Contact-dependent killing by Caulobacter crescentus via cell surface-associated, glycine zipper proteins
- Massachusetts Institute of Technology, United States
- Howard Hughes Medical Institute, Massachusetts Institute of Technology, United States
Figures
Figure 1 with 1 supplement
The cdz gene cluster is very highly expressed in stationary phase.
(A) Genome-wide RNA-seq profiles of C. crescentus CB15N grown in rich medium to exponential (top) or stationary (bottom) phase. Expression levels (RPM) for both strands combined are plotted as a …
Figure 1—figure supplement 1
Growth curve for cells expressing a PcdzC-YFP reporter; see Fig.
https://doi.org/10.7554/eLife.24869.003
Figure 2 with 1 supplement
The cdz gene cluster encodes a two-peptide bacteriocin, secreted via a type I secretion system, and a small transmembrane immunity protein.
(A) Co-culture competition of wild-type C. crescentus and ∆cdzABCDI in rich medium. Strains were mixed 1:1 in exponential phase and co-cultured, with CFUs of each strain measured up to 14 hr. (B) …
Figure 2—figure supplement 1
Control experiments for the genetic dissection of the Cdzc system.
(A) Co-culture competition of a wild-type producer and ∆cdzABCDI indicator showing that the antibiotic selection marker does not affect the outcome of the competition. (B) Co-culture competition of …
Figure 3 with 1 supplement
CdzC and CdzD kill an indicator strain in co-culture.
(A) Microscopy analysis of aliquots taken from co-culture competitions of a wild-type producer synthesizing CFP (false colored blue) and a ∆cdzCDI indicator synthesizing YFP (false colored green), …
Figure 3—figure supplement 1
Control experiments for the microscopy-based analysis of Cdz-dependent killing.
(A) Phase and epifluorescence microscopy of aliquots taken from a co-culture competition of a wild-type producer synthesizing YFP (green) and a ∆cdzCDI indicator synthesizing CFP (blue), with the …
Figure 4 with 1 supplement
Cdz-mediated killing requires cell-cell contact between a producer and an indicator.
(A) Survival of a ∆cdzABCDI indicator when treated for 16 hr with live producer cells, producer culture supernatant, producer culture supernatant concentrated 2000X by trichloroacetic acid …
Figure 4—figure supplement 1
Control experiments for the analysis of contact-dependent inhibition.
(A) Western blot of HA-EdtC in concentrated (TCA-precipitated) culture supernatant. (B) Flow cytometry analysis of stationary phase cells expressing Venus or TdTomato, as indicated, either in pure …
Figure 5 with 1 supplement
CdzC and CdzD localize to the outer membranes of cells, while CdzI is an inner membrane protein.
(A) Western blots of the indicated cell fractions from a culture of cells producing HA-tagged CdzC and grown to stationary phase. RpoA, McpA, and RsaFa serve as controls for cytoplasmic, inner …
Figure 5—figure supplement 1
Epitope-tagging of CdzC and CdzD does not affect their toxicity.
(A) Competitions in which the producer strain was ∆cdzCDI harboring a low copy number plasmid expressing epitope-tagged CdzC or CdzD, as indicated, with the rest of the operon untagged. The …
Figure 6 with 2 supplements
CdzC aggregates on the surface of producer cells.
Immunogold labelling and transmission electron microscopy of the stationary phase producer cells indicated, each expressing an epitope-tagged CdzC. Middle and right images on each row are zoomed in …
Figure 6—figure supplement 1
CdzC aggregates on the surface of wild-type producer cells.
(A) Immunogold labelling and transmission electron microscopy of stationary phase wild-type producer cells expressing an epitope-tagged CdzC. Middle and right images are zoomed in regions noted on …
Figure 6—figure supplement 2
Immunofluorescence of the producer cells indicated, each expressing an epitope-tagged CdzC.
Stationary phase cells of (A) a ∆manB ∆rsaA producer strain and (B) a ∆cdzAB ∆manB ∆rsaA strain.
Figure 7 with 1 supplement
Phylogenetic distribution of putative Cdz-like systems in bacteria.
(A) Multiple sequence alignment used as the seed in an iterative computational search for Cdz-like systems (see Materials and methods). The presumed leader peptide of each sequence is indicated. …
Figure 7—figure supplement 1
Competitions between a wild-type or ∆cdzABCDI Caulobacter crescentus producer and the indicator species shown: ∆cdz Caulobacter crescentus, Caulobacter segnis, Brevundimonas subvibrioides, Asticcacaulis excentricus, Rhodobacter sphaeroides, Sinorhizobium meliloti, Agrobacterium tumefaciens, or E. coli.
In each panel, data points represent the mean of at least three independent experiments; error bars indicate S.D. Tree on the left illustrates phylogenetic relationship between groups, branch …
Additional files
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Supplementary file 1
Cdz-like peptides identified in UniprotKB.
- https://doi.org/10.7554/eLife.24869.017
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Supplementary file 2
Oligonucleotides.
- https://doi.org/10.7554/eLife.24869.018
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Supplementary file 3
Strains.
- https://doi.org/10.7554/eLife.24869.019
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Supplementary file 4
Plasmids.
- https://doi.org/10.7554/eLife.24869.020
