in the assembly of protein complexes [25]. Rv3876 was found to have a fold similar to ParA family proteins, which are known to function as suppressors of par operon involved in chromosome partitioning [26]. Analysis with Rv3877 predicted the N-terminal region of the protein to possess a ubiquitin-like domain. Ubiquitin- like domains are known to direct proteins either for degradation or to locations where they control other proteins and cell mecha- nisms. The 3D fold analysis of EspB predicted a helix-turn-helix fold at the N-terminal of the protein, similar to that adopted by the PE protein of a previously studied PE/PPE protein complex [27]. Interestingly, the C terminal end of EspB was predicted to have a fold similar to that adopted by the PPE counterpart of this complex, with a WEG tripeptide motif at the position 176–178. This tripeptide motif is similar to the WXG motif observed for the ESAT-6 like family of proteins. The N-terminal region of Rv3879c exhibited a fold similar to fibrinogen, with helical regions followed by coiled-coil regions. In addition, a WXG motif was present in the coil connecting the first two helices. These observations suggest that the N-terminal regions of EspB and Rv3879c together probably form a four-helix-bundle similar to CFP-10/ESAT-6 pair. Interestingly, the 3D fold analysis on EspC predicted this protein to adopt a fold similar to that of ESAT-6 like proteins (i.e. helix-turn-helix structure). The N-terminal region of EspA exhibited helical regions with WXG motif present in the linker region of the first two helices. This is similar to that observed for CFP-10, ESAT-6, EspB and Rv3879c. The positions of the conserved WXG motifs in EspB, Rv3879c and EspA are shown in Figure S2. The C-terminal region of EspA, on the other hand, showed a triple-helix conformation similar to that adopted by collagen molecules. The 3D folds predicted for each of these ESX- 1 components are further summarized in Table S2. Signal peptide search Bacterial secretion systems II, IV and V have been shown to translocate proteins with N-terminal signal sequences, which are recognized by the Sec or Tat translocons [28–30]. However, the known substrates of ESX-1 secretion machinery (ESAT-6, CFP- 10, EspA, EspC, EspB) have been reported to lack such signal sequences [4]. Besides the secreted proteins, many of the known bacterial outer membrane proteins also possess N-terminal signal sequence. Such proteins are known to contribute to the formation of the outer membrane pore apparatus of other secretion systems [28,29]. Signal peptide search revealed that among the various ESX-1 components, the protease Rv3883c (MycP1) and the transmem- brane protein Rv3882c contained N-terminal signal peptides (Table S2). Since these two proteins are not known to be secreted, it is likely that they either contribute to the mycomembrane or periplasmic component of the secretion machinery or are probably involved in the regulation of secretion (either in the periplasm-like space or in the mycomembrane). Prediction of glycosylation sites Proteins with glycosylation sites are likely to be localized in the mycomembrane or periplasm-like space. Detection of such proteins may help to identify components that may contribute to the formation of the mycomembrane pore or may act as gates for the regulation of secretion. The present study predicted glycosyl- ation sites in two ESX-1 components, namely, Rv3869 and Rv3873 (PPE68). The glycosylation of PPE68 was also experi- mentally observed by a previous study [31]. Interestingly, a series of glycosylation sites were predicted around residue 150 of Model of Type VII Secretion System Rv3869. This region corresponds to the long portion of this protein in the periplasm-like space (earlier described under the section ‘Prediction of transmembrane helices’), indicating that this region of the protein is likely to interact with the mycomembrane of the bacteria. The positions of the glycosylation sites for both these proteins are provided in Table S2. Identification of proteolytic cleavage sites ESX-1 region is known to contain a serine protease Rv3883c (MycP1). This protease was experimentally shown to cleave one of the secreted proteins, namely Rv3881c (EspB) at at least two sites [18]. Ohol et al. (2010) identified the cleavage site to be a tetrapeptide motif with a conserved proline residue at the last position. It was observed that, more than 60% of the maximum activity of MycP1 could be retained with motifs having a profile ‘[ALV][X][ALR]P’. Thus, presence of this tetrapeptide motif in any of other ESX-1 component would probably tag that as a novel substrate of MycP1. In the present study, a ‘LVLP’ motif was identified at the N- terminal of the ubiquitin-like domain across all orthologs of Rv3877. This suggests that, Rv3877 could be a probable substrate for MycP1. The location of the cleavage motif in Rv3877 is shown in Figure S2. Prediction of protein-protein association Analysis of protein-protein associations revealed direct or indirect linkage among the ESX-1 components. This indicates that, the genes of the ESX-1 region are functionally linked to each other and probably participate in the same pathway. The analysis indicated that, most of the MCE proteins, namely Rv0170 (Mce1B), Rv0171 (Mce1C), Rv0174 (Mce1F) and Rv0177, had probable functional associations with Rv3869, Rv3870 and Rv3882c. Interestingly, these MCE proteins were also identified to have similar phylogenetic profiles as the ESX-1 components. In addition, the latter three MCE proteins (Mce1C, Mce1F and Rv0177) showed a high probability of association with few other proteins belonging to ESX-1 region (Rv3871, ESAT-6, Rv3876 and Rv3877). These results suggest that, the above proteins of MCE cluster 1 probably function together with ESX-1 compo- nents and participate in the ESX-1 secretion pathway. Detection of secretion islands It is known that genes responsible for secretion of bacterial virulence factors are mostly located in genomic islands having an oligonucleotide composition distinct from the rest of the genome [32,33]. The present study identified a compositionally distinct region spanning from Rv3874 to Rv3902c (a total of 33 genes). Interestingly, the entire ESX-1 gene cluster was observed to be located in this genomic island (Table S3). This suggests that the ESX-1 region corresponds to distinct secretion island similar to that seen in the other bacterial secretion systems. Furthermore, the same compositionally distinct region was also observed to contain ESX-2 gene cluster (Rv3884c–Rv3896c). In addition, a portion of the MCE Cluster 1 region (which also possessed similar phylogenetic profiles as ESX-1 components) was also predicted to be located in a compositionally distinct region of M. tuberculosis genome, spanning the genes Rv0152c to Rv0174 (Table S3). Discussion The analysis of the ESX-1 region presented here in conjunction with the existing published data has been used to predict functional roles for components of the ESX-1 secretion system. Based on these insights, we propose a model for the secretion of PLoS ONE | www.plosone.org 4 November 2011 | Volume 6 | Issue 11 | e27980