Model of Type VII Secretion System Figure 1. Relationships between the phylogenetic profiles of the probable proteins involved in ESX-1 secretion pathway. Each node (represented as an oval) denotes proteins having identical phylogenetic profiles. Edge between two nodes is equivalent to one bit difference between the phylogenetic profiles of the corresponding nodes. The bit difference between any two nodes is equal to the number of edges required to travel between the two nodes. Proteins which lie outside the ESX-1 region, but were identified to have similar phylogenetic profiles to one or more of the ESX-1 components are highlighted in bold. doi:10.1371/journal.pone.0027980.g001 The present study identified additional proteins, not belonging to the ESX-1 region, but having phylogenetic profiles either identical or similar to the profile of some of the ESX-1 components. For example, Rv3862c (WhiB6) had phylogenetic profile identical to that of Rv3866. Apart from this, five proteins, namely, Rv0167 (YrbE1A), Rv0168 (YrbE1B), Rv0170 (Mce1B), Rv0171 (Mce1C) and Rv0174 (Mce1F), corresponding to Mammalian Cell Entry cluster 1 (MCE 1), had phylogenetic profiles similar to that of some of the ESX-1 components. Similarly, two MCE 1-associated proteins (Rv0176 and Rv0177) also showed similarity in their phylogenetic profiles with those of ESX-1 components. The above observations suggest that these MCE and MCE-associated proteins probably have functional relationships with the ESX-1 components and may serve as accessory components of the ESX-1 secretion pathway. Domain search Identification of known functional domains in hypothetical proteins may help in providing valuable information about their functional roles. The search of the ESX-1 components against protein domain databases revealed new information about two hypothetical proteins, namely, Rv3866 and Rv3876 (Table S2). Rv3866 was found to possess a DNA binding domain, suggesting its probable function in transcriptional regulation. Rv3876 was identified to have an FlhG domain. FlhG domain containing proteins have been earlier reported to be involved in negative regulation of flagellar biogenesis in V. cholerae [24]. These results indicate a probable role of Rv3866 and Rv3876 in the regulation of the secretion machinery. Prediction of transmembrane helices Identification of ESX-1 components with probable transmem- brane (TM) regions is likely to aid in the detection of proteins forming the inner membrane or the mycomembrane transolocon. Identification of TM regions may also help to demarcate the domains of some of the ESX-1 components that are located in the cytoplasmic, periplasmic and/or mycomembrane regions. This may help to gain further insights into the probable mechanism of these components. The cytoplasmic and periplasmic domains of some of the ESX-1 components were identified based on the position and orientation of the TM helices predicted for them. These details are listed in Table S2. The orientation of the TM regions detected in some of the ESX-1 components, namely, Rv3869, Rv3870, Rv3877, Rv3883c (MycP1) and Rv3882c, are in accordance with what has been reported earlier [13]. Rv3869 (480 amino acids) was predicted to contain a single TM helix approximately from residues 40–60, and thus, had a long portion (,420 aa) in periplasm-like space. Rv3870 was identified with two N-terminal TM helices with a short periplasmic loop. The inner membrane pore forming protein, Rv3877, was predicted to contain either ten or eleven TM helices. Analysis of MycP1 showed a single C-terminal TM helix preceded by a long N-terminal region (,420 aa) in the periplasm-like space. Rv3882c was predicted to possess two N-terminal TM helices with a short cytoplasmic loop. Prediction of 3D folds The three dimensional structure or fold of a protein is known to provide insights into its probable function. Proteins with similar function often tend to adopt similar folds even when they have very low sequence similarity. The results obtained from the 3D fold analysis indicate probable novel functional roles for seven ESX-1 components, namely, Rv3868, Rv3876, Rv3877, Rv3879c and the secreted proteins Rv3881c (EspB), Rv3616c (EspA) and Rv3615c (EspC). The regions containing these predicted 3D folds (for each of the above components) are illustrated in Figure S1. Rv3868 was identified to possess an N-terminal TPR (tetra- tricopeptide repeat) domain. Such TPR domains are known to aid PLoS ONE | www.plosone.org 3 November 2011 | Volume 6 | Issue 11 | e27980