Dominant Vibrio cholerae phage exhibits lysis inhibition sensitive to disruption by a defensive phage satellite
- Department of Plant and Microbial Biology, University of California, United States
- Chan Zuckerberg Biohub, United States
Figures
Figure 1
Characterizing lysis inhibition.
Schematic of T4 infection of E. coli. (A) At low multiplicities of infection (MOI), available E. coli are readily infected by T4 (red). Under these conditions, an infected cell is hijacked to …
-
Figure 1—source data 1
This spreadsheet contains the data used to create Figure 1C and D.
- https://cdn.elifesciences.org/articles/53200/elife-53200-fig1-data1-v1.xlsx
Figure 2
Identification and characterization of ICP1’s holin TeaA.
(A) Schematic of canonical T4 lysis in E. coli. Lysis occurs in four steps with a potential delay caused by lysis inhibition. Step one: Phage encoded proteins including holins, endolysins, and …
-
Figure 2—source data 1
Fluorescence Source Data.
This spreadsheet contains the data used to create Figures 2C and 4C.
- https://cdn.elifesciences.org/articles/53200/elife-53200-fig2-data1-v1.xlsx
-
Figure 2—source data 2
DNP Source Data.
This spreadsheet contains the data used to create Figures 2D and 4D.
- https://cdn.elifesciences.org/articles/53200/elife-53200-fig2-data2-v1.xlsx
Figure 3 with 1 supplement
Antiholin ArrA identification and characterization.
(A) Initial plaquing of wild type ICP1 on CRISPR-Cas (+) V. cholerae targeting arrA yielded a mixture of plaque phenotypes. (B) Plaques with clear edges can be found in populations of phage that …
-
Figure 3—source data 1
This spreadsheet contains the data used to create Figure 3D.
- https://cdn.elifesciences.org/articles/53200/elife-53200-fig3-data1-v1.xlsx
Figure 3—figure supplement 1
Proteins with similarity to TeaA.
This unrooted tree shows proteins identified with BLASTP that share 30% identity with TeaA over 85% of the query. The source of the sequence is shown in the label on the tree while colored blocks …
Figure 4 with 3 supplements
Accelerated lysis by PLE and LidI.
(A) The optical density (OD600) of ICP1 MOI = 5 infections was followed in PLE (-), PLE 1, and PLE 1 ΔlidI V. cholerae strains as well as V. cholerae strains containing induced empty vector (EV) and …
-
Figure 4—source data 1
This spreadsheet contains the data used to create Figure 4A and E.
- https://cdn.elifesciences.org/articles/53200/elife-53200-fig4-data1-v1.xlsx
Figure 4—figure supplement 1
PRALINE alignment of LidI homologs.
LidI homologs cluster neatly in two groups: (A) LidIPLE 1 and LidIPLE 2 are short 66 amino acid predicted proteins with four synonymous SNPs between them, while (B) lidIPLE 3, lidIPLE 4, and lidIPLE …
Figure 4—figure supplement 2
Complete Western Blot.
Tagged LidIPLE 1 was visualized via western blot. EV and FLAG-LidIPLE 1 expressed in trans served as controls showing FLAG-LidIPLE 1 specificity as well as the presence of background bands. In PLE 1 …
Figure 4—figure supplement 3
Redundancy in the PLE.
(A) PLE one and PLE 1 ΔlidI were infected with ICP1 (MOI = 1) followed by superinfection (MOSI = 5; arrow) showing lysis inhibition is collapsed by PLE in the face of exogenously added phage. Every …
Figure 5
LidI functions through lysis inhibition disruption.
(A) Optical density (D600) of empty vector (EV) or LidIPLE 1 expressing cultures after infection with different initial multiplicities of infection (highest MOI top to lowest MOI bottom). Data …
-
Figure 5—source data 1
This spreadsheet contains the data used to create Figure 5.
- https://cdn.elifesciences.org/articles/53200/elife-53200-fig5-data1-v1.xlsx
Figure 6 with 4 supplements
LidI puts a bottleneck on phage populations.
(A and B) Phage infection yields measured in plaque forming units per mL (PFU/mL) were determined from V. cholerae with empty vector (EV), lidIPLE 1, and lidIPLE 4 constructs induced at the …
-
Figure 6—source data 1
This spreadsheet contains the data used to create Figure 6.
- https://cdn.elifesciences.org/articles/53200/elife-53200-fig6-data1-v1.xlsx
Figure 6—figure supplement 1
Accelerated lysis and phage yield after low MOI infections.
(A, B and C) Optical density (OD600) of empty vector (EV) or LidIPLE 1-expressing cultures after infection at MOI = 0.001 (A), MOI = 0.0001 (B), and MOI 0.00001 (C). Data points represent the …
-
Figure 6—figure supplement 1—source data 1
This spreadsheet contains the data used to create Figure 6—figure supplement 1.
