FIG 4.

Removal of aTc leads to the loss of dCas9. McCoy cells were infected with dCas9 transformants with an incA-targeting gRNA. Transformants were induced at 4 hpi with 10 nM aTc and allowed to proceed until 16 hpi (A). At 16 hpi a subset of the samples was washed with Dulbecco’s phosphate-buffered saline (DPBS) and fixed with 100% MeOH (B). For the next subset of samples, the medium was aspirated and the cells washed with DPBS. Fresh medium without any aTc was added, and the infection was allowed to proceed for an additional 4 h and then fixed with 100% MeOH. All fixed samples were stained using primary antibodies to major outer membrane protein (MOMP), IncA, and dCas9. All images were acquired on an Axio Imager.Z2 microscope with ApoTome.2 at ×100 magnification. Bars, 10 μm. (C and D). Transcript levels for incA and clpP2 were measured at the indicated times from the indicated culture conditions as described in the legend of Fig. 2. Student’s t test was used to compare induced to uninduced samples following log₁₀ transformation, *, P < 0.05.