Project description:The heat-shock response is a cellular protection mechanism against sudden temperature upshifts extensively studied in Escherichia coli. However, the effects of thermal evolution on this response remain largely unknown. In this study, we investigated the early and late physiological and transcriptional responses to temperature upshift in a thermotolerant strain under continuous culture conditions. Adaptive laboratory evolution was performed on a metabolically engineered E. coli strain (JU15), designed for D-lactic acid production, to enable cellular growth and fermentation of glucose at 45 °C in batch cultures. The resulting homofermentative strain, ECL45, successfully adapted to 45 °C in a glucose-mineral medium at pH 7 under non-aerated conditions. The thermal-adapted ECL45 retained the parental strain’s high volumetric productivity and product/substrate yield. Genomic sequencing of ECL45 revealed eight mutations, including one in a non-coding region and six within the coding regions of genes associated with metabolic, transport, and regulatory functions. Transcriptomic analysis comparing the evolved strain with its parental counterpart under early and late temperature upshifts indicated that the adaptation involved an inactive stringent response. This mechanism likely contributes to the strain’s ability to maintain growth capacity at high temperatures.
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: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:Transcription profile of Escherichia coli cells in biofilms under static batch culture was compared to that of E. coli cells in planktonic cultures. Both E. coli biofilm and planktonic cultures were cultivated for 18 h in 10% Luria-Bertani broth at room temperature (20 degree Celsius). Biofilms were grown in static batch culture in petri dishes. Both planktonic culture and biofilms were homogenized and run through a separated protocol.
Project description:This Series involves two studies: 1) The gene expression of E. coli K-12 BW25113 ompA mutant strain vs. wild type strain glasswool biofilm cells and E. coli K-12 BW25113 ompA mutant vs. wild type polystyrene biofilm cells. 2) The gene expression of E. coli BW25113 ompA/pCA24N_ompA vs. ompA/pCA24N suspension cells. Strains: E. coli K-12 BW25113 wild type, ompA mutant Medium: LB Cell type: Biofilm cells grown on glasswool and polystyrene surfaces Time: 15 h Temperature: 37C Strains: BW25113 ompA/pCA24N_ompA and ompA/pCA24N Medium: LB Time: 7 h Temperature: 37C Cell type: suspension cells, induced by 0.1 mM IPTG
Project description:YbjN, an enterobacteria-specific protein, is a multicopy suppressor of ts9 temperature sensitivity in Escherichia coli. Microarray study revealed that the expression level of ybjN was inversely correlated with the expression of flagellar, fimbrial and acid resistance genes. Over-expression of ybjN significantly down-regulated genes involved in the citric acid cycle, glycolysis, the glyoxylate shunt, oxidative phosphorylation, and amino acid and nucleotide metabolism. On the other hand, over-expression of ybjN up-regulated toxin-antitoxin modules, the SOS responsive pathway, cold shock proteins and starvation-induced transporter genes. Our results collectively suggest that YbjN may play important roles in regulating bacterial multicellular behaviors, metabolism and survival under various stress conditions in Es. coli. A total of 8 samples were analyzed: E. coli wild type strain (2 replicates); E. coli ybjN mutant strain (3 replicates); E. coli ybjN over-expression strain (3 replicates).
Project description:Responses of Escherichia coli W3110gyrb234 as they are upshifted to 42C Escherichia coli W3110gyrb234 cells sampled at several time points (2,5, 10, 40 min) as they are shifted to 42 C in LB, vs 0 min before upshift in LB
Project description:Escherichia coli (E. coli) amine oxidase (ECAO) encoded by tynA gene has been one of the model enzymes to study the mechanism of oxidative deamination of amines to the corresponding aldehydes by amine oxidases. The biological roles of ECAO have been less addressed. Therefore we have constructed a gene deletion Escherichia coli K-12 strain, E. coli tynA-, and used the microarray technique to address its function by comparing the total RNA gene expression to the one of the wt. Our results suggest that tynA is a reserve gene for stringent environmental conditions and its gene product ECAO a growth advantage compared to other bacteria due to H2O2 production.
Project description:2D-LC/MS/MS analysis was used to examine time-dependent changes in proteome of E. coli O157:H7 strain Sakai upon an abrupt downshift in temperature (i.e., from 35°C to 14°C). Cell cultures were harvested at 30, 90, 160, and 330 min post-temperature downshift. It also should be noted that these time points were chosen with the aims to characterize the physiology of E. coli during dynamic changes in growth kinetics induced by an abrupt temperature downshift. Specifically, the samples taken at time 30 and 90 min were obtained during adaptation period, whereas the samples at time 160 and 330 min reflected the physiological state of E. coli during growth after the shift. MS/MS data obtained from each protein sample were processed by the Computational Proteomics Analysis System (CPAS), a web-based system built on the LabKey Server (v9.1, released 02.04.2009). The experimental mass spectra produced were subjected to a semi-tryptic search against the combined databases of E. coli O157:H7 Sakai (5,318 entries in total) downloaded from the National Center for Biotechnology Information (NCBI, https://www.ncbi.nlm.nih.gov/, downloaded 25.11.2008) using X!Tandem v2007.07.01. These databases included the E. coli O157:H7 Sakai database (5230 entries, NC_002695.fasta) and two E. coli O157:H7 Sakai plasmid databases, plasmid pO157 (85 entries, NC_002128.fasta) and plasmid pOSAK1 (three entries, NC_002127.fasta). The parameters for the database search were as follows: mass tolerance for precursor and fragment ions: 10 ppm and 0.5 Da, respectively; fixed modification: cysteine cabamidomethylation (+57 Da); and no variable modifications. The search results were then analyzed using the PeptideProphet and ProteinProphet algorithms from the Trans Proteomic Pipeline v3.4.2. All peptide and protein identifications were accepted at PeptideProphet and ProteinProphet of ≥0.9, corresponding to a theoretical error rate of ≤2%.