P. aeruginosa and P. aeruginosa delta PA4101 (bfmR) were grown in a flow-through bioflm on minimal medium with 10 mM glutamate for 6 days.
Extracted molecule
total RNA
Extraction protocol
Cells were treated with RNAprotect (Qiagen), and total RNA was extracted using an RNeasy mini purification kit (Qiagen) per the manufacturer’s instructions. RNA quality and the presence of residual DNA were checked on an Agilent Bioanalyzer 2100 electrophoretic system pre- and post-DNase treatment.
Label
biotin
Label protocol
Ten micrograms of total RNA was used for cDNA synthesis, fragmentation, and labeling according to the Affymetrix GeneChip P. aeruginosa genome array expression analysis protocol. End labeled with biotin-ddUTP with use of the Enzo BioArray Terminal Labeling kit (Affymetrix).
Hybridization protocol
Affymetrix GeneChip P. aeruginosa genome array expression analysis protocol.
Scan protocol
Affymetrix GeneChip P. aeruginosa genome array expression analysis protocol.
Data processing
Microarray data were generated using Affymetrix protocols as previously described (Frisk et al., 2004, Lizewski et al., 2004, Morici et al., 2007). Absolute expression transcript levels were normalized for each chip by globally scaling all probe sets to a target signal intensity of 500. Three statistical algorithms (detection, change call, and signal log ratio) were then used to identify differential gene expression in experimental and control samples. The detection metric (presence, absence, or marginal) for a particular gene was determined using default parameters in MAS software (version 5.0; Affymetrix). Batch analysis was performed in MAS to make pairwise comparisons between individual experimental and control GeneChips in order to generate change calls and a signal log ratio for each transcript. These data were imported into Data Mining Tools (version 3.0; Affymetrix). Transcripts that were absent under both control and experimental conditions were eliminated from further consideration. Statistical significance of signals between the control and experimental conditions (P < 0.05) for individual transcripts was determined using the t test. We defined a positive change call as one in which greater than 50% of the transcripts had a call of increased (I) or marginally increased (MI) for up-regulated genes and decreased (D) or marginally decreased (MD) for down-regulated genes. Finally, the mean value of the signal log ratios from each comparison file was calculated. Only those genes that met the above criteria and had a mean signal log ratio of greater than or equal to 1 for up-regulated transcripts and less than of equal to 1 for down-regulated transcripts were kept in the final list of genes. Signal log ratio values were converted from log2 and expressed as fold changes.
Frisk, A., J. R. Schurr, G. Wang, D. C. Bertucci, L. Marrero, S. H. Hwang, D. J. Hassett & M. J. Schurr, (2004) Transcriptome analysis of Pseudomonas aeruginosa after interaction with human airway epithelial cells. Infect. Immun. 72: 5433-5438. Lizewski, S. E., J. R. Schurr, D. W. Jackson, A. Frisk, A. J. Carterson & M. J. Schurr, (2004) Identification of AlgR-regulated genes in Pseudomonas aeruginosa by use of microarray analysis. J. Bacteriol. 186: 5672-5684. Morici, L. A., A. J. Carterson, V. E. Wagner, A. Frisk, J. R. Schurr, K. H. zu Bentrup, D. J. Hassett, B. H. Iglewski, K. Sauer & M. J. Schurr, (2007) Pseudomonas aeruginosa AlgR represses the Rhl quorum-sensing system in a biofilm-specific manner. J. Bacteriol. 189: 7752-7764.
