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. 2019 Jun 10;14(6):e0218241. doi: 10.1371/journal.pone.0218241

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© 2019 Liu et al

This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

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Fig 4 — (A) The ermR gene was amplified from R. anatipestifer CH-1 and cloned into the plasmid pBAD24 to generate pBAD24::ermR. The wild-type rpsL of R. anatipestifer ATCC11845 was cloned into pBAD24::ermR to generate pBAD24::ermR-rpsL. Subsequently, the upstream and downstream regions of the RA0C_1534 gene were amplified and cloned into pBAD24::ermR-rpsL to generate pBAD24::RA0C_1534 upstream-ermR-rpsL-RA0C_1534 downstream. RA0C_1534 upP1* and RA0C_1534 downP2* were used to amplify the RA0C_1534 upstream-ermR-rpsL-RA0C_1534 downstream fragments. (B) Schematic depiction of markerless gene deletion based on natural transformation. (C) a, The clones after the first homologous recombination were verified by PCR and agarose gel electrophoresis. Lane 1 and lane 2: the ermR-rpsL cassette (1323 bp) sequence was amplified from R. anatipestifer ATCCs and Erm-resistant clones, respectively. Lane 3 and lane 4: the 16S rDNA (525 bp) sequence was amplified from R. anatipestifer ATCCs and Erm-resistant clones, respectively. b, PCR identification of the deletion mutant after the second homologous recombination. Lane 1 and lane 2: the ermR-rpsL cassette (1323 bp) sequence was amplified from R. anatipestifer ATCCs and transformants, respectively. Lanes 3 and 4: RA0C_1534 gene containing the native promoter region was amplified from R. anatipestifer ATCCs (828 bp) and transformants (approximately 250 bp). Lane 5 and lane 6: the 16S rDNA (525 bp) sequence was amplified from R. anatipestifer ATCCs and transformants, respectively. M indicates the BM5000 DNA marker.