Fig. 8: Mutational study of the relaxosome and Mechanistic model of conjugation. | Nature Communications

Fig. 8: Mutational study of the relaxosome and Mechanistic model of conjugation.

From: Cryo-EM Structure of the relaxosome, a complex essential for bacterial mating and the spread of antibiotic resistance genes

Fig. 8

a–d In each panel, the structure of the region of TraI where the mutations were introduced is shown in ribbon, with mutated residues in sphere representation colour-coded in blue or grey depending on whether they result in increased conjugation or not, respectively. Also reported is the log 10 bar graph of the conjugation rates observed for each of the mutant derivatives as described in the methods section. Data are presented as mean values +/− SD (n = 4 for wild-type, n = 3 for mutants). Colour-coding of bars is green, grey and blue for wild-type, mutants with no impact on conjugation, and mutants displaying increased conjugation, respectively. Ordinary one-way ANOVA with Holm-Šídák correction evaluated statistical significance between wild-type conjugation frequencies and complementation by mutant derivatives affecting each interface hub. P-values for each statistically significant difference (P < 0.05) is shown. Exact P-values for every comparison are indicated in Supplementary Table 3. Inset for each panel: zoom-out showing the entire relaxosome structure in the same orientation as main panel. a, TraI VH2B/2B-like interaction with DNA. b, TraI VH2B/2B-like interaction with TraY1. c TraI VH2B/2B-like interaction with IHF. d TraI VH2B/2B-like interaction with TraI TE. Source data are provided as a Source Data file. e The molecular details of conjugation in the donor cell. Shown are the outer and inner membrane (OM and IM, respectively), the T4SS (light blue), TraD (red), the pilus (yellow) and the relaxosome proteins TraM (magenta), TraY1−3 (pale yellow, yellow and olive green, respectively), IHF heterodimer (pink and red) and TraITE/TraIhelicase (orange for TE, green for VH and violet for AH domain). The plasmid DNA is coloured light blue for the R-strand and dark blue for T-strand. ‘nic’ site is indicated by an orange filled triangle. TE domain of TraITE is shown in transparent orange to indicate conformational flexibility (not bound) or in solid orange (bound and ordered). (1) formation of a quiescent relaxosome, (2) relaxosome recruitment to the T4SS, (3) activating signal, (4) formation of ssDNA bubble on the T-strand, (5) loading of TraIhelicase, (6) DNA unwinding, (7) DNA is driven through the T4SS and the pilus, and injected into the recipient cell Created in BioRender80.

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