strain: BLOB22 genome/variation: sigB mutant medium: Spizzien minimal medium
Treatment protocol
SMM; (ii) SMM with 1 mM glycine betaine, (iii) SMM with 0.7 M NaCl and (iv) SMM with 0.7 M NaCl and 1 mM glycine betaine.
Growth protocol
The Bacillus subtilis sigB mutant strain BLOB22 was grown in Spizzien minimal medium (SMM) to an OD 578nm of 1.0.
Extracted molecule
total RNA
Extraction protocol
Total RNA was isolated from nitrogen frozen cells using acidic phenol/chloroforn/Isoamyl alcohol extraction.
Label
[alpha-33P]dCTP
Label protocol
For cDNA synthesis, 2 µg of total RNA was mixed with 4 µl of a commercially available primer mix (Sigma-Genosys Ltd.) and 3 µl of 10× hybridization buffer (100 mM Tris [pH 7.9], 10 mM EDTA, 2.5 M KCl) in a total volume of 30 µl. The sample was heated to 95°C for 10 min and subsequently cooled to 42°C for primer annealing. Reverse transcription was performed in a total volume of 60 µl with SuperScript II reverse transcriptase and [alpha -33P]dCTP in the appropriate buffer for 1 h (Life Technologies, GmbH, Karlsruhe, Germany). After addition of 2 µl of 1% sodium dodecyl sulfate (SDS), 2 µl of 0.5 M EDTA (pH 8.0), and 6 µl of 3 M NaOH, the remaining RNA was hydrolyzed by incubation at 65°C for 30 min and at room temperature for 15 min. Prior to ethanol precipitation, the cDNA solution was neutralized with 20 µl of 1 M Tris (pH 8.0) and 6 µl of 2 N HCl. After a wash with 70% ethanol the pellet was carefully dried and resolved in 100 µl of distilled water. Labeling efficiency was determined with a liquid scintillation counter. This study was performed with Panorama B. subtilis gene arrays from Sigma-Genosys Ltd., which carry duplicate spots of PCR products representing 4,107 currently known B. subtilis genes.
Hybridization protocol
Prehybridization was carried out in 5 ml of hybridization solution (5x Denhardt solution, 5x SSC [1x SSC is 0.15 M NaCl plus 0.015 M sodium citrate], 2% sodium dodecyl sulfate [SDS], 100 µg of denatured herring sperm DNA [Sigma]/ml) for 2 h at 65°C. Hybridization was performed for 20 h at 65°C in 5 ml of hybridization solution containing the labeled cDNA probe that had been heated to 98°C for 2 min. After hybridization, arrays were washed twice with 200 ml of 2x SSC and 0.1% (wt/vol) SDS for 5 min at room temperature and twice with 200 ml of 0.2x SSC and 0.1% (wt/vol) SDS for 20 min at 65°C.
Scan protocol
The arrays were exposed for 2 and 4 days to storage phosphor screens (Molecular Dynamics, Sunnyvale, Calif.) and scanned with a Storm 840/860 PhosphorImager (Molecular Dynamics) at a resolution of 50 µm and a color depth of 16 bits.
Description
SMM_1_raw
Data processing
Data analysis followed a three-step procedure. First, ArrayVision software (version 6.1; Imaging Research, St. Catherine's, Ontario, Canada) was used for the quantification of the hybridization signals after direct import of the phosphorimager files. The analysis yielded the artifact-removed volumes and background values, calculated from the median of lines surrounding each group of eight spots on the array. These data were then used in a second step in Microsoft Excel, calculating for every spot on the array a quality score that reflected the ratio between the signal intensity and the background intensity. This quality score was utilized to identify the hybridization signals close to the detection limit, thereby avoiding artificially high induction ratios for those genes. Data normalization and analysis were performed in a third step using GeneSpring (version 7.3.1; Agilent Technologies).
