CO2 Responsive Regulon in Bordetella Figure 4. Duplication in the bvgS gene in strain JC100. The DNA and protein sequences of bvgS gene from JC100 are aligned against those of bvgS gene from RB50 and 761. The red line indicates the 29 amino acid duplication in the bvgS locus in JC100 compared to RB50 and 761. doi:10.1371/journal.pone.0047635.g004 BvgAS. The CO2-responsive regulon appears to contain genes that are Bvg-regulated, as well as genes that are not, suggesting a regulatory mechanism that functions independently or coopera- tively with BvgAS rather than subordinate to it. CO2 Responsiveness is Not Conferred by Differences in the bvgAS Loci between Strains Three additional B. bronchiseptica strains, JC100, 308 and 448, were also observed to be hemolytic only when grown in 5% CO2, but not in normal atmospheric conditions. Changes in virulence factor expression have previously been attributed to variation in bvgAS and since some CO2-responsive genes are Bvg-regulated, the bvg locus of these strains was analyzed revealing that strain JC100 carries a 29 amino acid duplication in the region of the bvgS gene encoding the periplasmic domain (Fig. 4). To determine if this duplication is involved in the CO2/ACT dependent phenotype in JC100, the bvgAS locus from JC100 was expressed in a RB50 knockout of bvgAS (RB55::pBvgASJC100). This strain was hemolytic in the absence of 5% CO2, indicating that transfer of the bvgAS locus does not confer the CO2-dependence for ACT production (Table S5). The reverse was also true; when a plasmid carrying the bvgAS locus from RB50 was introduced into JC100 (MLJC114::pEG100), ACT production remained dependent on growth in 5% CO2 (Table S5). Furthermore, the bvgS gene of 761 did not have this duplication (Fig. 4). These data indicate that the duplication in bvgS in JC100 is neither necessary nor sufficient for the CO2 requirement for hemolysis. CO2 Responsiveness in the B. bronchiseptica Bvg2 State Since some virulence genes known to be Bvg-regulated were responsive to 5% CO2 conditions, we sought to determine if they are differentially regulated in response to 5% CO2 in the absence of BvgAS. Transcription of six genes responsive to CO2, cyaA, fhaB, bopD, bopB, cheZ and flgB, were analyzed in B. bronchiseptica RB50 and RB54, a Bvg–phase locked derivative of strain RB50, grown in normal atmospheric or 5% CO2 conditions (Fig. 5). For RB50, addition of 5% CO2 increased transcription of cyaA, fhaB, bopD and bopB (Fig. 5A–D), but decreased transcription of cheZ and flgB (Fig. 5 E,F). In RB54, transcription of bopD and bopB was not increased in response to addition of 5% CO2 (Fig. 5C,D), and transcription of cheZ and flgB was not decreased (Fig. 5 E,F). Therefore, the differential transcription of bopD, bopB, cheZ and flgB in response to 5% CO2 is dependent on BvgS. However, in the bvgS mutant RB54, the transcription of genes cyaA and fhaB was increased in response to addition of 5% CO2 (Fig. 5 A,B), indicating that some gene regulation in response to 5% CO2 is independent of BvgS. To determine whether differential transcription results in differential accumulation of antigens in the absence of BvgS, Western blots were performed. Lysates from RB54 grown in normal atmospheric or 5% CO2 conditions were probed with serum antibodies from mice convalescent from RB50 infection PLOS ONE | www.plosone.org 6 October 2012 | Volume 7 | Issue 10 | e47635