Metabolomics,Unknown,Transcriptomics,Genomics,Proteomics

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Gene expression of E. coli in continuous culture during adaptation to artificial sunlight

ABSTRACT: E. coli growing in continuous culture under continuous UVA irradiation exhibits growth inhibition with a subsequent adaptation to the stress. Transcriptome analysis was performed during transient growth inhibition and in the UVA light-adapted growth state. The results indicate that UVA light induces stringent response and an additional response that includes the upregulation of the synthesis of valine, isoleucine, leucine, phenylalanine, histidine and glutamate. The induction of several SOS response-genes strongly points to DNA damage as a result of UVA exposure. The involvement of oxidative stress was observed with the induction of ahpCF. Taken together it supports the hypothesis of the production of reactive oxygen species by UVA light. In the UVA-adapted cell population strong repression of the acid tolerance response was found. We identified the enzyme chorismate mutase as a possible chromophore for UVA light-inactivation and found strong repression of the pyrBI operon and the gene mgtA encoding for an ATP dependent Mg2+ transporter. Furthermore, our results indicate that the role of RpoS may not be as important in the adaptation of E. coli to UVA light as it was implicated by previous results with starved cells, but that RpoS might be of crucial importance for the resistance under transient light exposure. Slide microarrays were purchased from MWG-Biotech AG (Ebersberg, Germany). The MWG E. coli Array contains 4,288 gene specific oligonucleotide probes representing the complete E. coli K12 genome. Microarray slides were scanned using the Affymetrix 428TM Array Scanner (High Wycombe, UK). Spot intensities and corresponding background signals were quantified with the Affymetrix JaguarTM software version 2.0. Further data analysis was performed with the program GeneSpring from Silicon Genetics (Redwood City, CA, USA). Induction factors (IF) were calculated from the Cy3 and Cy5 signal intensities of the spot. Spots with signal intensity below a value of 50 were excluded from the analysis and the minimal induction factor was set to 0.01. The normalization was performed with the 50th percentile distribution of remaining spots after background correction. The mean value of the IF of a specific gene was calculated from three replicates. Biological experiments were carried out three times, which provided three biological repeats. Dye swapping was performed for one replicate and did not alter the result.

ORGANISM(S): Escherichia coli

SUBMITTER: Michael Berney

PROVIDER: E-GEOD-4569 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Gene expression of Escherichia coli in continuous culture during adaptation to artificial sunlight.

Berney Michael M Weilenmann Hans-Ulrich HU Egli Thomas T

Environmental microbiology 20060901 9

Escherichia coli growing in continuous culture under continuous UVA irradiation exhibits growth inhibition with a subsequent adaptation to the stress. Transcriptome analysis was performed during transient growth inhibition and in the UVA light-adapted growth state. The results indicate that UVA light induces stringent response and an additional response that includes the upregulation of the synthesis of valine, isoleucine, leucine, phenylalanine, histidine and glutamate. The induction of several ...[more]

PMID: 16913923

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Project description:The faecal indicator bacterium Escherichia coli K12 was used to study the effects of the global regulators RpoS and cAMP at the transcription level using microarray technology during short-term (physiological) adaptation to slow growth under limited nutrient supply. Effects due to the absence of one global regulator were assessed by comparing the mRNA levels isolated from rpoS or cya mutants under glucose-limited continuous culture at a dilution rate of 0.3 h-1 for the rpoS mutant or 0.16 h-1 for the cya mutant with those from wt E. coli grown under the same conditions. Wild-type Escherichia coli K12 MG1655 (ATCC 700926), E. coli delta-rpoS (MG1655 delta-rpoS::Tn10; Wick et al. (2002)) and AMS2 (MG1655 delta-cya-854; Schultz et al. (1988)) were grown at 37°C in mineral medium. Steady-state with respect to optical density was reached approximately 20 hours after switching on the medium feed. Samples for catabolic pathway assays and transcriptome analysis were taken 40 hours after switching to the continuous culture mode, and each experiment was done in triplicate. 800 ml of culture liquid was withdrawn from the chemostat, immediately cooled down on crushed ice and cells were collected by centrifugation at 4°C. Total RNA from E. coli cells was isolated (Sambrook et al., 1989). RNA in samples was quantified spectrophotometrically by measuring extinction at 260 nm, and purity (free from DNA) was checked by gel electrophoresis. Synthesis of cDNA from RNA was performed with the CyScribe First-Strand cDNA Labelling Kit (Amersham Bioscience, Little Chalfont, England). The purified cDNA was concentrated to 5 ul and was mixed with 120 ul of hybridization buffer (MWG-Biotech AG), heated to 95°C for 3 min and cooled down on ice for 3 min. The hybridization mixture was then added to the microarray slide and covered with a coverslip. The hybridization slide was incubated overnight at 42°C. After the hybridization step, the slide was washed three times, the first time for 5 min in 2x (times concentrated) SSC - 0.1 % SDS, the second time for 5 min in 1x SSC, and finally for 5 min in 0.1x SSC. SSC buffer was prepared as 20x solution containing 0.3M Na-citrate and 3M NaCl at pH 7.0. The slides were dried by centrifugation at room temperature for 2 min at 500g. Microarray slides were scanned using the Affymetrix 428TM Array Scanner (High Wycombe, UK). Spot intensities and corresponding background signals were quantified with the Affymetrix JaguarTM software version 2.0. Further data analysis was performed with the program GeneSpring from Silicon Genetics (Redwood City, USA). Induction factors were calculated from the Cy3 and Cy5 signal intensities of the spot. Spots with signal intensity below a value of 50 were excluded from the analysis and the minimal induction factor was set to 0.01. The normalization was performed with the 50th percentile distribution of remaining spots after background correction. The mean value of the induction factors of a specific gene was calculated from three replicates. Biological experiments were carried out three times, which provided three biological repeats. Data from the independent experiment were combined, genes that were differently regulated ?3 and ?-3 (t-test, p ? 0.2) were defined as being statistically significant.

