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Supplementary Information for “De novo morphogenesis in L-forms via
geometric control of cell growth”
Gabriel Billings1, Nikolay Ouzounov2, Tristan Ursell5, Samantha M. Desmarais5,
Joshua Shaevitz3,4, Zemer Gitai2*, and Kerwyn Casey Huang5,6*
1Department
of Physics, Stanford University, Stanford, CA 94305, USA
of Molecular Biology, Princeton University, Princeton, NJ 08544, USA
3Department of Physics, Princeton University, Princeton, NJ 08544, USA
4Lewis-Sigler Institute for Integrative Genomics, Princeton University, Princeton, NJ
08544, USA
5Department of Bioengineering, Stanford University, Stanford, CA 94305, USA
6Department of Microbiology and Immunology, Stanford University School of
Medicine, Stanford, CA 94305, USA
2Department
Keywords: Bacterial cell wall, MreB, curvature-dependent localization, L-forms
*Corresponding
authors:
Kerwyn Casey Huang
Stanford University, Department of Bioengineering
318 Campus Drive
James H. Clark Center, Room S325
Stanford, CA 94305, USA
Phone: (650) 804-1694
Email: kchuang@stanford.edu
Zemer Gitai
Princeton University, Department of Molecular Biology
Washington St.
Princeton, NJ 08544
Phone: (609) 258-9420
Email: zgitai@princeton.edu
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Supplementary Figure S1: Three-dimensional morphology of an L-form on an
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agarose pad. Deconvolved z-stack of an L-form labeled with the membrane dye
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FM4-64. On an agarose pad, the cell has an approximately spherical three-
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dimensional geometry rather than a squashed morphology. Scale-bar: 5 µm.
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Supplementary Figure S2: Growth rate of rod-shaped E. coli MG1655 cells
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under L-form conditions. Growth curves of rod-shaped E. coli MG1655 cells in
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LFLB at 30 °C. Each colored line represents one of 12 samples; the black curve
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shows the average fit to the Gompertz equation, revealing a doubling time t2 of
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1.00±0.02 h (mean ± standard error of mean for n = 12 samples).
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Supplementary Figure S3: Chromatographic analyses of muropeptides in
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reverting L-forms. Baseline-subtracted UV chromatograms of purified
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muropeptides from an overnight culture of L-forms (A), from various time points in
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the reversion process (B: 80 min, C: 135 min, D: 170 min, E: 215 min, F: 260 min),
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and from rod-shaped MG1655 growing exponentially in LB (G). Injection volumes
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in (A-F) are 10 µl; for (G), an injection volume of 5 µl was used in order to operate
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within the linear regime of the detector. mAU = milli-absorbance units.
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Supplementary Figure S4: Temporal cross-correlation of MreB and curvature
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in reverting L-forms. MreB is associated with negative curvature, thus the cross-
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correlation is negative. The minimum cross-correlation occurs at a time delay of 0±1
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min, suggesting that neither signal appears before the other.
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Supplementary Movie Legends
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Movie S1: Generation and reversion of L-forms in a microfluidic device. The
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movie depicts the same cells in Fig. 1. Scale bar: 5 µm.
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Movie S2: Cell shape dynamics during L-form reversion. An L-form reverting on
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an agarose pad without A22. The movie depicts the same cells in Fig. 2A. Scale bar: 5
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µm.
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Movie S3: Cell segmentation during L-form reversion. The movie depicts the
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same cells in Fig. 2A and Movie S2; blue outlines denote a segmented cell lineage.
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Cell tracking at higher cell densities proved challenging, thus the movie terminates
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earlier than Movie S2. Scale bar: 5 µm.
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Movie S4: Cell shape dynamics during L-form reversion in the presence of the
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MreB inhibitor A22. An L-form reverting on an agarose pad containing 10 µg/ml
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A22. The movie depicts the same cells in Fig. 2B. Scale bar: 5 µm.