FIG 5.
TaeI is the immunity protein that prevents TafE from killing A. baumannii by direct interactions. (A) Coexpression of TaeI blocks the yeast toxicity of TafE. Top, a plasmid constitutively expressing HA-TaeI by the GPD promoter was introduced into the yeast strain harboring Flag-TafE driven by Pgal. Serially diluted cells of the yeast strains were spotted onto medium containing glucose or galactose, and the growth was assessed after 3 days of incubation. Bottom, the expression of Flag-TafE and HA-TaeI was probed by immunoblotting using antibodies specific for Flag and hemagglutinin (HA), respectively. (B, C) Yeast cells expressing TaeI are resistant to killing by A. baumannii. Yeast strains harboring the HA-TaeI-expressing plasmid or empty vector were used as prey for the indicated A. baumannii strains. (B) Top, survival of yeast after mixing with A. baumannii was determined by spotting serially diluted cells onto selective medium. Images were acquired after 4 days of incubation at 30°C. Bottom, the expression of TaeI in yeast was probed by immunoblotting with HA-specific antibody, and PGK was probed as the loading control. (C) The rates of survival were determined by enumerating the CFU of yeast. The results shown are the mean values for prey survival (CFU) ± standard deviations. Similar results were obtained in three independent experiments. **, P < 0.01; ns, not significant. (D) TaeI inhibits the DNase activity of TafE. GST or GST-TaeI was mixed with TafE, and DNA was added to the reaction mixtures. The integrity of the DNA was evaluated by agarose gel electrophoresis. Note that the DNA remained intact in reaction mixtures receiving both His6-TafE and GST-TaeI. (E) TaeI directly interacts with TafE. GST or GST-TaeI was mixed with His6-TafE, and the protein complex was captured by GST beads. Note that His6-TafE was pulled down by the beads in the presence of GST-TaeI.