- https://cdn.elifesciences.org/articles/53200/elife-53200-fig6-figsupp1-data1-v1.xlsx
Figure 6—figure supplement 2
Diversity of progeny phage from infections as measured by phage escape from CRISPR-Cas – expanded schematic.
1. V. cholerae strains with and without LidIPLE 1 (top/purple and bottom/grey, respectively) were exposed to ICP1 (yellow). 2. 90 minutes post-infection, infected cultures were mechanically lysed to …
Figure 6—figure supplement 3
Successful recombination of progeny phage from infections as measured by phage-encoded CRISPR-Cas overcoming PLE – expanded schematic.
A We engineered strains of ICP1 encoding a Type I-F CRISPR-Cas system to lack nuclease activity by deleting Cas2-3 in the CRISPR-Cas* phage (yellow) and disrupt targeting by deleting the spacers and …
Figure 6—figure supplement 4
EOP of evolution experiments in the presence of LidIPLE 1.
(A) Efficiency of plaquing (EOP) was determined between plaque forming units (PFUs) from LidIPLE 1V. cholerae infections and PFUs from EV V. cholerae infections plaqued on CRISPR-Cas (+) V. cholerae …
-
Figure 6—figure supplement 4—source data 1
This spreadsheet contains the data used to create Figure 6—figure supplement 4.
- https://cdn.elifesciences.org/articles/53200/elife-53200-fig6-figsupp4-data1-v1.xlsx
Tables
Table 1
Acronyms.
All the acronyms used in this work are listed in alphabetical order.
| Acronym | Meaning |
|---|---|
| DiOC2(3) | 3,3’-diethloxacarbocyanine iodide |
| DNP | 2,4-dinitrophenol |
| EOP | efficiency of plaquing |
| EV | empty vector |
| gp | gene product |
| IM | inner membrane |
| LIN | lysis inhibition |
| MGE | mobile genetic element |
| MOI | multiplicity of infection |
| MOSI | multiplicity of superinfection |
| OD | optical density |
| OM | outer membrane |
| ORF | open reading frame |
| PG | peptidoglycan |
| PLE | phage-inducible chromosomal island-like element |
| SaPI | Staphylococcus aureus pathogenicity island |
| WT | wild type |
Key resources table
| Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional information |
|---|---|---|---|---|
| Gene (Vibrio cholerae) | lidIPLE 1 (PLE 1 ORF20.1) | * | ||
| Gene (Vibrio cholerae) | lidIPLE 2 (PLE 2 ORF24.1) | * | ||
| Gene (Vibrio cholerae) | lidIPLE 3 (PLE 3 ORF24.1) | * | ||
| Gene (Vibrio cholerae) | lidIPLE 4 (PLE 4 ORF26) | (O'Hara et al., 2017) | ||
| Gene (Vibrio cholerae) | lidIPLE 5 (PLE 5 ORF26) | (O'Hara et al., 2017) | ||
| Gene (bacteriophage ICP1) | teaAICP1 (gp137) | (Angermeyer et al., 2018),* | ||
| Gene (bacteriophage ICP1) | arrAICP1 (gp138) | (Angermeyer et al., 2018),* | ||
| Strain (Vibrio cholerae) | PLE (-) V. cholerae (E7946) | (Levine et al., 1982) | KDS 6 | |
| Strain (Vibrio cholerae) | PLE 1 V. cholerae (PLE 1 E7946) | (O'Hara et al., 2017) | KDS 36 | |
| Strain (Vibrio cholerae) | ΔlacZ::Ptac-EV (E7946) | (McKitterick and Seed, 2018) | KDS 116 | |
| Strain (Vibrio cholerae) | ΔlacZ::Ptac-lidIPLE 1 (E7946) | * | KDS 139 | |
| Strain (Vibrio cholerae) | ΔlacZ::Ptac-lidIPLE 4 (E7946) | * | KDS 267 | |
| Strain (Vibrio cholerae) | PLE 1 FLAG-LidIPLE 1 (E7946) | * | KDS 268 | |
| Strain (Vibrio cholerae) | PLE 1 ΔlidI (E7946) | * | KDS 170 | |
| Strain (Vibrio cholerae) | PLE 4 ΔlidI (E7946) | * | KDS 269 | |
| Strain (Vibrio cholerae) | CRISPR-Cas (+) (E7946) | (Box et al., 2016) | KDS 112 | Inducible Cas Proteins |
| Recombinant DNA reagent (plasmid) | Ptac-Empty Vector (pKL06 in E7946) | (McKitterick and Seed, 2018) | KDS 196 | Empty Vector Control |
| Recombinant DNA reagent (plasmid) | Ptac-lidIPLE 1 (plasmid in E7946) | * | KDS 219 | Inducible lidIPLE 1 |
| Recombinant DNA reagent (plasmid) | Ptac-lidIPLE 4 (plasmid in E7946) | * | KDS 270 | Inducible lidIPLE 4 |
| Recombinant DNA reagent (plasmid) | Ptac-teaAICP1 (plasmid in E7946) | * | KDS 271 | Inducible teaAICP1 |
| Recombinant DNA reagent (plasmid) | Ptac-arrAICP1 (plasmid in E7946) | * | KDS 272 | Inducible arrAICP1 |
