In recent years, considerable progress has been made in topologically and functionally characterizing integral outer membrane proteins (OMPs) of Treponema pallidum subspecies pallidum, the syphilis spirochete, and identifying its surface-exposed β-barrel domains. Extracellular loops in OMPs of Gram-negative bacteria are known to be highly variable. We examined the sequence diversity of β-barrel-encoding regions of tprC, tprD, and bamA in 31 specimens from Cali, Colombia; San Francisco, California; and the Czech Republic and compared them to allelic variants in the 41 reference genomes in the NCBI database. To establish a phylogenetic framework, we used T. pallidum 0548 (tp0548) genotyping and tp0558 sequences to assign strains to the Nichols or SS14 clades. We found that (i) β-barrels in clinical strains could be grouped according to allelic variants in T. pallidum subsp. pallidum reference genomes; (ii) for all three OMP loci, clinical strains within the Nichols or SS14 clades often harbored β-barrel variants that differed from the Nichols and SS14 reference strains; and (iii) OMP variable regions often reside in predicted extracellular loops containing B-cell epitopes. On the basis of structural models, nonconservative amino acid substitutions in predicted transmembrane β-strands of T. pallidum repeat C (TprC) and TprD2 could give rise to functional differences in their porin channels. OMP profiles of some clinical strains were mosaics of different reference strains and did not correlate with results from enhanced molecular typing. Our observations suggest that human host selection pressures drive T. pallidum subsp. pallidum OMP diversity and that genetic exchange contributes to the evolutionary biology of T. pallidum subsp. pallidum They also set the stage for topology-based analysis of antibody responses to OMPs and help frame strategies for syphilis vaccine development.IMPORTANCE Despite recent progress characterizing outer membrane proteins (OMPs) of Treponema pallidum, little is known about how their surface-exposed, β-barrel-forming domains vary among strains circulating within high-risk populations. In this study, sequences for the β-barrel-encoding regions of three OMP loci, tprC, tprD, and bamA, in T. pallidum subsp. pallidum isolates from a large number of patient specimens from geographically disparate sites were examined. Structural models predict that sequence variation within β-barrel domains occurs predominantly within predicted extracellular loops. Amino acid substitutions in predicted transmembrane strands that could potentially affect porin channel function were also noted. Our findings suggest that selection pressures exerted within human populations drive T. pallidum subsp. pallidum OMP diversity and that recombination at OMP loci contributes to the evolutionary biology of syphilis spirochetes. These results also set the stage for topology-based analysis of antibody responses that promote clearance of T. pallidum subsp. pallidum and frame strategies for vaccine development based upon conserved OMP extracellular loops.

Centro Internacional de Entrenamiento e Investigaciones Medicas Cali Colombia

Department of Biology Faculty of Medicine Masaryk University Brno Czech Republic

Department of Genetic and Genome Sciences UConn Health Farmington Connecticut USA

Department of Immunology UConn Health Farmington Connecticut USA

Department of Medicine UConn Health Farmington Connecticut USA

Department of Molecular Biology and Biophysics UConn Health Farmington Connecticut USA

Department of Pediatrics UConn Health Farmington Connecticut USA

Division of Pediatric Infectious Diseases Connecticut Children's Medical Center Hartford Connecticut USA

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Patton ME, Su JR, Nelson R, Weinstock H, Centers for Disease Control and Prevention . 2014. Primary and secondary syphilis—United States, 2005–2013. MMWR Morb Mortal Wkly Rep 63:402–406. PubMed PMC

Peeling RW, Mabey D, Kamb ML, Chen XS, Radolf JD, Benzaken AS. 2017. Syphilis. Nat Rev Dis Prim 3:17073. doi: 10.1038/nrdp.2017.73. PubMed DOI PMC

Gottlieb SL, Deal CD, Giersing B, Rees H, Bolan G, Johnston C, Timms P, Gray-Owen SD, Jerse AE, Cameron CE, Moorthy VS, Kiarie J, Broutet N. 2016. The global roadmap for advancing development of vaccines against sexually transmitted infections: update and next steps. Vaccine 34:2939–2947. doi: 10.1016/j.vaccine.2016.03.111. PubMed DOI PMC

Lithgow KV, Cameron CE. 2017. Vaccine development for syphilis. Expert Rev Vaccines 16:37–44. doi: 10.1080/14760584.2016.1203262. PubMed DOI PMC

Salazar JC, Hazlett KR, Radolf JD. 2002. The immune response to infection with PubMed DOI

