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Series GSE4941

Query DataSets for GSE4941

Status

Public on Jun 01, 2006

Title

Expression data from untreated and IAA treated cells

Platform organism

Escherichia coli

Sample organism

Escherichia coli K-12

Experiment type

Expression profiling by array

Summary

We investigated the physiological changes induced by indoleacetic acid (IAA) treatment in the totally sequenced Escherichia coli K-12 MG1655.
We used DNA macroarrays to measure the mRNA levels for all the 4,290 E. coli protein-coding genes and observed that 50 genes (1.1 %) exhibited significantly different expression profiles. In particular, genes involved in the tricarboxylic acid cycle (TCA), glyoxylate shunt, and amino acid biosynthesis (leucine, isoleucine, valine and proline) were up-regulated, whereas the fermentative adhE gene was down-regulated. To confirm the indications obtained from the macroarray analysis we measured the activity of 33 enzymes involved in the central metabolism and found an activation of the TCA cycle and the glyoxylate shunt.
Keywords: treatment

Overall design

Gene expression levels for control and IAA-treated cells were monitored by using DNA macroarray membranes containing the whole E. coli K-12 MG1655 genome.To identify ORFs whose expression was altered by addition of IAA, the two growth conditions (with and without IAA addition) were compared by determining the ratio of the corresponding intensities of each pair of ORF-specific spots on two blots. These ratios represent the relative transcript levels of each E. coli ORF under the same growth condition. Each ORF-specific spot was present in duplicate, and the mean of the normalised intensity values of the duplicate spots of each gene was used for further analysis.

Cells were grown aerobically at 37 °C in M9 minimal medium containing 0.4 % L-arabinose as carbon source. Exponentially growing E. coli K-12 (MG1655) cultures (OD600 = 0.6) were split into two aliquots; to the first one, an IAA solution was added to a final concentration of 0.5 mM and the other was left untreated (control). After two hours (OD600 = 1.2 for both cultures) different cell batches, taken from IAA-treated and untreated cells, were aliquoted, frozen in liquid nitrogen for 5 min and stored at –80 °C for use in experiments. After RNA purification, probe synthesis, labelling, arrays (Panorama E. coli gene arrays) hybridisation and washing steps were performed using the protocol provided by the manufacturer (Sigma-Genosys Biotechnologies Inc.).
The exposed phosphorimager screens were scanned and were analysed to determine the signal intensities for each spot by summing the value of each pixel within the boundaries of the spot using Image-Quant (version 3.0) software (Molecular Dynamics). Background values were measured in the surrounding region of the four corner grids containing E. coli genomic DNA, as a positive control, and empty spots. The intensity values for each spot were exported from ImageQuant to Microsoft Excel spreadsheet. Each ORF-specific spot was present in duplicate, and the mean of the normalised intensity values of the duplicate spots (a and b) of each gene was used for further analysis. To avoid extreme intensity ratio for genes close to or below the detection limit, signal intensity values corresponding to a signal-to-background (S/B) ratio < 2.0 were scaled up to a value corresponding to the normalised background means. To identify ORFs whose expression was altered by addition of IAA, the two growth conditions (with and without IAA addition) were compared by determining the ratio of the corresponding intensities of each pair of ORF-specific spots on two blots. These ratios represent the relative transcript levels of each E. coli ORF under the same growth condition. Ratios were calculated such that the log2 of the absolute value of the expression ratio was positive for intensities that were higher in IAA-treated cells and negative for intensities that were higher in control cells. A gene was considered significantly regulated when the relative intensity was least two times greater than the background expression threshold and the log2 expression ratio was greater than or equal to 0.8 (up-regulated) or less than or equal to -0.8 (down-regulated) and greater than 4 times standard deviation from the mean.

Contributor(s)

Bianco C, Imperlini E, Calogero R, Senatore B, Pucci P, Defez R

Citation(s)

16849805

Submission date

May 30, 2006

Last update date

Mar 16, 2012

Contact name

Roberto Defez

E-mail(s)

[email protected]