Global phenotypic profiling identifies a conserved actinobacterial cofactor for a bifunctional PBP-type cell wall synthase
- Department of Microbiology, Harvard Medical School, United States
- Howard Hughes Medical Institute, United States
Figures
Figure 1 with 1 supplement
Phenotypic profiling of a Corynebacterium glutamicum transposon mutant library.
(A) Overview of the phenotypic profiling procedure. A transposon mutagenized library of Cglu MB001 was exposed to sub-MIC concentrations of the indicated antibiotics or to the listed stress …
Figure 1—figure supplement 1
Example growth curves for Cglu treated with selected drugs.
(A) Cultures of wild-type Cglu were grown for 24–36 hr from a starting OD600 of 2.5 × 10−6 in BHI with the addition of the indicated concentration of drug. The culture OD600 was measured every 5 min …
Figure 2 with 1 supplement
Clustering analysis of phenotypic profiles identifies genes with related functions.
(A) Heatmap showing clustered phenotypic profiles for all non-essential genes in Cglu. The test conditions are oriented along the abscissa and are ordered by the stress or the physiological process …
-
Figure 2—source data 1
Phenotypic profiling results.
The gene fitness for each gene under each growth condition is listed.
- https://cdn.elifesciences.org/articles/54761/elife-54761-fig2-data1-v2.xlsx
Figure 2—figure supplement 1
A genetic locus with a potential role in undecaprenol biogenesis or utilization.
(A) Shown is the transposon insertion profile for the cgp_3012–3020 genomic locus for untreated (top, -vanc) and vancomycin-treated (+vanc, 0.4 µg/ml, bottom) samples. (B) Phenotypic profiles for …
Figure 3 with 1 supplement
CofA is required for PBP1a accumulation.
(A) Phenotypic profiles of cofA (cgp_0016), ponA (cgp_0336, encoding PBP1a), and ponB (cgp_3313, encoding PBP1b) displayed as in Figure 2. Note that cofA and ponA clustered tightly together in the …
Figure 3—figure supplement 1
Functionality of mScar fusion proteins and localization of catalytically defective PBP1a variants.
(A) Cultures of wild-type, ΔponA, and ΔcofA strains harboring the indicated mScar fusion under theophylline-inducible control were grown and plated as in Figure 3. The concentration of agar …
Figure 4
CofA specifically interacts with PBP1a.
Shown are results from the POLAR two-hybrid assay with proteins expressed in E. coli cells. Bait proteins were fused with GFP and the H3H4 peptide to target them to polar assemblies of the PopZ …
Figure 5 with 1 supplement
Interaction with CofA is required for polar localization of PBP1a.
(A) Bocillin labeling of mScar-PBP1a in the indicated strains. Production of the mScar fusions was induced with 0.3 mM theophylline as in Figure 3. For this gel, 7.5 μg of total protein was loaded …
Figure 5—figure supplement 1
CofA does not interact with PBP1a with a heterologous transmembrane domain.
(A) POLAR two-hybrid assay assessing the interaction of Cglu mScar-CofA prey with GFP-EcTM-PBP1a bait. Results are displayed as in Figure 4.
Figure 6 with 1 supplement
The CofA-PBP1a interaction is conserved.
(A) Phylogenetic tree showing the distribution of CofA-like proteins containing the DUF3566 domain. (B) Schematics showing the genomic organization of loci encoding cofA-like genes in representative …
Figure 6—figure supplement 1
Interaction of full length MtbCofA with PonA2.
(A) POLAR two-hybrid assay assessing the interaction of GFP-MtbPonA2-TM or GFP-MtbPonA1-TM baits with full-length mScar-MtbCofA prey. Results are displayed as in Figure 4.
Tables
Key resources table
| Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional information |
|---|---|---|---|---|
| Strain, strain background (Escherichia coli) | DH5α(λpir) | Gibco BRL | F– hsdR17 deoR recA1 endA1 phoA supE44 thi-1 gyrA96 relA1 Δ(lacZYA-argF)U169 ϕ80dlacZΔM15 ****add pir | |
| Strain, strain background (Escherichia coli) | TB28 | (Bernhardt and de Boer, 2003) | MG1655 ΔlacIZYA::frt | |
| Strain, strain background (Corynebacterium glutamicum) | MB001 | (Baumgart et al., 2013) | ATCC 13032 ΔCGP1 (cg1507-cg1524) ΔCGP2 (cg1746-cg1752) ΔCGP3 (cg1890-cg2071) | |
| Strain, strain background (Corynebacterium glutamicum) | HL18 | This work | MB001 ΔponA | Bernhardt lab (MB001/pHCL86, see Materials and methods) |
| Strain, strain background (Corynebacterium glutamicum) | JS5 | This work | MB001 Δcgp_3012-cgp_3020 | Bernhardt lab (MB001/pJWS1, see Materials and methods) |
| Strain, strain background (Corynebacterium glutamicum) | JS6 | This work | MB001 Δcgp_3019 | Bernhardt lab (MB001/pJWS2, see Materials and methods) |
| Strain, strain background (Corynebacterium glutamicum) | JS7 | This work | MB001 Δcgp_3018 | Bernhardt lab (MB001/pJWS3, see Materials and methods) |
| Strain, strain background (Corynebacterium glutamicum) | JS8 | This work | MB001 Δcgp_0016 | Bernhardt lab (MB001/pJWS4, see Materials and methods) |
