H37Rv. The ESX gene clusters have been shown to have evolved through gene duplication events in the order: ESX-4, ESX-1, ESX-3, ESX-2 and ESX-5 [13]. Several studies have provided evidences for the involvement of one or more of these regions (especially ESX-1 and ESX-5) in pathogenesis and macrophage escape [2–4,7,14,15]. A majority of these studies have focused specifically on the ESX-1 region. One of the first characterized substrates of the ESX-1 secretion system was the CFP-10 (Rv3874)/ESAT-6 (Rv3875) pair [2]. The 3D structure of the complex of these two substrates showed that, both CFP-10 and ESAT-6 adopt helix-turn-helix folds and interact with each other forming a tight four-helix-bundle [16]. Five such CFP-10/ESAT-6 gene pairs were identified in the 5 ESX regions [13]. Apart from these five pairs (one pair in each ESX region), six additional CFP- 10/ESAT-6 like gene pairs were also identified outside the ESX regions [13]. Interestingly, protein pairs with similar properties were observed not only in other Actinobacterial species, but also in distant phyla like Firmicutes [5]. The proteins constituting these pairs had certain conserved structural properties like length of approximately 100 residues forming probable helix-turn-helix structures and containing a conserved WXG motif in the region linking the two helices. Such pairs were subsequently referred to as belonging to the WXG100 superfamily [5]. Previous studies have analyzed one or more components of ESX-1 region (covering genes Rv3866-Rv3883c) and predicted probable models for the translocation of proteins [4,15,17,18]. The overall model of the mechanism emerging from these studies is as follows. The C-terminal end of CFP-10 contains a probable signal sequence, that interacts with the FtsK domain containing protein Rv3871 [3,7,19,20]. Subsequently, Rv3871 interacts with Rv3870 and recruits the CFP-10/ESAT-6 pair to the probable inner membrane pore (Rv3877), which then translocates this pair through the inner membrane. Two other proteins, namely, EspA (Rv3616c) and EspC (Rv3615c), have been associated with this secretion pathway. These two proteins have been shown to be secreted along with CFP-10/ESAT-6 pair in a mutually dependent manner [8,21]. In addition, a few genes located inside ESX-1 have also been shown to be essential for the expression or secretion of these proteins. These include Rv3866, Rv3868, Rv3869, PE35 (Rv3872) and MycP1 (Rv3883c) [2,7,22]. Howev- er, the probable mechanism by which these components aid or modulate the secretion is still ill defined. The focus of most of the above studies has been on the inner membrane translocation machinery. However, in addition to the inner membrane, mycobacterial species have a complex mycolic acid rich outer membrane, referred to as the mycomembrane. The components of the ESX-1 secretion machinery, constituting the translocon through the mycomembrane are still unknown. In this study, we have performed a detailed in silico characterization of the proteins encoded by the genes located in ESX-1 cluster (from 14 different mycobacterial species) using a combination of bioinformatics analysis and known information from previously reported studies (experimental and computation- al). Our analysis suggests functional roles for various components associated with the ESX-1 secretion pathway. Based on these results, we also propose a probable working mechanism of Type VII secretion system in M. tuberculosis. Results Orthology search and analysis of phylogenetic profiles Proteins interacting with each other or belonging to same biochemical pathway are known to be conserved across all bacterial species possessing that particular pathway. In order to Model of Type VII Secretion System identify the interaction patterns between ESX-1 components and also to identify additional proteins that may act as regulators or as accessory components of this secretion machinery, we adopted the phylogenetic profile based approach. For this purpose, orthologs of each protein belonging to M. tuberculosis H37Rv were first identified across other mycobacterial species. The orthology search of ESX-1 components of M. tuberculosis H37Rv against 13 mycobacterial species identified three species, which did not have orthologs corresponding to any of the 17 ESX-1 components. These species corresponded to Mycobacterium avium, Mycobacterium ulcerans and Mycobacterium abscessus. The remaining ten species were observed to contain orthologs corresponding to at least nine out of the 17 ESX-1 components. Table S1 gives the number of orthlogs corresponding to the ESX-1 components identified in the various mycobacterial species. While M. bovis had orthologs corresponding to all the 17 components, other species lacked orthologs corresponding to Rv3876 and Rv3879c. In addition, orthologs of Rv3881c (EspB) were observed to be present only in M. bovis and Mycobacterium marinum. These results suggest that the three proteins (Rv3876, Rv3879c and EspB) may not be an integral or essential components of the secretion machinery and probably perform organism specific functions. Phylogenetic profiles represent the presence/absence of ortho- logs corresponding to a set of proteins in organisms under study. Proteins involved in the same pathway show co-evolution and are thus likely to possess similar phylogenetic profiles [23]. Hence, we have used the phylogenetic profile based analysis in order to identify putative structural or regulatory proteins involved in ESX- 1 secretion pathway. Comparison of the phylogenetic profiles of ESX-1 components indicated eight components having identical profiles. Apart from this major cluster, two of the ESX-1 components were observed to be located in the neighborhood of this cluster, suggesting their functional relatedness. The neighbor- hood of a protein (or a protein cluster) can be defined in terms of bit difference. The bit difference between any two proteins (or protein clusters) is defined as the number of positions where their phylogenetic profiles differ. In other words, this denotes the number of species wherein one of the proteins (or protein clusters) has an ortholog whereas the other does not. The bit difference thus provides a quantitative measure of the degree of similarity in the co-evolution trends of the compared proteins (or protein clusters). In the current study, the set of proteins constituting the neighborhood of the major cluster were identified as those whose phylogenetic profiles had a bit difference of less than or equal to three with that corresponding to the major cluster. Details of the proteins present in the major cluster as well as those located in its neighborhood are discussed below and are summarized in Figure 1. Out of the 17 ESX-1 components, the major cluster contained eight components (present in 11 organisms including M. tuberculosis H37Rv), namely, Rv3868, Rv3869, Rv3870, Rv3871, Rv3875 (ESAT-6), Rv3877, Rv3882c and Rv3883c (MycP1). While the phylogenetic profiles of Rv3866 and Rv3874 (CFP-10) differed from the major cluster by a bit difference of one, their phylogenetic profiles differed from each other by a bit difference of two (Figure 1). Orthologs of Rv3866 and CFP-10 were absent in Mycobacterium lepreae and Mycobacterium vanbaalenii, respectively. The remaining seven components of ESX-1, namely, Rv3872 (PE35), Rv3873 (PPE68), Rv3876, Rv3879c, EspB, Rv3615c (EspC) and Rv3616c (EspA), did not belong to the neighborhood space of the major cluster. Furthermore, none of them had phylogenetic profiles identical to each other. These results indicate that the above seven ESX-1 components probably have organism-specific functions. PLoS ONE | www.plosone.org 2 November 2011 | Volume 6 | Issue 11 | e27980