A toxin-mediated policing system in Bacillus optimizes division of labor via penalizing cheater-like nonproducers
- Jiangsu Provincial Key Lab of Solid Organic Waste Utilization, Jiangsu Collaborative Innovation Center of Solid Organic Wastes, Nanjing Agricultural University, China
- State Key Laboratory of Efficient Utilization of Arid and Semi-arid Arable Land in Northern China, Institute of Agricultural Resources and Regional Planning, Chinese Academy of Agricultura Sciences, China
- National Engineering Research Center for Efficient Utilization of Soil and Fertilizer Resources, College of Resources and Environment, Shandong Agricultural University, China
Figures
Figure 1 with 1 supplement
Expression of ECM production and BAs biosynthesis/immunity were located in the same subpopulation.
Fluorescence emission patterns of double-labeled strains. Colony cells of different double-labeled strains were visualized using CLSM to monitor the distribution of fluorescence signals from …
Figure 1—figure supplement 1
Quantification of fluorescence emission patterns of double-labeled strains.
(A) Flow cytometry monitoring the subfractions of extracellular polysaccharides (EPS)-producing cells (Peps-mCherry) and BAs-producing cells (PbnaF-gfp). Peps-mCherry: strain harboring only the Peps-…
Figure 2 with 5 supplements
ECM and BAs producing subpopulations eliminated the nonproducing cheaters.
The time-lapse experiment for observing the source and distribution of dead cells. Colony cells of different gfp-labeled strains were stained with propidium iodide (PI, a red-fluorescent dye for …
Figure 2—figure supplement 1
Quantification of the source and location of dead cells newly appeared during 3 hr observation.
The statistical objects were newly emerged dead cells during 3 hr observation. Proportion of dead cells that originated from gfp or non-gfp expressing cells are shown on the left panel, proportion …
Figure 2—video 1
Dynamic observation of extracellular polysaccharides (EPS)-producing cells and dead cells in B. velezensis SQR9 community during biofilm formation.
Colony cells of SQR9-Peps-gfp was stained with propidium iodide (PI, a red-fluorescent dye for labeling dead cell) for 15 min, and then visualized by a CLSM to monitor the distribution of …
Figure 2—video 2
Dynamic observation of TasA fibers-producing cells and dead cells in B. velezensis SQR9 community during biofilm formation.
Colony cells of SQR9-PtapA-gfp was stained with PI for 15 min, and then visualized by a CLSM to monitor the distribution of fluorescence signal from the reporter and the PI dye. The video consists …
Figure 2—video 3
Dynamic observation of BAs-producing cells and dead cells in B. velezensis SQR9 community during biofilm formation.
Colony cells of SQR9-PbnaF-gfp was stained with PI for 15 min, and then visualized by a CLSM to monitor the distribution of fluorescence signal from the reporter and the PI dye. The video consists …
Figure 2—video 4
Dynamic observation of BAs-immunity cells and dead cells in B. velezensis SQR9 community during biofilm formation.
Colony cells of SQR9-PbnaAB-gfp was stained with PI for 15 min, and then visualized by a CLSM to monitor the distribution of fluorescence signal from reporter and the PI dye. The video consists of …
Figure 3 with 2 supplements
Spo0A is the co-regulator for triggering ECM production and BAs synthesis/immunity.
(A) Oxford cup assay. Inhibition of the lawn of B. velezensis FZB42 by the BAs extract of wild-type SQR9, its different mutants altered in ECM production, and complementary strain Δspo0A/spo0A. (B) …
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Figure 3—source data 1
Related to Figure 3B.
- https://cdn.elifesciences.org/articles/84743/elife-84743-fig3-data1-v2.xlsx
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Figure 3—source data 2
Related to Figure 3D.
- https://cdn.elifesciences.org/articles/84743/elife-84743-fig3-data2-v2.xlsx
Figure 3—figure supplement 1
The biofilm formation and EPS production were seriously impaired in Δspo0A.
Pellicle formation (A) and extracellular polysaccharides (EPS) production (B) by wild-type SQR9, Δspo0A, and Δspo0A/spo0A. Columns with different letters are statistically different according to the …
Figure 3—figure supplement 2
Expression level of eps, tapA, bnaF, and bnaAB in the colony cells of wild-type SQR9, Δspo0A, and Δspo0A/spo0A, as monitored by using gfp reporters fused to the corresponding promoters.
(A) Colony fluorescence. Colonies were observed under both bright field and GFP channels, to monitor the fluorescence of Peps-gfp, PtapA-gfp, PbnaF-gfp, and PbnaAB-gfp reporters in different …
Figure 4 with 5 supplements
Spo0A activates ACC for BAs synthesis and self-immunity.
(A) Involvement of ACC in the biosynthesis of BAs in B. velezensis SQR9. ACC catalyzes acetyl-CoA to generate malonyl-CoA, which is transformed to malonyl-ACP under the catalyzation of ACP …
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Figure 4—source data 1
Related to Figure 4B.
- https://cdn.elifesciences.org/articles/84743/elife-84743-fig4-data1-v2.xlsx
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Figure 4—source data 2
Related to Figure 4D.
- https://cdn.elifesciences.org/articles/84743/elife-84743-fig4-data2-v2.xlsx
Figure 4—figure supplement 1
Interaction between the purified protein Spo0A and the promoter of bnaF (PbnaF) as determined by Biolayer interferometry data (BLI).
