[33–35]; however the Bvg+ phase has been shown to be necessary and sufficient for host colonization [36]. Although BvgAS appears to be sufficient for regulation of virulence factors the ability to respond to multiple signal inputs to differentially regulate transcriptional networks likely allows for adaptation to different microenvironments within the host. Bordetella species have multiple putative transcription factors within their genomes, indicating that gene regulation is likely to be a more complex regulatory system than is currently appreciated [16]. Here we identify, through screening of a collection of B. bronchiseptica isolates, strains that only produce ACT in response to growth in elevated CO2 conditions. Both strain 761 and the sequenced laboratory reference strain RB50 increased transcrip- tion of cyaA and production of ACT when grown in 5% CO2 conditions, although only strain 761 was dependent on 5% CO2 for efficient expression. Several other virulence factor genes were increased in transcription in response to growth in elevated CO2. BvgAS was required for ACT production, but cyaA and fhaB were transcriptionally increased in response to 5% CO2 conditions in the absence of BvgS. Together this indicates that an additional regulatory system increases production of ACT and other virulence factors in various Bordetella species. Materials and Methods Ethics Statement This study was carried out in strict accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health. The protocol was approved by the Institutional Animal Care and Use Committee at The Pennsylvania State University at University Park, PA (#31297 Bordetella-Host Interactions). All animals were anesthetized using isoflourane or euthanized using carbon dioxide inhalation to minimize animal suffering. Bacterial Strains and Growth B. bronchiseptica strain RB50 is an isolate from a rabbit [36]. RB54 and RB50DbscNDcyaA are previously described derivatives of strain RB50 [33,37]. B. bronchiseptica strain 761, 448, and 308 were obtained from the CDC in Atlanta, Georgia and have been previously described [15,38,39]. B. bronchiseptica strain JC100 has been previously described [15]. B. parapertussis strain 12822 was isolated from German clinical trials and has been previously described [40,41]. B. pertussis strain 536 is a streptomycin resistant derivative of Tohama I [42] and strain 18323 has been previously described [43]. B. pertussis strain CHOC 0012 was isolated on Regan-Lowe media by the Eunice Kennedy National Insitute of Child Health and Human Development (NICHD) Collaborative Pediatric Critical Care Research Network from a child displaying severe Pertussis. Bacteria were maintained on Bordet-Gengou agar (Difco, Sparks, MD) containing 10% sheep blood (Hema Resources, Aurora OR) and 20 mg/mL streptomycin (Sigma Aldrich, St. Louis, MO). Liquid cultures were grown at 37uC overnight in a shaker to mid-log phase (O.D. 0.7–1.0) in Stainer- Scholte (SS) broth. Bacteria were grown overnight with constant shaking (250 rpm) in standard glass test tubes in either atmospheric concentrations of oxygen and carbon dioxide (atmospheric conditions) or in atmospheric levels of oxygen with the constant controlled addition of 5% carbon dioxide into a sealed incubator 37uC (5% CO2 conditions). cAMP Assay Murine macrophage-like cell line, J774, was cultured in Dulbecco modified Eagle medium (DMEM) with 10% fetal CO2 Responsive Regulon in Bordetella bovine serum (FBS) (Hyclone Laboratories, Inc., Logan, UT). Cells were grown to approximately 80% confluency, and bacteria were added at a multiplicity of infection (MOI) of 1. After a 5 minute centrifugation at 2506g, the mixture was incubated for 30 minutes at 37uC. cAMP was measured with a cyclic AMP ELISA system (Tropix, Bedford, MA) according to the manufacturer’s instructions. Results were analyzed using analysis of variance with a Tukey simultaneous test, and a P value of ,0.05 was considered significant. Animal Experiments C57BL/6 mice were obtained from Jackson Laboratories (Bar Harbor, ME). Mice were bred in our Bordetella-free, specific pathogen-free breeding rooms at The Pennsylvania State Univer- sity. All animal experiments were performed in accordance with institutional animal care and use committee (IACUC) guidelines. 4 to 6 week old mice were lightly sedated with 5% isoflurane (IsoFlo, Abbott Laboratories) in oxygen and 56105 CFU were pipetted in 50 ul of phosphate-buffered saline (PBS) (Omnipur, Gibbstown, NJ) onto the external nares. This method reliably distributes the bacteria throughout the respiratory tract [43]. To obtain serum, blood from inoculated or vaccinated mice was obtained 28 days post-inoculation and serum was separated from the blood by centrifugation at 5006g for 5 minutes. Western Immunoblots Western blots were performed on whole cell extracts of B. bronchiseptica, B. pertussis and B. parapertussis grown to mid-log phase in SS broth as described previously [38,39]. Lysates were prepared by resuspending 16109 CFU in 100 ml of Laemmli sample buffer; total cellular protein content were quantitated using the BCA assay to equalize protein content between samples. 16108 CFU (10 ml) were run on an 8% sodium dodecyl sulfate-polyacrylamide electrophoresis gels in denaturing conditions and transferred to a polyvinylidene difluoride membrane (Millipore, Bedford, MA). Membranes were probed with pooled serum from mice inoculated with B. bronchiseptica, B. pertussis, B. parapertussis, or a monoclonal antibody against ACT (anti-ACT) at the following dilutions, 1:1000, 1:500, 1:1000 and 1:1000, respectively. A 1:10,000 dilution of goat anti-mouse Ig HRP conjugated antibody (Southern Biotech, Birmingham, AL) was used as the detector antibody. Membranes were visualized with ECL Western blotting detection reagents (Amersham Biosciences, Piscataway, NJ) and quantified using Image J software [44]. RNA Isolation RNA was isolated from three independent biological replicates of B. bronchiseptica strains RB50, 761, RB54, 536 and 12822 grown in SS broth overnight. Bacteria were subcultured at a starting OD600 of 0.1 into 5 ml of SS broth and grown at 37uC while shaking in either atmospheric or 5% CO2 conditions until the OD600 reached 0.75. Bacteria were harvested and total RNA was extracted using a RNAeasy Kit (Qiagen, Valencia, CA) and treated with RNase-free DNase I (Invitrogen, Carlsbad, CA) according to the manufacturer’s instructions. Preparation of Labeled cDNA and Microarray Analysis RNA isolated from strains RB50 and 761 were used in microarray experiments. A 2-color hybridization format was used for the microarray analysis. For each biological replicate, RNA extracted from cells grown in 5% CO2 conditions was used to generate Cy5-labeled cDNA and RNA extracted from cells grown in atmospheric conditions was used to generate PLOS ONE | www.plosone.org 2 October 2012 | Volume 7 | Issue 10 | e47635