Lafond RE, Lukehart SA. 2006. Biological basis for syphilis. Clin Microbiol Rev 19:29–49. doi: 10.1128/CMR.19.1.29-49.2006. PubMed DOI PMC

Radolf JD, Tramont EC, Salazar JC. 2014. Syphilis (

Lukehart SA. 2008. Scientific monogamy: thirty years dancing with the same bug: 2007 Thomas Parran Award Lecture. Sex Transm Dis 35:2–7. doi: 10.1097/OLQ.0b013e318162c4f2. PubMed DOI

Hawley KL, Cruz AR, Benjamin SJ, La Vake CJ, Cervantes JL, LeDoyt M, Ramirez LG, Mandich D, Fiel-Gan M, Caimano MJ, Radolf JD, Salazar JC. 2017. IFNγ enhances CD64-potentiated phagocytosis of PubMed DOI PMC

Koebnik R, Locher KP, Van Gelder P. 2000. Structure and function of bacterial outer membrane proteins: barrels in a nutshell. Mol Microbiol 37:239–253. doi: 10.1046/j.1365-2958.2000.01983.x. PubMed DOI

Nikaido H. 2003. Molecular basis of bacterial outer membrane permeability revisited. Microbiol Mol Biol Rev 67:593–656. doi: 10.1128/MMBR.67.4.593-656.2003. PubMed DOI PMC

Chevalier S, Bouffartigues E, Bodilis J, Maillot O, Lesouhaitier O, Feuilloley MGJ, Orange N, Dufour A, Cornelis P. 2017. Structure, function and regulation of PubMed DOI

Wimley WC. 2003. The versatile β-barrel membrane protein. Curr Opin Struct Biol 13:404–411. doi: 10.1016/S0959-440X(03)00099-X. PubMed DOI

Hobbs MM, Alcorn TM, Davis RH, Fischer W, Thomas JC, Martin I, Ison C, Sparling PF, Cohen MS. 1999. Molecular typing of PubMed DOI

Haake DA, Suchard MA, Kelley MM, Dundoo M, Alt DP, Zuerner RL. 2004. Molecular evolution and mosaicism of leptospiral outer membrane proteins involves horizontal DNA transfer. J Bacteriol 186:2818–2828. doi: 10.1128/JB.186.9.2818-2828.2004. PubMed DOI PMC

Cody AJ, Maiden MJ, Dingle KE. 2009. Genetic diversity and stability of the PubMed DOI PMC

Stenkova AM, Isaeva MP, Shubin FN, Rasskazov VA, Rakin AV. 2011. Trends of the major porin gene (ompF) evolution: insight from the genus PubMed DOI PMC

Matthias KA, Strader MB, Nawar HF, Gao YS, Lee J, Patel DS, Im W, Bash MC. 2017. Heterogeneity in non-epitope loop sequence and outer membrane protein complexes alters antibody binding to the major porin protein PorB in serogroup B PubMed DOI

Lin J, Huang S, Zhang Q. 2002. Outer membrane proteins: key players for bacterial adaptation in host niches. Microbes Infect 4:325–331. doi: 10.1016/S1286-4579(02)01545-9. PubMed DOI

McClean S. 2012. Eight stranded beta-barrel and related outer membrane proteins: role in bacterial pathogenesis. Protein Pept Lett 19:1013–1025. PubMed

Mikula KM, Kolodziejczyk R, Goldman A. 2012. PubMed DOI PMC

Confer AW, Ayalew S. 2013. The OmpA family of proteins: roles in bacterial pathogenesis and immunity. Vet Microbiol 163:207–222. doi: 10.1016/j.vetmic.2012.08.019. PubMed DOI

Haake DA, Zückert WR. 2015. The leptospiral outer membrane. Curr Top Microbiol Immunol 387:187–221. doi: 10.1007/978-3-662-45059-8_8. PubMed DOI PMC

Seidman D, Hebert KS, Truchan HK, Miller DP, Tegels BK, Marconi RT, Carlyon JA. 2015. Essential domains of PubMed DOI PMC

Wu Z, Periaswamy B, Sahin O, Yaeger M, Plummer P, Zhai W, Shen Z, Dai L, Chen SL, Zhang Q. 2016. Point mutations in the major outer membrane protein drive hypervirulence of a rapidly expanding clone of PubMed DOI PMC

