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Transcriptional analysis of E. coli whole genome during heat inactivation processes related to industrial cooking

ABSTRACT: This study was conducted to determine the impact of extreme temperatures, aiming at controlling microorganisms by cooking, on the physiology and the transcriptome of Escherichia coli. ABSTRACT: Because the genome of Escherichia coli K12 is well understood, this organism was used as a model to investigate physiological and molecular changes during cell adaptation and survival to cooking temperatures used in the food industry. Bacteria grown at 37°C to stationary phase in Brain Heart Infusion (BHI) broth, were heated at 58°C or 60°C to reach a process lethality value (F) of 2 and 3, or until an internal core temperature of 71°C was reached (control cooking temperature). Both growth recovery and cell membrane integrity were evaluated immediately after heating and a global transcription analysis was performed by using gene expression microarrays. Only cells heated at 58°C F = 2 were still able to grow on liquid or solid BHI after heat treatment. However, their transcriptome did not differ significantly compared to those of bacteria heated at 58°C F = 3 (P-FDR > 0.01) where no growth recovery was observed post treatment. Genome-wide transcriptomic data obtained at 71°C were distinct from the other treatments. Quantification of heat-shock genes expression by real-time PCR revealed that dnaK and groEL mRNA levels were significantly lower at 71°C than at 58°C and 60°C (P < 0.0001). Furthermore, despite of similar cell inactivation but growth on BHI media after heating, 132 and 8 genes were differentially expressed at 71°C when compared to 58°C and 60°C at F = 3 respectively (P-FDR < 0.01). Among them, genes like aroA, citE, glyS, oppB and asd, whose expression was up-regulated at 71°C, may be considered as good biomarkers to determine more accurately the efficiency of heat treatments.

ORGANISM(S): Escherichia coli O157:H7 str. Sakai Escherichia coli str. K-12 substr. MG1655 Escherichia coli CFT073 Escherichia coli O157:H7 str. EDL933

PROVIDER: GSE40557 | GEO | 2012/09/15

SECONDARY ACCESSION(S): PRJNA174293

REPOSITORIES: GEO

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Project description:This study was conducted to determine the impact of extreme temperatures, aiming at controlling microorganisms by cooking, on the physiology and the transcriptome of Escherichia coli. ABSTRACT: Because the genome of Escherichia coli K12 is well understood, this organism was used as a model to investigate physiological and molecular changes during cell adaptation and survival to cooking temperatures used in the food industry. Bacteria grown at 37Â°C to stationary phase in Brain Heart Infusion (BHI) broth, were heated at 58Â°C or 60Â°C to reach a process lethality value (F) of 2 and 3, or until an internal core temperature of 71Â°C was reached (control cooking temperature). Both growth recovery and cell membrane integrity were evaluated immediately after heating and a global transcription analysis was performed by using gene expression microarrays. Only cells heated at 58Â°C F = 2 were still able to grow on liquid or solid BHI after heat treatment. However, their transcriptome did not differ significantly compared to those of bacteria heated at 58Â°C F = 3 (P-FDR > 0.01) where no growth recovery was observed post treatment. Genome-wide transcriptomic data obtained at 71Â°C were distinct from the other treatments. Quantification of heat-shock genes expression by real-time PCR revealed that dnaK and groEL mRNA levels were significantly lower at 71Â°C than at 58Â°C and 60Â°C (P < 0.0001). Furthermore, despite of similar cell inactivation but growth on BHI media after heating, 132 and 8 genes were differentially expressed at 71Â°C when compared to 58Â°C and 60Â°C at F = 3 respectively (P-FDR < 0.01). Among them, genes like aroA, citE, glyS, oppB and asd, whose expression was up-regulated at 71Â°C, may be considered as good biomarkers to determine more accurately the efficiency of heat treatments. Bacteria were submitted to four different heat-treatments. Biological replicates: 5 Controls grown to an optimal temperature of 37Â°C (and used to prepare a common reference for each array hybridization), 5 Treatment 1 (58Â°C F = 2), 4 Treatment 2 (58Â°C F = 3), 4 Treatment 3 (60Â°C F = 3), 5 Treatment 4 (core temperature of 71Â°C). Treatment replicates were randomly hybridized on 3 microarray slides Agilent 8x15K (total of 24 array areas). Equal amounts of the labeled control cDNA pool and labeled cDNA from one of the heated groups were mixed and hybridized on each array.

