraceae had established. Although on-site geochemistry links degradation to sulphidogenic processes, dominating catabolic (benzylsuccinate synthase alpha-subunit, bssA) genes detected in situ appeared more related to those of Geobacter spp. Therefore, a stable isotope probing (SIP) incubation of sediment samples with 13C7-toluene and comparative electron acceptor amendment was performed. We introduce pyrosequencing of templates from SIP microcosms as a powerful new strategy in SIP gradient interpretation (Pyro-SIP). Our results reveal the central role of Desulfobulbaceae for sulphidogenic toluene degradation in situ, and affiliate the detected bssA genes to this lineage. This, and the absence of 13C-labelled DNA of Geobacter spp. in SIP gradients preclude their relevance as toluene degraders in situ. In contrast, Betaproteobacteria related to Georgfuchsia spp. became labelled under iron-reducing conditions. Furthermore, secondary toluene degraders belonging to the Peptococcaceae detected in both treatments suggest the possibility of functional redundancy amongst anaerobic toluene degraders on site. 2 samples examined from the different electron-acceptors (sulphate or ferric iron) incubates at the time point of maximal toluene degradation.
Project description:Bioavailability of electron acceptors is probably the most limiting factor in the restoration of anoxic, contaminated environments. The oxidation of contaminants such as aromatic hydrocarbons, particularly in aquifers, often depends on the reduction of ferric iron or sulphate. We have previously detected a highly active fringe zone beneath a toluene plume at a tar-oil contaminated aquifer in Germany, where a specialized community of contaminant degraders co-dominated by Desulfobulbaceae and Geobacteraceae had established. Although on-site geochemistry links degradation to sulphidogenic processes, dominating catabolic (benzylsuccinate synthase alpha-subunit, bssA) genes detected in situ appeared more related to those of Geobacter spp. Therefore, a stable isotope probing (SIP) incubation of sediment samples with 13C7-toluene and comparative electron acceptor amendment was performed. We introduce pyrosequencing of templates from SIP microcosms as a powerful new strategy in SIP gradient interpretation (Pyro-SIP). Our results reveal the central role of Desulfobulbaceae for sulphidogenic toluene degradation in situ, and affiliate the detected bssA genes to this lineage. This, and the absence of 13C-labelled DNA of Geobacter spp. in SIP gradients preclude their relevance as toluene degraders in situ. In contrast, Betaproteobacteria related to Georgfuchsia spp. became labelled under iron-reducing conditions. Furthermore, secondary toluene degraders belonging to the Peptococcaceae detected in both treatments suggest the possibility of functional redundancy amongst anaerobic toluene degraders on site.
Project description:Toluene is an industrial raw material and solvent that can be found abundantly in our daily life products. The amount of toluene vapor is one of the most important measurements for evaluating air quality. The evaluation of toluene scavenging ability of different plants has been reported, but the mechanism of plant response to toluene is only partially understood. In this study, we performed RNA sequencing (RNA-seq) analysis to detect differential gene expression in toluene-treated and untreated leaves of Ardisia pusilla. A total of 88,444 unigenes were identified by RNA-seq analysis, of which 49,623 were successfully annotated and 4,101 were differentially expressed. Gene ontology analysis revealed several sub-categories of genes related to toluene response, including cell part, cellular process, organelle, and metabolic processes. We mapped the main metabolic pathways of genes related to toluene response and found that the differentially expressed genes were mainly involved in glycolysis/gluconeogenesis, starch and sucrose metabolism, glycerophospholipid metabolism, carotenoid biosynthesis, phenylpropanoid biosynthesis, and flavonoid biosynthesis. In addition, 53 transcription factors belonging to 13 transcription factor families were identified. We verified 10 differentially expressed genes related to metabolic pathways using quantitative real-time PCR and found that the results of RNA-seq were positively correlated with them, indicating that the transcriptome data were reliable. This study provides insights into the metabolic pathways involved in toluene response in plants.
Project description:We report the application of a high-throughput technique, RNA-seq, to study the transcriptomic response of P. putida KT2440 and DOT-T1E in the presence of toluene with the aim to study in more detail the mechanisms involved in toluene response in a toluene-sensitive and a toluene-tolerant strains.
Project description:The effects of the aromatic hydrocarbons benzene and toluene on Nitrosomonas europaea, a nitrifying bacterium that plays an important role in the removal of nitrogen from wastewater treatment plants, were studied in batch reactors. Exposure to 20 M toluene and 40 M benzene resulted in a 50% reduction in nitrifying activity after 1 h. However, Affymetrix microarray experiments detected no significant changes in gene expression in toluene exposed cells. Cells exposed to benzene were found to up-regulate a gene cluster (NE 1545 - NE 1551). This gene cluster appears to be involved with fatty-acid metabolism, lipid and membrane protein biosynthesis. TEM experiments reveal that cells exposed to benzene decrease the thickness of their membrane and the membrane becomes more structured. Keywords: stress response, benzene, toluene
Project description:The physiological and transcriptional response of Nitrosomonas europaea biofilms to phenol and toluene was examined and compared to suspended cells. Biofilms were grown in Drip Flow Biofilm Reactors under continuous flow conditions of growth medium containing ammonia as growth substrate. The responses of N. europaea biofilms to the aromatic hydrocarbons phenol and toluene were determined during short-term (3 h) additions of each compound to the biofilms. Ammonia oxidation in the biofilms was inhibited 50% by 60 uM phenol and 100 uM toluene. These concentrations were chosen for microarray analysis of phenol- and toluene-exposed N. europaea biofilms. Liquid batch cultures of exponentially growing N. europaea cells were harvested alongside the biofilms to determine differential gene expression between attached and suspended growth of N. europaea.