Cox DL. 1994. Culture of PubMed DOI

Norris SJ, Cox DL, Weinstock GM. 2001. Biology of PubMed

Penn CW, Cockayne A, Bailey MJ. 1985. The outer membrane of PubMed DOI

Cox DL, Chang P, McDowall AW, Radolf JD. 1992. The outer membrane, not a coat of host proteins, limits antigenicity of virulent PubMed PMC

Radolf JD, Norgard MV, Schulz WW. 1989. Outer membrane ultrastructure explains the limited antigenicity of virulent PubMed DOI PMC

Walker EM, Zampighi GA, Blanco DR, Miller JN, Lovett MA. 1989. Demonstration of rare protein in the outer membrane of PubMed DOI PMC

Fraser CM, Norris SJ, Weinstock GM, White O, Sutton GG, Dodson R, Gwinn M, Hickey EK, Clayton R, Ketchum KA, Sodergren E, Hardham JM, McLeod MP, Salzberg S, Peterson J, Khalak H, Richardson D, Howell JK, Chidambaram M, Utterback T, McDonald L, Artiach P, Bowman C, Cotton MD, Fujii C, Garland S, Hatch B, Horst K, Roberts K, Sandusky M, Weidman J, Smith HO, Venter JC. 1998. Complete genome sequence of PubMed DOI

Radolf JD, Kumar S 29 August 2017. The PubMed DOI PMC

Desrosiers DC, Anand A, Luthra A, Dunham-Ems SM, LeDoyt M, Cummings MA, Eshghi A, Cameron CE, Cruz AR, Salazar JC, Caimano MJ, Radolf JD. 2011. TP0326, a Treponema pallidum β-barrel assembly machinery A (BamA) orthologue and rare outer membrane protein. Mol Microbiol 80:1496–1515. doi: 10.1111/j.1365-2958.2011.07662.x. PubMed DOI PMC

Anand A, Luthra A, Dunham-Ems S, Caimano MJ, Karanian C, LeDoyt M, Cruz AR, Salazar JC, Radolf JD. 2012. TprC/D (Tp0117/131), a trimeric, pore-forming rare outer membrane protein of Treponema pallidum, has a bipartite domain structure. J Bacteriol 194:2321–2333. doi: 10.1128/JB.00101-12. PubMed DOI PMC

Anand A, LeDoyt M, Karanian C, Luthra A, Koszelak-Rosenblum M, Malkowski MG, Puthenveetil R, Vinogradova O, Radolf JD. 2015. Bipartite topology of PubMed DOI PMC

Luthra A, Anand A, Hawley KL, LeDoyt M, La Vake CJ, Caimano MJ, Cruz AR, Salazar JC, Radolf JD. 2015. A homology model reveals novel structural features and an immunodominant surface loop/opsonic target in the PubMed DOI PMC

Noinaj N, Kuszak AJ, Gumbart JC, Lukacik P, Chang H, Easley NC, Lithgow T, Buchanan SK. 2013. Structural insight into the biogenesis of β-barrel membrane proteins. Nature 501:385–390. doi: 10.1038/nature12521. PubMed DOI PMC

Centurion-Lara A, Castro C, Barrett L, Cameron C, Mostowfi M, Van Voorhis WC, Lukehart SA. 1999. PubMed DOI PMC

Centurion-Lara A, Giacani L, Godornes C, Molini BJ, Brinck Reid T, Lukehart SA. 2013. Fine analysis of genetic diversity of the PubMed DOI PMC

Anand A, Luthra A, Edmond ME, Ledoyt M, Caimano MJ, Radolf JD. 2013. The major outer sheath protein (Msp) of PubMed DOI PMC

Puthenveetil R, Kumar S, Caimano MJ, Dey A, Anand A, Vinogradova O, Radolf JD. 2017. The major outer sheath protein forms distinct conformers and multimeric complexes in the outer membrane and periplasm of PubMed DOI PMC

Cruz AR, Pillay A, Zuluaga AV, Ramirez LG, Duque JE, Aristizabal GE, Fiel-Gan MD, Jaramillo R, Trujillo R, Valencia C, Jagodzinski L, Cox DL, Radolf JD, Salazar JC. 2010. Secondary syphilis in Cali, Colombia: new concepts in disease pathogenesis. PLoS Negl Trop Dis 4:e690. doi: 10.1371/journal.pntd.0000690. PubMed DOI PMC