| Recombinant DNA reagent (plasmid) | Ptac-tT4 (plasmid in E7946) | * | KDS 273 | Inducible tT4 |
| Recombinant DNA reagent (plasmid) | Ptac-anti-gp138 spacer (plasmid in E7946) | * | KDS 274 | CRISPR array containing anti-gp138 spacer |
| Recombinant DNA reagent (plasmid) | Ptac-anti-gp138 spacer and repair template | * | KDS 275 | CRISPR array containing anti-gp138 spacer and repair template |
| Recombinant DNA reagent (plasmid) | Ptac-FLAG-lidIPLE 1 | * | KDS 276 | Inducible FLAG-tagged blot control |
| Recombinant DNA reagent (plasmid) | Ptac-none (CRISPR array with no spacers against WT ICP1) | (McKitterick et al., 2019b) | KDS 277 | Spacer control |
| Recombinant DNA reagent (plasmid) | Ptac-spacer A | * | KDS 278 | Spacer A against ICP1 |
| Recombinant DNA reagent (plasmid) | Ptac-spacer B | * | KDS 279 | Spacer B against ICP1 |
| Recombinant DNA reagent (plasmid) | Ptac-spacer C | * | KDS 280 | Spacer C against ICP1 |
| Strain (bacteriophage ICP1) | ICP1 (ICP1 2006E ΔCRISPR ΔCas) | (McKitterick and Seed, 2018) | ||
| Strain (bacteriophage ICP1) | ΔarrA ICP1 (ICP1 2006E ΔarrA/gp137) | * | SGH Φ 61 | |
| Strain (bacteriophage ICP1) | CRISPR*-Cas ICP1 (ICP1 2011A Δspacer2-9 Cas1D244A) | (McKitterick et al., 2019b) | ACM Φ 232 | |
| Strain (bacteriophage ICP1) | CRISPR-Cas* ICP1 (ICP1 2011A Δcas2-3) | * | SGH Φ 62 | |
| Chemical compound | Isopropyl-beta-D-thiogalactoside (IPTG) | GoldBio | 12481C5 | |
| Chemical compound | Theophylline | Sigma-Aldrich | T1633-100G | |
| Chemical compound | 2,4-Dinitrophenol (DNP) | Sigma-Aldrich | D198501-100G | |
| Chemical compound | 3,3-Diethyloxacarbocyanine iodide (DiOC2(3)) | Sigma-Aldrich | 320684–1G | |
| Antibody | Rabbit anti-FLAG polyclonal antibody | Sigma-Aldrich | RRID:SAB4301135 | |
| Antibody | Goat anti-Rabbit IgG antibody, peroxidase conjugated | Sigma-Aldrich | RRID:AP132P |
-
*Identified or created in this work.
Additional files
-
Source data 1
This spreadsheet contains the data used to create Supplementary files 2 and 3.
- https://cdn.elifesciences.org/articles/53200/elife-53200-data1-v1.xlsx
-
Supplementary file 1
ICP1_2006_E gene product (gp) GenBank References.
The gene products referred to in this work relate to open reading frames (ORFs) as noted in the ‘Locus Tag Note’.
- https://cdn.elifesciences.org/articles/53200/elife-53200-supp1-v1.docx
-
Supplementary file 2
TeaA homologs.
BLASTP was used to find homologs that share 30% identity with TeaA over 85% of the query. The GenBank ID, description, number of transmembrane domains (TMD) as predicted by TMHMM Server 2.0, and organism is listed for each homolog. Whether or not an ArrA homolog was found in the same organism is noted in the ‘ArrA’ column. Additionally, the adjacent upstream and downstream genes were analyzed for TMDs. GenBank descriptions are color coded. Due to the number of homologs analyzed, this table is only available as a spreadsheet as Source data 1.
- https://cdn.elifesciences.org/articles/53200/elife-53200-supp2-v1.docx
-
Supplementary file 3
ArrA homologs.
BLASTP was used to find proteins with 20% identity to ArrA over 75% of the query. The GenBank ID, description, number of transmembrane domains (TMD) as predicted by TMHMM Server 2.0, and organism is listed for each homolog. Whether or not a TeaA homolog was found in the same organism is noted in the ‘TeaA’ column. Additionally, the adjacent upstream and downstream genes of each homolog were analyzed for TMDs. GenBank descriptions are color coded. The source data for this table is available in Source data 1.
- https://cdn.elifesciences.org/articles/53200/elife-53200-supp3-v1.docx
-
Supplementary file 4
Primer Table.
Primers used in this work are provided with a description, identifier, and sequence.
- https://cdn.elifesciences.org/articles/53200/elife-53200-supp4-v1.xlsx
-
Transparent reporting form
- https://cdn.elifesciences.org/articles/53200/elife-53200-transrepform-v1.docx
