Dynamic response of P. putida S12 and S12ΔtrgI to sudden addition of toluene
ABSTRACT: 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. 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 S12ΔtrgI. 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:To elucidate any observable metabolic alterations during interactions of several strains of Pseudomonas putida (DOT-T1E, and its mutants DOT-T1E-PS28 and DOT-T1E-18) with the aromatic hydrocarbon toluene, metabolomic approaches were employed. Initially, Fourier-transform infrared (FT-IR) spectroscopy, which provided a rapid, high-throughput metabolic fingerprint of P. putida strains, was used to investigate any phenotypic changes resulting from exposure to toluene. Principal component discriminant function analysis (PC-DFA) allowed the differentiation between different conditions of toluene on bacterial cells, which indicated phenotypic changes associated with the presence of the solvent within the cell. Examination of PC-DFA loading plots suggested that the protein and fatty acids groups were responsible for discrimination of responses by P. putida strains to toluene. To identify metabolites of interest, the polar extracts of P. putida cells were analysed using gas chromatography-mass spectrometry (GC-MS) and 15 metabolites of P. putida central metabolic pathways were detected. Multi-block principal component analysis (MB-PCA) indicated that P. putida cultures challenged with toluene were differentially clustered away from the non-challenged cells. Investigation of MB-PCA loading plots and N-way ANOVA for condition | strain×time blocking (dosage of toluene) suggested ornithine as the most significant compound that increased upon solvent exposure. Ornithine presents itself as a major feature which may have important functions in toluene stress tolerance mechanisms.
Project description:Obesity is risk factor for development of fatty liver. Analysis of altered gene expression gives better understading about the mechanisms involved/alterted in the development of obesity-induced fattyliver in this new obese rat model. We used Microarrays to delinate the alted gene expression in liver of WNIN/Ob obese rats Liver was collected from 4 month old WNIN/Ob lean and obese rats for RNA extraction and hybridization on Affymatrix Rat gene 1ST arrays. Four RNA samples from each phenotype were pooled and used for the study.
Project description:In this study we profile expression of target genes downstream of auxin signaling in Arabidopsis hypocotyls elongating in response to auxin. The synthetic auxin picloram is used in order to take advantage of the picloram-resistant auxin receptor mutant afb5-5, which fails to elongate in response to picloram. A 4-plex chip was used for analyzing the transcriptome in hypocotyls dissected from wild-type seedlings following a 30min and 2hr picloram or solvent control treatment. Three biological replicate samples were analyzed, each containing roughly 400 hypocotyls. A 12-plex chip was used for analyzing mRNA from hypocotyls dissected from wild-type and afb5-5 (SALK_110643) mutant seedlings following a 2hr picloram or solvent control treatment. Three biological replicates were analyzed, each containing roughly 700 hypocotyls.
Project description:B-methylthiolation of the Escherichia coli Ribosomal Protein S12 Regulates Anaerobic Gene Expression. B-methylthiolation is a unique post-translational modification (PTM) that maps to a conserved Asp 88 of the bacterial ribosomal protein S12. This modification is phylogenetically conserved in several bacteria yet has not been identified on other proteins. Overall design: We use microarrays to delineate the association of prokaryotic ribosomal protein PTM to the regulation of genes.
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. Overall design: P. putida KT2440 and DOT-T1E mRNA profiles in the presence of control condition (M9), M9 plus toluene in vapor phase and M9 plus a shock of butanol at exponential phase
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 Overall design: 40 uM benzene caused 50 % inhibition in physiological response following 1 hour incubation. Transcriptional levels of the cells inhibited by benzene were compared with the cells under control conditions (ie, no benzene). 20 uM toluene also caused 50 % inhibition in physiological response following 1 hour incubation. Transcriptional levels of the cells inhibited by toluene were compared with the cells under control conditions (ie, no toluene).
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. Overall design: 2 samples examined from the different electron-acceptors (sulphate or ferric iron) incubates at the time point of maximal toluene degradation.