Cruz AR, Ramirez LG, Zuluaga AV, Pillay A, Abreu C, Valencia CA, La Vake C, Cervantes JL, Dunham-Ems S, Cartun R, Mavilio D, Radolf JD, Salazar JC. 2012. Immune evasion and recognition of the syphilis spirochete in blood and skin of secondary syphilis patients: two immunologically distinct compartments. PLoS Negl Trop Dis 6:e1717. doi: 10.1371/journal.pntd.0001717. PubMed DOI PMC

Katz KA, Pillay A, Ahrens K, Kohn RP, Hermanstyne K, Bernstein KT, Ballard RC, Klausner JD. 2010. Molecular epidemiology of syphilis—San Francisco, 2004–2007. Sex Transm Dis 37:660–663. doi: 10.1097/OLQ.0b013e3181e1a77a. PubMed DOI

Flasarová M, Pospíšilová P, Mikalová L, Vališová Z, Dastychová E, Strnadel R, Kuklová I, Woznicová V, Zákoucká H, Šmajs D. 2012. Sequencing-based molecular typing of PubMed DOI

Grillová L, Pětrošová H, Mikalová L, Strnadel R, Dastychová E, Kuklová I, Kojanová M, Kreidlová M, Vaňousová D, Hercogová J, Procházka P, Zákoucká H, Krchňáková A, Vašků V, Šmajs D. 2014. Molecular typing of PubMed DOI PMC

Nechvátal L, Pětrošová H, Grillová L, Pospíšilová P, Mikalová L, Strnadel R, Kuklová I, Kojanová M, Kreidlová M, Vaňousová D, Procházka P, Zákoucká H, Krchňáková A, Smajs D. 2014. Syphilis-causing strains belong to separate SS14-like or Nichols-like groups as defined by multilocus analysis of 19 PubMed DOI

Mikalová L, Strouhal M, Čejková D, Zobaníková M, Pospíšilová P, Norris SJ, Sodergren E, Weinstock GM, Šmajs D. 2010. Genome analysis of Treponema pallidum subsp. pallidum and subsp. pertenue strains: most of the genetic differences are localized in six regions. PLoS One 5:e15713. doi: 10.1371/journal.pone.0015713. PubMed DOI PMC

Arora N, Schuenemann VJ, Jäger G, Peltzer A, Seitz A, Herbig A, Strouhal M, Grillová L, Sánchez-Busó L, Kühnert D, Bos KI, Davis LR, Mikalová L, Bruisten S, Komericki P, French P, Grant PR, Pando MA, Vaulet LG, Fermepin MR, Martinez A, Centurion Lara A, Giacani L, Norris SJ, Šmajs D, Bosshard PP, González-Candelas F, Nieselt K, Krause J, Bagheri HC. 2016. Origin of modern syphilis and emergence of a pandemic PubMed DOI

Marra C, Sahi S, Tantalo L, Godornes C, Reid T, Behets F, Rompalo A, Klausner JD, Yin Y, Mulcahy F, Golden MR, Centurion-Lara A, Lukehart SA. 2010. Enhanced molecular typing of PubMed DOI PMC

Pillay A, Liu H, Chen CY, Holloway B, Sturm AW, Steiner B, Morse SA. 1998. Molecular subtyping of PubMed DOI

Eitinger T, Suhr J, Moore L, Smith JA. 2005. Secondary transporters for nickel and cobalt ions: theme and variations. Biometals 18:399–405. doi: 10.1007/s10534-005-3714-x. PubMed DOI

Gallo Vaulet L, Grillová L, Mikalová L, Casco R, Rodríguez Fermepin M, Pando MA, Šmajs D. 2017. Molecular typing of PubMed DOI PMC

Turner TB, Hollander DH. 1957. Biology of the treponematoses. World Health Organization, Geneva, Switzerland. PubMed

Pinto M, Borges V, Antelo M, Pinheiro M, Nunes A, Azevedo J, Borrego MJ, Mendonça J, Carpinteiro D, Vieira L, Gomes JP. 2016. Genome-scale analysis of the non-cultivable PubMed DOI

Zhang J, Nielsen R, Yang Z. 2005. Evaluation of an improved branch-site likelihood method for detecting positive selection at the molecular level. Mol Biol Evol 22:2472–2479. doi: 10.1093/molbev/msi237. PubMed DOI

Randall A, Cheng J, Sweredoski M, Baldi P. 2008. TMBpro: secondary structure, β-contact and tertiary structure prediction of transmembrane beta-barrel proteins. Bioinformatics 24:513–520. doi: 10.1093/bioinformatics/btm548. PubMed DOI

