Bioinformatic characterization of TcpAh-encoding gene and protein in A. hydrophila JBN2301 strain. (A) Localization of TcpAh-encoding gene in JBN2301 genome. The encoding sequence of TcpAh is indicated in red arrow and att means attachment site. (B) Sequence alignment of TIR domains using Jalview/ClustalW and ClustalX programs. Conserved functional motifs and amino acid residues are shaded in different colors. Secondary structures were predicted by Jalview/Jpred program. Gray boxes, gray arrows, and black lines denote alpha helices, beta-sheet areas, and connecting loops, respectively. (C) Prediction of the 3D structures of TIR domains using Phyre server and PyMol program. The TcpAh TIR domain (38–184 aa) was modeled with 100% confidence by the single highest scoring template as the TIR domain of TLR5 (PDB c3j0aA). Box 1 (purple), box 2 and BB loop (black), and box 3 and DD loop (hot pink) are indicated within the TIR domains. (D) Phylogenetic trees of the amino acid sequences of TcpAh and its homologs in other species constructed by neighbor-joining method using MEGA 7.0. The tree was drawn to scale, and branch lengths are in the same units as those of the evolutionary distances used to infer the phylogenetic tree. The evolutionary distances were computed using the p-distance method. The GenBank accession numbers of the sequences are as follows: H. sapiens MyD88, AAC50954.1; D. rerio MyD88, AAQ91324.1; H. sapiens TLR1, NP_891549.1; D. rerio TLR1, AAI63271.1; H. sapiens TLR2, AAY85648.1; D. rerio TLR2, NP_997977.1; H. sapiens TLR3, NP_003256.1; D. rerio TLR3, AAI07956.1; H. sapiens TLR4, NP_003257.1; D. rerio TLR4, NP_001315534.1; H. sapiens TLR5, NP_003259.2; D. rerio TLR5, NP_001124067.2; H. sapiens TRIF, AAH09860.2; D. rerio TRIF, NP_001038224.1; H. sapiens TRIAP, NP_001034750.1; D. rerio TRIAP, XP_002667158.2; H. sapiens SARM, NP_055892.2; D. rerio SARM, NP_001124068.1; Y. pestis YpTdp, WP_198249346.1; A. hydrophila TcpAh, WP_043158960; P. aeruginosa PumA, WP_012075302.1; S. aureus TirS, WP_000114516.1; E. coli CFT073 TcpC, WP_000282336.1; S. enterica TlpA, WP_079786625.1; P. denitrificans PdTLP, QAR27511.1; B. melitensis TcpB, EPZ76643.1.
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