Phage resistance profiling identifies new genes required for biogenesis and modification of the corynebacterial cell envelope
- Department of Microbiology, Harvard Medical School, United States
- Howard Hughes Medical Institute, United States
Figures
Figure 1 with 1 supplement
The Corynebacterium glutamicum (Cglu) envelope and adaptation of corynephages to the lab strain.
(A) Cartoon of the multi-layered cell envelope of Cglu and other members of the Corynebacteriales. The mycomembrane is composed of mycolic acids covalently attached to the arabinogalactan layer or …
-
Figure 1—source data 1
Single nucleotide polymorphisms (SNPs) detected in phages adapted to MB001.
This table contains the whole-genome sequencing data for the lab-adapted phages.
- https://cdn.elifesciences.org/articles/79981/elife-79981-fig1-data1-v2.docx
-
Figure 1—source data 2
Adsorption source data.
This spreadsheet contains the data used to create Figure 1C.
- https://cdn.elifesciences.org/articles/79981/elife-79981-fig1-data2-v2.xlsx
Figure 1—figure supplement 1
Adaptation of Cog phage to lab Corynebacterium glutamicum (Cglu) strain MB001 and genome sequencing.
(A) Cog forms plaques on its environmental corynebacterial host LP-6 (top left plate) but fails to form a plaque on the lab Cglu strain MB001 grown on brain-heart infusion (BHI) medium (middle plate …
Figure 2
CL31 and Cog have distinct requirements for host envelope components.
The heatmap shows genes that displayed a log2 fold change in transposon insertion reads of >5 in the transposon sequencing (Tn-seq) analysis following challenge with the indicated phage. Each phage …
-
Figure 2—source data 1
Phage challenge transposon sequencing (Tn-Seq) metadata.
This table contains the metadata associated with the Tn-Seq runs.
- https://cdn.elifesciences.org/articles/79981/elife-79981-fig2-data1-v2.docx
-
Figure 2—source data 2
Transposon sequencing results.
This spreadsheet contains the data acquired from the transposon sequencing experiments.
- https://cdn.elifesciences.org/articles/79981/elife-79981-fig2-data2-v2.xlsx
-
Figure 2—source data 3
Transposon enrichment source data.
This spreadsheet contains the data used to create Figure 2.
- https://cdn.elifesciences.org/articles/79981/elife-79981-fig2-data3-v2.xlsx
Figure 3 with 1 supplement
Cog requires genes implicated in arabinogalactan modification to infect Corynebacterium glutamicum (Cglu).
(A) Transposon sequencing (Tn-Seq) insertion profiles for a locus encoding genes with potential functions in the modification of the Cglu arabinogalactan with galactosamine (GalN). Genes enriched …
-
Figure 3—source data 1
Phenotypic profiling data.
This spreadsheet contains the data from Sher et al., 2020 used to make Figure 3C.
- https://cdn.elifesciences.org/articles/79981/elife-79981-fig3-data1-v2.xlsx
Figure 3—figure supplement 1
Transposon sequencing (Tn-Seq) insertion profiles for the cgp_0658 locus with and without Cog challenge.
The cgp_0658 gene encodes a putative glycosyltransferase involved in the galactosamine (GalN) modification of arabinogalactan.
Figure 4
Mycomembrane proteins are required for Cog infection.
(A,D) Transposon sequencing (Tn-Seq) insertion profile for the protX locus and the porAH locus, respectively. (B,E) Validation of the Tn-Seq results. Ten-fold serial dilutions of lysates for the …
-
Figure 4—source data 1
Adsorption and COI data.
This spreadsheet contains the data used to make Figure 4C and D.
- https://cdn.elifesciences.org/articles/79981/elife-79981-fig4-data1-v2.xlsx
Figure 5 with 1 supplement
Mycolic acids are required for CL31 infection.
