Project description:Bacteria respond to osmotic stress by a substantial increase in the intracellular osmolality, adjusting their cell turgor for altered growth conditions. Using E. coli as a model organism we demonstrate here that bacterial responses to hyperosmotic stress specifically depend on the nature of osmoticum used. We show that increasing acute hyperosmotic NaCl stress above ~1.0 Os kg-1 causes a dose-dependent K+ leak from the cell, resulting in a substantial decrease in cytosolic K+ content and a concurrent accumulation of Na+ in the cell. At the same time, isotonic sucrose or mannitol treatment (non-ionic osmotica) results in a gradual increase of the net K+ uptake. Ion flux data is consistent with growth experiments showing that bacterial growth is impaired by NaCl at the concentration resulting in a switch from net K+ uptake to efflux. Microarray experiments reveal that about 40% of up-regulated genes shared no similarity in their responses to NaCl and sucrose treatment, further suggesting specificity of osmotic adjustment in E. coli to ionic- and non-ionic osmotica The observed differences are explained by the specificity of the stress-induced changes in the membrane potential of bacterial cells highlighting the importance of voltage-gated K+ transporters for bacterial adaptation to hyperosmotic stress.
Project description:The present work was devoted to a multi-level characterization of E. coli exposed to Ag+-mediated stress using for the first time an approach of integrative biology, based on the combination of physiological, biochemical and transcriptomic data sets. Bacterial growth and survival after Ag+ exposure were first quantified and related to the accumulation of intracellular silver, as detected by Nano Secondary Ion Mass Spectroscopy (NanoSIMS) at high lateral resolution. The whole transcriptomic response of E. coli cells under ionic silver-mediated stress was then characterized. Clear correlations were established between (i) cell physiology, (ii) variations in the biochemical characteristics of cell fatty acids and proteins, and (iii) regulation of gene expression. This challenging approach allowed determining key genetic markers of the E. coli response to ionic silver. In particular, we identified Ag+-mediated regulations of gene expression in correlation with growth (e.g. genes of transporters, transcriptional regulators, ribosomal proteins), necessary for ionic silver transport and detoxification (e.g. copA, cueO, mgtA, nhaR) and to cope with various stress (dnaK, pspA, metA,R, oxidoreductase genes). Regulation of gene expression after Ag+ exposure was also correlated to macromolecular modifications, such as acyl chain length (e.g. fadL, lpxA, arnA), protein secondary structure (e.g. dnaJ, htpX, degP) and cell morphology (e.g. ycfS, ycbB).
Project description:Bacteria respond to osmotic stress by a substantial increase in the intracellular osmolality, adjusting their cell turgor for altered growth conditions. Using E. coli as a model organism we demonstrate here that bacterial responses to hyperosmotic stress specifically depend on the nature of osmoticum used. We show that increasing acute hyperosmotic NaCl stress above ~1.0 Os kg-1 causes a dose-dependent K+ leak from the cell, resulting in a substantial decrease in cytosolic K+ content and a concurrent accumulation of Na+ in the cell. At the same time, isotonic sucrose or mannitol treatment (non-ionic osmotica) results in a gradual increase of the net K+ uptake. Ion flux data is consistent with growth experiments showing that bacterial growth is impaired by NaCl at the concentration resulting in a switch from net K+ uptake to efflux. Microarray experiments reveal that about 40% of up-regulated genes shared no similarity in their responses to NaCl and sucrose treatment, further suggesting specificity of osmotic adjustment in E. coli to ionic- and non-ionic osmotica The observed differences are explained by the specificity of the stress-induced changes in the membrane potential of bacterial cells highlighting the importance of voltage-gated K+ transporters for bacterial adaptation to hyperosmotic stress. Experiment Overall Design: Two biological replicates per treatment with microarray analysis using the Affymetrix GeneChip E. coli Genome array. Treatments used included: Experiment Overall Design: Control - E. coli Frag1, grown to early stationary growth phase in a mineral salts medium with 0.1% glucose at 25 C Experiment Overall Design: Sucrose hyperosmotic treatment - 1.25 M sucrose added to control culture Experiment Overall Design: for 10 min. Experiment Overall Design: NaCl hyperosmotic treatment - 1.37 M NaCl added to control culture Experiment Overall Design: For microarray data comparisons the sucrose and NaCl hyperosmotic treatment data was compared to the no treatment control data separately. The sucrose to control and NaCL to control comparison data tables are linked below.
Project description:A genome reduced E. coli strain MDS42ΔgalK::Ptet-gfp-kan were applied for the comparative transcriptome analysis. Genome-wide transcriptional changes under high osmotic prresure, high temperature condition and starvation were evaluated.
