IS481-insertion mutant: a 3.2-kbp DNA fragment (bteA+IS481) containing the bteA 59-UTR and IS481a was amplified with the B1-bteA and B2-bteA primers (Table S1) using B. pertussis BP155 genomic DNA as the template. pABB-bteA+IS481 was constructed from pDONR-bteA+IS481 and transconjugated into SM-resistant B. pertussis nonvaccine-type BP157 via E. coli SM10lpir. The resultant mutant strain was designated +IS481-BP157. Statistical analysis The Student’s t-test was employed. A value of P,0.05 was considered statistically significant. Nucleotide sequence accession number The IS481a sequence was deposited in the DDBJ/EMBL/ GenBank nucleotide sequence databases under accession number AB473880. Supporting Information Figure S1 High secretion of BteA protein in Bordetella bronchiseptica. B. bronchiseptica (BB R05), B. pertussis BP155 (vaccine-type) and BP157 (nonvaccine-type) were cultured in modified SS medium for 24 h. Total protein extracted from the bacterial cells (Cell) and culture supernatants (CS) was separated by SDS-PAGE followed by silver staining (left panel). Immuno- blots were incubated with anti-BteA, anti-BtcA or anti-BopD antiserum (right panel). For BteA detection, 0.5 mg of total protein (for Cell) and 5 ml of CS were loaded in the indicated lanes. The amount of total protein loaded was one-twentieth of that in Figure 2, and the loaded CS volume was one-fortieth of that in Figure 3. (TIF) References 1. Mattoo S, Cherry JD (2005) Molecular pathogenesis, epidemiology, and clinical manifestations of respiratory infections due to Bordetella pertussis and other Bordetella subspecies. Clin Microbiol Rev 18: 326–382. 2. Andrews R, Herceg A, Roberts C (1997) Pertussis notifications in Australia, 1991 to 1997. Commun Dis Intell 21: 145–148. 3. de Melker HE, Schellekens JF, Neppelenbroek SE, Mooi FR, Ru¨mke HC, et al. (2000) Reemergence of pertussis in the highly vaccinated population of the Netherlands: observations on surveillance data. Emerg Infect Dis 6: 348–357. 4. Hellenbrand W, Beier D, Jensen E, Littmann M, Meyer C, et al. (2009) The epidemiology of pertussis in Germany: past and present. BMC Infect Dis 9: 22. 5. Tanaka M, Vitek CR, Pascual FB, Bisgard KM, Tate JE, et al. (2003) Trends in pertussis among infants in the United States, 1980-1999. JAMA 290: 2968–2975. 6. Bottero D, Gaillard ME, Fingermann M, Weltman G, Ferna´ndez J, et al. (2007) Pulsed-field gene electrophoresis, pertactin, pertussis toxin S1 subunit polymor- phisms, and surfaceome analysis of vaccine and clinical Bordetella pertussis strains. Clin Vaccine Immunol 14: 1490–1498. 7. Fry NK, Neal S, Harrison TG, Miller E, Matthews R, et al. (2001) Genotypic variation in the Bordetella pertussis virulence factors pertactin and pertussis toxin in historical and recent clinical isolates in the United Kingdom. Infect Immun 69: 5520–5528. 8. Kodama A, Kamachi K, Horiuchi Y, Konda T, Arakawa Y (2004) Antigenic divergence suggested by correlation between antigenic variation and pulsed-field gel electrophoresis profiles of Bordetella pertussis isolates in Japan. J Clin Microbiol 42: 5453–5457. 9. Litt DJ, Neal SE, Fry NK (2009) Changes in genetic diversity of the Bordetella pertussis population in the United Kingdom between 1920 and 2006 reflect vaccination coverage and emergence of a single dominant clonal type. J Clin Microbiol 47: 680–688. 10. Tsang RSW, Lau AKH, Sill ML, Halperin SA, Van Caeseele P, et al. (2004) Polymorphisms of the fimbria fim3 gene of Bordetella pertussis strains isolated in Canada. J Clin Microbiol 42: 5364–5367. 