(A) Cartoon of the mycolic acid synthesis pathway. Proteins outlined in burgundy were enriched for transposon insertions following CL31 challenge. (1) Long-chain fatty acids are synthesized by FadD2 …
Figure 5—figure supplement 1
CL31 phage requires genes involved in mycolic acid to infect Corynebacterium glutamicum (Cglu).
(A–C) Transposon sequencing (Tn-Seq) insertion profiles with and without challenge with the indicated phage for the ostA genetic locus.
Figure 6
CL31 likely requires a post-translationally modified protein for efficient infection.
(A) Transposon sequencing (Tn-Seq) insertion profiles of cmt1 following phage challenge. (B) Ten-fold serial dilutions of lysates for the indicated phages were prepared and 3 µL of each dilution …
-
Figure 6—source data 1
Adsorption data.
This spreadsheet contains the data used to make Figure 6C and D.
- https://cdn.elifesciences.org/articles/79981/elife-79981-fig6-data1-v2.xlsx
Figure 7 with 1 supplement
An uncharacterized gene, actinobacterial helix-grip fold A (ahfA) (cgp_0475), is required for CL31 adsorption.
(A) Transposon sequencing (Tn-Seq) insertion profile for the ahfA (cgp_0475) locus. (B) Ten-fold serial dilutions of lysates for the indicated phages were prepared and 3 µL of each dilution were …
-
Figure 7—source data 1
Single nucleotide polymorphisms (SNPs) in Corynebacterium glutamicum (Cglu) following challenge with WTCL31.
This table contains the whole-genome sequencing results from MB001 survivors following WTCL31 challenged.
- https://cdn.elifesciences.org/articles/79981/elife-79981-fig7-data1-v2.docx
-
Figure 7—source data 2
Adsorption data.
This spreadsheet contains the data used to make Figure 7D.
- https://cdn.elifesciences.org/articles/79981/elife-79981-fig7-data2-v2.xlsx
Figure 7—figure supplement 1
Actinobacterial helix-grip fold A (AhfA) is required for efficient CL31 infection and has homologs in other mycolic acid synthesizing bacteria.
(A) Diagram of the ahfA locus in Corynebacterium glutamicum (Cglu), Mycobacterium tuberculosis (Mtb), and Mycobacterium smegmatis (Msmeg). Notably, there are two ahfA alleles encoded in this locus …
Figure 8
Actinobacterial helix-grip fold A (AhfA) is required for mycolic acid synthesis.
(A) Wild-type (WT) cells expressing gfp or cells of the indicated strains lacking a fluorescent protein marker were grown to an OD600 between 0.15 and 0.4 depending on strain fitness, stained with …
-
Figure 8—source data 1
Uncropped microscopy images.
Wild-type (WT) cells expressing gfp or unlabeled Δpks (left) or ΔahfA (right) cells lacking a fluorescent protein marker were grown to an OD600 between 0.15 and 0.4 depending on strain fitness, stained with 6-TMR-Tre for 30 minutes, washed, mixed together, and then applied to an agarose pad for visualization by fluorescence microscopy. Scale bar represents 10 µm and white box indicates area cropped for enlarged figure.
- https://cdn.elifesciences.org/articles/79981/elife-79981-fig8-data1-v2.pdf
-
Figure 8—source data 2
6-TAMRA-Trehalose (6-TMR-Tre) data.
This spreadsheet contains the data used to make Figure 8B.
- https://cdn.elifesciences.org/articles/79981/elife-79981-fig8-data2-v2.xlsx
-
Figure 8—source data 3
TLC analysis of lipids extracted with chloroform/methanol from the indicated Corynebacterium glutamicum (Cglu) strains.
The plate was developed in chloroform:methanol:water (30:8:1), dipped in primuline, and spots visualized with UV. Key species are labeled: TMM, trehalose monomycolate; TDM, trehalose dimycolate; P-lipids, phospholipids. The strains in panels B and C contained an empty vector (EV) or the indicated gene constitutively expressed from the PSOD promoter.
