Total RNA was extracted from the wild-type strain (Bacillus subtilis, 168) and phoR and sigB-null mutants before, during (T0) and after transition to phosphate-limited growth. Cell harvesting, preparation of RNA, synthesis of radioactively labeled cDNA and hybridization of B. subtilis macroarrays (Sigma-Genosys, The Woodlands, Tex., USA) were performed as described by Eymann and co-workers (Eymann, C., H. Mach, C. R. Harwood, and M. Hecker. 1996. Phosphate-starvation-inducible proteins in Bacillus subtilis: a two-dimensional gel electrophoresis study. Microbiology 142:3163-70.). Each analysis was carried out twice, using two independently isolated RNA preparations and two different array batches. The arrays were exposed to a phosphorescent screen which was subsequently scanned with a Storm 860 PhosphorImager (Molecular Dynamics, Sunnyvale, CA, USA) at a resolution of 50 µm and a 16-bit color depth. For quantification of the hybridization signals and background subtraction, the ArrayVision software (Version 5.1, Imaging Research, Onteria, Canada) was used. Calculation of normalized intensity values of the individual spots was performed using the over-all-spot-normalization function of ArrayVision. To avoid extreme expression ratios for genes close to or below the detection limit, genes with signal intensity values corresponding to less that twice the background were not counted in the analysis. Subsequently, the average of the normalized intensity values of the duplicate spots of each gene was used to calculate the expression level ratios. Data analysis (statistical analysis, visualization and the generation of lists) was performed using the GeneSpring software (Version 4.2.12, Silicon Genetics, Redwood City, CA). Keywords = phosphate starvation Keywords = PhoP regulon Keywords = signal transduction Keywords = DNA-array Keywords = gene expression
