File S1 - Functional Domain Analysis of the Cell Division Inhibitor EzrA

Figure S1, (A) Quantitative immunoblot of lysates from wild type and ezrA TM mutant cells. All constructs were expressed from the native promoter with the exception of ezrAΔTM, which was expressed from the IPTG inducible Pspachy promoter. FtsZ loading control on bottom. Primary rabbit antisera raised against EzrA or FtsZ was detected using secondary anti-rabbit serum conjugated to horse radish peroxidase. (B) Immunoblot of membrane fractionation of wild type and ezrA TM mutant lysates. As expected, wild type EzrA and TM helix chimeras are concentrated in the membrane fraction. The TM-less mutant is only in the cytoplasmic fraction. Figure S2, (A) Quantitative immunoblot of coiled-coil deletion constructs. FtsZ loading control on bottom. Note faint TM-QNR-GFP band on far right, consistent with degradation. (B) (Top three panels) Representative immunoblots of membrane fractionations from coiled-coil deletion strains. Three independent experiments are presented. Degradation over the course of the two-day assay, the latter steps of which were performed in the absence of protease inhibitors, led to variation in protein levels between experiments. Molecular weight marker is visible on far left of all three blots (whitish band). (Bottom) Soluble control (FtsZ). (C) Immunoblot of soluble fractions from coiled-coil deletion strains probed with anti-GFP sera (top) or anti-FtsZ sera (bottom). No GFP was visible in soluble fractions, consistent with membrane retention of all CC deletion mutants. Primary rabbit antibody against GFP (Genscript) or FtsZ was detected using secondary anti-rabbit serum conjugated to HRP. Figure S3, (A) Micrographs of GFP fusions to wild type EzrA, the full length EzrA(R510D) mutant, and an EzrA deletion mutant [ezrAΔ(31–499)] that includes EzrA's native transmembrane helix and QNR patch but is missing all four coiledcoils. Note absence of medial localization in both the ezrA(R510D) and ezrAΔ(31–499) images. (B) FtsZ localization by immunofluorescence microscopy. Note the presence of polar FtsZ rings in the ezrAΔ(31–499) images. (A and B). Thick arrows indicate medial EzrA and FtsZ localization. Thin arrows indicate EzrA localization at septa. Arrowheads indicate polar FtsZ rings. Exposure times are equivalent for each fluorophore. Bars  = 5 µm. (C) Consistent with loss of medial localization, the ezrAΔ(31–499) allele is equivalent to an ezrA null with regard to its ability to suppress the lethality associated with overexpression of the MinCD inhibitor or the heat sensitivity of the ftsZts allele. Bars equal standard deviation from three repeated experiments. Table S1, Bacterial Strains Used in this Study. Table S2, Plasmids used in this study. Table S3, Primers used in this study.
(PDF)