The mycobacterial ImuA'-ImuB-DnaE2 mutasome: composition and recruitment in live cells
- University of Cape Town, South Africa
- Howard Hughes Medical Institute, United States
- Leiden University Medical Center, Netherlands
- University of Saskatchewan, Canada
- Umeå University, Sweden
- Helmholtz Institute for Pharmaceutical Research Saarland, Germany
- Swiss Federal Institute of Technology in Lausanne, Switzerland
- Eunice Kennedy Shriver National Institute of Child Health and Human Development, United States
- Vilnius University, Lithuania
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.
-
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
Meindert Hugo Lamers
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.
Reviewing Editor
- Christina L Stallings, Washington University School of Medicine, United States
Version history
- Received: November 16, 2021
- Preprint posted: November 17, 2021 (view preprint)
- Accepted: August 1, 2023
- Accepted Manuscript published: August 2, 2023 (version 1)
- Version of Record published: August 11, 2023 (version 2)
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.
Metrics
-
- 825
- views
-
- 143
- downloads
-
- 4
- citations
Views, downloads and citations are aggregated across all versions of this paper published by eLife.
Download links
A two-part list of links to download the article, or parts of the article, in various formats.
Downloads (link to download the article as PDF)
Open citations (links to open the citations from this article in various online reference manager services)
Cite this article (links to download the citations from this article in formats compatible with various reference manager tools)
The mycobacterial ImuA'-ImuB-DnaE2 mutasome: composition and recruitment in live cells
eLife 12:e75628.
https://doi.org/10.7554/eLife.75628
Further reading
-
- Biochemistry and Chemical Biology
- Cell Biology
Secreted chemokines form concentration gradients in target tissues to control migratory directions and patterns of immune cells in response to inflammatory stimulation; however, how the gradients are formed is much debated. Heparan sulfate (HS) binds to chemokines and modulates their activities. In this study, we investigated the roles of HS in the gradient formation and chemoattractant activity of CCL5 that is known to bind to HS. CCL5 and heparin underwent liquid–liquid phase separation and formed gradient, which was confirmed using CCL5 immobilized on heparin-beads. The biological implication of HS in CCL5 gradient formation was established in CHO-K1 (wild-type) and CHO-677 (lacking HS) cells by Transwell assay. The effect of HS on CCL5 chemoattractant activity was further proved by Transwell assay of human peripheral blood cells. Finally, peritoneal injection of the chemokines into mice showed reduced recruitment of inflammatory cells either by mutant CCL5 (lacking heparin-binding sequence) or by addition of heparin to wild-type CCL5. Our experimental data propose that co-phase separation of CCL5 with HS establishes a specific chemokine concentration gradient to trigger directional cell migration. The results warrant further investigation on other heparin-binding chemokines and allows for a more elaborate insight into disease process and new treatment strategies.
-
- Biochemistry and Chemical Biology
- Evolutionary Biology
The emergence of new protein functions is crucial for the evolution of organisms. This process has been extensively researched for soluble enzymes, but it is largely unexplored for membrane transporters, even though the ability to acquire new nutrients from a changing environment requires evolvability of transport functions. Here, we demonstrate the importance of environmental pressure in obtaining a new activity or altering a promiscuous activity in members of the amino acid-polyamine-organocation (APC)-type yeast amino acid transporters family. We identify APC members that have broader substrate spectra than previously described. Using in vivo experimental evolution, we evolve two of these transporter genes, AGP1 and PUT4, toward new substrate specificities. Single mutations on these transporters are found to be sufficient for expanding the substrate range of the proteins, while retaining the capacity to transport all original substrates. Nonetheless, each adaptive mutation comes with a distinct effect on the fitness for each of the original substrates, illustrating a trade-off between the ancestral and evolved functions. Collectively, our findings reveal how substrate-adaptive mutations in membrane transporters contribute to fitness and provide insights into how organisms can use transporter evolution to explore new ecological niches.