- https://cdn.elifesciences.org/articles/79981/elife-79981-fig8-data3-v2.pdf
Tables
Key resources table
| Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional information |
|---|---|---|---|---|
| Strain, strain background (Echerichia coli) | F– hsdR17 deoR recA1 endA1 phoA supE44 thi-1 gyrA96 relA1 Δ(lacZYA-argF)U169 Φ80dlacZΔM15 ****add pir | Gibco BRL | DH5α | |
| Strain, strain background (Cglu, MB001) | MB001 (ATCC 13032 ΔCGP1 (cg1507-gp1524) ΔCGP2 (cg1746-1752) ΔCGP3 (cg1890-cg2071)) | Baumgart et al., 2013 | H60 | |
| Strain, strain background (Cglu) | Environmental Cglu isolate ATCC 15990 | Félix d'Hérelle Reference Center for Bacterial Viruses | ATCC 15990 | |
| Strain, strain background (Cglu) | Environmental Cglu isolate LP-6 | Félix d'Hérelle Reference Center for Bacterial Viruses | LP-6 | |
| Strain, strain background (Cglu, MB001) | ΔRM (Δcgp_0844) | This paper | H722 | Bernhardt Lab |
| Strain, strain background (Cglu, MB001) | ΔppgS (Δcgp_0394) | This paper | H2258 | Bernhardt Lab |
| Strain, strain background (Cglu, MB001) | Δcgp_0658 | This paper | H2259 | Bernhardt Lab |
| Strain, strain background (Cglu, MB001) | Δcgp_0396 | This paper | H2260 | Bernhardt Lab |
| Strain, strain background (Cglu, MB001) | ΔprotX (Δcgp_2875) | This paper | H1111 | Bernhardt Lab |
| Strain, strain background (Cglu, MB001) | ΔporBC (Δcgp_1108c) | This paper | H1239 | Bernhardt Lab |
| Strain, strain background (Cglu, MB001) | ΔporAH (Δcgp_3008–3009) | This paper | H1152 | Bernhardt Lab |
| Strain, strain background (Cglu, MB001) | ΔporH (Δcgp_3009) | This paper | H1241 | Bernhardt Lab |
| Strain, strain background (Cglu, MB001) | ΔporA (Δcgp_3008) | This paper | H1247 | Bernhardt Lab |
| Strain, strain background (Cglu, MB001) | ΔotsA (Δcgp_2907) | This paper | H1666 | Bernhardt Lab |
| Strain, strain background (Cglu, MB001) | Δpks (Δcgp_3178) | This paper | H2261 | Bernhardt Lab |
| Strain, strain background (Cglu, MB001) | Δcmt1 (Δcgp_0413) | This paper | H1664 | Bernhardt Lab |
| Strain, strain background (Cglu, MB001) | ΔahfA (Δcgp_0475) | This paper | H2262 | Bernhardt Lab |
| Strain, strain background (Cglu, MB001) | WT PSOD-ahfA | This paper | H2272 | pK-PIM derivative encoding PSOD-ahfA isothermally assembled and directly transformed into Cglu. |
| Strain, strain background (Cglu, MB001) | ΔahfA PSOD-ahfA | This paper | H2273 | pK-PIM derivative encoding PSOD-ahfA isothermally assembled and directly transformed into Cglu. |
| Strain, strain background (Bacteriophage) | envCL31 (phage isolated on Cglu ATCC 15990) | Félix d'Hérelle Reference Center for Bacterial Viruses Trautwetter et al., 1987 | HER#229 | |
| Strain, strain background (Bacteriophage) | envCog (phage isolated on Cglu LP-6) | Félix d'Hérelle Reference Center for Bacterial Viruses (Sonnen et al., 1990) | HER#360 | |
| Strain, strain background (Bacteriophage) | WTCL31 (T19334G) | This paper | ACMΦ2 | Bernhardt Lab |
| Strain, strain background (Bacteriophage) | CPCL31 (T19334G, T24296A) | This paper | ACMΦ9 | Bernhardt Lab |
| Strain, strain background (Bacteriophage) | Cog (A19202T) | This paper | ACMΦ8 | Bernhardt Lab |
