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. 2021 Feb 28;49(6):3584–3598. doi: 10.1093/nar/gkab126

Figure 2.

Figure 2.

Real-time visualization of E. coli killing after acquisition of TAP. (A) Upper panel: diagram of the fluorescent reporter system allowing microcopy visualization of TAP transfer and subsequent nucleoid disorganization in transconjugants. Donor cells exhibit diffuse green fluorescence due to sfGFP production from TAP; HU-mCherry recipients exhibit red nucleoid associated fluorescence; transconjugants are identified by the production of both green and red fluorescence. Lower panel: time-lapse microscopy images performed in a microfluidic chamber. D (donor), recipient (R), and transconjugant (T) cells are indicated. Scale bar 1 μm. Donors TAP-Cas9-nsp (LY1371) or TAP-Cas9-lacZ2 (LY1380); recipient HU-mCherry lac+ (LY248). (B, C) Single-cells time-lapse quantification of transconjugants (B) bacterial and (C) nucleoid lengths. Average and SD are indicated (n cells analysed). The time of TAP acquisition (red dashed line at 0 min) corresponds to a 15% increase in the green fluorescence in the transconjugant cells. (D) Upper panel: diagram of the fluorescent reporter system. Donor cells exhibit diffuse red fluorescence from the mCherry produced by TAP; recipients exhibit diffuse RecA-GFP fluorescence; transconjugants are identified by the production of red fluorescence followed by RecA-GFP polymerization. Lower panel: time-lapse microscopy images performed in a microfluidic chamber. Donor (D), recipient (R), and transconjugant (T) cells are indicated. Scale bar 1 μm. Donors TAP-Cas9-nsp (LY1537) or TAP-Cas9-lacZ2 (LY1538), recipient RecA-GFP (LY844). (E, F) Single-cells time lapse quantification of transconjugants (E) cell length and (F) skewness of RecA-GFP fluorescence signal. Average and SD are indicated (n cells analysed). The time of TAP acquisition (red dashed line at 0 min) corresponds to a 30% increase in the green fluorescence in the transconjugant cells. (B–C and E–F) Multiple t-test were performed corrected with Holm–Sidak method. Stars indicate the time with significant difference (P-value < 0.05). Significant difference was observed from this point until the end of the analysis.