Vp-PAI Gene Expression Figure 3. Genome-wide transcriptional analysis of crude bile-responsive genes in V. parahaemolyticus. A. Gene expression was determined by comparing cDNA generated from WT (blue line) or a vtrA mutant strain (red line) grown in the presence of 0.04% crude bile with that generated from the WT grown in the absence of crude bile. Data are expressed as the average of triplicate experiments. The Vp-PAI (VPA1310- VPA1369) region on chromosome 2 is indicated by a bold line. Gray dotted lines indicate the boundaries for a fourfold change and the identities of genes that were significantly upregulated or downregulated ($ fourfold difference, P,0.05) in response to crude bile in the WT strain are indicated. B. Hierarchical cluster plot showing the relative expression of V. parahaemolyticus genes that were upregulated or downregulated in the presence of crude bile. Green indicates repression and red indicates induction of at least fourfold relative to the untreated samples. The scale bar indicates the mean of the log ratio. Gene identities are listed on the right. doi:10.1371/journal.pone.0013365.g003 results suggest that bile acids are essential for inducing transcription of Vp-PAI genes. We then examined the ability of nine individual bile salts that are present in the intestinal tract to induce vtrB expression. The results (Fig. 4C) revealed that these bile salts could be classed into three groups with respect to their transcription-inducing activity: those with high inducing activity (taurodeoxycholate, TDC; and glycodeoxycholate, GDC), those with intermediate inducing activity (deoxycholate, DC; tauroche- nodeoxycholate, TCDC; glycochenodeoxycholate, GCDC; tau- rocholate, TCA; and glycocholate, GCA) and those lacking inducing activity (chenodeoxycholate, CDC; and cholate, CA). These results were confirmed by immunoblotting analysis of bile salts that had the highest level of VtrB induction and those that had no VtrB-inducing activity (Fig. 4D). In addition, the extent to which individual bile salts induced vtrB expression was strongly correlated with the level of production of TDH and T3SS2- related proteins; TDC and GDC were the strongest inducers, whereas CDC and CA lacked inducing activity (Fig. S3A and B). Therefore, these four bile salts were used in subsequent experiments. To confirm that the loss of the ability of cholestyramine-treated crude bile to induce Vp-PAI genes was caused by depletion of bile acids, we examined the effect of addition of supplementary bile salts to cholestyramine-treated crude bile on vtrB-lacZ transcriptional activity (Fig. 4E). The diminished ability of cholestyramine-treated crude bile to induce vtrB-lacZ transcription was fully restored not only by addition of crude bile but also by the addition of TDC or GDC, whereas neither CDC nor CA addition overcame the loss of induction activity. Similar results were obtained for VtrB protein immuno- blotting: production of VtrB was induced only when crude bile, TDC or CA were added to cholestyramine-treated crude bile (Fig. 4F). These results suggest that bile acids in crude bile, especially TDC and GDC, are major transcription-inducing substances for Vp-PAI genes. PLoS ONE | www.plosone.org 5 October 2010 | Volume 5 | Issue 10 | e13365