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. 2021 Oct 6;12:5845. doi: 10.1038/s41467-021-26101-5

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© The Author(s) 2021

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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Fig. 4 — a Sequence alignments of circular pGO1-derivative plasmids were performed using Easyfig software (v2.2.2). Genes are coloured according to their sequence and function. Black arrows indicate relevant restriction sites. b Plasmid sequences were affirmed using Southern blotting of total DNA digested with SpeI, BamHI and PstI, with probes against the aacA-aphD [‘G’] and nes regions encoding gentamicin resistance and the relaxase, respectively. Expected plasmid fragment sizes following triple digestion were as follows: pGO1, G: 30,498 bp, nes: 14,103 bp; pGO1evol_A-B, G: 41,026 bp, nes: 41,026 bp; pGO1evol_F, G: 27,634 bp, nes: 14,103 bp; pGO1evol_{E, H-I}, G: 16,796 bp, nes: 14,103 bp; and pGO1evol, G: 13,471 bp, nes: 14,103 bp. c Sequence alignments of SaPIbov2 bap::tetM orf7ΩpGO1evol_C and SaPIbov2 bap::tetM orf7ΩpGO1evol_J hybrid species were performed using Easyfig software (v2.2.2). Genes are coloured according to their sequence and function. Black arrows indicate relevant restriction sites. d SaPIbov2 bap::tetM orf7ΩpGO1evol sequences were affirmed using Southern blotting of total DNA digested with BamHI and NruI, with probes against the aacA-aphD region encoding gentamicin resistance [G] in pGO1, and the tetM cassette in SaPIbov2 bap::tetM [T], respectively. Expected fragment sizes following double digestion were as follows: pGO1, G: 54,000 bp, T: no band; SaPIbov2 bap::tetM, G: no band, T: 6,488 bp; SaPIbov2 bap::tetM orf7ΩpGO1evol_C, G and T both 22,317 bp; and SaPIbov2 bap::tetM orf7ΩpGO1evol_J, G and T both 25,584 bp. See Supplementary Data 1 for details of recombination events and Table S2 for details of mobility mechanisms utilised by the variant pGO1 plasmids. See Supplementary Data 1 for details of recombination events, Table S2 for details of mobility mechanisms utilised by the variant pGO1 plasmids, and Table S3 for details of relative fitness of the variant pGO1 plasmids compared to the parental pGO1. Panels b and d are representative of data obtained from three independent experiments (n = 3) yielding similar results.