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Sequential assembly of the septal cell envelope prior to V snapping in Corynebacterium glutamicum

Nature Chemical Biology volume 15, pages 221–231 (2019)Cite this article

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Abstract

Members of the Corynebacterineae, including Corynebacterium and Mycobacterium, have an atypical cell envelope characterized by an additional mycomembrane outside of the peptidoglycan layer. How this multilayered cell envelope is assembled remains unclear. Here, we tracked the assembly dynamics of different envelope layers in Corynebacterium glutamicum and Mycobacterium smegmatis by using metabolic labeling and found that the septal cell envelope is assembled sequentially in both species. Additionally, we demonstrate that in C. glutamicum, the peripheral peptidoglycan layer at the septal junction remains contiguous throughout septation, forming a diffusion barrier for the fluid mycomembrane. This diffusion barrier is resolved through perforations in the peripheral peptidoglycan, thus leading to the confluency of the mycomembrane before daughter cell separation (V snapping). Furthermore, the same junctional peptidoglycan also serves as a mechanical link holding the daughter cells together and undergoes mechanical fracture during V snapping. Finally, we show that normal V snapping in C. glutamicum depends on complete assembly of the septal cell envelope.

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Fig. 1: The septal cell envelope is sequentially assembled during cytokinesis in C. glutamicum.
Fig. 2: The mycomembrane of C. glutamicum becomes confluent before V snapping.
Fig. 3: The mycomembrane is confluent between physically touching neighbor cells in C. glutamicum.
Fig. 4: Peripheral PGL in C. glutamicum forms a diffusion barrier for lipids.
Fig. 5: Peripheral PGL in C. glutamicum undergoes mechanical fracture during V snapping.
Fig. 6: V snapping in C. glutamicum depends on complete assembly of the septal cell envelope.

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All MATLAB scripts used to analyze microscopy images are available upon request.

Data availability

All data generated or analyzed during this study are included in this published article (and its supplementary information files) or are available from the corresponding author on reasonable request.

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Acknowledgements

We thank B. Swarts (Central Michigan University) for providing O-alkTMM and J. Ngo (University of California, San Diego) for DBF-N3; J. Perrino, L. Joubert, and M. Footer (Stanford University) for assistance with the contrast-enhanced TEM sample preparation; A. Straight (Stanford University) for access to the microscope used for FRAP experiments; J. Frunzke (Institute of Bio- and Geosciences, IBG-1: Biotechnology, Forschungszentrum Jülich GmbH) for strain MB001; the Research Institute of Innovative Technology for the Earth in Kyoto Japan for plasmid pCRD206; M. Tsuchida (Open Imaging) for help with the laser ablation experiment; and E. Koslover (University of California, San Diego) for helpful discussions. X.Z. was supported by a Stanford University Interdisciplinary Graduate Fellowship. F.P.R.-R. was supported by a Ford Foundation Predoctoral Fellowship and a University of California Chancellor’s Fellowship. H.C.L. was supported by a Simons Foundation Life Science Research Foundation fellowship. J.C.B. was supported by an NIH Ruth Kirchstein National Research Service Award (F32GM116338). This work was supported by National Institutes of Health grants AI036929 (to J.A.T.), GM058867 (to C.R.B.), and AI051622 (to C.R.B.); Stanford Center for Systems Biology grant P50-GM107615 (to J.A.T.); and the Howard Hughes Medical Institute (to J.A.T. and C.R.B.) and an HHMI-Simons Faculty Scholar Award (to T.G.B.). The transmission electron microscope used in this project was supported in part by ARRA award 1S10RR026780-01 from the National Center for Research Resources (NCRR). The manuscript’s contents are solely the responsibility of the authors and do not necessarily represent the official views of the NCRR or the National Institutes of Health.

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  1. Julie A. Theriot

    Present address: Department of Biology, University of Washington, Seattle, WA, USA

Authors and Affiliations

  1. Department of Biochemistry, Stanford University School of Medicine, Stanford, CA, USA

    Xiaoxue Zhou, Jason C. Bell & Julie A. Theriot

  2. Department of Chemistry, Stanford University, Stanford, CA, USA

    Frances P. Rodriguez-Rivera & Carolyn R. Bertozzi

  3. Department of Microbiology & Immunobiology, Harvard Medical School, Boston, MA, USA

    Hoong Chuin Lim & Thomas G. Bernhardt

  4. Howard Hughes Medical Institute, Stanford University, Stanford, CA, USA

    Carolyn R. Bertozzi & Julie A. Theriot

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Contributions

X.Z., F.P.R.-R., C.R.B., and J.A.T. conceived the project and designed experiments. X.Z. performed the experiments and analyzed data. F.P.R.-R. synthesized and characterized the fluorescent probes. H.C.L. and T.G.B. constructed bacterial strains. J.C.B. contributed expertise to the FRAP experiments and analysis. X.Z. and J.A.T. wrote the manuscript. All authors discussed the results and interpretation of the data, and read and commented on the final version of the manuscript.

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Correspondence to Julie A. Theriot.

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Supplementary information

Supplementary Text and Figures

Supplementary Table 1 and Supplementary Figures 1–14

Reporting Summary

Supplementary Video 1

Cell envelope assembly dynamics in C. glutamicum.

Supplementary Video 2

Confluency of mycomembrane in C. glutamicum.

Supplementary Video 3

Cell envelope assembly dynamics and delayed V snapping in ∆cgp_1735.

Supplementary Video 4

Perturbed cell envelope assembly and V snapping in EMB treated C. glutamicum

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Zhou, X., Rodriguez-Rivera, F.P., Lim, H.C. et al. Sequential assembly of the septal cell envelope prior to V snapping in Corynebacterium glutamicum. Nat Chem Biol 15, 221–231 (2019). https://doi.org/10.1038/s41589-018-0206-1

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