Different concentrations of Spo0A (100, 250, 500, and 1000 nM) were chosen to test interaction with PbnaF.
Figure 4—figure supplement 2
Expression of accDA in the colony cells of wild-type SQR9, Δspo0A, and Δspo0A/spo0A, as monitored by using gfp reporters fused to the promoter of accDA.
(A) Colony fluorescence. Colonies were observed under both bright field and GFP channel, to monitor the fluorescence of PaccDA-gfp in different strains. The bar represents 1 mm. (B) Quantification …
Figure 4—figure supplement 3
Quantification of the inhibition zone is shown in Figure 4E.
Sensitivity of wild-type SQR9 and SQR9-Pxyl-accDA (as the lawn) to the BAs extract of SQR9 (100 μL (1x) or 200 μL (2x)), with the addition of different concentrations of xylose (0%, 0.1%, and 0.2%). …
Figure 4—figure supplement 4
Quantification of bnaAB and bnaF in strain SQR9 and SQR9-Pxyl-accDA.
Expression level of bnaAB (A, C) and bnaF (B, D) in the wild-type SQR9 and SQR9-Pxyl-accDA, with the addition of different concentrations of xylose (0%, 0.1%, and 0.2%). (AB) Quantification of …
Figure 4—figure supplement 5
Production of ACC and BAs immunity were located in the same subpopulation.
(A) Fluorescence emission patterns of double-labeled strains. Colony cells of different double-labeled strains were visualized using a CLSM to monitor the distribution of fluorescence signal from …
Figure 5 with 4 supplements
The co-regulation policing system optimizes the division of labor and enhances population fitness.
(A) Flow cytometry monitoring the expression of Peps-gfp and PtapA-gfp reporters in wild-type SQR9, SQR9ΔbnaV, and SQR9-P43-bnaAB. (B) Quantification of (A). The proportion of the active cells (%) …
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Figure 5—source data 1
Related to Figure 5B.
- https://cdn.elifesciences.org/articles/84743/elife-84743-fig5-data1-v2.xlsx
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Figure 5—source data 2
Related to Figure 5D.
- https://cdn.elifesciences.org/articles/84743/elife-84743-fig5-data2-v2.xlsx
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Figure 5—source data 3
Related to Figure 5F.
- https://cdn.elifesciences.org/articles/84743/elife-84743-fig5-data3-v2.xlsx
Figure 5—figure supplement 1
Growth curves of wild-type SQR9, SQR9ΔbnaV, and SQR9-P43-bnaAB.
Figure 5—figure supplement 2
Pellicle morphology.
Dynamic pellicle formation of wild-type SQR9, SQR9ΔbnaV, and SQR9-P43-bnaAB in MSgg medium under different stressed conditions.
Figure 5—figure supplement 3
Quantification of pellicles.
Dynamic pellicle weight of wild-type SQR9, SQR9ΔbnaV, and SQR9-P43-bnaAB in MSgg medium under different stressed conditions.
Figure 5—figure supplement 4
Quantification of neutral/alkaline protease activity and siderophore production by wild-type SQR9, SQR9ΔbnaV, and SQR9-P43-bnaAB.
Columns with different letters are statistically different according to the Duncan’s multiple range test (n=3, p<0.05).
Figure 6
A working model and ecological significance of the co-regulation policing system in B. velezensis.
In certain conditions (e.g. environmental or self-produced clues, surface attachments, etc.), Bacillus cells can differentiate into Spo0A-ON (~moderate phosphorylated) and Spo0A-OFF …
Author response image 1
Tables
Key resources table
| Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional information |
|---|---|---|---|---|
| Strain, strain background (Bacillus velezensis) | SQR9 | Lab strain | CGMCC accession No. 5808 | |
| Strain, strain background (Bacillus velezensis) | FZB42 | Chen et al., 2007 | BGSC accession no. 10A6 | |
| Strain, strain background (Escherichia coli) | Top 10 | Invitrogen | Host for plasmids | |
| Strain, strain background (Escherichia coli) | BL21 (DE3) | Invitrogen | For recombinant protein expression | |
| Recombinant DNA reagent | pNW33n (plasmid) | Zhou et al., 2018 | B. subtilis-E. coli shuttle vector | |
| Gene (Bacillus velezensis) | spo0A | GenBank | V529_25300 | |
| Gene (Bacillus velezensis) | bnaA | GenBank | V529_06410 | |
| Gene (Bacillus velezensis) | bnaB | GenBank | V529_06420 | |
| Gene (Bacillus velezensis) | bnaV | GenBank | V529_06620 | |
| Software, algorithm | FlowJo V10 | FlowJo V10 | ||
| Software, algorithm | SPSS | SPSS | ||
| Other | Propidium iodide | Invitrogen | L7012 | (20 mM) |
Additional files
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Supplementary file 1
Strains, plasmids, and primers used in this study.
(a) List of strains and plasmids used in this study. (b) List of primers used in this study.
- https://cdn.elifesciences.org/articles/84743/elife-84743-supp1-v2.docx
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MDAR checklist
- https://cdn.elifesciences.org/articles/84743/elife-84743-mdarchecklist1-v2.docx