| Recombinant DNA reagent (plasmid) | PSOD riboE EV, inducible (KanR, pK-PIM derivative encoding PSOD-riboE1 empty vector) | This paper | pACM185 | Bernhardt Lab, |
| Recombinant DNA reagent (plasmid) | PSOD riboE cmt1, inducible (KanR, pK-PIM derivative encoding PSOD-riboE1-cmt1) | This paper | pACM64 | Bernhartdt Lab, |
| Recombinant DNA reagent (plasmid) | KanR, pK-PIM derivative encoding PSOD-riboE1-cmt1 S189V (PSOD riboE cmt1 S189V, inducible) | This paper | pACM66 | Bernhardt Lab |
| Recombinant DNA reagent (plasmid) | PSOD EV, constitutive (KanR, pK-PIM derivative encoding PSOD-empty vector) | This paper | pEMH7 | Bernhardt Lab |
| Recombinant DNA reagent (plasmid) | PTAC-gfp, inducible (KanR, PTAC-eGFP, pGA1 mini replicon) | Ravasi et al., 2012 | pTGR5 | |
| Recombinant DNA reagent (plasmid) | KanR, pCRD206 derivative containing an insert covering upstream and downstream of cgp_0844 | This paper | pJWS51 | Bernhardt Lab |
| Recombinant DNA reagent (plasmid) | KanR, pCRD206 derivative containing an insert covering upstream and downstream of cgp_0394 (ppgS) | This paper | pACM285 | Bernhardt Lab |
| Recombinant DNA reagent (plasmid) | KanR, pCRD206 derivative containing an insert covering upstream and downstream of cgp_0658 | This paper | pACM286 | Bernhardt Lab |
| Recombinant DNA reagent (plasmid) | KanR, pCRD206 derivative containing an insert covering upstream and downstream of cgp_0396 | This paper | pACM284 | Bernhardt Lab |
| Recombinant DNA reagent (plasmid) | KanR, pCRD206 derivative containing an insert covering upstream and downstream of cgp_2875 (protX) | This paper | pEMH1 | Bernhardt Lab |
| Recombinant DNA reagent (plasmid) | KanR, pCRD206 derivative containing an insert covering upstream and downstream of cgp_1108-cgp_1109 (porBC) | This paper | pEMH52 | Bernhardt Lab |
| Recombinant DNA reagent (plasmid) | KanR, pCRD206 derivative containing an insert covering upstream and downstream of cgp_3008–3009 (porAH) | This paper | pEMH10 | Bernhardt Lab |
| Recombinant DNA reagent (plasmid) | KanR, pCRD206 derivative containing an insert covering upstream and downstream of cgp_3009 (porH) | This paper | pEMH9 | Bernhardt Lab |
| Recombinant DNA reagent (plasmid) | KanR, pCRD206 derivative containing an insert covering upstream and downstream of cgp_3008 (porA) | This paper | pEMH16 | Bernhardt Lab |
| Recombinant DNA reagent (plasmid) | KanR, pCRD206 derivative containing an insert covering upstream and downstream of cgp_2907 (otsA) | This paper | pACM30 | Bernhardt Lab |
| Recombinant DNA reagent (plasmid) | KanR, pCRD206 derivative containing an insert covering upstream and downstream of cgp_3178 (pks) | This paper | pACM175 | Bernhardt Lab |
| Recombinant DNA reagent (plasmid) | KanR, pCRD206 derivative containing an insert covering upstream and downstream of cgp_0413 (cmt1) | This paper | pACM20 | Bernhardt Lab |
| Recombinant DNA reagent (plasmid) | KanR, pCRD206 derivative containing an insert covering upstream and downstream of cgp_0475 (ahfA) | This paper | pACM230 | Bernhardt Lab |
| Chemical compound. drug | 6-TAMRA-Trehalose (6-TMR-Tre) | Tocris | 6802 |