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. A precise rpoS deletion mutant of EDL933 was constructed and employed in this study. EDL933 wild type and rpoS mutants were inoculated in triplicate into LB media at a starting OD of 0.0001 and grown aerobically at 37C. Cultures were harvested at OD600 = 0.3 in exponential phase and OD600=1.5 in stationary phase. For RNA extraction, cultures were mixed directly with a boiling lysis buffer containing SDS and EDTA followed by acidic hot phenol to minimize RNA degradation. RNA samples were hybridized to Affymetrix E. coli Genome 2.0 Array according to Affymetrix's standard protocols.

Project description:The faecal indicator bacterium Escherichia coli K12 was used to study the cellular events that take place at the transcription level using the microarray technology during short-term (physiological) and long-term (genetic) adaptation to slow growth under limited nutrient supply. Short-term and long-term adaptation were assessed by comparing the mRNA levels isolated after 40 or 500 hours of glucose-limited continuous culture at a dilution rate of 0.3 h-1 with those from batch culture with glucose excess. Wild-type Escherichia coli K12 MG 1655 (ATCC 700926) was grown at 37 Â°C in mineral medium. Growth was followed by measuring OD546 in regular intervals to confirm that cells were growing at ï?max (0.63 Â± 0.02 h-1). A sample for transcriptome analysis was taken from the batch exponential phase at an OD546 of ï?¾ 0.4, and immediately after sampling the medium flow was started for continuous cultivation with a dilution rate of 0.3 h-1. Steady-state with respect to biomass and residual glucose concentrations was reached approximately 15 hours after this manipulation. 40 and 500 hours after switching to the glucose-limited continuous culture mode 800 ml of culture liquid was withdrawn from the chemostat, immediately cooled down on crushed ice and cells were collected by centrifugation at 4 Â°C. Total RNA from E. coli cells was isolated as described earlier (Sambrook et al., 1989). RNA in samples was quantified spectrophotometrically by measuring extinction at 260 nm and purity (free from DNA) was checked by gel electrophoresis. Synthesis of cDNA from RNA was performed with the CyScribe First-Strand cDNA Labelling Kit (Amersham Bioscience, Little Chalfont, England). The purified cDNA was concentrated to 5 Î¼l and was mixed with 120 Î¼l of hybridization buffer (MWG-Biotech AG), heated to 95 Â°C for 3 min and cooled down on ice for 3 min. The hybridization mixture was then added to the microarray slide and covered with a coverslip. The hybridisation slide was incubated over night at 42 Â°C. After the hybridization step the slide was washed three times, the first time for 5 min in 2x (times concentrated) SSC - 0.1 % SDS, the second time for 5 min in 1x SSC, and finally for 5 min in 0.1x SSC. SSC buffer was prepared as 20x solution containing 0.3M Na-citrate and 3M NaCl at pH 7.0. The slides were dried by centrifugation at room temperature for 2 min at 500g. Microarray slides were scanned using the Affymetrix 428TM Array Scanner (High Wycombe, UK). Spot intensities and corresponding background signals were quantified with the Affymetrix JaguarTM software version 2.0. Further data analysis was performed with the program GeneSpring from Silicon Genetics (Redwood City, USA). Induction factors were calculated from the Cy3 and Cy5 signal intensities of the spot. Spots with signal intensity below a value of 50 were excluded from the analysis and the minimal induction factor was set to 0.01. The normalization was performed with the 50th percentile distribution of remaining spots after background correction. The mean value of the induction factors of a specific gene was calculated from three replicates. Biological experiments were carried out three times, which provided three biological repeats. Data from the independent experiments were combined, genes that were differentially regulated â?¥ 2 and â?¤ 0.5 (t-test, p â?¤ 0.05) were defined as being statistically significant.

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Dataset Information

Gene expression of E. coli in continuous culture during adaptation to artificial sunlight

Publications

Gene expression of Escherichia coli in continuous culture during adaptation to artificial sunlight.

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