Antibody production against recombinant BteA, BtcA and ACT The BteA gene (NCBI accession: NP_879352) was amplified by PCR from B. pertussis Tohama DNA using BteA-F and BteA-R primers, and cloned into the XmnI/HindIII sites of pMal-c2X (New England Biolabs, Beverly, MA) to generate a maltose binding protein (MBP) fusion with BteA. Production of this fusion protein was induced in E. coli BL21 with 0.5 mM isopropyl-b-D- thiogalactopyranoside (IPTG) and subsequently purified using amylose resin (New England Biolabs) and Resource Q (Amersham Pharmacia Biotech, Uppsala, Sweden) columns. A two-step PCR was carried out to amplify recombinant BtcA (NCBI accession: NP_879351). The first PCR was performed using the BtcA-BteA- F3 and BtcA-BteA-R3 primers (Table S1), which amplified the region between positions 165122 and 167190 of the B. pertussis Tohama genome (GenBank accession: BX640412). In the second PCR, btcA was amplified from the first PCR product with the 5- BtcA and 3-BtcA primers (Table S1) and cloned into the NdeI/ HindIII sites of pCold II DNA (TAKARA Bio Inc.). His-tagged BtcA was induced in E. coli BL21 with 0.5 mM IPTG at 15uC and purified using the HisTrap FF Crude Kit (GE Healthcare UK Ltd.). A recombinant catalytic domain of B. pertussis adenylate cyclase toxin (ACT) was a gift from Mineo Watanabe. Antibodies against MBP-BteA, BtcA and ACT were generated in mice at Nippon Biotest Laboratories, Inc. (Tokyo, Japan). The MBP-BteA antiserum was pre-absorbed with MBP2 protein (New England BioLabs) and the resulting antiserum was used. Immunoblot analysis B. pertussis clinical strains were inoculated in modified SS medium with a starting optical density of 0.2 at 600 nm, and further cultured with shaking at 36uC. Total protein was extracted with SDS-lysis buffer, and culture supernatant (CS) proteins were precipitated with 10% TCA. Protein samples were subjected to SDS-PAGE, transferred to nitrocellulose membranes (Bio-Rad) and incubated with anti-BteA, anti-BtcA, anti-BopD [32], anti- ACT, anti-FHA, or anti-PT antiserum. Antigen-antibody com- plexes were visualized using horseradish peroxidase (HRP)- conjugated secondary antibody (Bio-Rad, Hercules, CA) and ECL Western Blotting Detection Reagents (GE Healthcare). DNA sequencing The region between the btcA and bteA gene corresponding to positions 165122 to 168021 of B. pertussis Tohama (GenBank accession: BX640412) was amplified in vaccine-type and non- vaccine-type clinical strains with the appropriate primers and sequenced. Sequencing reactions were carried out with the BigDye Terminator v3.1 Cycle Sequencing Kit (Applied Biosystems, Foster City, CA), and the products were sequenced on an ABI PRISM 3130xl Genetic Analyzer (Applied Biosystems). Transcriptional analyses Total RNA was isolated using the RNeasy Mini Kit (QIAGEN, Hilden, Germany) and treated with RNase-free DNase (Promega) to degrade contaminating DNA. Reverse transcriptase-PCR (RT- PCR) was performed with bteA RT-R and btcA RT-R primers (Table S1) using the TAKARA One Step RNA PCR Kit (AMV, TAKARA Bio Inc.). PCR was performed with the following conditions: one cycle of 50uC for 30 min, 95uC for 2 min; 25 cycles of 95uC for 30 s, 58uC for 30 s, 72uC for 1 min; and a final incubation at 72uC for 10 min. Primer sets, bteA RT-F/bteA RT- R and btcA RT-F/btcA RT-R, were used for bteA and btcA amplification, respectively (Table S1). Products were analyzed by Differential Expression of BteA in B. Pertussis electrophoresis on a 1.5% agarose gel. Reverse transcriptase was omitted from the negative control