Figure 1. Double-stranded DNA deaminase A (DddA) expression leads to nucleoid degradation in E. coli wild-type cells and uracil accumulation in E. coli Δung.

(A) Fluorescence microscopy of the indicated E. coli strains expressing DddA_tox or carrying an empty vector (Control). DAPI staining (DNA) is shown in cyan. Top: Representative micrographs for each condition, scale bar = 10 μm. Bottom: representative images of cells with intact or degraded nucleoids. (B) Quantification of nucleoid state in cells shown in A (n ≈100–200 cells per condition). (C) Agarose gel electrophoresis analysis of total genomic DNA isolated from the indicated E. coli strains expressing DddA_tox, DddA_tox^E1347A, or carrying an empty vector (Control) after induction for the time period shown. (D) Fluorescence microscopy indicating genomic uracil incorporation (red) of E. coli strains expressing DddA_tox or carrying an empty vector (Control), scale bar = 10 μm. (E) Quantification of uracil labeling signal from cells shown in D (n ≈50 cells per condition). Values and error bars reflect mean ± s.d. of n = 2 independent biological replicates. p-Values derive from unpaired two-tailed t-test.

Figure 1—figure supplement 1. Expression of DddA_tox leads to nucleoid degradation in E. coli.

Full field view of fluorescence micrograph shown in Figure 1 (A) depicting E. coli strains expressing DddA_tox or carrying an empty vector (Control). DAPI staining (DNA) is shown in cyan. Scale bar = 10 µM.

Figure 1—figure supplement 2. DddA_tox induction leads to genomic uracil accumulation in E. coli.

(A,B) Fluorescence microscopy indicating genomic uracil incorporation (red) in the noted E. coli strains. Scale bar = 10 µM. (A) Staining of a positive control strain for uracil incorporation, E. coli CJ236 [dut−, ung−]. This strain accumulates ~500 times more uracil than wild type. (B) Complete fields of view used to generate fluorescence micrographs depicted in Figure 1D.