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. 2019 Mar 12;9:4235. doi: 10.1038/s41598-019-40682-8

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© The Author(s) 2019

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.

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Construction of plasmids pTraG-VirD4 and pTEM-1. (A) pTraG-VirD4 map. The synthesized DNA fragment encoding chimeric T4CP, TraG-VirD4 was cloned into the pMal-c5X between MfeI and BamHI sites, followed by the antibiotic resistance gene swapping from ampicillin resistance gene (Amp^R) to Streptomycin resistance gene (Sm^R), leading to the generation of pTraG-VirD4 with the length of 6743 base pairs (bp). The expression of TraG-VirD4 is under control of tac promoter. (B) pTEM-1 map. The synthesized DNA fragment including tac promoter, 6xHistidine, T7 tag, TEM-1(bla(M))-encoding sequences, multiple cloning sites, and transcription terminator (rrnB T1 and rrnB T2) was cloned into plasmid pACYCDuet-1 between EcoNI and Bsu36I sites, leading to the generation of pTEM-1 with the length of 4786 bp. The expression of TEM-1 fused with 6 × His and T7 tags is under control of tac promoter. (C) The schematic diagram of TraG of RP4 plasmid (TraG_RP4, molecular mass:69.9 kDa), VirD4 of A. phagocytophilum T4SS (VirD4_Ap, molecular mass:84.9 kDa), and the chimeric T4CP (TraG-VirD4, molecular mass: 84.2 kDa). TraG-VirD4 was constructed by fusion of the N-terminal transmembrane domain (NTD) of TraG_RP4 (1^st–102^nd amino acids (aa)) with the cytoplasmic domain of VirD4 of A. phagocytophilum (103^rd–740^th amino acids, molecular mass: 73.1 kDa), which contains nucleotide binding domain, all-alpha-domain, and C-terminal domain. (D) The multiple cloning sites in pTEM-1. The DNA sequence is showed in top with endonuclease cleavage sites, while the deduced amino acid sequence is presented in bottom, including partial TEM-1 (bla(M)) encoding sequence. *Stop codon.