GNAT toxins evolve toward narrow tRNA target specificities

Abstract

Type II toxin-antitoxin (TA) systems are two-gene modules widely distributed among prokaryotes. GNAT toxins associated with the DUF1778 antitoxins represent a large family of type II TAs. GNAT toxins inhibit cell growth by disrupting translation via acetylation of aminoacyl-tRNAs. In this work, we explored the evolutionary trajectory of GNAT toxins. Using LC/MS detection of acetylated aminoacyl-tRNAs combined with ribosome profiling, we systematically investigated the in vivo substrate specificity of an array of diverse GNAT toxins. Our functional data show that the majority of GNAT toxins are specific to Gly-tRNA isoacceptors. However, the phylogenetic analysis shows that the ancestor of GNAT toxins was likely a relaxed specificity enzyme capable of acetylating multiple elongator tRNAs. Together, our data provide a remarkable snapshot of the evolution of substrate specificity.

Figure 1.

GNAT toxins acetylate different sets of aminoacylated tRNAs. (A) Schematic outline of experimental design. tRNA extracted from toxin-overexpressing cells was treated with Fmoc-OSu to stabilize the unmodified aminoacyl moieties and digested by RNase I to generate the Acetyl- and Fmoc-derivatives of aminoacylated 3′-terminal adenosine of aa-tRNAs. The resulting derivatized aa-A were quantified by LC/MS. (B) Fractions of acetylated aminoacyl-tRNAs isoacceptors in cells expressing AtaT, ItaT, TacT3, and AtaT2 toxins. Fractions of each Ac-aa-A were calculated using the formula shown above the graph. S, the area under the peak of eluted ion; RRF_i, the relative response factors experimentally determined for Fmoc derivatives (see Materials and Methods and Supplementary Table S5). Analysis was performed for 5 min (top) and 45 min (bottom) time points of toxin induction. Error bars show the standard deviation values obtained in three independent experiments.

Figure 2.

Expression of GNAT toxins does not abolish translation initiation. (A) Relative fMet-tRNA^fMet abundances in control and toxin-expressing cells after 45 min of induction taken from the experiment shown in Figure 1B. Peak areas of fMet-Ade ([M + H]⁺ at m/z 427.13) were normalized to that of Fmoc-Pro-Ade ([M + H]⁺ at m/z 587.22) in each sample. (B). Peptidyl tRNA Hydrolase (Pth) neutralizes the toxicity of AtaT and ItaT overexpressed in E. coli. Each panel shows the growth curves of bacteria expressing toxin or Pth alone or together with the toxin with Pth, using an empty vector as control. Data points show the mean of three biological replicates ± standard deviation.

Figure 3.

GNAT toxin activity causes ribosome stalling at specific codons. (A) Distribution of ribosomes over ORFs in cells harboring pBAD, pBAD-tacT3, and pBAD-itaT. Each coding region was divided into ten equal bins from 5′- to 3′-end, and the fraction of ribosomes mapped to each bin was calculated. The plot shows the geometric means of the ribosome density for each bin. (B) Heatmaps of MPS (the mean ribosome density at a particular codon normalized to the mean density across the given ORF) for each codon located in A, P, and E sites of ribosomes, during TacT3 or ItaT intoxication, or in a negative control sample. (C) Mean pause scores for individual Gly and Ile codons located in the ribosomal A-site in the cells expressing TacT3 and ItaT toxins, respectively. Black horizontal lines represent mean pause scores in cells with an empty pBAD vector.

Figure 4.

GNAT toxins evolve toward narrow substrate specificity. (A) The cladogram of GNAT toxin homologs. Transfer Bootstrap Expectation (values ≥ 40%) is shown as grey circles. The segmented colored outer ring indicates antitoxin genes adjacent to the GNAT toxin genes: grey, canonical antitoxins containing DUF1778; and red, novel antitoxins containing DUF4065. Clades are colored and labeled with a letter. Toxins experimentally studied in this work are indicated outside of the ring. (B) LC/MS determined tRNA target specificity of GNAT toxins. Stacked bars represent the total LC/MS signal for all N-acetylated aa-tRNAs detected, grouped by amino acid, for cells expressing the indicated GNAT toxins. Toxins were expressed in exponential cultures of S. Typhimurium (TacT, TacT2, TacT3) or E. coli (AtaT, KacT, AcaT, ItaT, SonT, VcaT, TacT_Sce, GmvT, TacT_Bcn, TacT_Ret) for 4 h, RNA was extracted and hydrolyzed by Nuclease P1, and the resulting acetylated aminoacyl-adenylates (Ac-aa-A) were quantified by LC/MS.