FIGURE 2.

Depletion of EzrA results in severe growth, size, and shape defects. EzrA depletion in untagged-strain, IU8799 (ΔezrA//bgaA::P_Zn-ezrA⁺) in (A) and (B) or tagged-strain, IU9572 (ΔezrA//bgaA::P_Zn-ezrA-FLAG³) in (C). (A) Representative growth curves after resuspension in BHI broth containing or lacking ZnCl₂ show EzrA-depletion results in bacteriostatic-like growth. IU8799 and IU1945 (WT parent) were grown exponentially in BHI broth supplemented with 0.5 mM ZnCl₂ and 0.05 mM MnSO₄ to OD₆₂₀ ≈ 0.1, spun, and resuspended to OD₆₂₀ ≈ 0.001 for this growth curve. (B) Representative PCm images of wild-type (IU1945) or EzrA depletion strain (IU8799) after resuspension in BHI broth supplemented with, or lacking supplemented ZnCl₂ for 1, 2, 3, or 4 h. Resuspension volumes were adjusted in BHI broth supplemented with or without 0.5 mM ZnCl₂ and 0.05 mM MnSO₄ to OD₆₂₀ ≈ 0.36 (for T = 1 h time-point) or 0.012 (for T = 2, 3, and 4 h time-points). Experiments were performed three times with similar results. See Supplementary Figure 8 for quantitation. (C) Representative western blots of gels containing extracts of IU1945 (wild-type), IU5456 (ezrA-FLAG³), or IU9572 (ΔezrA//P_Zn-ezrA-FLAG³). Western blotting using anti-FLAG antibody or anti-FtsZ are described in section “Materials and Methods” and Supplementary Materials. Protein amounts of ZnCl₂-induced and ZnCl₂-depleted strains relative to IU5456 (ezrA-FLAG³ native) for EzrA-FLAG³ levels or IU1945 (untagged parent isolate) for FtsZ, levels were quantified and are from at least 3 independent biological replicates experiments (± standard deviations).