Figure 3. Defining the CO2 responsive regulon in B. bronchi- septica. Changes in gene expression of B. bronchiseptica in response to 5% CO2 are analyzed by MeV analysis [48]. Several known virulence factor genes reported to be regulated by BvgAS in prototypical B. bronchiseptica strain RB50 are shown for strain RB50 (left) and strain 761 (right), with yellow representing increased transcription and blue indicative of decreased transcription in growth in 5% CO2 conditions, compared to growth in normal atmospheric conditions. doi:10.1371/journal.pone.0047635.g003 affected by additiona of 5% CO2 (Figure S1.), although other indirect effects are possible. To more directly assess the production of ACT, lysates of strains RB50 and 761 grown in liquid cultures (mid-log phase, O.D. 0.7– 0.9) in normal atmospheric conditions or 5% CO2 conditions were probed with a monoclonal antibody to the cyaA protein product, ACT. RB50 produced more ACT when grown in 5% CO2 CO2 Responsive Regulon in Bordetella conditions than in normal atmospheric conditions (Fig. 1E). Strain761 grown in normal atmospheric conditions produced no detectable ACT, while 761 in 5% CO2 conditions did produce ACT (Fig. 1E). To determine if strain 761 produces a functional ACT, cyclic-AMP (cAMP) was measured in murine macrophages stimulated with bacteria grown in either normal or 5% CO2 conditions. Cells were stimulated for 30 minutes with strains RB50 or 761 to assess their effects on cAMP levels. Both strains grown in 5% CO2 induced significantly more cAMP than the same strains grown in normal atmospheric conditions (Fig. 1F, G). Together, these data demonstrate by different measures that the prototypical B. bronchiseptica strain, RB50, and strain 761 increase production of functional ACT when grown in 5% CO2 conditions, but only strain 761 appears to be dependent on 5% CO2 conditions for production of ACT. Since ACT production was increased in 5% CO2, we hypothesized that other antigens might also be differentially regulated in response to these conditions. To test this, Western blot analysis was performed by probing RB50 and 761 lysates, grown in ambient air or in 5% CO2 conditions, with serum antibodies from animals convalescent from RB50 infection. No antigens appeared to be produced in greater amounts in RB50 grown in ambient air, while strain 761 produced antigens of between 72 and 95 kDa in greater amounts in 5% CO2 growth conditions (Fig. 2, arrowheads). Antigens greater than 130 kDa were produced in greater amounts when RB50 was grown in 5% CO2 conditions compared to ambient air (Fig. 2, arrows). Additionally, both 761 and RB50 produced antigens between 36 and 55 kDa in greater amounts when grown in elevated CO2 concentrations than in ambient air (Fig. 2). These data indicate that additional antigens besides ACT are differentially regulated in response to 5% CO2. Defining a CO2 Responsive Regulon in B. bronchiseptica To determine which genes are differentially regulated in response to different CO2 concentrations, microarray analyses were performed comparing RB50 grown in atmospheric concen- trations of CO2 to growth in elevated CO2 conditions. Transcript abundance of 35 genes increased in RB50 in response to 5% CO2, based on SAM analysis (Table S1, Fig. 3), including genes encoding ACT, as well as genes encoding other known virulence factors such as members of the TTSS locus (bscE, bscF, bopD), FHA (fhaC, fhaB, fhaD, fhaA), fimbriae (fimA, fim3), and Prn, (Fig. 3). Expression of 452 genes were decreased when RB50 was grown in 5% CO2, many of which are known to be expressed in the Bvg2 phase including, flaA, cheW, cheB, wbmD, flgH, fliS, and cheD (Fig. 3). A similar trend was observed for strain 761; genes encoding known virulence factors were increased in 5% CO2 growth conditions while genes for flagellar assembly and chemotaxis were decreased in these conditions (Table S2, Fig. 3). Expression of 6 genes, including cyaA, was increased in both strains and expression of 41 genes decreased in both strains when grown in 5% CO2 conditions (Table S4). qPCR of 12 genes confirmed the microarray results (Table S3). Overall, these data indicate there is a CO2 responsive regulon in B. bronchiseptica that includes several virulence factors, suggesting a role during infection. Among the 35 genes in strain RB50 increased in expression in 5% CO2 conditions, 19 genes were reported to be positively regulated under Bvg+ conditions, 4 genes negatively regulated by BvgAS, and 13 genes not previously known to be regulated by BvgAS, based on previous analysis [46,51]. Similarly, of the 452 genes negatively regulated by 5% CO2, 252 were known to be negatively regulated under Bvg+ conditions, 19 were positively regulated and 181 were not previously known to be regulated by PLOS ONE | www.plosone.org 5 October 2012 | Volume 7 | Issue 10 | e47635