Project description:Understanding the response processes in cellular systems to external perturbations is a central goal of large-scale molecular profiling experiments. We investigated the molecular response of yeast to increased and lowered temperatures relative to optimal reference conditions across two levels of molecular organization: the transcriptome using a whole yeast genome microarray and the metabolome applying the GC/MS technology with in-vivo stable-isotope labeling for accurate relative quantification of a total of 50 different metabolites. The molecular adaptation of yeast to increased or lowered temperatures relative control conditions at both the metabolic and transcriptional level is dominated by temperature-inverted differential regulation patterns of transcriptional and metabolite responses and the temporal response observed to be biphasic. The set of previously described general environmental stress response (ESR) genes showed particularly strong temperature-inverted response patterns. Among the metabolites measured, trehalose was detected to respond strongest to the temperature stress and with temperature-inverted up and downregulation relative to control, mid-temperature conditions. Although associated with the same principal environmental parameter, the two different temperature regimes caused very distinct molecular response patterns at both the metabolite and the transcript level. While pairwise correlations between different transcripts and between different metabolites were found generally preserved under the various conditions, substantial differences were also observed indicative of changed underlying network architectures or modified regulatory relationships. Gene and associated gene functions were identified that are differentially regulated specifically under the gradual stress induction applied here compared to abrupt stress exposure investigated in previous studies, including genes of as of yet unidentified function and genes involved in protein synthesis and energy metabolism. Reference: Strassburg K, Walther D, Takahashi H, Kanaya S, and Kopka J. Dynamic transcriptional and metabolic responses in yeast adapting to temperature stress. OMICS JIB, 2010, 14(3), in press. Time-series with 8 time points for three different ambient temperatures. Single replicate per time point and temperature series.

Project description:Understanding the response processes in cellular systems to external perturbations is a central goal of large-scale molecular profiling experiments. We investigated the molecular response of yeast to increased and lowered temperatures relative to optimal reference conditions across two levels of molecular organization: the transcriptome using a whole yeast genome microarray and the metabolome applying the GC/MS technology with in-vivo stable-isotope labeling for accurate relative quantification of a total of 50 different metabolites. The molecular adaptation of yeast to increased or lowered temperatures relative control conditions at both the metabolic and transcriptional level is dominated by temperature-inverted differential regulation patterns of transcriptional and metabolite responses and the temporal response observed to be biphasic. The set of previously described general environmental stress response (ESR) genes showed particularly strong temperature-inverted response patterns. Among the metabolites measured, trehalose was detected to respond strongest to the temperature stress and with temperature-inverted up and downregulation relative to control, mid-temperature conditions. Although associated with the same principal environmental parameter, the two different temperature regimes caused very distinct molecular response patterns at both the metabolite and the transcript level. While pairwise correlations between different transcripts and between different metabolites were found generally preserved under the various conditions, substantial differences were also observed indicative of changed underlying network architectures or modified regulatory relationships. Gene and associated gene functions were identified that are differentially regulated specifically under the gradual stress induction applied here compared to abrupt stress exposure investigated in previous studies, including genes of as of yet unidentified function and genes involved in protein synthesis and energy metabolism. Reference: Strassburg K, Walther D, Takahashi H, Kanaya S, and Kopka J. Dynamic transcriptional and metabolic responses in yeast adapting to temperature stress. OMICS JIB, 2010, 14(3), in press.

Project description:Zymomonas mobilis ZM4 produces near theoretical yields of ethanol with high specific productivity and recombinant strains are able to ferment both C-5 and C-6 sugars. However, the genetic and physiological basis of the ZM4 response to various industrially-relevant stresses is poorly understood. In this study, the dynamics of ZM4 oxygen stress responses were elucidated by characterizing the transcriptomic and metabolomic profiles of aerobic and anaerobic fermentations using whole-genome microarray analysis and gas chromatography-mass spectrometry. In the absence of oxygen, ZM4 consumed glucose more rapidly, had a higher growth rate, and ethanol was the major end-product. Under aerobic conditions, much less ethanol was observed with other end-products produced, including acetate, lactate, and acetoin. In the early exponential phase, no genes were detected as being significantly differentially expressed between aerobic and anaerobic conditions via microarray analysis. However, microarray analysis of the stationary phase cultures revealed that 166 genes were significantly differentially expressed by more than two-fold. Quantitative-PCR validated the expression values for seventeen genes from different categories of stationary phase microarray data. Transcripts for Entner-Doudoroff pathway genes and gene pdc, encoding a key enzyme leading to the ethanol production, were at least 30-fold more abundant under anaerobic conditions at stationary phase based on quantitative-PCR results. Expression of stress response genes was found to be greater under oxygen stress conditions. GC-MS analysis of stationary phase intracellular metabolites indicated that ZM4 under anaerobic conditions contained lower levels of amino acids, glucose and ED pathway intermediates, whereas metabolites such as ribitol, trehalose, myristic acid, glyceric acid, glucose 6-phosphate, mannose 6-phosphate, and 4-hydroxybutanoic acid were more abundant than under aerobic conditions. Transcriptomic and metabolomic data were consistent with faster glucose consumption and greater ethanol production under anaerobic conditions and suggested gene targets for deletion and improved fermentation. Keywords: Time course/ Stress Response

Project description:RpoS is a conserved stress regulator that plays a critical role in survival under stress conditions in Escherichia coli and other γ-proteobacteria. RpoS is also involved in virulence of many pathogens including Salmonella and Vibrio species. Though well characterized in non-pathogenic E. coli K12 strains, the effect of RpoS on transcriptome expression has not been examined in pathogenic isolates. E. coli O157:H7 is a serious human enteropathogen, possessing a genome 20% larger than that of E. coli K12, and many of the additional genes are required for virulence. The genomic difference may result in substantial changes in RpoS-regulated gene expression. To test this, we compared the transcriptional profile of wild type and rpoS mutants of the E. coli O157:H7 EDL933 type strain. The rpoS mutation had a pronounced effect on gene expression in stationary phase, and more than 1,000 genes were differentially expressed (two-fold, p<0.05). By contrast, we found 11 genes expressed differently in exponential phase. Western blot analysis revealed that, as expected, RpoS level was low in exponential phase and substantially increased in stationary phase. The defect in rpoS resulted in impaired expression of genes responsible for stress response (e.g., gadA, katE and osmY), arginine degradation (astCADBE), putrescine degradation (puuABCD), fatty acid oxidation (fadBA and fadE), and virulence (ler, espI and cesF). For EDL933-specific genes on O-islands, we found 50 genes expressed higher in wild type EDL933 and 49 genes expressed higher in the rpoS mutants. The protein levels of Tir and EspA, two LEE-encoded virulence factors, were elevated in the rpoS mutants under LEE induction conditions. Our results show that RpoS has a profound effect on global gene expression in the pathogenic strain O157:H7 EDL933, and the identified RpoS regulon, including many EDL933-specific genes, differs substantially from that of laboratory K12 strains. In this study, we characterized the RpoS regulon of E. coli O157:H7 strain EDL933 using microarray analysis.

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Time resolved response of E. coli to 5 different stress conditions

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