| Strain, strain background (Corynebacterium glutamicum) | JS10 | This work | MB001 Δcgp_0016 ΔponA | Bernhardt lab (JS8/pHCL86, see Materials and methods) |
| Recombinant DNA reagent | pJWS1 | This work | KanR, pCRD206 derivative containing an insert covering upstream and downstream of cgp_3012-cgp_3020. | Bernhardt lab (see Supplementary file 2) |
| Recombinant DNA reagent | pJWS2 | This work | KanR, pCRD206 derivative containing an insert covering upstream and downstream of cgp_3019. | Bernhardt lab (see Supplementary file 2) |
| Recombinant DNA reagent | pJWS3 | This work | KanR, pCRD206 derivative containing an insert covering upstream and downstream of cgp_3018. | Bernhardt lab (see Supplementary file 2) |
| Recombinant DNA reagent | pJWS4 | This work | KanR, pCRD206 derivative containing an insert covering upstream and downstream of cgp_0016 (cofA). | Bernhardt lab (see Supplementary file 2) |
| Recombinant DNA reagent | pJWS18 | This work | KanR, pK-PIM derivative encoding Psod-riboE1-mscar-cgp_0016 (cofA) | Bernhardt lab (see Supplementary file 2) |
| Recombinant DNA reagent | pJWS19 | This work | KanR, pK-PIM derivative encoding Psod-riboE1-mscar-cgp_0336 (ponA) | Bernhardt lab (see Supplementary file 2) |
| Recombinant DNA reagent | pJWS97 | This work | KanR, pK-PIM derivative encoding Psod-riboE1-mscar-C. gluponA (E. coliTM) | Bernhardt lab (see Supplementary file 2) |
| Recombinant DNA reagent | pJWS102 | This work | KanR, pK-PIM derivative encoding Psod-riboE1-mscar-cgp_0336 (ponA) GT- (E97A) | Bernhardt lab (see Supplementary file 2) |
| Recombinant DNA reagent | pJWS103 | This work | KanR, pK-PIM derivative encoding Psod-riboE1-mscar-cgp_0336 (ponA) TP- (S393A) | Bernhardt lab (see Supplementary file 2) |
| Recombinant DNA reagent | pJWS104 | This work | KanR, pK-PIM derivative encoding Psod-riboE1-mscar-cgp_0336 (ponA) GT- and TP- (E97A and S393A) | Bernhardt lab (see Supplementary file 2) |
| Recombinant DNA reagent | pHCL86 | (Lim et al., 2019) | KanR, pCRD206 derivative containing an insert covering upstream and downstream of cgp_0336 (ponA). | |
| Recombinant DNA reagent | pHCL149 | (Lim and Bernhardt, 2019) | CmR, Para-popZ-rbs-H3H4-msfGFPN-tmponB. | |
| Recombinant DNA reagent | pHCL152 | (Lim and Bernhardt, 2019) | TetR, lacI-Plac-mscar. | |
| Recombinant DNA reagent | pJWS29 | This work | CmR, Para-popZ-rbs-H3H4-msfGFP-ponA | Bernhardt lab (see Supplementary file 2) |
| Recombinant DNA reagent | pJWS41 | This work | TetR, lacI-Plac-mscar-cgp_0016 | Bernhardt lab (see Supplementary file 2) |
| Recombinant DNA reagent | pJWS70 | This work | TetR, lacI-Plac-mscar-jk0012 | Bernhardt lab (see Supplementary file 2) |
| Recombinant DNA reagent | pJWS73 | This work | CmR Para-popZ-rbs-H3H4-msfGFP-ponATM | Bernhardt lab (see Supplementary file 2) |
| Recombinant DNA reagent | pJWS75 | This work | CmR, Para-popZ-rbs-H3H4-msfGFP-ponASwap | Bernhardt lab (see Supplementary file 2) |
| Recombinant DNA reagent | pJWS78 | This work | CmR, Para-popZ-rbs-H3H4-jk1977TM | Bernhardt lab (see Supplementary file 2) |
| Recombinant DNA reagent | pJWS80 | This work | CmR, Para-popZ-rbs-H3H4-msfGFP-ponB | Bernhardt lab (see Supplementary file 2) |
| Recombinant DNA reagent | pJWS81 | This work | CmR, Para-popZ-rbs-H3H4-ponA2TM | Bernhardt lab (see Supplementary file 2) |
| Recombinant DNA reagent | pJWS83 | This work | TetR, lacI-Plac-mscar-rv0007FL | Bernhardt lab (see Supplementary file 2) |
| Recombinant DNA reagent | pJWS88 | This work | CmR, Para-popZ-rbs-H3H4-ponA1TM | Bernhardt lab (see Supplementary file 2) |
| Recombinant DNA reagent | pJWS90 | This work | CmR, Para-popZ-rbs-H3H4-jk2069TM | Bernhardt lab (see Supplementary file 2) |
| Recombinant DNA reagent | pJWS114 | This work | TetR, lacI-Plac-mscar-rv0007ΔN | Bernhardt lab (see Supplementary file 2) |
| Recombinant DNA reagent | pJWS119 | This work | CmR, Para-popZ-rbs-H3H4-C. gluponA (E. coliTM) | Bernhardt lab (see Supplementary file 2) |
| Chemical compound | Bocillin | ThermoFisher Scientific | BOCILLIN FL Penicillin, Sodium Salt |
Additional files
-
Supplementary file 1
Growth conditions for the phenotypic profiling.
The different growth conditions used for the profiling analysis are listed. Samples 1 g_A, B, and C, correspond to those sequenced to analyze the transposon insertion profile in the original library following one generation of growth. Similarly, 11 g_A and 11 g_B correspond to samples grown for 11 generations without treatment.
- https://cdn.elifesciences.org/articles/54761/elife-54761-supp1-v2.docx
-
Supplementary file 2
Plasmid construction methods.
- https://cdn.elifesciences.org/articles/54761/elife-54761-supp2-v2.docx
-
Transparent reporting form
- https://cdn.elifesciences.org/articles/54761/elife-54761-transrepform-v2.docx
