Processing of C. abortus Pmp18D Figure 4. Bayesian dendrogram showing evolutionary relatedness of PmpD amino acid sequences originating from the nine Chlamydia species. Accession numbers of the amino acid sequences are indicated to the right of the strain name. The posterior probability of discrete groupings is indicated by the number at the node. The 3 PmpD clusters (cluster 1: C. abortus, C. psittaci, C. caviae and C. felis; cluster 2: C. pneumoniae and C. pecorum; and cluster 3: C. trachomatis, C. suis and C. muridarum) that were identified in this study are indicated to the right of the dendrogram. doi:10.1371/journal.pone.0049190.g004 within the Chlamydiaceae, with few conserved motifs between species in processing regions. Further studies are required to elucidate the function of PmpD and investigate whether this function is conserved across the different Chlamydia spp. Supporting Information Figure S1 Western blot demonstrating the solubility of the Pmp18D N-terminal passenger domain in 2% sarkosyl (using the Mid-Pmp18D antibody). Lane 1 Whole C. abortus Elementary bodies (EBs); Lane 2 Soluble fraction after treatment of whole EBs with 2% sarkosyl; Lane 3 Soluble fraction after treatment of sarkosyl insoluble pellet with 1mM DTT/2% Sarkosyl; Lane 4 Sarkosyl insoluble material. (PDF) References 1. Longbottom D, Coulter LJ (2003) Animal chlamydioses and zoonotic implications. J Comp Pathol 128: 217–244. 2. Henderson IR, Navarro-Garcia F, Desvaux M, Fernandez RC, a’Aldeen D (2004) Type V protein secretion pathway: the autotransporter story. Microbiol Mol Biol Rev 68: 692–744. 3. Henderson IR, Lam AC (2001) Polymorphic proteins of Chlamydia spp. - autotransporters beyond the Proteobacteria. Trends Microbiol 9: 573–578. 4. Longbottom D, Russell M, Jones GE, Lainson FA, Herring AJ (1996) Identification of a multigene family coding for the 90 kda proteins of the ovine abortion subtype of Chlamydia psittaci. FEMS Microbiol Lett 142: 277–281. 5. Longbottom D, Russell M, Dunbar SM, Jones GE, Herring AJ (1998) Molecular cloning and characterization of the genes coding for the highly immunogenic cluster of 90-kilodalton envelope proteins from the Chlamydia psittaci subtype that causes abortion in sheep. Infect Immun 66: 1317–1324. Figure S2 Peptide coverage of Pmp18D protein cleavage products as determined by LC-ESI-MS-MS and MALDI ToF MS. (PDF) Figure S3 Region alignments of predicted cleavage sites in C. abortus Pmp18D. (DOC) Author Contributions Conceived and designed the experiments: NW DGES DL. Performed the experiments: NW KA ML KM NI. Analyzed the data: NW MS FW KM NI . Contributed reagents/materials/analysis tools: FW NI ML DL. Wrote the paper: NW MS DGES DL. 6. Stephens RS, Kalman S, Lammel C, Fan J, Marathe R, Aravind L, et al. (1998) Genome sequence of an obligate intracellular pathogen of humans: Chlamydia trachomatis. Science 282: 754–759. 7. Read TD, Brunham RC, Shen C, Gill SR, Heidelberg JF, et al. (2000) Genome sequences of Chlamydia trachomatis MoPn and Chlamydia pneumoniae AR39. Nucleic Acids Res 28: 1397–1406. 8. Thomson NR, Yeats C, Bell K, Holden MT, Bentley SD, et al. (2005) The Chlamydophila abortus genome sequence reveals an array of variable proteins that contribute to interspecies variation. Genome Res 15: 629–640. 9. Read TD, Myers GS, Brunham RC, Nelson WC, Paulsen IT, et al. (2003) Genome sequence of Chlamydophila caviae (Chlamydia psittaci GPIC): examining the role of niche-specific genes in the evolution of the Chlamydiaceae. Nucleic Acids Res 31: 2134–2147. 10. Azuma Y, Hirakawa H, Yamashita A, Cai Y, Rahman MA, et al. (2006) Genome sequence of the cat pathogen, Chlamydophila felis. DNA Res 13: 15–23. PLOS ONE | www.plosone.org 6 November 2012 | Volume 7 | Issue 11 | e49190