FIGURE 5.

Cell viability and morphology phenotypes upon overexpression of WT ZapD or ZapD mutant variants. a, spot viability assays of wild type (MG1655) strain bearing pTrc99a vector alone, WT ZapD, ZapD(W77A), ZapD(D84A), ZapD(L91A), or ZapD(L174A). The 10-fold dilutions of cells cultured as described under “Experimental Procedures” were spotted on LB plates with ampicillin at the indicated concentrations of IPTG and grown at 37 °C for ∼16 h. The experiment was repeated three times, and a representative image is shown. b, cell morphology phenotypes of wild type (MG1655) strain bearing pTrc99a vector alone (panel i), WT ZapD (panel ii), ZapD(W77A) (panel iii), ZapD(D84A) (panel iv), ZapD(L91A) (panel v), or ZapD(L174A) (panel vi). Cells were grown and imaged under conditions described under “Experimental Procedures.”. Bar, 3 μm. c, quantitative immunoblots of FtsZ, WT ZapD, and ZapD mutant protein levels. Whole cell proteins were harvested from cells at mid-log grown under the same conditions described in the legend to b. Samples were normalized to optical densities and separated by SDS-PAGE. Western analysis was conducted using anti-FtsZ rabbit polyclonal antibody (GenScript) at 1:10,000 and anti-ZapD rabbit polyclonal antibody (GenScript) at 1:1000. Protein bands were normalized to total proteins (BLOT-FastStain, G-Biosciences) as loading and transfer controls. Bands were visualized using a LI-COR Odyssey CLx imager, and intensities were measured using ImageStudio software (LI-COR). Three independent experiments were conducted, and a representative blot with average band intensities ± S.D. is shown. Std indicates molecular size standards, and na indicates not available.