Figure 5. Predicted deaminase toxins from BaDTF2 and BaDTF3 clades exhibit mutagenic activity and a BaDTF2 representative targets ssDNA.

(A) Dendogram indicated evolutionary history of clades within the deaminase superfamily their predicted substrates (colored dots), modified from Iyer et al., 2011. Predicted toxins with unknown substrates, yellow boxes; deaminases toxins with defined biochemical activity, pink boxes. (B) Toxicity of representative BaDTF2 and BaDTF3 toxins as indicated by the proliferation (fold change in colony-forming unit [cfu] recovered) of E. coli after 1 hr expressing the toxins or the empty vector (Control). Values represent means ± s.d., and p-values derive from unpaired two-tailed t-test. (C and D) Representation of single-nucleotide variants (SNVs) by chromosomal position, frequency, and density in E. coli Δung after 1 hr expression of representative BaDTF2 (C) or BaDTF3 (D) toxins. (E and F). Distribution of different nucleotides substitutions among SNVs detected in E. coli Δung expressing representative BaDTF2 (E) or BaDTF3 (F) toxins. (G and H) Probability sequence logo of the region flanking mutated cytosines from E. coli Δung intoxicated with representative BaDTF2 (G) or BaDTF3 (H) toxins. (I and J). In vitro cytidine deamination assays for BaDTF2 toxin single-strand DNA deaminase toxin A (SsdA) using a single-stranded (I) or double-stranded (J) FAM-labeled DNA substrate 'S' with cytidines in the contexts CC, TC, AC, and GC. Cytidine deamination leads to products 'P' with increased mobility. A3A, APOBEC3A (control for activity on ssDNA). DddA_tox was used as a control for activity toward dsDNA. K. In vitro cytidine deamination assays for SsdA_tox or DddA_tox using a single-stranded or double-stranded FAM-labeled DNA substrate with a single cytidine in the context indicated at top. Gels shown in I-K are representative of two replicates. Data in B represent means ± s.d., p-values derive from unpaired two-tailed t-test (n = 3).

Figure 5—figure supplement 1. SsdA_tox exhibits cytidine deaminase activity toward RNA in vitro, but not in vivo.

(A) Poisoned primer extension assay to detect deamination of cytidine in single-stranded RNA substrates. Deamination activity generates a 26-mer product. Image is representative of n = 2 independent replicates. (B–C) Representation of single-nucleotide variants (SNVs) by chromosomal position, frequency, and density detected with RNA-seq analysis of total cDNA collected from E. coli 60 min after induction of SsdA_tox (B) or an empty vector (Control) (C). The high-frequency SNVs detected near position 3 × 10⁶ are the result of reads from highly expressed genes mapping incorrectly to homologous but not identical regions of the genome.