Investigating the composition and recruitment of the mycobacterial ImuA′–ImuB–DnaE2 mutasome
- SAMRC/NHLS/UCT Molecular Mycobacteriology Research Unit, DSI/NRF Centre of Excellence for Biomedical TB Research, Department of Pathology, University of Cape Town, South Africa
- Institute of Infectious Disease and Molecular Medicine, University of Cape Town, South Africa
- Laboratory of Microbiology and Microsystems, School of Life Sciences, Swiss Federal Institute of Technology in Lausanne (EPFL), Switzerland
- Advanced Imaging Center, Howard Hughes Medical Institute, United States
- Department of Cell and Chemical Biology, Leiden University Medical Center, Netherlands
- Department of Integrative Biomedical Sciences, University of Cape Town, South Africa
- Confocal and Light Microscope Imaging Facility, Department of Human Biology, University of Cape Town, South Africa
- Helmholtz Centre for Infection Research, Helmholtz Institute for Pharmaceutical Research Saarland, Germany
- German Centre for Infection Research (DZIF), Partner Site Hannover-Braunschweig, Germany
- Laboratory of Genomic Integrity, Eunice Kennedy Shriver National Institute of Child Health and Human Development, United States
- Wellcome Centre for Infectious Diseases Research in Africa, University of Cape Town, South Africa
- Institute of Biotechnology, Vilnius University, Lithuania
Figures
Figure 1 with 1 supplement
Visualization of the mycobacterial mutasome components.
Representative stills from fluorescence microscopy experiments of M. smegmatis expressing translational reporters of the different mutasome components in their respective knockout backgrounds. …
Figure 1—figure supplement 1
Design and representative fluorescent images of the mutasome translational reporters.
(A) Construction of the reporter mutants, indicating: (i) the genomic organization of the wild-type M. smegmatis imuA′–imuB operon; (ii) the PSOS(imuA′)-egfp-imuB construct encoding G-ImuB; (iii) …
Figure 2 with 1 supplement
Functional validation of translational reporters.
(A) N-terminally tagged fluorescence reporter mutants of M. smegmatis ImuA′, ImuB, and DnaE2 retain function in DNA damage-induced mutagenesis. Cultures of M. smegmatis deletion mutants and …
Figure 2—figure supplement 1
V-ImuA’ and G-ImuB fluorescent bioreporters restore ultra-violet (UV)-induced DNA damage sensitivities of the corresponding ΔimuA’ and ΔimuB deletion mutants.
For this assay, a 10-fold dilution series was spotted on standard, antibiotic-free 7H10 plates, allowed to dry, and then exposed to UV at the indicated doses, after which plates were incubated at …
Figure 3 with 2 supplements
Representative time-lapse series of single cells of M. smegmatis expressing the mutasome reporters in combination with mCherry-DnaN.
(A) G-ImuB (green) and mCherry-DnaN (magenta), (B) V-ImuA′ (green) and mCherry-DnaN (magenta), and (C) G-DnaE2 (green) and mCherry-DnaN (magenta). Overlapping signals are viewed as white. The cells …
Figure 3—figure supplement 1
mCherry-DnaN and G-ImuB focus formation following DNA damage treatment.
(A) Frequency of mCherry-DnaN (top panel) and G-ImuB (bottom panel) foci following different DNA damage treatments. (B) Population analysis of cells with foci showing the proportion of cells …
Figure 3—figure supplement 2
Formation of fluorescent EGFP-ImuB foci in the absence of functional mutasome components.
Each panel represents the expression of EGFP-ImuB foci following 1× MIC mitomycin C (MMC) treatment in different genetic backgrounds: (A) ΔimuA′, (B) dnaE2AIA, and (C) ΔdnaE2. Scale bars, 5 μm.
Figure 4 with 1 supplement
ImuB and ImuA′–ImuB interact with DnaN and these interactions are disrupted by griselimycin (GRS).
(A) Gel filtration profiles of M. smegmatis (i) ImuA′B-DnaN and (ii) ImuB-DnaN complexes in the absence or presence of 15 μM GRS. For these experiments, 5 μM DnaN was added to 10 μM of (i) ImuA′B or …
Figure 4—figure supplement 1
Biochemical confirmation of stable ImuA′–ImuB complex formation and griselimycin (GRS)-binding DnaN.
(A) M. smegmatis ImuA′ and ImuB form a stable complex. Gel filtration profiles of the ImuA′B complex at 14 μM (solid line) and 0.4 μM (dashed line) show identical elution volumes, indicating that …
Figure 5 with 1 supplement
Disrupting the ImuB–β clamp interaction.
(A) Representative images of G-ImuB exposed to 2× MIC mitomycin C (MMC) for 4 hr (top panel) or 2× MIC MMC plus griselimycin (GRS) for 4 hr (center panel), and the G-ImuBAAAAGG mutant exposed to 2× …
Figure 5—figure supplement 1
Comparison of M. smegmatis and M. tuberculosis β sliding clamps bound to griselimycin (GRS) crystal structures.
(A) M. smegmatis β clamp bound to GRS (PDB id: 5AH2). Each DnaN monomer is represented by a different color and each monomer is bound to GRS which is represented in blue and green. (B) Superimposed …
Videos
Video 1
Time-lapse microscopy of G-ImuB and mCherry-DnaN dual reporter.
Representative time-lapse movie of the reporter strain expressing G-ImuB and mCherry-DnaN. Bacteria were imaged on fluorescence and phase channels for up to 36 hr at 10-min intervals. Treatment with …
Video 2
Time-lapse microscopy of V-ImuA′ and mCherry-DnaN dual reporter.
Representative time-lapse movie of the reporter strain expressing V-ImuA' and mCherry-DnaN. Bacteria were imaged on fluorescence and phase channels for up to 36 hr at 10-min intervals. Treatment …
Video 3
Time-lapse microscopy of G-DnaE2 and mCherry-DnaN dual reporter.
Representative time-lapse movie of the reporter strain expressing G-DnaE2 and mCherry-DnaN. Bacteria were imaged on fluorescence and phase channels for up to 36 hr at 10-min intervals. Treatment …
Additional files
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Supplementary file 1
DnaN contact data.
- https://cdn.elifesciences.org/articles/75628/elife-75628-supp1-v2.xlsx
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Supplementary file 2
Key reagents.
- https://cdn.elifesciences.org/articles/75628/elife-75628-supp2-v2.xlsx
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Transparent reporting form
- https://cdn.elifesciences.org/articles/75628/elife-75628-transrepform1-v2.pdf