reaction mixtures. For quantitative RT-PCR (qRT-PCR), 5 mg of RNA was reverse transcribed into cDNA using the SuperScript First-Strand Synthesis System (Invitrogen, Carlsbad, CA) with random hexamer primers. Relative levels of total bteA and recA transcripts were determined using TaqMan probes (bteA- and recA-probes, Table S1) and Premix Ex TaqTM (Perfect Real Time, TAKARA Bio Inc.) with the ABI PRISM 7500 Sequence Detection System (Applied Biosystems). The qRT-PCR conditions were 30 s at 95uC, followed by 40 cycles of 95uC for 15 s and 60uC for 1 min. The expression of recA was used as an internal control [45]. All samples were run in triplicate and bteA transcript (P1 + P2 transcripts) was normalized to the recA transcript for each sample. The bteA IS481a-promoter transcript (P2 transcript) was deter- mined using a TaqMan probe (IS481-bteA probe). The qRT-PCR conditions were 30 s at 95uC, followed by 40 cycles of 95uC for 15 s and 55uC for 1 min. The ratio of P2 transcript to total bteA transcript (P2 transcript/P1 + P2 transcripts) was estimated from four independent experiments. The regions amplified by qRT- PCR are shown in Figure 6A. Mapping transcriptional start sites 59 rapid amplification of cDNA ends (59-RACE) was performed using 59-Full RACE Core Set (TAKARA Bio Inc.) according to the manufacturer’s instructions. Reverse transcription was execut- ed at 55uC using a 59 phosphorylated RT primer (bteA-RT, Table S1). The first PCR used primers bteA-S1 (S1) and bteA-A1 (A1) primers, and bteA-S2 (S2) and bteA-A2 (A2) for the second (Table S1). PCR products were cloned into the pT7Blue T-vector (Novagen, Madison, Wis.) and transformed into E. coli XL1-Blue, which were plated on LB agar plates. Several clones were sequenced. The transcription start site of btcA was located using 59- RACE with five primers, btcA-RT (59 phosphorylated primer), btcA-S1 (S1), btcA-A1 (A1), btcA-S2 (S2) and btcA-A2 (A2) (Table S1). Generation of BteA mutants Four BteA mutants, DbteA-BP155, DbteA-BP157, DIS481-BP155 and +IS1481-BP157, were constructed by homologous recombi- nation as described previously with minor modifications [30] (Figure 6A). BteA-deficient mutants: A 2.2-kbp DNA fragment containing a 59 portion of the bteA gene was amplified by PCR with the B1-bteA and B2-bteA primers (Table S1) using the B. pertussis Tohama genomic DNA as the template. The PCR product was cloned into the pDONR221 vector (Invitrogen) to obtain pDONR-bteA by means of adaptor PCR and site-specific recombination techniques with the Gateway Cloning System (Invitrogen). Inverse PCR was then carried out with R1-bteA and R2-bteA primers (Table S1) using circular pDONR-bteA as the template. The R1-bteA and R2-bteA primers contained a BamHI site. The resulting PCR product was digested with BamHI and self-ligated to obtain pDONR-DbteA, which contained a 178-bp deletion around the 59 region of bteA. pDONR-DbteA was mixed with pABB-CRS2 [46] to obtain pABB-DbteA using the Gateway Cloning System. pABB- DbteA was then introduced into E. coli SM10lpir and transconju- gated into streptomycin (SM)-resistant B. pertussis BP155 (vaccine- type) and BP157 (nonvaccine-type) clinical strains. The resultant mutant strains were designated DbteA-BP155 and DbteA-BP157. IS481-deletion mutant: pABB-bteA was constructed from pDONR- bteA. pABB-bteA was introduced into E. coli SM10lpir and transconjugated into SM-resistant B. pertussis vaccine-type BP155. The resultant mutant strain was designated DIS481-BP155. PLoS ONE | www.plosone.org 8 March 2011 | Volume 6 | Issue 3 | e17797