Project description:Plants respond to stress by using multiple gene regulatory mechanisms including the post-transcriptional regulation of gene expression. The stresses suffered by plants under salinity include osmotic stress and ion stress. In this study, three cotton small RNA libraries were constructed and sequenced under normal consideration, osmotic and ionic stress. The length distribution of obtained small RNAs was significantly different between libraries. A total of 228 cotton miRNAs were identified. Of them 24 were novel miRNAs. There were 88 and 75 miRNAs with different expression in response to the influence of osmotic and ionic factors of saline stress, respectively. The identification of these small RNAs as well as elucidating their functional significance broadens our understanding of the post-transcriptional gene regulations in response to salt stress.
Project description:Expression level of whole genome genes in Escherichia coli CC72 at early-exponential phase, and 10 min, 20 min and 45 min after osmotic stress. Venus was fused to the 3' end of rpoC in E. coli MG1655 to localize RNA polymerase as reported previously (C. Cagliero and D. J. Jin, 2013).
Project description:A genome reduced E. coli strain MDS42M-NM-^TgalK::Ptet-gfp-kan were applied for the comparative transcriptome analysis. Genome-wide transcriptional changes under high osmotic prresure, high temperature condition and starvation were evaluated. Exponentially growing cells from recoverying stress response were collected for the transcriptome analysis. High osmotic prresure, high temperature condition and starvation were chosen as stressor. In each kind of stress, three level of stresses were applied. Every 3 biological replications were performed.
Project description:In a previous study we adopted an integrated transcriptomic and proteomic approach to determine the physiological response of E. coli O157:H7 Sakai during exponential phase growth under steady-state conditions relevant to low temperature and water activity conditions experienced during meat carcass chilling in cold air (Kocharunchitt et al., 2012). The findings of that study provide a baseline of knowledge of the physiology of this pathogen, with the response of E. coli O157:H7 to steady-state conditions of combined cold and osmotic stress. To provide an insight into the genetic systems enabling this organism to adapt to growth at low temperature, we extended the aforementioned study to investigate the growth kinetics of E. coli O157:H7 Sakai during abrupt temperature downshift from 35 degrees C to 14 degrees C and, examined time-dependent global alterations in its genome expression upon cold shock from 35 degrees C to 14 degrees C. The genome-wide expression response of E. coli was analysed by both cDNA microarray (transcriptome response) and 2D-LC/MS/MS analysis (proteome response). Differences in gene and protein expression patterns in E. coli before and after cold shock were analysed through quantitative and comparative analysis of time series changes in both mRNA and proteins levels.
Project description:In a previous study we adopted an integrated transcriptomic and proteomic approach to determine the physiological response of E. coli O157:H7 Sakai during exponential phase growth under steady-state conditions relevant to low temperature and water activity conditions experienced during meat carcass chilling in cold air (Kocharunchitt et al., 2012). The findings of that study provide a baseline of knowledge of the physiology of this pathogen, with the response of E. coli O157:H7 to steady-state conditions of cold and osmotic stress. To provide an insight into the genetic systems enabling this organism to adapt to growth at low water activity, we extended the aforementioned study to investigate the growth kinetics of E. coli O157:H7 Sakai during abrupt water activity downshift from 0.993 to 0.967 and, examined time-dependent global alterations in its genome expression upon water activity downshift from 0.993 to 0.967. The genome-wide expression response of E. coli was analysed by both cDNA microarray (transcriptome response) and 2D-LC/MS/MS analysis (proteome response). Differences in gene and protein expression patterns in E. coli before and after water activity downshift were analysed through quantitative and comparative analysis of time series changes in both mRNA and proteins levels.
Project description:Microbes exhibit short and long term responses when exposed to challenging environmental conditions. To what extent these responses are correlated, what their evolutionary potential is and how they translate to cross-stress fitness is still unclear. In this study, we comprehensively characterized the response of Escherichia coli populations to four abiotic stresses (n-butanol, osmotic, acidic, and oxidative) and their combinations by performing genome-scale transcriptional analysis and growth profiling. We performed an analysis of their cross-stress behavior which identified 15 cases of cross- protection and one case of cross vulnerability. To elucidate the evolutionary potential of stress responses to individual stresses and stress combinations, we re-sequenced E. coli populations evolved in those four environments for 500 generations. We developed and applied a network-driven method that integrates mutations and differential expression to identify core and stress-specific gene communities that are likely to have a phenotypic impact. Our results suggest that beyond what is expected from the general stress response mechanisms, cross-stress behavior arises both from common pathways, several including metal ion binding and glycolysis/gluconeogenesis, and stress-specific expression programs. The stress-specific dependences uncovered, argue that cross-stress behavior is ubiquitous and central to understanding microbial physiology under stressful conditions.