A Tad-like apparatus is required for contact-dependent prey killing in predatory social bacteria
- Aix-Marseille Université - CNRS UMR 7283, Institut de Microbiologie de la Méditerranée and Turing Center for Living Systems, France
- Aix-Marseille Université - CNRS UMR 7288, Institut de Biologie du Développement de Marseille and Turing Center for Living Systems, France
- Aix-Marseille Université - CNRS UMR 7255, Institut de Microbiologie de la Méditerranée, France
Figures
Figure 1
A-motility is required for invasion of prey colonies.
Figure 2 with 8 supplements
A-motile cells kill prey cells by contact.
(a) Prey (E. coli) colony invasion by an ‘arrowhead formation’. Activity of the A-motility complex is followed by monitoring Myxococcus cells expressing the bFA-localized AglZ-YFP protein. Upper …
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Figure 2—source data 1
E. coil loss of fluorescence during contact-dependent lysis (Figure 2c).
- https://cdn.elifesciences.org/articles/72409/elife-72409-fig2-data1-v2.xlsx
Figure 2—figure supplement 1
A-motility appears to be essential for predation.
WT M. xanthus and the different motility mutant strains were mixed with E. coli and spotted on CF 1.5% agar plates (+0.07% glucose). After 24 hr of incubations pictures were taken, showing that WT …
Figure 2—figure supplement 2
Contact-dependent killing by an A-S- motility mutant (∆aglQ ∆pilA).
Growth of E. coli cells leads to contact with non-motile Myxococcus cells and rapid lysis. Example cell reflects events observed for n=20 events. Scale bar = 2 µm.
Figure 2—figure supplement 3
Contact-dependent killing by a ∆t6ss mutant.
E. coli prey cells are labeled with GFP to monitor contact-dependent lysis. Example cell reflects events observed for n=20 events. Scale bar = 2 µm.
Figure 2—figure supplement 4
T6SS VipA sheath assembly in Myxococcus cells during predation.
Several assembly patterns are observed as described in other bacteria. Stretched: extended T6SS sheaths. Contracted: retracted T6SS sheath. Scale bars = 2 µm.
Figure 2—figure supplement 5
Prey contact-dependent lysis is not correlated to T6SS sheath contraction.
Contact-dependent lysis and VipA-GFP dynamics were observed simultaneously. Contraction and lysis at the contacted site were only marginally observed (correlated) suggesting that T6SS intoxication …
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Figure 2—figure supplement 5—source data 1
Contact-dependent lysis and VipA-GFP dynamics.
- https://cdn.elifesciences.org/articles/72409/elife-72409-fig2-figsupp5-data1-v2.xlsx
Figure 2—figure supplement 6
Contact-dependent lysis in liquid cultures.
E. coli lysis is detected as extracellular release of the β-galactosidase allows hydrolysis of CPRG which becomes colored after 24 hr. Lysis is not observed when Myxococcus (WT or ∆t6ss) and E. coli …
Figure 2—figure supplement 7
CPRG colorimetric assay performed on motility and T6SS mutant strains.
β-galactosidase activities of the cell lysates (n=4, expressed in Miller Units) were measured for each strain. After 24 hr incubation, only WT, ∆t6ss and A-S+ strains had the ability to lyse E. coli …
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Figure 2—figure supplement 7—source data 1
CPRG assay.
- https://cdn.elifesciences.org/articles/72409/elife-72409-fig2-figsupp7-data1-v2.xlsx
Figure 2—figure supplement 8
Crystal violet assay.
After 24 hr incubation, wells containing the different M. xanthus strains mixed with E. coli were stained with a crystal violet solution to revealed biofilm formation. The ∆pilA strains appeared to …
Figure 3 with 3 supplements
A Tad-like apparatus is required for prey recognition and contact-dependent killing.
(a) Model structure of the Kil system following bioinformatics predictions. Annotated cluster 1 and cluster 2 genes are shown together with the possible localization of their protein products. Dark …
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Figure 3—source data 1
CPRG assay (Figure 3b).
- https://cdn.elifesciences.org/articles/72409/elife-72409-fig3-data1-v2.xlsx
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Figure 3—source data 2
Counting percentage of contacts with a prey leading to motility pauses and prey cell lysis (Figure 3c and d).
- https://cdn.elifesciences.org/articles/72409/elife-72409-fig3-data2-v2.xlsx
Figure 3—figure supplement 1
Bioinformatics analyses of Kil proteins.
(a, b) Structural models of the putative KilC secretin (a) and KilF hexameric ATPase (b). KilC Secretin: tertiary and quaternary structural models were based on the structure of E. coli type II …
Figure 3—figure supplement 2
The kil genes are expressed during starvation.
RNA-seq analysis of kil gene expression in rich medium, starvation medium and starvation medium with live prey cells extracted and computed from data by Livingstone et al., 2018. For each gene and …
Figure 3—figure supplement 3
CPRG colorimetric assay.