Project description:Studying the effect of varying durations (1, 7 and 14 days) of locally applied heat stress on grape berries at three developmental stages (middle green, veraison and middle ripening). Among various environmental factors, temperature is a major regulator affecting plant growth, development and fruit composition. Reproductive development of grapevine and berry composition, are both highly influenced by temperature. To date, the molecular mechanisms involved in grapevine berries tolerance to high temperatures (HS) are poorly understood. Although recent data addressed the consequences on berry development of elevated temperatures applied at the whole plant level, the present work particularly focuses on the fruit responses triggered by direct exposure to heat stress. In the context of climate change, this work focused on microclimate effect is of particular interest to better understand the consequences of leaf removal (a common viticultural practice) on berry development. HS (+ 8°C) was locally applied to clusters from Cabernet Sauvignon fruiting cuttings at three different developmental stages (middle green, véraison and middle ripening). Samples were collected 1, 7 and 14 days after treatment and used for metabolic and transcriptomic analyses. The results showed dramatic and specific biochemical and transcriptomic changes in heat exposed berries, depending on the developmental stage and the stress duration. When applied at the herbaceous stage, HS delayed the onset of véraison. Heating also strongly altered the berry concentration of amino acids and organic acids (e.g. PHE, GABA, and malate) and decreased the anthocyanin content at maturity. These physiological alterations could be partly explained by the deep remodelling of transcriptome in heated berries. More than 7000 genes were deregulated in at least one of the nine experimental conditions. The most affected processes belong to the categories “stress responses”, “protein metabolism” and “secondary metabolisms”, highlighting the intrinsic capacity of grape berries to perceive heat stress and to build adaptive responses. Additionally, important changes in processes related to “transport”, “hormone” and “transcription” might contribute to the postponing of véraison. Finally, opposite effects depending on heating duration were observed for genes encoding enzymes of the general phenylpropanoid pathway, suggesting that the HS-induced decrease in anthocyanin content may result from a combination of transcript abundance and product degradation.

Project description:We hypothesized that broad-scale expression profiling would provide insight into the regulatory pathways that control gene expression in response to stress, and potentially identify novel heat-responsive genes. HEp2 cells were heated at 37 to 43 Â°C for 60 min to gauge the heat shock response, using as a proxy inducible HSP-70 quantified by western blot analysis. Based on these results, microarray experiments were conducted at 37, 40, 41, 42 and 43Â°C (3 replicates/temperature x 5 groups = 15 U95Aver2 GeneChips). Using linear modeling, we compared the sets of microarrays at 40, 41, 42 and 43Â°C with the 37Â°C baseline temperature and took the union of the genes exhibiting differential gene expression signal to create two sets of âheat shock responseâ genes, each set reflecting either increased or decreased RNA abundance. Leveraging human and mouse orthologous alignments, we used the two lists of co-expressed genes to predict transcription factor binding sites in silico, including those for heat shock factor 1 (HSF1) and heat shock factor 2 (HSF2) transcription factors. We discovered HSF1 and HSF2 binding sites in 15 genes not previously associated with the heat shock response. We conclude that microarray experiments coupled with upstream promoter analysis can be used to identify novel genes that respond to heat shock. Additional experiments are required to validate these putative heat shock proteins and facilitate a deeper understanding of the mechanisms involved during the stress response. Experiment Overall Design: Cell Culture: HEp2 cells were acquired from American Type Culture Cell (Rockville, MD) and cultured in EMEM supplemented with 10% fetal bovine serum (Gibco Laboratories, Grand Island, NY), 1% nonessential amino acids (Gibco), and 1.5g/L sodium bicarbonate. The cell cultures were maintained at 37Â°C in a humidified incubator in an atmosphere of 5% CO2 â 95% air and passaged at a 1:3 ratio with trypsin every 5-7 days. Experiment Overall Design: Heat shock and RNA extraction: After growing to confluency, cells were heated for 60 min in an incubator at 37, 38, 39, 40, 41, 42, or 43Â°C as measured by a sterile probe placed into each plateâs media. After heating, media was removed and ice cold PBS was immediately added and cells were scraped from plates and put into 1.5mL centrifuge tubes and pelletted. PBS was removed and replaced with 1 mL of TRIzolÂ® and vortexed vigorously (Invitrogen, Carlsbad, CA) for 5min. RNA was then extracted as per manufactures protocol. RNA integrity was assessed by gel electrophoresis and the Agilent Bioanalyzer 2100 machine (Agilent Technologies, Waldbronn, Germany). Experiment Overall Design: Affymetrix cRNA target generation: RNA purification (RNAeasy, Qiagen), cDNA synthesis (Invitrogen, Carlsbad, CA) and biotinylated cRNA (Invitrogen, Carlsbad, CA) were all performed in accordance with manufacturerâs instructions (Affymetrix, Inc., Santa Clara, CA). Fragmented target cRNA was hybridized with U95v2 GeneChipï£ª using AffymetrixÂ® equipment at the Alvin J. Siteman Cancer Center Genechip Facility. Fluorescent signal was collected using an Affmetrix ï£ª G2500A GeneArray Scanner (Hewlett-Packard, Inc., Cupertino, CA). Experiment Overall Design: GeneChip microarray data were generated at cellular temperatures of 37, 40, 41, 42, and 43Â°C (U95Av2 GeneChipsÂ®, ~10,000 genes per microarray; 3 technical replicates per temperature = 15 GeneChipsÂ®).

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Transcriptional analysis of E. coli whole genome during heat inactivation processes related to industrial cooking

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