Differential Expression of BteA in B. Pertussis Figure 5. Physical maps of the btcA2bteA region of B. pertussis vaccine-type and nonvaccine-type strains. (A) The vaccine-type clinical strain BP155. The location of IS481a is represented by a gray arrow on the physical map. The recognition sequence of IS481a is underlined. The two mapped transcriptional start sites (P1 and P2) of bteA are shown by arrows. Region amplified by qRT-PCR to determine the IS481a-promoter (P2) and total (P1 + P2) transcripts are shown by two-headed arrows below the physical map. (B) The nonvaccine-type clinical strain BP159. The mapped transcriptional start sites of bteA (P1) and btcA [P (btcA)] are shown by arrows. The single nucleotide polymorphism (ARG) at 2207 bp from the bteA translation start codon is indicated by an asterisk. doi:10.1371/journal.pone.0017797.g005 IS481 belongs to the recently defined IS481 family [38], and 238 copies of IS481 are present in the B. pertussis Tohama genome [39]. In B. pertussis clinical strains, IS481 is also present in multiple copies on the chromosome and it plays a critical role in B. pertussis evolution [26,40]. Many IS elements have been shown to activate the expression of neighboring genes. IS481 contains an outward- facing promoter that is located in close proximity to the left terminal inverted repeat, and this promoter is responsible for the transcription of katA in certain B. pertussis strains [34]. Here we identified an IS481a insertion in the bteA 59-UTR in B. pertussis vaccine-type clinical strains and detected a high level of bteA transcripts from the IS481a promoter (P2) compared with its own promoter (P1). However, the vaccine-type strains showed a low level of BteA protein expression, suggesting that insertion of IS481a represses P1 promoter activity, and that P2 transcript has a low translational efficiency from the additional nucleotide sequence (79 nucleotides) at its 59 end. Use of a cell-free coupled transcription-translation system revealed that the additional nucleotide sequence is involved in down-regulation of transcrip- tion and/or translation (Figure S2). The 59-UTR of bacterial mRNAs can bear regulatory elements that are involved in down- or up-regulation of translation [41]. The regulatory mechanisms in this region are controlled by RNA-binding proteins, small noncoding RNAs and structural rearrangements with the 59- UTR. In addition, a 59 stem-loop structure that sequesters the ribosomal binding site has been shown to be involved in translational regulation. Bioinformatic analysis uncovered a predicted stem-loop structure in the bteA 59-UTR of P2 transcript (Figure S2). In this study, the 59-UTRs of five B. pertussis vaccine-type clinical strains were sequenced; all had an insertion of an IS481a in the bteA 59-UTR, both transcribed in the same direction. In one of the vaccine-type strains, BP155, the major bteA mRNA was tran- scribed from P2 in the IS481a-promoter. These observations raise the possibilities that (i) the P2 transcript is translated into BteA under certain environmental conditions, and (ii) the P2 transcript is translated into another novel protein by translational frame- shifting. BteA is known to be regulated by the BvgAS system and an extracytoplasmic function (ECF) sigma factor BtrS in B. bronchiseptica [18,29]. In B. pertussis, it has been suggested that expression of the T3SS translocon Bsp22 is blocked by post- transcriptional regulation [18]. However, the molecular details of the regulatory mechanism are still unclear. Further studies are needed to determine the down-regulation of BteA protein in B. pertussis vaccine-type clinical strains. In conclusion, B. pertussis vaccine-type strains have been replaced with the nonvaccine-type strains in many countries, and the resurgence of pertussis has been observed in several PLoS ONE | www.plosone.org 6 March 2011 | Volume 6 | Issue 3 | e17797