All strains were grown until late exponential phase (OD600 0.9) in the defined rich medium MCDB 202 (CryoBioSystem), at 37oC. Pulsed Field Gel Electrophoresis was performed for molecular characterization of the strains. Bacterial cells were grown overnight in BHI medium (Difco) and inoculated (10%) in 10 ml MCDB 202 medium for total RNA extraction. For each strain, two overnight cultures were used.
Extracted molecule
total RNA
Extraction protocol
Cells were harvested by centrifugation at 12000 g for 4 min at 4oC, and immediately frozen. Pellets were resuspended in 400 ul of buffer (10% glucose, 12.5 mM Tris pH 7.6, 5 mM EDTA) and 60 ul of EDTA 0.5 M, and cells were mechanically disrupted in a FastPrep apparatus after the addition of 500 ul of acid phenol (pH 4.5) and 0.4 g of glass beads (Sigma). After a centrifugation step, the aqueous phase was transferred to new tubes, 1 ml of Trizol (Invitrogen) was added and samples were incubated for 5 min at room temperature. 200 ul of chlorophorm/isoamylic alcohol were added to each sample, followed by centrifugation for 5 min. This step was repeated twice and to the final aqueous supernatant 500 ul of 2-propanol were added for total RNA precipitation. The mixture was left at -20oC for 15 min and centrifuged for 30 min at 4oC. Pellets were then washed with 70% ethanol and dried at room temperature prior to being dissolved in 50 ul of diethylpyocarbonate (DEPC)-treated water. The RNA concentration was estimated using a spectrophotometer and RNA quality was checked on agarose gel containing ethidium bromide.
Label
33P
Label protocol
Total RNA (1ug) was mixed with 6 ul of 5X AMV reverse transcriptase buffer (Roche), 3 ul of a mixture of dATP, dGTP, dTTP (10 mM), 5 ug of random hexamers (Primer Random p(dN)6, Roche) and DEPC-treated water to a final volume of 21 ul. The mixture was heated at 90oC for 2 min and cooled to 42oC before addition of 3 ul of alpha-33P dCTP (20 uCi) and 2 ul (50U/ul) of AMV reverse transcriptase (Roche). The mixture was incubated for 2 h at 42oC. A column was used for the removal of unincorporated nucleotides according to the manufacturer’s instructions (QIAquick Nucleotide Removal Kit, QIAgen). The obtained cDNA was denaturated by heating at 99oC for 5 min just before hybridization.
Hybridization protocol
Macroarrays were pre-hybridized in roller bottles with 10 ml of a hybridization solution containing 5X SSPE (1X SSPE is 0.18 M NaCl, 10 mM NaH2PO4 and 1 mM EDTA pH7.7), 2% SDS, 1X Denhardt’s reagent (Sigma), 100 ug/ml of sonicated salmon sperm DNA for 1h at 65oC. Hybridizations were performed overnight at 65oC with 5 ml of fresh hybridization solution containing the labeled cDNA. After hybridization macroarrays were washed twice for 5 min at room temperature and twice for 20 min at 65oC with a washing solution (0.5X SSPE, 0.2% SDS).
Scan protocol
The macroarrays were then exposed to a phosphoimager screen and scanned with the Typhoon phosphoimager (Pharmacia-Amersham). Two hybridizations for each independent RNA extraction were performed. In total, a set of 4 macroarrays was used for each strain. Hybridized arrays were scanned using the Typhoon 9400 phosphoimager (Amersham Biosciences). The signal intensity was quantified using the ArrayVision software (Imaging Research). The intensity was measured in arbitrary units using the ARMdens measure.
Description
see above.
Data processing
Normalized by a multiplicative constant. The correction factor is obtained setting arbitrarily the median of log10(intensities) to ZERO.
