Volume 27, Issue 22, Pages 3419-3429.e4 (November 2017) Deep Phenotypic Mapping of Bacterial Cytoskeletal Mutants Reveals Physiological Robustness to Cell Size  Handuo Shi, Alexandre Colavin, Marty Bigos, Carolina Tropini, Russell D. Monds, Kerwyn Casey Huang  Current Biology  Volume 27, Issue 22, Pages 3419-3429.e4 (November 2017) DOI: 10.1016/j.cub.2017.09.065 Copyright © 2017 Elsevier Ltd Terms and Conditions

Current Biology 2017 27, 3419-3429.e4DOI: (10.1016/j.cub.2017.09.065) Copyright © 2017 Elsevier Ltd Terms and Conditions

Figure 1 Flow-Cytometry-Based Sorting Enriches Cell-Size Mutants (A) Left: cellular dimensions of three strains (n > 2,000 cells for each strain). Middle and right: wider cells exhibited higher FSC signals. Red dotted lines are schematics of FSC gating. Actual gates were set by selecting 2%–3% or 0.5% of the wild-type population. (B) Schematics of library construction and flow-cytometry-based sorting. (I) Construction of complementing parental strains. (II) Wild-type plasmids were exchanged for mutagenized mreB/mrdA plasmids. (III) Cells bearing different mutant plasmids were pooled. (IV) The resulting population was sorted for wider (as shown) or thinner mutants, and single cells were isolated for further characterization. (C) Mean cell width and length measured via single-cell microscopy formed a U shape across mreB and mrdA libraries. Each data point is mean ± SD, with n > 100 cells. (D) Lag time positively correlated with cell length across cell-size mutants (n = 133 strains). (E) Single-cell growth rates across selected cell-size mutants did not vary with cell width (n = 19 strains). Each data point is mean ± SEM, with n > 100 cells. See also Figure S1. Current Biology 2017 27, 3419-3429.e4DOI: (10.1016/j.cub.2017.09.065) Copyright © 2017 Elsevier Ltd Terms and Conditions

Figure 2 Locations of Mutations Are Unbiased across Proteins, and Cell Shape Variation Modes Are Correlated (A) Locations of MreB and MrdA mutations were unbiased. Left: the conservation score (STAR Methods) of each residue (gray curves) and the residue positions with missense mutations in our libraries (blue/red stripes). Right: histograms of conservation scores across all residues (gray bars) and those with mutations (blue/red curves). Bars and curves were not statistically different (two-sample Kolmogorov-Smirnov test). (B) Mutated residues in MreB were distributed across all four structural subdomains. Gray, protein backbone; colored spheres, mutated residues; yellow, ATP. (C) Mutated residues in MreB were distributed across many functional subdomains. Colors are as in (B). (D) Modes of shape variations in the cell body and the cell tip were calculated using PCA of single-cell contours. The dominant modes in cell body shape were length/width variations and bending, and the dominant modes in cell tips were width variation, tip tapering, and tip asymmetry. (E) Longer cells exhibited greater bending (n > 100), regardless of cell width. Two representative cells from mutants with different cell mode 3 projection values and cell lengths are shown. (F) Tip modes 2 and 3 were highly correlated (n > 100), and wider cells tended to have more blunt and asymmetric tips. Two representative cells are shown. See also Figure S2. Current Biology 2017 27, 3419-3429.e4DOI: (10.1016/j.cub.2017.09.065) Copyright © 2017 Elsevier Ltd Terms and Conditions

Figure 3 DNA and FtsZ Concentrations Remain Constant across Cell Sizes (A) Typical fluorescence images of DAPI-stained cells. (B) Simulated models were scaled to fit DAPI fluorescence in selected mutants, suggesting that DNA concentration remains constant for different cell widths (green fit). Each data point is mean ± SEM, with n > 78 cells. (C) Phase (top) and fluorescence (bottom) images of a representative cell with FtsZ-mVenus localizing to midcell. (D) Typical fluorescence images of FtsZ-mVenus fusions in mreB mutants. (E) Simulated models were scaled to fit FtsZ-mVenus fluorescence in selected mreB mutants, suggesting that FtsZ remains at constant concentration across cell sizes (green fit). Each data point is mean ± SEM, with n > 40 cells. (F) Schematics defining FtsZ ring width (wz) and FtsZ intensity within a Z-ring. For clarity, only half of the cell contour is shown on the right. (G) FtsZ intensity was higher in wider cells than in thinner cells (n = 16 strains). Each data point is mean ± SEM, with n > 40 cells. See also Figure S3. Current Biology 2017 27, 3419-3429.e4DOI: (10.1016/j.cub.2017.09.065) Copyright © 2017 Elsevier Ltd Terms and Conditions

Figure 4 A22 Sensitivity Is Strongly Width Dependent (A) Schematic of the assay of antibiotic sensitivity (STAR Methods). (B) Determination of IC50 to A22 for several strains. (C) IC50 for A22 in all mutants. Both libraries exhibited a strong width dependence in A22 sensitivity (n = 96 mutants for mreB and n = 38 for mrdA). Each data point is mean ± SEM. (D) IC50 for mecillinam in all mutants. Although there was a weak width dependence for the mreB library (n = 96 mutants), the IC50 range was small. Each data point is mean ± SEM. (E) Cell widths and A22 sensitivity in combined mutants. Mutations altered cell width in an approximately additive fashion, and cell widths were highly correlated with A22 IC50 (n = 7 strains). See also Figure S4. Current Biology 2017 27, 3419-3429.e4DOI: (10.1016/j.cub.2017.09.065) Copyright © 2017 Elsevier Ltd Terms and Conditions

Figure 5 MreB Enrichment Profile Is Width Dependent (A and B) Mean width (A) and length (B) of the wild-type MreB-msfGFP strain in bulk culture as a function of time. Cell length and width both changed actively throughout the experiment (blue); however, with further dilutions at 60 and 150 min (red arrows), cell morphology remained steady after 120 min. Data points are mean ± SEM, with n > 100 cells. (C) In a culture without repeated dilutions, MreB enrichment profile continued to shift as cells changed morphologies. (D) With repeated dilutions, during the first 120 min, the MreB enrichment profile dynamics resembled those in (C). After 120 min, the MreB enrichment reached a steady state. (E) Mean cell width and length for selected MreB-msfGFP mutants after repeated dilutions were highly correlated. Data points are mean ± SEM, with n > 100 cells. (F) MreB localization profiles for selected mutants and mecillinam-treated MreBWT-msfGFP cells at steady state. In the mutants, wider cells exhibited flatter profiles (smaller slopes) that shifted to the left (solid lines). Mecillinam-treated cells also had profiles shifted to the left (dashed lines). Inset: the mean curvature C1 negatively correlated with cell width (n = 12 mutants or conditions). See also Figure S5. Current Biology 2017 27, 3419-3429.e4DOI: (10.1016/j.cub.2017.09.065) Copyright © 2017 Elsevier Ltd Terms and Conditions