Kringelum JV, Lundegaard C, Lund O, Nielsen M. 2012. Reliable B cell epitope predictions: impacts of method development and improved benchmarking. PLoS Comput Biol 8:e1002829. doi: 10.1371/journal.pcbi.1002829. PubMed DOI PMC

Centurion-Lara A, Sun ES, Barrett LK, Castro C, Lukehart SA, Van Voorhis WC. 2000. Multiple alleles of PubMed DOI PMC

Cameron CE, Lukehart SA, Castro C, Molini B, Godornes C, Van Voorhis WC. 2000. Opsonic potential, protective capacity, and sequence conservation of the PubMed DOI

Pětrošová H, Zobaníková M, Čejková D, Mikalová L, Pospíšilová P, Strouhal M, Chen L, Qin X, Muzny DM, Weinstock GM, Šmajs D. 2012. Whole genome sequence of PubMed DOI PMC

Tamura K, Stecher G, Peterson D, Filipski A, Kumar S. 2013. MEGA6: molecular evolutionary genetics analysis version 6.0. Mol Biol Evol 30:2725–2729. doi: 10.1093/molbev/mst197. PubMed DOI PMC

Peng RR, Wang AL, Li J, Tucker JD, Yin YP, Chen XS. 2011. Molecular typing of PubMed DOI PMC

Ho EL, Lukehart SA. 2011. Syphilis: using modern approaches to understand an old disease. J Clin Invest 121:4584–4592. doi: 10.1172/JCI57173. PubMed DOI PMC

Soucy SM, Huang J, Gogarten JP. 2015. Horizontal gene transfer: building the web of life. Nat Rev Genet 16:472–482. doi: 10.1038/nrg3962. PubMed DOI

van der Ley P, Heckels JE, Virji M, Hoogerhout P, Poolman JT. 1991. Topology of outer membrane porins in pathogenic PubMed PMC

Azghani AO, Idell S, Bains M, Hancock RE. 2002. PubMed DOI

Gray RR, Mulligan CJ, Molini BJ, Sun ES, Giacani L, Godornes C, Kitchen A, Lukehart SA, Centurion-Lara A. 2006. Molecular evolution of the PubMed DOI

Hollingshead S, Jongerius I, Exley RM, Johnson S, Lea SM, Tang CM. 2018. Structure-based design of chimeric antigens for multivalent protein vaccines. Nat Commun 9:1051. doi: 10.1038/s41467-018-03146-7. PubMed DOI PMC

Staudová B, Strouhal M, Zobaníková M, Cejková D, Fulton LL, Chen L, Giacani L, Centurion-Lara A, Bruisten SM, Sodergren E, Weinstock GM, Smajs D. 2014. Whole genome sequence of the PubMed DOI PMC

Mikalová L, Strouhal M, Oppelt J, Grange PA, Janier M, Benhaddou N, Dupin N, Šmajs D. 2017. Human PubMed DOI PMC

Centurion-Lara A, LaFond RE, Hevner K, Godornes C, Molini BJ, Van Voorhis WC, Lukehart SA. 2004. Gene conversion: a mechanism for generation of heterogeneity in the PubMed DOI

Smith SG, Mahon V, Lambert MA, Fagan RP. 2007. A molecular Swiss army knife: OmpA structure, function and expression. FEMS Microbiol Lett 273:1–11. doi: 10.1111/j.1574-6968.2007.00778.x. PubMed DOI

Maruvada R, Kim KS. 2011. Extracellular loops of the PubMed DOI PMC

Fairman JW, Noinaj N, Buchanan SK. 2011. The structural biology of β-barrel membrane proteins: a summary of recent reports. Curr Opin Struct Biol 21:523–531. doi: 10.1016/j.sbi.2011.05.005. PubMed DOI PMC

Radolf JD, Deka RK, Anand A, Šmajs D, Norgard MV, Yang XF. 2016. PubMed DOI PMC

Zhang Q, Meitzler JC, Huang S, Morishita T. 2000. Sequence polymorphism, predicted secondary structures, and surface-exposed conformational epitopes of PubMed DOI PMC

Alcalá B, Salcedo C, Arreaza L, Abad R, Enríquez R, De La Fuente L, Uría MJ, Vázquez JA. 2004. Antigenic and/or phase variation of PorA protein in non-subtypable PubMed DOI

