Comment[ArrayExpressAccession] E-GEOD-3591
Public Release Date 2005-11-21
Investigation Title A reassesment of the FNR regulon and the effects of nitrate,nitrite, narXL and narQP
Comment[Submitted Name] A reassesment of the FNR regulon and the effects of nitrate,nitrite, narXL and narQP
Experiment 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.
Date of Experiment
Term Source Name EFO
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Term Source File http://www.ebi.ac.uk/efo/efo.owl
Person Last Name Constantinidou Constantinidou Overton Hobman Griffiths Patel Penn Cole
Person First Name Chrystala Chrystala Timothy Jon Lesley Mala Charles Jeff
Person Mid Initials L D w A
Person Email C.Constantinidou@bham.ac.uk
Person Affiliation University of Birmingham
Person Phone +44 (0)121 414 5564
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Person Address School of Biosciences, University of Birmingham, , Birmingham, United Kingdom
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Protocol Name P-GSE3591-2 P-GSE3591-1 P-GSE3591-3
Protocol Description ID_REF =
CH1_MEAN = Cy5 Mean Foreground
CH1_SD = Cy5 Foreground SD
CH1_BKD_MEAN = Cy5 Mean Background
CH1_BKD_SD = Cy5 Background SD
CH2_MEAN = Cy3 Mean Foreground
CH2_SD = Cy3 Foreground SD
CH2_BKD_MEAN = Cy3 Mean Background
CH2_BKD_SD = Cy3 Background SD
VALUE = Log ratio (log2 of PRE_VALUE)
PRE_VALUE = (Cy5 Foreground - Background) / (Cy3 Foreground - Background) ID_REF =
CH1_MEAN = Cy5 Mean Foreground
CH1_SD = Cy5 Foreground SD
CH1_BKD_MEAN = Cy5 Mean Background
CH1_BKD_SD = Cy5 Background SD
CH2_MEAN = Cy3 Mean Foreground
CH2_SD = Cy3 Foreground SD
CH2_BKD_MEAN = Cy3 Mean Background
CH2_BKD_SD = Cy3 Background SD
VALUE = Log ratio (log2 of PRE_VALUE)
PRE_VALUE = (Cy5 Foreground – Background) / (Cy3 Foreground – Background) Raw, not normalised
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Protocol Type bioassay_data_transformation bioassay_data_transformation feature_extraction
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Experimental Factor Name GENETIC BACKGROUND GROWTH CONDITIONS
Experimental Factor Type Genetic background Growth conditions
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Publication Title Comparative genomic hybridization detects secondary chromosomal deletions in Escherichia coli K-12 MG1655 mutants and highlights instability in the flhDC region. A reassessment of the FNR regulon and transcriptomic analysis of the effects of nitrate, nitrite, NarXL, and NarQP as Escherichia coli K12 adapts from aerobic to anaerobic growth.
Publication Author List Hobman JL, Patel MD, Hidalgo-Arroyo GA, Cariss SJ, Avison MB, Penn CW, Constantinidou C Constantinidou C, Hobman JL, Griffiths L, Patel MD, Penn CW, Cole JA, Overton TW
PubMed ID 17921306 16377617
Publication DOI 10.1128/JB.00977-07 10.1074/jbc.M512312200
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Comment[SecondaryAccession] GSE3591
Comment[GEOLastUpdateDate] 2012-03-16
Comment[AEExperimentType] transcription profiling by array
Comment[GEOReleaseDate] 2005-11-21
Comment[ArrayExpressSubmissionDate] 2005-11-10
SDRF File E-GEOD-3591.sdrf.txt