In a 96-well plate, the different kil strains transformed with pSWU19-EV (Empty Vector) or complemented (comp) with a pSWU19 carrying the different kil genes were incubated with E. coli in liquid. …
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Figure 3—figure supplement 3—source data 1
CPRG assay.
- https://cdn.elifesciences.org/articles/72409/elife-72409-fig3-figsupp3-data1-v2.xlsx
Figure 4 with 4 supplements
The Kil Tad-like system assembles upon contact and causes prey cell lysis.
(a) NG-KilF clusters form in contact with the prey and their formation precedes cell lysis. Scale bar = 2 µm. See associated Video 5 for the full time lapse. (b) KilG-NG forms clusters at the …
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Figure 4—source data 1
Counting percentage of contacts with a prey leading to NG-KilD foci formation and counting percentage of NG-KilD foci associated with motility pause and prey cell lysis (Figure 4e, f and g).
- https://cdn.elifesciences.org/articles/72409/elife-72409-fig4-data1-v2.xlsx
Figure 4—figure supplement 1
The strains expressing Neon Green (NG) fusions of KilD or KilF have predation phenotypes similar to wild-type in a CPRG colorimetric assay.
This experiment was independently performed twice. Error bars represent the standard deviation to the mean.
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Figure 4—figure supplement 1—source data 1
CPRG assay.
- https://cdn.elifesciences.org/articles/72409/elife-72409-fig4-figsupp1-data1-v2.xlsx
Figure 4—figure supplement 2
The ∆kilG strain expressing KilG-NG is defective in predation.
In a 96-well plate, ∆kilG transformed with pSWU19-EV (Empty Vector), pSWU19-PpilA-kilG (comp) or pSWU19-PpilA-kilG-NG was incubated with E. coli in liquid. After 24 hr incubation, a CPRG …
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Figure 4—figure supplement 2—source data 1
CPRG assay.
- https://cdn.elifesciences.org/articles/72409/elife-72409-fig4-figsupp2-data1-v2.xlsx
Figure 4—figure supplement 3
Time to lysis after cluster formation.
Time to lysis was determined by first monitoring cluster formation and then loss of contrast by the prey cell. The measurements were performed over two biological replicates (n=61). The median is …
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Figure 4—figure supplement 3—source data 1
Lysis time.
- https://cdn.elifesciences.org/articles/72409/elife-72409-fig4-figsupp3-data1-v2.xlsx
Figure 4—figure supplement 4
Stable expression of NG-KilD in different mutant backgrounds.
NG-KilD is detected at the expected molecular weight by the anti-neon Green antibody. (-) NG: DZ2 Myxococcus cell extracts that do not express neon Green. Dotted line indicates gel splicing.
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Figure 4—figure supplement 4—source data 1
Western Blot.
- https://cdn.elifesciences.org/articles/72409/elife-72409-fig4-figsupp4-data1-v2.zip
Figure 5 with 2 supplements
The kil genes are required for M.xanthus nutrition over prey cells.
(a) The Kil system is essential for predation. Core deletion mutants in Tad-like genes, ∆kilC (Secretin), ∆kilF (ATPase), ∆kilH (IM platform), and ∆kilG (IM platform) were mixed with E. coli and …
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Figure 5—source data 1
Flow cytometry (Figure 5c and d).
- https://cdn.elifesciences.org/articles/72409/elife-72409-fig5-data1-v2.xlsx
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Figure 5—source data 2
M. xanthus growth during prey colony invasion (Figure 5e).
- https://cdn.elifesciences.org/articles/72409/elife-72409-fig5-data2-v2.xlsx
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Figure 5—source data 3
Increase in M. xanthus cell length during predation (Figure 5f).
- https://cdn.elifesciences.org/articles/72409/elife-72409-fig5-data3-v2.xlsx
Figure 5—figure supplement 1
Growth and motility of WT and ∆kilACF strains on agar supporting both A- and S-motility (1.5%) and S-motility only (0.5%).
Scale bar = 2 mm.
Figure 5—figure supplement 2
Growth curves of WT and ∆kilACF in CYE-rich medium.
The measurements were performed over three biological replicates. Error bars represent the standard deviation to the mean.
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Figure 5—figure supplement 2—source data 1
Growth curves.
- https://cdn.elifesciences.org/articles/72409/elife-72409-fig5-figsupp2-data1-v2.xlsx
Figure 6
The Kil system mediates killing against diverse bacterial species.
(a) The kil genes are predation determinants against various species. To evaluate if M. xanthus kil mutant had lost the ability to lyse by direct contact different preys, prey-cell suspensions were …
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Figure 6—source data 1
Prey CFU counts during predation (Figure 6b,c,d,e,f).
- https://cdn.elifesciences.org/articles/72409/elife-72409-fig6-data1-v2.xlsx
Figure 7 with 1 supplement
The Kil system is conserved in predatory delta-proteobacteria.
Phylogenetic tree of the Type-IV filamentous system that gave rise to the M. xanthus Kil system. Only the four well-conserved Kil system components were used for constructing the phylogenetic tree. …
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Figure 7—source data 1
Supermatrix alignment.
