Fig. 4 | Nature Communications

Fig. 4

From: Activity of acetyltransferase toxins involved in Salmonella persister formation during macrophage infection

Fig. 4

TacT2SEn acetylates aminoacyl-tRNAs more efficiently than TacT2STm. a TacT2 polymorphism. Electrostatic potential of the surface of the modelled TacT2SEn (top panels) or TacT2STm (lower panel) dimer showing positive potential in blue and negative in red. Arrow points toward the polymorphic amino acid. A 45° rotation about the y-axis from the first view of TacT2SEn showing the negative electrostatic potential of E29 in TacT2STm. b Extracted tRNA molecules from S. Typhimurium 12023 ΔtacATtacAT2tacAT3 were treated with equivalent amounts of TacT2STm or TacT2SEn as assessed by western blotting. [14C]Ac-CoA was added to all samples from the onset of the assay and the pH of the reactions was set over a 5.9–7.5 range. Samples were analysed by acid-urea PAGE, tRNAs revealed by methylene blue staining (MB) (top panel) and acetylation tracked by autoradiography (AR) (lower panel). Quantification of acetylation of tRNAs in all conditions is reported in the bar chart, where data represent the mean ± SEM (n = 3). c TacT2SEn (red) K29 and TacT (grey) K31 form two hydrogen bonds between the epsilon amine group of the lysine and carbonyl oxygens from peptide bonds on a neighbouring chain. In a model of TacT2STm (blue), no hydrogen bond can be formed with E29. In TacT3 (orange), a stabilizing hydrogen bond is formed between Q32 and the side chain of the neighbouring R17. Growth curves of S. Typhimurium 12023 ΔtacAT expressing from pBAD33, tacT (+TacT), point mutant toxin (+TacTK31E) or carrying the empty vector (−TacT), or ΔtacAT2 expressing from pBAD33 the Enteritidis wild-type toxin (+TacT2SEn) or carrying the empty vector (−TacT2). All cultures were supplemented with arabinose in fresh rich medium during lag phase. Data represent the mean ± SEM (n ≥ 3). d Binding of the two isoforms of TacT2 to tRNA molecules. Equivalent amounts of purified His-tagged inactive toxins TacT2STmY137F and TacT2SEnY137F were incubated with tRNA molecules extracted from S. Typhimurium 12023 ΔtacATtacAT2tacAT3 and subsequently pulled down with anti-His antibody. The input and output tRNA molecules were analysed by acid-urea PAGE and revealed by methylene blue staining (MB—top panels). Equivalent pulldown of the two toxin isoforms was tested by SDS-PAGE and Coomassie staining (CM—lower panel). Quantification of the amount of tRNA molecules pulled down by the toxins is reported in the bar chart where data represent the mean ± SEM (n ≥ 3)

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