Isaeva MP, Stenkova AM, Guzev KV, Bystritskaya EP, Shubin FN, Rasskazov VA, Rakin A. 2012. Diversity and adaptive evolution of a major porin gene ( PubMed DOI

Davies RL, Lee I. 2004. Sequence diversity and molecular evolution of the heat-modifiable outer membrane protein gene ( PubMed DOI PMC

McKnew DL, Lynn F, Zenilman JM, Bash MC. 2003. Porin variation among clinical isolates of PubMed DOI

Unemo M, Vorobieva V, Firsova N, Ababkova T, Leniv I, Haldorsen BC, Fredlund H, Skogen V. 2007. PubMed DOI

Nunes A, Borrego MJ, Nunes B, Florindo C, Gomes JP. 2009. Evolutionary dynamics of ompA, the gene encoding the PubMed DOI PMC

Gomes JP, Bruno WJ, Nunes A, Santos N, Florindo C, Borrego MJ, Dean D. 2007. Evolution of PubMed DOI PMC

Cameron CE, Lukehart SA. 2014. Current status of syphilis vaccine development: need, challenges, prospects. Vaccine 32:1602–1609. doi: 10.1016/j.vaccine.2013.09.053. PubMed DOI PMC

Izac JR, Oliver LD Jr, Earnhart CG, Marconi RT. 2017. Identification of a defined linear epitope in the OspA protein of the Lyme disease spirochetes that elicits bactericidal antibody responses: implications for vaccine development. Vaccine 35:3178–3185. doi: 10.1016/j.vaccine.2017.04.079. PubMed DOI PMC

Oliver LD Jr, Earnhart CG, Virginia-Rhodes D, Theisen M, Marconi RT. 2016. Antibody profiling of canine IgG responses to the OspC protein of the Lyme disease spirochetes supports a multivalent approach in vaccine and diagnostic assay development. Vet J 218:27–33. doi: 10.1016/j.tvjl.2016.11.001. PubMed DOI

Earnhart CG, Marconi RT. 2007. Construction and analysis of variants of a polyvalent Lyme disease vaccine: approaches for improving the immune response to chimeric vaccinogens. Vaccine 25:3419–3427. doi: 10.1016/j.vaccine.2006.12.051. PubMed DOI PMC

Cox DL, Luthra A, Dunham-Ems S, Desrosiers DC, Salazar JC, Caimano MJ, Radolf JD. 2010. Surface immunolabeling and consensus computational framework to identify candidate rare outer membrane proteins of Treponema pallidum. Infect Immun 78:5178–5194. doi: 10.1128/IAI.00834-10. PubMed DOI PMC

Chen CY, Chi KH, George RW, Cox DL, Srivastava A, Rui Silva M, Carneiro F, Lauwers GY, Ballard RC. 2006. Diagnosis of gastric syphilis by direct immunofluorescence staining and real-time PCR testing. J Clin Microbiol 44:3452–3456. doi: 10.1128/JCM.00721-06. PubMed DOI PMC

Yang Z. 1997. PAML: a program package for phylogenetic analysis by maximum likelihood. Comput Appl Biosci 13:555–556. doi: 10.1093/bioinformatics/13.5.555. PubMed DOI

Yang Z. 2007. PAML 4: phylogenetic analysis by maximum likelihood. Mol Biol Evol 24:1586–1591. doi: 10.1093/molbev/msm088. PubMed DOI

Xu B, Yang Z. 2013. PAMLX: a graphical user interface for PAML. Mol Biol Evol 30:2723–2724. doi: 10.1093/molbev/mst179. PubMed DOI

Yang Z, Nielsen R. 2002. Codon-substitution models for detecting molecular adaptation at individual sites along specific lineages. Mol Biol Evol 19:908–917. doi: 10.1093/oxfordjournals.molbev.a004148. PubMed DOI

Pettersen EF, Goddard TD, Huang CC, Couch GS, Greenblatt DM, Meng EC, Ferrin TE. 2004. UCSF Chimera—a visualization system for exploratory research and analysis. J Comp Chem 25:1605–1612. doi: 10.1002/jcc.20084. PubMed DOI

Abagyan R, Totrov M. 1994. Biased probability Monte Carlo conformational searches and electrostatic calculations for peptides and proteins. J Mol Biol 235:983–1002. doi: 10.1006/jmbi.1994.1052. PubMed DOI

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Evolutionary Processes in the Emergence and Recent Spread of the Syphilis Agent, Treponema pallidum

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