CO2 Responsive Regulon in Bordetella Figure 8. Differential expression of virulence factor genes in B. pertussis and B. parapertussis in response to 5% CO2 conditions. qRT- PCR analysis was performed on B. pertussis (A) and B. parapertussis (B) grown in atmospheric or elevated CO2 conditions. Fold-change expression (FCE) in B. pertussis and B. parapertussis grown in 5% CO2 was compared to expression in atmospheric levels of CO2 and expressed as the mean 6 standard deviation. Lysates from bacteria grown in either atmospheric conditions or 5% CO2 conditions were probed with serum from mice inoculated with either a B. pertussis strain 536 (C) or B. parapertussis strain 12822 (D). The ratio of band intensity between antigens produced in ambient air and 5% CO2 conditions is indicated in the margins: 1 = equal amounts produced in either conditions, ,1 = more produced in ambient air, .1 = more produced in 5% CO2 conditions. doi:10.1371/journal.pone.0047635.g008 the ability to respond appears to be conserved the regulon may vary between species and strains. Collectively these data demonstrate that a CO2 response mechanism contributes to regulation of virulence factors in the classical bordetellae (Fig. 5A, B). This is the first description of a CO2 sensing mechanism that regulates virulence factor expression cooperatively with, or independently of, BvgAS. Our data support the idea that virulence factor gene expression can be fine-tuned in response to signals specific to different microenvironments within the respiratory tract or deeper tissues within the host. Supporting Information Figure S1 Growth and pH of B. bronchiseptica strains RB50 and 761 in ambient air or 5% CO2 conditions. The growth of strains RB50 (diamonds) and 761(squares) grown in normal atmospheric oxygen conditions (black) or in elevated 5% CO2 conditions (white) was measured. pH was assessed at the indicated timepoints expressed as the mean 6 standard deviation. (PDF) Table S1 (XLS) Table S2 (XLSX) B. bronchiseptica strain RB50 Expression Arrays 1 & 2. B. bronchiseptica strain 761 Expression Arrays 1 & 2. Table S3 qRT-PCR Data and Primers. (XLSX) PLOS ONE | www.plosone.org 10 October 2012 | Volume 7 | Issue 10 | e47635