FIGURE 8.

Recruitment of EzrA-rings to equators of future dividing cells is dependent on FtsZ. Wild-type parent strain IU7223 (ftsZ-Myc ezrA-HA) and FtsZ depletion strain IU8237 (ezrA-HA ΔftsZ//bgaA::P_Zn-ftsZ-Myc) were grown exponentially, and IU8237 was depleted of FtsZ-Myc by shifting cells to BHI broth not supplemented with additional ZnCl₂ and MnSO₄ as described in section “Materials and Methods.” Cells were obtained at appropriate time intervals and prepared for IFM as described in Materials and Methods. Data are from two independent biological replicates. (A) Averaged images with fluorescence intensity traces showing FtsZ-Myc and EzrA-HA localization during FtsZ-Myc depletion in IU8237 (ezrA-HA ΔftsZ//P_Zn-ftsZ-Myc). Cells were binned into division stages 1-4, and images from the indicated number of cells (n) from at least two independent biological replicates were averaged using IMA-GUI program as described in section “Materials and Methods.” (i) Row 1, cell shapes determined from phase-contrast images; row 2, nucleoid locations from DAPI labeling; row 3, EzrA-HA locations from IFM; row 4, FtsZ-Myc locations from IFM; row 5, normalized mean fluorescence intensity distributions along the horizontal cell axis for each channel (black, phase image; blue, DNA; green, EzrA; red, FtsZ). Arrows indicate equatorial ring where EzrA is recruited in +Zn condition but less in -Zn conditions. (B) Bar graph quantifying FtsZ-Myc and EzrA-HA-ring co-occurrence during FtsZ-Myc depletion. Bars are the averages of two separate biological replicates while error bars are the SD. (C) Schematic for the orchestration of divisome components (turquoise) by FtsZ (red). FtsZ is required to assemble division sites. In the absence of FtsZ, pre-formed divisome rings can still exist but recruitment of the divisome to equators of future dividing daughter cells ceases to exist. Diffuse unorganized PG synthesis at old division sites still occurs, which leads to enlargement of cells, cell death, and eventual cell lysis.