CO2 Responsive Regulon in Bordetella Figure 1. Differential production of ACT in B. bronchiseptica strains grown in 5% CO2. Strains RB50 (A, C) and 761 (B, D), grown in normal atmospheric oxygen conditions (A,B) or in elevated 5% CO2 conditions (C, D). (E) RB50 and 761 grown in atmospheric conditions or grown in 5% CO2 conditions, or recombinant ACT (2.5 ng) were probed with a monoclonal antibody to CyaA protein at a dilution of 1:1000. J774 murine macrophage cells were stimulated with media or media containing RB50 (F) or 761 (G) at an MOI of 1 for 30 minutes, and cAMP levels were assessed. * indicates a p-value less than 0.05. doi:10.1371/journal.pone.0047635.g001 Cy3-labeled cDNA. Additionally, dye-swap experiments were performed analogously, in which the fluorescent labels were exchanged to ensure that uneven incorporation did not confound our results. Fluorescently-labeled cDNA copies of the total RNA pool were prepared by direct incorporation of fluorescent nucleotide analogs during a first-strand reverse transcription (RT) reaction [39,45–47]. The two differentially labeled reactions were then combined and directly hybridized to a B. bronchiseptica strain RB50-specific long-oligonucleotide microarray [46]. Slides were then scanned using a GenePix 4000B microarray scanner and analyzed with GenePix Pro software (Axon Instruments, Union City, CA). Spots were assessed visually to identify those of low quality and arrays were normalized so that the median of ratio across each array was equal to 1.0. Spots of low quality were identified and were filtered out prior to analysis. Ratio data from the two biological replicates were compiled and normalized based on the total Cy3% intensity and Cy5% intensity to eliminate slide to slide variation. Gene expression data were then normalized to 16S rRNA. The statistical significance of the gene expression changes observed was assessed by using the significant analysis of microarrays (SAM) program [48]. A one-class unpaired SAM analysis using a false discovery rate of 0.001% was performed. Hierarchical clustering of microarray data using Euclidean Distance metrics and Average Linkage clustering was performed using MeV software from TIGR [49]. All microarray data are available in Tables S1 and S2 and have been deposited in ArrayExpress or ArrayExpress Archive under accession number E-MEXP-2875. Real-time qPCR (qPCR) qPCR was performed using a modified protocol previously described [46,47]. RNA was extracted as described, and 1 mg of RNA from each biological replicate was reverse transcribed using ImProm-II Reverse transcriptase and 0.5 mg of random oligonu- cleotide hexamers (Promega, Madison, WI). cDNA was diluted 1:1,000 and 1 ml was used in RT-qPCRs containing 300 nM primers designed with Primer Express software (Applied Biosys- tems, Foster City, CA, and Integrated DNA Technologies software, www.idtdna.com) (Primer sequences are listed in Table S3) and SYBR Green PCR master mix (Invitrogen, Carlsbad, CA). Samples without reverse transcriptase were included to confirm lack of DNA contamination and dissociation curve analysis was performed to determine cycle threshold (CT) for each reaction. Amplification of the recA RNA amplicon was used as an internal control and for data normalization. Change in transcript level was determined using the relative quantitative method (DDCT) [50]. Results were analyzed using analysis of variance with a Tukey simultaneous test, and a P value of ,0.05 was considered significant. PLOS ONE | www.plosone.org 3 October 2012 | Volume 7 | Issue 10 | e47635