- https://cdn.elifesciences.org/articles/72409/elife-72409-fig7-data1-v2.docx
Figure 7—figure supplement 1
Predation phenotype of a Myxococcus D,D-decarboxylase mutant (Zhang et al., 2020a).
Colony plate assays showing invasion of an E. coli prey colony 48 hr after plating by a dacB mutant. Scale bar = 2 mm.
Videos
Video 1
Invasion of an E. coli colony by WT Myxococcus cells.
This movie was taken at the interface between the two colonies during invasion. The movie is an 8x compression of an original movie that was shot for 10 hr with a frame taken every 30 s at ×40 …
Video 2
A-motility is required for prey invasion.
This movie was taken at the interface between the two colonies during invasion. The movie is an 8x compression of an original movie that was shot for 10 hr with a frame taken every 30 s at ×40 …
Video 3
Prey invasion by A-motile cells in ‘arrowhead’ formations.
Focal adhesions and thus active A-motility complexes were detected with an AglZ-Neon green fusion. The movie contains 51 frames taken every 30 s at ×100 magnification. Shown side-by-side are …
Video 4
A Myxococcus cell kills an E. coli cell by contact.
The Myxococcus cell expresses AglZ-NG and the E. coli cell expresses mCherry. Shown side-by-side are fluorescence images and MiSiC segmentation (Myxococcus: green, E. coli: magenta). The movie …
Video 5
NG-KilF cluster formation in contact with E. coli prey cells.
Shown is an overlay of the fluorescence and phase contrast images of a motile Myxococcus cell in predatory contact with E. coli cells. The movie was shot at x100 magnification objective for 30 min. …
Video 6
KilG-NG cluster formation in contact with E. coli prey cells.
Shown is an overlay of the fluorescence and phase contrast images of a motile Myxococcus cell in predatory contact with E. coli cells. The movie was shot at ×100 magnification objective for 9 min. …
Video 7
NG-KilD cluster formation in contact with E. coli prey cells.
Shown is an overlay of the fluorescence and phase contrast images of a motile Myxococcus cell in predatory contact with E. coli cells. The movie was shot at x100 magnification objective for 15 min. …
Video 8
NG-KilD clusters form in a ∆kilC mutant but no motility pauses and prey cell lysis can be observed.
Shown is an overlay of the fluorescence and phase contrast images of a motile Myxococcus cell in predatory contact E. coli cells. The movie was shot at ×100 magnification objective for 5.5 min. …
Video 9
A ∆kilACF still invades but does not kill E. coli prey cells.
This movie was taken at the interface between the two colonies during invasion. The movie is a 4x compression of an original movie that was shot for 4.5 hr with a frame taken every 30 s at ×40 …
Video 10
Predatory cells division and tracking during invasion of prey colony.
To follow cell growth and division at the single cell level during prey invasion, WT cells were mixed with a WT strain expressing the mCherry at a 50:1 ratio and imaged every 30 s at ×40 …
Video 11
NG-KilD cluster formation in contact with Caulobacter crescentus.
Shown is an overlay of the fluorescence and phase contrast images of a motile Myxococcus cell in predatory contact with a C. crescentus cell. The movie was shot at ×100 magnification objective for 7 …
Video 12
NG-KilD cluster formation in contact with Salmonella typhimurium.
Shown is an overlay of the fluorescence and phase contrast images of a motile Myxococcus cell in predatory contact with an S. enterica Typhimurium cell. The movie was shot at ×100 magnification …
Video 13
NG-KilD cluster formation in contact with Bacillus subtilis.
Shown is an overlay of the fluorescence and phase contrast images of a motile Myxococcus cell in predatory contact with a B. subtilis cell. The movie was shot at ×100 magnification objective for 30 …
Video 14
Pseudomonas aeruginosa is not lysed by Myxococcus and does not induce NG-KilD cluster formation.
Shown is an overlay of the fluorescence and phase contrast images of a motile Myxococcus cells mixed with Pseudomonas cells. The movie was shot at ×100 magnification objective for 30 min. Pictures …
Additional files
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Supplementary file 1
Table 1: Characteristics of Myxococcus xanthus proteins.
- https://cdn.elifesciences.org/articles/72409/elife-72409-supp1-v2.docx
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Supplementary file 2
Table 2: M. xanthus proteins with homologs identified in B. bacteriovorus HD100, C. crescentus CB15 and B. sediminis.
- https://cdn.elifesciences.org/articles/72409/elife-72409-supp2-v2.docx
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Supplementary file 3
Table 3: Strains.
- https://cdn.elifesciences.org/articles/72409/elife-72409-supp3-v2.docx
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Supplementary file 4
Table 4: Plasmids.
- https://cdn.elifesciences.org/articles/72409/elife-72409-supp4-v2.docx
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Supplementary file 5
Table 5: Primers.
- https://cdn.elifesciences.org/articles/72409/elife-72409-supp5-v2.docx
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Transparent reporting form
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