P. knackmussii B13, the original host of ICEclc, was cultivated in minimal medium (MM) based on the type 21C medium (Gerhardt et al. 1981). This MM was complemented with 10mM 3CBA and the bacteria were grown at 30°C. Bacterial growth was assessed from culture turbidity at 600 nm (OD600). Cells were recovered 48 h after the beginning of the stationary phase. For RNA isolation, 100 ml of culture was immediately harvested by centrifugation (at 15,000 × g for 1 min at 4°C) and the supernatant was decanted. Cell pellets were resuspended in 4 ml RNAprotect Bacteria Reagent (QIAGEN GmbH). After 5 min incubation, the suspensions were centrifuged again (at 5,000 × g for 5 min at room temperature); the supernatant was discarded and pellets were stored at -80°C.
Extracted molecule
total RNA
Extraction protocol
Prior to RNA extraction, pellets were slowly thawed, then resuspended in 0.5 ml TES buffer [10 mM Tris-HCl (pH 8.0), 1 mM EDTA, 100 mM NaCl], followed by addition of and mixing with 0.25 ml lysis solution [20 mM sodium acetate (pH 5.5), 1 mM EDTA, 0.5% SDS]. After that, the total RNA was further purified by the hot acid-phenol method as described previously [Baumann et al. 1996]. RNA samples were purified from contaminating DNA by treatment with 50 U of DNase I (RNase free; Roche) during 1 h at 37°C. Finally, the RNA was dissolved in 50 µl diethylpyrocarbonate (DEPC)-treated water and quantified by absorbance at 260 and 280 nm on a NanoDrop spectrophotometer (Witec AG). The integrity of RNA was determined by agarose gel electrophoresis and the absence of DNA was verified by PCR.
Label
Cy3
Label protocol
cDNA was synthesized from total RNA and directly labeled with cyanine-3-dCTP using a modification of a protocol described elsewhere (Charbonnier et al. 2005). Briefly, each 50-µL reaction contained 10 µg of total RNA, 1.25 µg of random hexanucleotide primers (Promega), 100 µM each of unlabeled dATP, dGTP, and dTTP (Invitrogen), 25 µM of unlabeled dCTP (Invitrogen), 25 µM of cyanine-3-labeled dCTP (Perkin-Elmer), 25 U SUPERase•In (Ambion), and 400 U Superscript II reverse transcriptase (Invitrogen). Reactions were performed by heating at 42ºC for 2 hours followed by 70ºC for 10 min. RNA was then removed by adding 100 mM NaOH, heating to 65ºC for 20 min, and neutralizing with 100 mM HCl and 300 mM sodium acetate (pH 5.2). Labeled cDNA products were purified using the MinElute PCR purification kit (Qiagen) and the quantity and incorporation frequency of cyanine-3-labeled dCTP were calculated using the MICROARRAY function on a NanoDrop Spectrophotometer.
Hybridization protocol
60 ng of labeled cDNA was then loaded onto each microarray, hybridized for 17 hours at 65ºC, and washed and scanned as described for labeled cRNA in the One-Color Microarray-Based Gene Expression Analysis Manual (Agilent). The fragmentation step (heating to 60ºC for 30 minutes) was omitted.
Scan protocol
Slides were scanned immediately after washing on the Agilent DNA Microarray Scanner (G2505B) using one color scan setting for 8x15k array slides (Scan Area 61x21.6 mm, Scan resolution 5um, Dye channel is set to Green and Green PMT is set to XDR: Hi:100% and XDR Lo:10%.
Description
Gene expression 48hours after entrance in stationary phase after growth on 10mM trichlorobenzaote (3CBA) as substrate. ICEclcB13 in Pseudomonas knackmussii strain B13
Data processing
The scanned images were analyzed with Feature Extraction Software 10.5.1.1 (Agilent) using default parameters (protocols GE1-v5_95_Feb07 or GE1_105_Dec08 and Grid: 020923_D_F_20080708) to obtain background subtracted and spatially detrended Processed Signal intensities. Data were then transported to GeneSpring GX v11.0, Generic Single Color experiment, thresholding 1.0, Quantile normalization, no baseline transformation performed. Data were grouped according to genotypes (wt, inrR-/-, rpoS), filtered on expression value (20-100th percentile), analysed for significance (Oneway ANOVA unequal variance, corrected p-value cut-off 0.05, assymptotic p-value computation, Benjamini-Hochberg multiple testing correction).
