The mycobacterial ImuA'-ImuB-DnaE2 mutasome: composition and recruitment in live cells
Abstract
A DNA damage-inducible mutagenic gene cassette has been implicated in the emergence of drug resistance in Mycobacterium tuberculosis during anti-tuberculosis (TB) chemotherapy. However, the molecular composition and operation of the encoded 'mycobacterial mutasome' - minimally comprising DnaE2 polymerase and ImuA′ and ImuB accessory proteins - remain elusive. Following exposure of mycobacteria to DNA damaging agents, we observe that DnaE2 and ImuB co-localize with the DNA polymerase III β subunit (β clamp) in distinct intracellular foci. Notably, genetic inactivation of the mutasome in an imuBAAAAGG mutant containing a disrupted β clamp-binding motif abolishes ImuB-β clamp focus formation, a phenotype recapitulated pharmacologically by treating bacilli with griselimycin and in biochemical assays in which this β clamp-binding antibiotic collapses pre-formed ImuB-β clamp complexes. These observations establish the essentiality of the ImuB-β clamp interaction for mutagenic DNA repair in mycobacteria, identifying the mutasome as target for adjunctive therapeutics designed to protect anti-TB drugs against emerging resistance.
Data availability
Source data for all figures contained in the manuscript and SI have been deposited in Dryad; see https://datadryad.org/stash/share/fjhwiXFEIIM5-6liMtXIQn0Ehq4NIKZ3690FiR8lWyI.
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Data from: The mycobacterial ImuA'-ImuB-DnaE2 mutasome: composition and recruitment in live cellsDryad Digital Repository, doi:10.5061/dryad.76hdr7szc.
Article and author information
Author details
Funding
Eunice Kennedy Shriver National Institute of Child Health and Human Development (U01HD085531)
- Roger Woodgate
- Digby F Warner
Norges Forskningsråd (261669)
- Digby F Warner
South African Medical Research Council (SHIP and Extramural Unit)
- Valerie Mizrahi
- Digby F Warner
National Research Foundation
- Valerie Mizrahi
- Digby F Warner
Howard Hughes Medical Institute (Senior International Research Scholars)
- Valerie Mizrahi
Leids Universitair Medisch Centrum (LUMC Fellowship)
- Meindert Hugo Lamers
National Research Foundation (104683)
- Michael Anton Reiche
David and Elaine Potter Foundation (PhD Fellowship)
- Zela Alexandria-Mae Martin
The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.
Copyright
This is an open-access article, free of all copyright, and may be freely reproduced, distributed, transmitted, modified, built upon, or otherwise used by anyone for any lawful purpose. The work is made available under the Creative Commons CC0 public domain dedication.
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Further reading
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- Biochemistry and Chemical Biology
- Microbiology and Infectious Disease
Teichoic acids (TA) are linear phospho-saccharidic polymers and important constituents of the cell envelope of Gram-positive bacteria, either bound to the peptidoglycan as wall teichoic acids (WTA) or to the membrane as lipoteichoic acids (LTA). The composition of TA varies greatly but the presence of both WTA and LTA is highly conserved, hinting at an underlying fundamental function that is distinct from their specific roles in diverse organisms. We report the observation of a periplasmic space in Streptococcus pneumoniae by cryo-electron microscopy of vitreous sections. The thickness and appearance of this region change upon deletion of genes involved in the attachment of TA, supporting their role in the maintenance of a periplasmic space in Gram-positive bacteria as a possible universal function. Consequences of these mutations were further examined by super-resolved microscopy, following metabolic labeling and fluorophore coupling by click chemistry. This novel labeling method also enabled in-gel analysis of cell fractions. With this approach, we were able to titrate the actual amount of TA per cell and to determine the ratio of WTA to LTA. In addition, we followed the change of TA length during growth phases, and discovered that a mutant devoid of LTA accumulates the membrane-bound polymerized TA precursor.
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- Biochemistry and Chemical Biology
- Computational and Systems Biology
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