Fig. 3: ButCD powered by TonB3-ExbBD system is essential for BfUbb transport. | Nature Communications

Fig. 3: ButCD powered by TonB3-ExbBD system is essential for BfUbb transport.

From: A highly conserved SusCD transporter determines the import and species-specific antagonism of Bacteroides ubiquitin homologues

Fig. 3

a–c Assays performed with wild type B. fragilis GS077 and isogenic mutants ΔTonB box (deletion of TonB box in butC) and TonB box mut (TonB box 115AAAAA119 in butC). a Agar spot assays to evaluate the inhibitory effect of BfUbb on the growth of B. fragilis GS077 isogenic sets. b Western blot to detect BfUbb accessibility in the cell lysates of B. fragilis GS077 isogenic sets, pre-incubated with BfUbb. c Confocal microscopy images to show the uptake of Cy3-labelled BfUbb (yellow) following a 4 h incubation with B. fragilis GS077 isogenic sets. Scale bar, 5 μm. d Western blot to detect BfUbb in the cell lysates of B. fragilis GS084 and isogenic mutants ΔtonB1-6, pre-incubated with BfUbb. e–h Assays performed with B. fragilis GS084 and B. fragilis GS077, isogenic deletion mutants ΔtonB3, and complemented isogenic mutant ΔtonB3::ptonB3 in both genetic backgrounds. e, g Western blot to detect BfUbb accessibility in the cell lysates of B. fragilis GS084 (e) and GS077 (g) isogenic sets, pre-incubated with BfUbb. f Confocal microscopy images to show the uptake of Cy3-labelled BfUbb (yellow) following a 4 h incubation with B. fragilis GS084 isogenic set. Scale bar, 5 μm. h Agar spot assays to evaluate the inhibitory effect of BfUbb on the growth of B. fragilis GS077 isogenic sets. i Schematic representation of the tonB3 chromosomal region in B. fragilis GS077. Functional annotation groups assigned as TonB (red), ExbB (yellow), or ExbD (blue) homologues are indicated by arrows filled with colour. PfkA (6-phosphofructokinase), LytB (putative isoprene biosynthesis related reductase), CmpK (CMP kinase), HP (conserved hypothetical protein), and TatD (DNase) are also indicated. j-k Assays performed with B. fragilis GS077, isogenic deletion mutants ΔexbB1B2, ΔexbD1D2, and complemented isogenic mutants ΔexbB1B2::pexbB1, ΔexbB1B2::pexbB2, ΔexbB1B2::pexbB1B2, ΔexbD1D2::pexbD1, ΔexbD1D2::pexbD2, ΔexbD1D2::pexbD1D2. j Western blot to detect BfUbb accessibility in the cell lysates of the B. fragilis GS077 isogenic set, pre-incubated with BfUbb. k Agar spot assays to evaluate the inhibitory effect of BfUbb on the growth of B. fragilis GS077 isogenic sets. Experiments were conducted at least three times with consistent results.

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