Detection of secretion islands The components of Type I–IV and Type VI secretion systems were previously found to be located in compositionally distinct genomic regions [32,33]. In order to check whether the ESX-1 region occurs in compositionally distinct islands, M. tuberculosis H37Rv genome was given as input to the INDeGenIUS program [61]. The program identifies regions in a genome possessing a deviant oligonucleotide composition as compared to the rest of the genome. Supporting Information Figure S1 Predicted 3D folds and their location in seven ESX-1 components. (PDF) Figure S2 Conserved motifs in some of the ESX-1 secreted proteins and components identified using multiple sequence alignments of the corresponding orthologs. These include the conserved WXG motif in (A) Rv3881c (EspB), (B) Rv3879c, (C) Rv3616c (EspA) and (D) conserved cleavage motif for Rv3883c (MycP1) in Rv3877. (PDF) Text S1 Approximation of the diameter of the four helix bundle formed by the CFP-10/ESAT-6 pair and its homologous structures available in the Protein Data Bank (PDB). (PDF) Text S2 Calculation of inner diameter of the cytoplas- mic membrane pore Rv3877. (PDF) References 1. Kostakioti M, Newman CL, Thanassi DG, Stathopoulos C (2005) Mechanisms of Protein Export across the Bacterial Outer Membrane. J of Bacteriol 187: 4306–4314. 2. Pym AS, Brodin P, Majlessi L, Brosch R, Demangel C, et al. (2003) Recombinant BCG exporting ESAT-6 confers enhanced protection against tuberculosis. Nature Medicine 9: 533–539. 3. Stanley SA, Raghavan S, Hwang WW, Cox JS (2003) Acute infection and macrophage subversion by Mycobacterium tuberculosis require a specialized secretion system. Proc Natl Acad Sci USA 100: 13001–13006. 4. Abdallah AM, Gey van Pittius NC, Champion PA, Cox J, Luirink J, et al. (2007) Type VII secretion- mycobacteria show the way. Nature Rev 5: 883–891. 5. 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Proc Natl Acad Sci USA 100: 12420–12425. Model of Type VII Secretion System Table S1 List of mycobacterial species and the number of orthologs of the ESX-1 components. (PDF) Table S2 Summary of the predicted domains, trans- membrane helices, 3D folds, presence of signal peptides and glycosylation sites in ESX-1 components. (PDF) Table S3 Genes constituting the compositionally dis- tinct islands that harbor (A) ESX-1 gene cluster and, (B) a part of the MCE Cluster 1 region. (PDF) Table S4 List of gene components experimentally identified to be involved in ESX-1 secretion pathway. The genes and the corresponding protein names are taken from the TubercuList database (http://tuberculist.epf1.ch/). (PDF) Acknowledgments We thank Shekhar C Mande and Shubhada R Hegde for helpful discussion and comments. Author Contributions Conceived and designed the experiments: CD TSG SSM. Performed the experiments: CD TSG. Analyzed the data: CD TSG SSM. Wrote the paper: CD TSG SSM. 15. 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