Abstract
Tailocins are phage tail-like bacteriocins produced by various bacterial species to kill kin competitors. Given that tailocin release is dependent upon cell lysis, regulation of tailocin production at the single-cell and population level remains unclear. Here we used flow cytometry, competition assays and structural characterization of tailocin production in a human bacterial pathogen, Listeria monocytogenes. We revealed that a specialized subpopulation, constituting less than 1% of the total bacterial population, differentiates to produce, assemble and store thousands of tailocin particles. Tailocins are packed in a highly ordered manner, clustered in a liquid crystalline phase that occupies a substantial volume of the cell. Tailocin production confers a competitive growth advantage for the rest of the population. This study provides molecular insights into tailocin production as a form of altruism, showing how cell specialization within bacterial populations can confer competitive advantages at the population level.
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Data availability
Example tomograms (Tomo2, Tomo5, Tomo10, Tomo12, Tomo15, Tomo16, Tomo23) and cryo-EM maps were uploaded to the Electron Microscopy Data Bank with accession code EMD-18416. Atomic coordinates of the tail tube were uploaded to the Protein Data Bank with accession code 8QHS. Structures of TTPs that were used in this study for comparison are available in the Protein Data Bank (PDB ID 6TUI, 6TBA, 7T2E, 6V8I, 6YQ5). Source data are provided with this paper.
Code availability
No custom code was used or developed for the analysis of data presented in this paper.
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Acknowledgements
This work was funded by an ERC Consolidator Grant (Co-Patho-phage, 817842) from the European Research Council awarded to A.A.H. and by Instruct-ERIC to A.A.H. via the Weizmann Institute of Science (PID 18811). The electron microscopy studies were partially supported by the Irving and Cherna Moskowitz Center for Nano and Bio-nano Imaging (Weizmann Institute of Science).
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N.S., R.L.-W., S.S. and G.A. performed the experiments. I.B. performed bioinformatic analysis. V.H. performed negative-stain EM, S.G.W. collected cryo-EM and cryo-ET data and performed tomogram reconstructions. N.E. collected single-particle data and performed data analysis of cryo-ET. R.Z., R.Z. and G.A.F. analysed cryo-EM and cryo-ET data and reconstructed the tail-tube structure. A.A.H. prepared the paper.
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Nature Microbiology thanks Andreas Peschel, Jordan Vacheron and the other, anonymous, reviewer(s) for their contribution to the peer review of this work. Peer reviewer reports are available.
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Extended data
Extended Data Fig. 1 Growth analysis of Listeria strains.
L. monocytogenes strain 10403 S and its related mutants, as well as L. monocytogenes Scott A strain were grown in the rich BHI medium at 30 °C. The data represent an average of three biological repeats and the error bars represent the standard deviation of the independent experiments.
Extended Data Fig. 2 Identification of striped bacteria by TEM.
a, Representative TEM micrographs of stained thin plastic sections of WT L. monocytogenes 10403 S bacteria grown without MMC. Arrows point to stripped bacteria. b, Representative TEM micrographs of stained thin plastic sections of WT L. monocytogenes 10403 S bacteria grown with MMC.
Extended Data Fig. 3 The striped phenotype is dependent on monocin production.
Representative TEM micrographs of stained thin plastic sections of (a) L. monocytogenes 10403 S bacteria ectopically expressing MpaR from the pPL2 plasmid using the tet promoter grown with MMC, (b) L. monocytogenes 10403 S bacteria deleted of the monocin structural and lysis genes (Δmon-struc-lys) ectopically expressing MpaR from the pPL2 plasmid using the tet promoter grown with MMC, and (c) Phage−cured L. monocytogenes 10403 S bacteria ectopically expressing MpaR from the pPL2 plasmid using the tet promoter grown with MMC.
Extended Data Fig. 4 Bacillus subtilis ectopically expressing the monocin genes form stripes like those of L. monocytogenes.
Representative TEM micrographs of stained thin plastic sections of B. subtilis bacteria expressing or not expressing the monocin gene cluster (without the holin and lysin genes), in strain sGL-157 (a) and in strain Δ8 (b)6.
Extended Data Fig. 6 Ordered monocin arrays in bacteria.
Slices through a representative tomogram of a monocin-producing cell (Tomo10, relates to Fig. 3). a, A slice through five layers of ordered arrays of monocins. The volume surrounding the arrays is devoid of ribosomes. (b-c, e) Views of the slicing planes designated by the violet, yellow and cyan lines in panel (a). (d) Focus on a five-slice averaged sub-region in panel (a), marked by the red frame (also shown in Fig. 3e). Scales in (a-c) and (e) are 100 nm, and 25 nm in (d).
Extended Data Fig. 7 Cryo-EM single particle analysis.
a. A representative cryo-EM micrograph. White arrows indicate the three types of particles observed (Scale bar represents 30 nm). b. 2D class averages of the three types of particles identified in the micrographs. Type 1 was manually selected for further processing. c, Fourier Shell Correlation (FSC) measured by the Gold-standard method. d, Cryo-EM map obtained by applying helical symmetry (optimized helical twist - 25.08°, optimized helical rise - 39.30 Å). The map is colored by local resolution, estimated by CryoSPARC43 and prepared in UCSF-Chimera47.
Extended Data Fig. 8 Intact monocins produced by the Δ15-mttP mutant.
A TEM image of stained free monocin particles produced by L. monocytogenes 10403 S bacteria expressing the Δ15-MttP mutated protein, treated with MMC. Bacterial lysates were filtered through 0.22 μm filters, and the monocin particles were further precipitated using PEG-8000. A representative image is shown.
Supplementary information
Supplementary Information
Supplementary Tables 1–5.
Supplementary Video 1
STEM tomography of a plastic-embedded bacterial section.
Supplementary Video 2
Cryo-ET tomogram of a monocin-producing cell showing ordered monocin clusters.
Supplementary Video 3
Cryo-ET tomogram of a monocin-producing cell showing ordered monocin clusters.
Supplementary Video 4
Cryo-ET tomogram of a monocin-producing cell showing ordered monocin clusters.
Supplementary Video 5
Cryo-ET tomogram of a monocin-producing cell showing ordered monocin clusters.
Supplementary Video 6
Cube segmentation of cryo-ET tomogram of an ordered monocin array.
Supplementary Video 7
A view of monocins organized in rows within the clusters.
Supplementary Video 8
Cryo-ET tomogram of a monocin-producing cell.
Supplementary Video 9
Cryo-ET tomogram of a monocin-producing cell showing semi-ordered clusters.
Supplementary Video 10
Cryo-ET tomogram of a monocin-producing cell showing isotropic distribution of monocins.
Supplementary Video 11
Cryo-ET tomogram of a monocin-producing cell with ordered monocin clusters showing the aligned tilt series.
Source data
Source Data Fig. 1
Statistical source data and gating strategy.
Source Data Fig. 2
Statistical source data.
Source Data Fig. 3
Statistical source data.
Source Data Fig. 5
Statistical source data.
Source Data Extended Data Fig. 1
Statistical source data.
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Sigal, N., Lichtenstein-Wolfheim, R., Schlussel, S. et al. Specialized Listeria monocytogenes produce tailocins to provide a population-level competitive growth advantage. Nat Microbiol 9, 2727–2737 (2024). https://doi.org/10.1038/s41564-024-01793-9
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DOI: https://doi.org/10.1038/s41564-024-01793-9
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