Unknown,Transcriptomics,Genomics,Proteomics

Dataset Information

The transcriptomic response of E. coli to triclosan exposure

ABSTRACT: The gene expression profile of E. coli K-12 MG1655 grown in minimal medium treated with 0.12 mg/L of the biocide triclosan has been analysed using whole genome oligonucleotide microarrays. "Control" RNA was isolated from three independently grown 50ml MOPS minimal media cultures of E. coli K-12 MG1655. âTestâ RNA was isolated from three independently grown 50ml MOPS minimal cultures of E. coli K-12 MG1655, to which was added 0.12 mg/L of triclosan after reaching mid-logarithmic growth phase (OD600 ~ 0.7 +/- 0.02). Keywords: dose response Cy5-labelled cDNA probes were synthesised from three independent biological extractions of RNA from E. coli MG1655 unexposed to triclosan. Cy3-labelled cDNA probes were synthesised from three independent biological extractions of RNA from E. coli MG1655 exposed to 0.12 mg/L of triclosan. A dye-swap control was also included, whereupon the control cDNA probes were labelled with Cy3 and the test cDNA probes labelled with Cy5. Probes were mixed to allow comparison of the control and test expression profiles and hybridised onto an E. coli spotted oligonucleotide array.

ORGANISM(S): Escherichia coli

SUBMITTER: Nick Coldham

PROVIDER: E-GEOD-15059 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Publications

Exposure of Escherichia coli and Salmonella enterica serovar Typhimurium to triclosan induces a species-specific response, including drug detoxification.

Bailey Andrew M AM Constantinidou Chrystala C Ivens Al A Garvey Mark I MI Webber Mark A MA Coldham Nick N Hobman Jon L JL Wain John J Woodward Martin J MJ Piddock Laura J V LJ

The Journal of antimicrobial chemotherapy 20090916 5

<h4>Objectives</h4>The use of triclosan within various environments has been linked to the development of multiple drug resistance (MDR) through the increased expression of efflux pumps such as AcrAB-TolC. In this work, we investigate the effect of triclosan exposure in order to ascertain the response of two species to the presence of this widely used biocide.<h4>Methods</h4>The transcriptomes of Salmonella enterica serovar Typhimurium SL1344 and Escherichia coli K-12 MG1655 after exposure to th ...[more]

PMID: 19759044

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Project description:Background: Glucocorticoids are important pharmaceutical agents in the treatment of acute lymphoblastic leukemia in children. Resistance or sensitivity to glucocorticoids is considered to be of crucial importance for disease prognosis. Prednisolone is a first-line chemotherapeutic agent in the treatment of acute lymphoblastic leukemia. Here, the effects of prednisolone on the resistant CCRF-CEM leukemic cell line were studied. Methods: Prednisolone’s cytotoxic and cell cycle effects were studied with flow cytometry. NF-κB translocation was studied with Western Blotting and differential gene expression was studied with cDNA microarrays. Results: Prednisolone exerted a delayed biphasic effect, necrotic at low doses and apoptotic at higher doses. At low doses, prednisolone exerted a pre-dominant mitogenic effect despite its induction on total cell death, while at higher doses, prednisolone’s mitogenic and cell death effects were counterbalanced. NF-κB was constitutively present in the nucleus. Early gene microarray analysis revealed 40 differentially expressed genes upon 4 hours of prednisolone’s exposure. Notable differences in gene regulation were observed between the lowest and the highest glucocorticoid doses. Prednisolone activated genes related to apoptosis/tumor suppression, cell cycle progression, metabolism and intra-/ extra-cellular signaling pathways. Conclusions: The mitogenic/biphasic effects of prednisolone are of clinical importance in the case of resistant leukemic cells. This approach might lead to the identification of gene candidates for future molecular drug targets in combination therapy with glucocorticoids, along with early markers for glucocorticoid resistance. Elucidation of the mechanisms of GC action may lead to identification of gene targets responsible for GC resistance. Key tools in this process are high-throughput technologies such as microarray-based gene expression analysis. For this purpose, the parental CCRF-CEM cell line was chosen as the system of study for the effects of prednisolone treatment. This is a T-cell leukemia cell line characterized by a mutation (L753F) on one GR gene allele that impairs ligand binding (Thompson and Johnson 2003). It is known that both the DNA and ligand binding domains of the GR are required in order to repress NF-κB transactivation (Wissink, van Heerde et al. 1997). Interestingly, concerning the question whether this mutation would affect GC resistance, it has been reported previously that both the GC-resistant as well as the GC-sensitive CCRF-CEM subclones express heterogeneous populations of the GR (GRwt/GRL753F) (Palmer and Harmon 1991; Powers, Hillmann et al. 1993). The CCRF-CEM cell line has been reported to be resistant to GCs, presumably due to the accumulation of more resistant variants after long periods of prolonged culture (Norman and Thompson 1977). It is possible that these cells are clonally inhomogeneous, as possibly the cells obtained in vivo by patients. Moreover, the large number of the CCRF-CEM subclone studies in the literature makes it difficult to choose an appropriate resistant cell model. In addition, the utilization of an in vitro system for this study offered reproducibility, an opportunity to closely examine intracellular signals and avoid interference from other in vivo-participating systems. Thus, the cell line used for this study was considered to be useful in studying GC action and resistance in leukemic cells. The aim of this work was to determine the cytotoxic, cell cycle phase distribution and early cancer-specific gene expression effects of prednisolone in CCRF-CEM cells, as an in vitro model of ALL resistance to glucocorticoids. The early gene expression profile allowed identification of genes initiating pivotal, early onset regulatory mechanisms activated by GC and excluded ensuing feedback responses and further downstream signals. Samples tested were in the form of a loop-design i.e. control vs. 10nM prednisolone (designated 0vs1) , 10nM prednisolone vs.700uM prednisolone (designated 1vs3) and control vs. 700uM prednisolone (designated 0vs3), the sum of the logarithms of the first two should equal the third. In other words if Rj,i is the ratio of the ith gene in the jth experiment then R0vs1,i+R1vs3,i=R0vs3,i. We tested this using a statistical test. We have applied an intensity-dependent z-score where the sum of the ratios was compared to the ratio of the third experiment. If the difference was significant genes, in a standard deviation threshold of ±1.5 in relative units, were rejected from further analysis (Kerr and Churchill 2001; Kerr and Churchill 2001; Altman and Hua 2006; Kerr and Churchill 2007).

