Unknown,Transcriptomics,Genomics,Proteomics

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The role of multi-copper oxidase CueO

ABSTRACT: The effect of a cueO mutation on global gene expression in E. coli has been analysed using whole genome oligonucleotide microarrays. A cueO mutant lacks a periplasmic multi-copper oxidase, an enzyme that is believed to be involved in the oxidation of cuprous and ferrous ions as well as phenolic compounds in the periplasm. Deletion renders the cell sensitive to copper and this toxicity is likely associated with increased oxidative stress in a cueO mutant. Triplicate cultures of E. coli MG1655 and the isogenic cueO mutant were grown to mid-exponential phase (OD600 0.8) in M9C supplemented with 2ïM FeSO4. RNA was extracted using Qiagen RNAprotect and RNeasy Mini Kit. Reference RNA was similarly purified from triplicate cultures of E. coli MG1655 grown on M9C supplemented with 2ïM FeCl3. RNA concentration and quality was assessed using an Aligent Bioanalyser 2100 NanoLabchip.

ORGANISM(S): Escherichia coli

SUBMITTER: Jon Hobman

PROVIDER: E-GEOD-5177 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Publications

The expression profile of Escherichia coli K-12 in response to minimal, optimal and excess copper concentrations.

Kershaw Christopher J CJ Brown Nigel L NL Constantinidou Chrystala C Patel Mala D MD Hobman Jon L JL

Microbiology (Reading, England) 20050401 Pt 4

The gene expression profile of Escherichia coli K-12 MG1655 grown in minimal medium supplemented with elevated copper concentrations (as copper-glycine) has been analysed using whole-genome oligonucleotide microarrays. At 750 muM copper-glycine, the expression of both the cue and cus copper-export systems is evident. At near-lethal copper concentrations (2 mM copper-glycine), the expression of these two regulons increases significantly. Other regulons with increased transcription in response to ...[more]

PMID: 15817786

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Project description:Escherichia coli O157 presents a number of specific problems in terms of food safety and public health. It has been found that E. coli O157 is more resistant to a number of the stresses encountered during food production such as heat, pH and osmotic shock. This greater resistance is thought to contribute to the low infectious dose of E. coli O157 (<100 organisms). Moreover, E. coli O157 is associated with debilitating conditions such as haemorrhagic colitis and haemoytic uraemic syndrome, particularly in children and the elderly. We have been studying the stress responses of E. coli O157:H7 (Sakai) and comparing with a commensal strain of E. coli K-12, MG1655. We found that E. coli O157 (Sakai) is more sensitive to oxidative stress than MG1655. A microarray study of these strains treated with sub-lethal concentrations (0.5mg/ml) of menadione revealed big differences in their responses. In E. coli O157 (Sakai), 540 genes responded significantly to the treatment compared to 121 genes in MG1655. One surprising finding from the microarray data was the observation that many iron-transport genes were up-regulated in E. coli O157 (Sakai) whereas relatively few were induced in MG1655 despite the fact that the bacteria were grown in a medium containing ample iron. We speculated that the induction of iron transport genes in an iron-rich medium might have contributed to the enhanced killing of E. coli O157 (Sakai) through triggering of a Fenton reaction. We speculated that the difference in sensitivity to oxidative stress might be due to differences in the intracellular iron content of E. coli O157 and MG1655. We found that E. coli O157 contains ~50% more iron than MG1655 and believe that during oxidative stress, this iron is released by damaged proteins. The greater levels of free iron in E. coli O157 will trigger a greater Fenton reaction that can damage the ferric uptake regulator (Fur), resulting in unregulated iron transport. In MG1655, the lower iron content results in a smaller Fenton reaction, enabling the cellular protection systems to limit damage and protect Fur. Overnight cultures of E. coli O157 (Sakai) and E. coli K-12 MG1655 were grown in Neidhardt's EZ Rich Defined Medium and diluted 1:100 in 50 ml fresh medium in 125 ml Ehrlenmeyer flasks. The cultures were shaken at 37C until the optical density (OD600) reached 0.4. Each culture was divided into 2 equal parts in identical flasks. One flask contained menadione bisulphite to a final concentration of 0.5 mg/ml; the other flask contained an equivalent volume of distilled water. The flasks were shaken for a further 10 mins and then treated with RNAprotect™ to stabilise the mRNA. The experiment was performed 3 times on different days. Six custom-made microarray slides were used in this study; each slide was hybridised with labelled cDNA made from mRNA taken from untreated and treated E. coli O157 (Sakai) or MG1655.

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.

Dataset Information

The role of multi-copper oxidase CueO

Publications

The expression profile of Escherichia coli K-12 in response to minimal, optimal and excess copper concentrations.

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