pairs have a preference to adopt a four-helix-bundle structure. While the CFP-10/ESAT-6 pair was previously reported to have such a structure, the present analysis suggests that the other secretory protein pairs (EspA/EspC and EspB/Rv3879c) probably also adopt similar structures. One of the crucial aspects of the ESX-1 secretion mechanism pertains to the translocation of secretory proteins through the mycomembrane. In spite of considerable experimental efforts to understand the ESX-1 secretion system, the components involved in protein translocation through the mycomembrane is currently not known. The identification of the probable candidates (Rv3869, Rv3882c and the MCE Cluster 1 proteins) constituting the mycomembrane translocation apparatus is thus one of the most important outcomes from the current analysis. Thus, combining the results of the present computational analysis with those known from literature, the current study suggests a much more comprehensive model of ESX-1 secretion system. Methods Protein sequences belonging to 14 completely sequenced mycobacterial species were downloaded from the NCBI database (http://www.ncbi.nlm.nih.gov/) (Table S1). These species belonged to diverse clades of the Mycobacterium genus. The protein sequences corresponding to the 17 gene components from M. tuberculosis H37Rv (Table S4), earlier identified experimentally to be involved in ESX-1 secretion pathway [5,7,22,38], were selected and subjected to various in silico analysis. Figure 3 describes the schematic workflow of various bioinformatics analysis performed on these proteins. Orthology search and phylogenetic profile In order to identify the pattern of conservation of the genes involved in ESX-1 secretion pathway, a phylogenetic profile based approach was adopted. First, orthologs of each of the 3989 proteins of M. tuberculosis H37Rv were identified in 13 other mycobacteria (taking one representative strain from each species) using a standalone version of the BLASTp program [49]. The orthology search was restricted to only mycobacteria since organisms outside the Mycobacterium genus mostly possess orthologs of the genes belonging to only ESX-4 [4]. For each protein, only those hits which had an e-value of less than e24 and alignment coverage of at least 60% of the length between the query and the subject proteins (in the BLASTp alignment) were retained as significant hits. This was done to ensure that the identified list of probable orthologous hits (for a given protein) retained the domain architectures of the query protein. Finally, the true ortholog for each protein was identified using the reverse BLAST approach [23]. A phylogenetic profile matrix was generated with all proteins of M. tuberculosis H37Rv. The matrix had 3989 rows and 14 columns, where rows represented proteins of M. tuberculosis H37Rv and columns represented the 14 mycobacterial species. A given element Hij in this matrix represented information about the presence/absence of the ortholog of an M. tuberculosis H37Rv protein ‘i’ in mycobacterial species ‘j’. Thus, 0 and 1 in the matrix corresponded to absence and presence of orthologs, respectively. It is to be noted that the presence or absence (of orthologs) have been termed with respect to the true orthologs (identified after the reverse BLAST step). Subsequently, proteins with identical profiles were clustered together. A subset of these clusters, containing one or more ESX-1 components, were identified for further analysis. All proteins having similar profiles to these clusters (defined as the neighbour- hood space) were then obtained, since they are expected to have functional linkage with the ESX-1 components (present in these Model of Type VII Secretion System clusters). Here, the similarity of phylogenetic profiles was quantified in terms of the bit difference as defined by Pellegrini et al. (1999). In the present analysis, all proteins having a bit difference of less than or equal to three to a given cluster (containing ESX-1 components) were identified to have a similar phylogenetic profile. Domain search In order to detect the presence of known functional domains, protein sequences corresponding to each of the 17 ESX-1 components of M. tuberculosis were queried against various domain databases. The databases used for domain search were PRODOM (http://prodom.prabi.fr/prodom/current/html/form.php) [50], CDD (http://www.ncbi.nlm.nih.gov/Structure/cdd/wrpsb.cgi) [51] and Interpro (http://www.ebi.ac.uk/Tools/InterProScan/) [52]. Prediction of transmembrane helices To detect the presence of transmembrane regions, protein sequences of each of the 17 ESX-1 components and the probable interacting proteins were submitted to three transmembrane prediction servers, namely, TMHMM (http://www.cbs.dtu.dk/ services/TMHMM/) [53], TMPRED (http://www.ch.embnet. org/software/TMPRED_form.html) [54] and SOSUI (http://bp. nuap.nagoya-u.ac.jp/sosui/sosui_submit.html) [55]. Proteins pre- dicted to have the transmembrane regions by at least two of these methods were identified. Prediction of 3D folds The HHPred web server (http://toolkit.Tuebingen.mpg.de/ hhpred/) [56] was used to predict the 3D folds in all the 17 ESX-1 components of M. tuberculosis. This tool predicts 3D fold based on pairwise comparison of profile hidden Markov models (HMMs). Signal peptide search In order to identify potential candidates forming the myco- membrane component of the ESX-1 secretion machinery in M. tuberculosis, a tool called SignalP (http://www.cbs.dtu.dk/services/ SignalP/) [57] was used to detect the presence of N-terminal signal sequences in the analyzed set of proteins. Prediction of glycosylation sites Besides searching for the presence of N-terminal signal peptides, the protein sequences under study were also submitted to the NetOglyc web server (http://www.cbs.dtu.dk/services/NetO- Glyc/) [58] for the detection of putative structural components localizing to the mycomembrane or periplasm-like space. The NetOGlyc server performs Neural Network based prediction of glycosylation sites in human protein sequences. However, it has been previously demonstrated that it can accurately predict the presence of glycosylation sites in mycobacterial proteins [59]. In order to increase the reliability of this analysis, the results generated by the tool were further filtered by considering only those proteins which had at least three glycosylation sites within a ten amino acid window. Identification of proteolytic cleavage sites Multiple sequence alignments for the orthologs of all ESX-1 components of M. tuberculosis were performed using ClustalW (http://www.ebi.ac.uk/Tools/clustalw/) [60]. Subsequently these alignments were searched for the presence of the tetrapeptide motif having the MycP1 cleavage signature ‘[ALV][X][ALR]P’ (as observed by Ohol et al. 2010). PLoS ONE | www.plosone.org 9 November 2011 | Volume 6 | Issue 11 | e27980