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Peptide surfactants with post-translational C-methylations that promote bacterial development

Nature Chemical Biology volume 21, pages 1069–1075 (2025)Cite this article

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Abstract

Bacteria produce a variety of peptides to mediate nutrient acquisition, microbial interactions and other physiological processes. Of special interest are surface-active peptides that aid in growth and development. Herein we report the structure and characterization of clavusporins, unusual and hydrophobic ribosomal peptides with multiple C-methylations at unactivated carbon centers, which help drastically reduce the surface tension of water and thereby aid in Streptomyces development. The peptides are synthesized by a previously uncharacterized protein superfamily, termed DUF5825, in conjunction with a vitamin B12-dependent radical S-adenosylmethionine metalloenzyme. The operon encoding clavusporins is widespread among actinomycete bacteria, suggesting a prevalent role for clavusporins as morphogens in erecting aerial hyphae and thereby advancing sporulation and proliferation.

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Fig. 1: Discovery and structure of clavusporins.
Fig. 2: Clavusporins reduce surface tension and promote aerial hyphae growth.
Fig. 3: Biosynthesis of clavusporins.
Fig. 4: The mpc-type BGCs are widespread in actinomycetes.

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Data availability

Experimental data supporting the conclusions of this study are available within the article and Supplementary Information. Protein sequences were retrieved from the NCBI Nonredundant Protein Database (http://www.ncbi.nlm.nih.gov/protein/) and NCBI accession numbers of proteins in the S. clavuligerus and S. ghanaensis BGCs are as follows: WP_003958126.1(MpcT), WP_003958125.1 (MpcP), WP_003958123.1 (MpcB), WP_003958122.1 (MpcC), WP_004993370.1 (MpgT), WP_004993372.1 (MpgP), WP_004993375.1 (MpgB), WP_004993376.1 (MpgC). Raw NMR data used to elucidate natural product structures as well as the other data in this paper are available from the corresponding author upon request. Source data are provided with this paper.

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Acknowledgements

We thank the Seyedsayamdost Lab members for helpful discussions, J. Schreiber (Princeton University) for assistance in SEM experiments, D. Potapenko (Princeton University) for guidance in surface tension measurement and I. Pelczer (Princeton University) for collecting NMR data. We thank the National Institutes of Health (R01 GM140034 and R35 GM152049 to M.R.S.) for financial support.

Author information

Authors and Affiliations

  1. Department of Chemistry, Princeton University, Princeton, NJ, USA

    Chen Zhang, Yuchen Li, Ellysia N. Overton & Mohammad R. Seyedsayamdost

  2. Department of Molecular Biology, Princeton University, Princeton, NJ, USA

    Mohammad R. Seyedsayamdost

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Contributions

C.Z. and M.R.S. conceived of the project. C.Z. designed and performed all experiments and analyzed data if not otherwise stated. Y.L. generated mpc cluster knockout and performed qPCR analysis. E.N.O. and C.Z. conducted chemical complementation experiments. C.Z. and M.R.S. wrote the paper with contributions from all authors.

Corresponding author

Correspondence to Mohammad R. Seyedsayamdost.

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Competing interests

M.R.S. is cofounder of Cryptyx Bioscience and a consultant to Merck Co. These entities played no role in the current study. The other authors declare no competing interests.

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Extended data

Extended Data Fig. 1 Structural elucidation of clavusporins.

a,b, UPLC-coupled HRMS analysis of extracts of WT S. clavuligerus and the deletion mutants indicated. Shown are extracted ion chromatograms of clavusporin A (a) and clavusporin B (b). The traces are offset on the y axis for clarity. Each experiment was repeated with at least three biological replicates, and representative results are shown. c, Key NMR correlations used for determination of post-translational methylations in clavusporin B. d, Key NMR correlations used for determination of the overall structure of clavusporin B.

Source data

Extended Data Fig. 2 Phenotypic differences of WT S. clavuligerus and mpc mutants.

a–c, Images of WT S. clavuligerus (a), ΔmpcB (b) and ΔmpcC (c) growing on GYM agar for 10 days. Note the green spores produced by the WT. d–f, SEM images of WT (d), ΔmpcB (e) and ΔmpcC (f), which confirm sporulation in WT, and minimal aerial hyphae growth in mutants. Each experiment was repeated in three biological replicates, and representative results are shown.

Source data

Extended Data Fig. 3 Phenotypic differences between WT S. ghanaensis and the mpgB mutant.

a, The orthologous mpg gene cluster in S. ghanaensis. The gene locus of mpgA is marked by a black circle, and its protein sequence is shown. b,c, Images of WT S. ghanaensis (b) and ΔmpgB S. ghanaensis (c) growing on SFM agar for 3 days. Note the robust production of dark gray spores by the WT. d,e, SEM images of WT (d) and ΔmpgB S. ghanaensis (e), which show typical formation of Streptomyces spore chains in WT. Each experiment was repeated in three biological replicates, and representative results are shown.

Source data

Extended Data Fig. 4 MpcP is a site-specific leader peptidase.

a, HPLC–HRMS analysis of the MpcP reaction with NusA-MpcA. Heat-inactivated MpcP and a no-enzyme reaction were used as controls. Shown are extracted ion chromatograms of MpcA1-21. b, HPLC–HRMS analysis of trypsin-digested NusA-MpcA as a positive control of MpcP assay. Shown are extracted ion chromatograms of two tryptic fragments of MpcA, MpcA1–13 (blue) and MpcA(−15)–(−1) (red). c, HPLC–HRMS analysis of MpcP-treated MBP-MpcA fusion, which was coexpressed with MpcBC in E. coli. Shown are extracted ion chromatograms of MpcA1–21 with different methylation states. The traces are offset on the y axis for clarity. Each experiment was repeated in three biological replicates, and representative results are shown.

Source data

Supplementary information

Supplementary Information

Supplementary Note, Tables 1–10 and Figs. 1–11.

Reporting Summary

Source data

Source Data Fig. 1

Unprocessed MS signal file.

Source Data Fig. 2

Raw surface tension data and uncropped images.

Source Data Fig. 3

Raw extracted ion chromatogram data.

Source Data Fig. 4

Precursor peptide sequences and uncropped logo plots.

Source Data Extended Data Fig. 1

Raw extracted ion chromatogram data.

Source Data Extended Data Fig. 2

Uncropped images.

Source Data Extended Data Fig. 3

Uncropped images.

Source Data Extended Data Fig. 4

Raw extracted ion chromatogram data.

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Zhang, C., Li, Y., Overton, E.N. et al. Peptide surfactants with post-translational C-methylations that promote bacterial development. Nat Chem Biol 21, 1069–1075 (2025). https://doi.org/10.1038/s41589-025-01882-8

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