Abstract
Septation and cell separation occur as distinct events during cell division in many Gram-positive bacteria. The process first involves synthesis of a complete, multilayered peptidoglycan (PG) septum dividing the cell, which is subsequently hydrolyzed to facilitate physical separation. Using fluorescent D-amino acids and high-resolution microscopy, we identify a previously unrecognized, post-septational wave of transpeptidation that crosslinks septal PG during cell separation in Bacillus subtilis. Notably, this activity does not involve new PG synthesis, but instead remodels pre-existing septal PG. The transpeptidase PBPH plays a key role in this process, and its activity and localization at the separating septum depend on PG hydrolysis by the endopeptidase LytF. Disruption of this interplay impairs cell separation. Our findings reveal a mechanism whereby the coordinated activities of PG hydrolysis and transpeptidation ensure successful cytokinesis. This work expands the current model of cell division by identifying post-septational transpeptidation as a key step in septal resolution and pole formation.
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Data supporting the findings of this study are included in the manuscript and its supplementary information. Source data are available in the figshare repository https://doi.org/10.6084/m9.figshare.30723914.
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Acknowledgements
We thank past and present members of the Brun lab including Maxime Jacq, Kelley Gallagher, Gregory Whitfield, David Kysela, and Amelia Randich for their careful reading of the manuscript and valuable feedback. We also thank Velocity Hughes (Synthesis by Velocity, Malmö, Sweden) for critical reading of the manuscript and editorial assistance. We are grateful to Sven VanTeeffelen for thoughtful review of the manuscript and insightful comments. We thank the Garner lab (Harvard University), the Helmann lab (Cornell University), and the VanTeeffelen lab (University of Montreal) for generously providing strains and plasmids. We also thank Georgia Squyres for the MATLAB script used in data analysis. This research was supported by NIH grants 5R01GM113172 to M.S.V.; R35GM122556 to Y.V.B. and R35GM131783 to D.B.K. Y.V.B. is also supported by the Canada 150 Research Chairs Program in Bacterial Cell Biology.
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V.P., Y.P.H, D.B.K., M.S.V., and Y.V.B. designed the project. V.P. and Y.P.H. performed all the experiments. V.P., Y.P.H., and M.D. carried out data analysis and interpretation, supervised by M.S.V. and Y.V.B. The manuscript was written by V.P., Y.P.H., and Y.V.B. Funding was obtained by D.B.K., M.S.V., and Y.V.B.
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Patel, V., Hsu, YP., Debnath, M. et al. Cell wall hydrolysis promotes a second wave of transpeptidation to achieve cell separation following septation in Bacillus subtilis. Nat Commun (2026). https://doi.org/10.1038/s41467-026-69404-1
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DOI: https://doi.org/10.1038/s41467-026-69404-1