11. van Amersfoorth SCM, Schouls LM, van der Heide HGJ, Advani A, Hallander HO, et al. (2005) Analysis of Bordetella pertussis populations in European countries with different vaccination policies. J Clin Microbiol 43: 2834–2843. 12. Borisova O, Kombarova SY, Zakharova NS, van Gent M, Aleshkin VA, et al. (2007) Antigenic divergence between Bordetella pertussis clinical isolates from the Moscow, Russia, and vaccine strains. Clin Vaccine Immunol 14: 234–238. Differential Expression of BteA in B. Pertussis Figure S2 In vitro transcription-translation analysis of a bteA 59-UTR deletion series. (A) bteA 59-UTR deletion genes were PCR-amplified using B. pertussis BP155 (vaccine-type) as the template. Proteins were synthesized using the WakoPURE System (Wako Pure Chemical Industries, Ltd.). The 59-UTR deletion genes harbored the T7 promoter at their 59 end. (B) Expression of BteA protein in an in vitro transcription-translation system (WakoPURE System). The synthesized product was analyzed with immunoblots using anti-BteA antiserum. NC, negative control. (C) A predicted stem-loop structure in the 59-UTR of bteA mRNA (P2 transcript). The RNA secondary structure was analyzed by CentroidFold (http://www.ncrna.org/centroidfold). The schematic shows a simplified map. TIR, translation initiation region. (TIF) Primers and probes in this study. Table S1 (XLS) Acknowledgments We would like to thank Yuko Sasaki for her assistance with the LC-MS/ MS analysis and Jun-ichi Wachino for his technical advice in 59-RACE mapping. We also thank Mineo Watanabe (Kitasato University) for his kind gift of recombinant ACT protein. Author Contributions Conceived and designed the experiments: H-JH KK. Performed the experiments: H-JH KK. Analyzed the data: H-JH KK. Contributed reagents/materials/analysis tools: AK AA YA. Wrote the paper: KK. 13. Cassiday P, Sanden G, Heuvelman K, Mooi F, Bisgard KM, et al. (2000) Polymorphism in Bordetella pertussis pertactin and pertussis toxin virulence factors in the United States, 1935-1999. J Infect Dis 182: 1402–1408. 14. He Q, Ma¨kinen J, Berbers G, Mooi FR, Viljanen MK, et al. 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Yuk MH, Harvill ET, Miller JF (1998) The BvgAS virulence control system regulates type III secretion in Bordetella bronchiseptica. Mol Microbiol 28: 945–959. 20. Coburn B, Sekirov I, Finlay BB (2007) Type III secretion system and disease. Clin Microbiol Rev 20: 535–549. 21. Ghosh P (2004) Process of protein transport by the type III secretion system. Microbiol Mol Biol Rev 68: 771–795. 22. Fennelly NK, Sisti F, Higgins SC, Ross PJ, van der Heide H, et al. (2008) Bordetella pertussis expresses a functional type III secretion system that subverts protective innate and adaptive immune responses. Infect Immun 76: 1257–1266. 23. Nagamatsu K, Kuwae A, Konaka T, Nagai S, Yoshida S, et al. (2009) Bordetella evades the host immune system by inducing IL-10 through a type III effector, BopN. J Exp Med 206: 3073–3088. 24. Medhekar B, Shrivastava R, Mattoo S, Gingery M, Miller JF (2009) Bordetella Bsp22 forms a filamentous type III secretion system tip complex and is immunoprotective in vitro and in vivo. Mol Microbiol 71: 492–504. 25. Caro V, Bouchez V, Guiso N (2008) Is the sequenced Bordetella pertussis strain Tohama I representative of the species? J Clin Microbiol 46: 2125–2128. 26. Heikkinen E, Kallonen T, Saarinen L, Sara R, King AJ, et al. (2007) Comparative genomics of Bordetella pertussis reveals progressive gene loss in Finnish strains. PLoS ONE 2: e904. 27. King AJ, van Gorkom T, Pennings JL, van der Heide HG, He Q, et al. (2008) Comparative genomic profiling of Dutch clinical Bordetella pertussis isolates using PLoS ONE | www.plosone.org 9 March 2011 | Volume 6 | Issue 3 | e17797