Project description:The fumarate and nitrate reductase regulator protein, FNR, is a global transcription factor that regulates major biochemical changes as Escherichia coli adapts from aerobic to anaerobic growth. The ability of an fnr mutant to grow anaerobically in the presence of trimethylamine-N-oxide (TMAO) as the terminal electron acceptor was exploited in microarray experiments designed to determine a minimum number of Escherichia coli K-12 MG1655 operons that are regulated directly by FNR. In an anaerobic glycerol-TMAO-fumarate medium, the fnr mutant grew as well as the parental strain, enabling us to reveal the response of the E. coli transcriptome to oxygen, nitrate and nitrite in the absence of glucose repression or artefacts due to variations in growth rate. Many of the discrepancies between previous microarray studies of the E. coli FNR regulon were resolved in this study. First data for 43 previously characterised FNR-dependent operons were analysed. The current microarray data confirmed 32 of these 43 assignments, but alone did not confirm FNR-activation of 5 operons (adhE, glpTQ, cydDC, hlyE and arcA), or FNR repression of 6 operons (hemA, narXL, tpx, yeiL, norVW or ubiCA). Thirty-six operons not previously known to be included in the FNR regulon were activated by FNR and a further 26 operons appeared to be repressed. For each of these operons, an excellent match to the consensus FNR-binding site sequence was identified. The FNR regulon therefore minimally includes at least 94, and possibly as many as 105, operons. Many FNR-activated promoters are also regulated by one or both of two nitrate- and nitrite-responsive two-component regulatory systems, NarX-NarL and NarQ-NarP. Comparison of transcripts in the parental strain and a narXL deletion mutant revealed that transcription of 51 operons is activated, directly or indirectly, by NarL in response to nitrate, and a further 41 are repressed. As phosphorylated NarL can bind to the NarP DNA target sequence, the narP gene was also deleted from the narXL mutant to reveal the extent of regulation by phosphorylated NarP. Fourteen promoters were more active in the narP+ strain than in the mutant, and a further 37 were strongly repressed. This is the first report that NarP might function as a global repressor as well as a transcription activator. The data also revealed possible new biochemical defence mechanisms against reactive nitrogen species. Keywords: genetic modification, growth conditions An fnr mutant is either unable to grow anaerobically in the presence of most terminal electron acceptors and a non-fermentable carbon source such as glycerol or lactate, or grows far more slowly than the parental strain. Under such conditions, any differences in the transcriptomes of an fnr mutant and its parental strain would be due to both direct effects of FNR, and to differences in growth rate. As glucose represses expression from some FNR-activated promoters replacement of glucose by a less repressing fermentable carbohydrate would decrease effects due to glucose repression, but to an unknown extent. We therefore exploited the fact that fnr mutants can be grown anaerobically in the presence of the non-fermentable and non-repressing carbon source, glycerol, in the presence of trimethylamine-N-oxide (TMAO) in addition to fumarate as the terminal electron acceptor. Furthermore, the presence of TMAO has a minimal effect on NarX-NarL or NarQ-NarP-dependent induction or repression. Under these conditions, the fnr mutant grows as well as the parental strain and the use of the glycerol-TMAO-fumarate medium enables us to reveal the response of the E. coli transcriptome to nitrate, nitrite and the two-component regulator system, NarX-NarL. In each large set of experiments a common pool of reference RNA isolated from bacteria that had been grown anaerobically, and in which FNR-activated genes were expressed at a significant level. A potential disadvantage of this approach was the risk that some promoters repressed by FNR would be expressed at such a low level that the microarray signals would be too low to yield reliable data. To check for this artefact, further experiments were completed in which the reference RNA was a pool of samples isolated from bacteria in the early exponential phase of aerobic growth. “Reference” RNA was isolated from at least four independent cultures grown to OD 0.5 to 0.6. “Test” RNA was isolated from three independent cultures grown to OD 0.5 to 0.6.

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Dataset Information

The transcriptomic response of E. coli to triclosan exposure

Publications

Exposure of Escherichia coli and Salmonella enterica serovar Typhimurium to triclosan induces a species-specific response, including drug detoxification.

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