Spatiotemporal expression of endospore appendages and cryo-EM insights into Ena1C-mediated S-ENA anchoring in Bacillus paranthracis

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Spatiotemporal expression of endospore appendages and cryo-EM insights into Ena1C-mediated S-ENA anchoring in Bacillus paranthracis

Scientific Reports , Article number:  (2026) Cite this article

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Abstract

The endospores of many Bacillus cereus group species are decorated with highly resilient fibrous structures known as endospore appendages (ENAs), whose precise biological functions remain poorly understood. Structural and genetic studies have identified ena1A, ena1B, and ena1C as essential for forming the longer, thicker, and most abundant staggered (S)-ENA fibers in Bacillus paranthracis, whereas ena3A encodes the major subunit of the shorter, thinner, ladder-like (L)-ENAs. Here, we investigated the spatiotemporal expression dynamics of S- and L-ENA proteins and the specific role of Ena1C in S-ENA biogenesis. Using time-lapse fluorescence microscopy, we observed strict temporal regulation of ena gene expression, with no detectable ENA subunit production before spores became phase-bright. ENAs expression peaked during late sporulation phase, with fluorescence localized around the developing spore until its release; notably, S-ENA subunit expression began approximately one hour earlier than that of L-ENA subunits. Combining cryo-EM, negative-stain transmission electron microscopy, and genetic analyses, we show that Ena1C forms a nonameric ring-like structure required for tethering S-ENA to the spore surface. These findings provide new insights into the regulation of ENAs’ expression during fiber biogenesis and highlight their temporal coordination with spore coat and exosporium development.

Data availability

The datasets generated during and/or analysed during the current study are available from the corresponding author(s) on reasonable request.

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Acknowledgements

We gratefully acknowledge the Department of Molecular Genetics, University of Groningen, the Netherlands, for hosting E.D.Z and Lene Cecilie Hermansen (NMBU Imaging Center) for assistance with TEM imaging.

Funding

M.A. acknowledges funding from the Norwegian Research Council (NFR), grant number 335029- FORSKER22, and internal funding of NMBU. E.D.Z gratefully acknowledges financial support from the internal funding scheme at Norwegian University of Life Sciences (project no. 1211130114), which financed the international research stay at the University of Groningen, the Netherlands.

Author information

Author notes

  1. Jingqi Chen

    Present address: Department of Chemistry and the Carl R. Woese Institute for Genomic Biology , University of Illinois at Urbana-Champaign , Urbana, USA

Authors and Affiliations

  1. Department of Paraclinical Sciences, Faculty of Veterinary Medicine , Norwegian University of Life Sciences (NMBU) , Ås, 1433, Norway

    Ephrem Debebe Zegeye, Unni Lise Jonsmoen, Yohannes Beyene Mekonnen & Marina Aspholm

  2. Structural Biology Brussels, Vrije Universiteit Brussel, Brussels, Belgium

    Mike Sleutel & Han Remaut

  3. Structural and Molecular Microbiology Structural Biology Research Center, VIB, Brussels, Belgium

    Mike Sleutel & Han Remaut

  4. Molecular Genetics Group Groningen Biomolecular Sciences and Biotechnology Institute , University of Groningen , Groningen, The Netherlands

    Jingqi Chen, Luiza P. Morawska & Oscar P. Kuipers

Authors

  1. Ephrem Debebe Zegeye
  2. Mike Sleutel
  3. Unni Lise Jonsmoen
  4. Jingqi Chen
  5. Luiza P. Morawska
  6. Yohannes Beyene Mekonnen
  7. Oscar P. Kuipers
  8. Han Remaut
  9. Marina Aspholm

Contributions

E.D.Z, M.A., M.S. O.K. and H.R. designed the project. E.D.Z designed and generated all *B. paranthracis* constructs, performed time-lapse microscopy experiments, nsTEM and conducted image analyses with assistance from J.C., L.P.M, and YBM. M.S. expressed recombinant Ena1C and performed cryo-EM imaging, Alphafold modelling and data processing. U.L.J conducted nsTEM imaging and analysis. E.D.Z. wrote the manuscript with input from all authors.

Corresponding authors

Correspondence to Ephrem Debebe Zegeye or Marina Aspholm.

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The authors declare no competing interests.

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Zegeye, E.D., Sleutel, M., Jonsmoen, U.L. et al. Spatiotemporal expression of endospore appendages and cryo-EM insights into Ena1C-mediated S-ENA anchoring in Bacillus paranthracis. Sci Rep (2026). https://doi.org/10.1038/s41598-026-38321-0

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  • DOI: https://doi.org/10.1038/s41598-026-38321-0

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