Peptidoglycan precursor synthesis along the sidewall of pole-growing mycobacteria

  1. Alam García-Heredia
  2. Amol A Pohane
  3. Emily S Melzer
  4. Caleb R Carr
  5. Taylor J Fiolek  Is a corresponding author
  6. Sarah R Rundell
  7. Hoong Chuin Lim
  8. Jeffrey C Wagner
  9. Yasu S Morita
  10. Benjamin M Swarts
  11. M Sloan Siegrist  Is a corresponding author
  1. University of Massachusetts, United States
  2. Central Michigan University, United States
  3. Harvard Medical School, United States
  4. Harvard TH Chan School of Public Health, United States

Abstract

Rod-shaped mycobacteria expand from their poles, yet d-amino acid probes label cell wall peptidoglycan in this genus at both the poles and sidewall. We sought to clarify the metabolic fates of these probes. Monopeptide incorporation was decreased by antibiotics that block peptidoglycan synthesis or l,d-transpeptidation and in an l,d-transpeptidase mutant. Dipeptides complemented defects in d-alanine synthesis or ligation and were present in lipid-linked peptidoglycan precursors. Characterizing probe uptake pathways allowed us to localize peptidoglycan metabolism with precision: monopeptide-marked l,d-transpeptidase remodeling and dipeptide-marked synthesis were coincident with mycomembrane metabolism at the poles, septum and sidewall. Fluorescent pencillin-marked d,d-transpeptidation around the cell perimeter further suggested that the mycobacterial sidewall is a site of cell wall assembly. While polar peptidoglycan synthesis was associated with cell elongation, sidewall synthesis responded to cell wall damage. Peptidoglycan editing along the sidewall may support cell wall robustness in pole-growing mycobacteria.

Data availability

Source data has been provided with manuscript submission. These data will be deposited to Open Science Framework prior to publication.

Article and author information

Author details

  1. Alam García-Heredia

    Molecular and Cellular Biology Graduate Program, University of Massachusetts, Amherst, United States
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-9573-4087
  2. Amol A Pohane

    Department of Microbiology, University of Massachusetts, Amherst, United States
    Competing interests
    The authors declare that no competing interests exist.
  3. Emily S Melzer

    Department of Microbiology, University of Massachusetts, Amherst, United States
    Competing interests
    The authors declare that no competing interests exist.
  4. Caleb R Carr

    Department of Microbiology, University of Massachusetts, Amherst, United States
    Competing interests
    The authors declare that no competing interests exist.
  5. Taylor J Fiolek

    Department of Chemistry and Biochemistry, Central Michigan University, Mount Pleasant, United States
    For correspondence
    fiole1tj@cmich.edu
    Competing interests
    The authors declare that no competing interests exist.
  6. Sarah R Rundell

    Department of Chemistry and Biochemistry, Central Michigan University, Mount Pleasant, United States
    Competing interests
    The authors declare that no competing interests exist.
  7. Hoong Chuin Lim

    Department of Microbiology and Immunobiology, Harvard Medical School, Boston, United States
    Competing interests
    The authors declare that no competing interests exist.
  8. Jeffrey C Wagner

    Department of Immunology and Infectious Diseases, Harvard TH Chan School of Public Health, Boston, United States
    Competing interests
    The authors declare that no competing interests exist.
  9. Yasu S Morita

    Molecular and Cellular Biology Graduate Program, University of Massachusetts, Amherst, United States
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-4514-9242
  10. Benjamin M Swarts

    Department of Chemistry and Biochemistry, Central Michigan University, Mount Pleasant, United States
    Competing interests
    The authors declare that no competing interests exist.
  11. M Sloan Siegrist

    Molecular and Cellular Biology Graduate Program, University of Massachusetts, Amherst, United States
    For correspondence
    siegrist@gmail.com
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-8232-3246

Funding

National Institutes of Health (New Innovator Award DP2 AI138238)

  • M Sloan Siegrist

National Science Foundation (CAREER 1654408)

  • Benjamin M Swarts

Simons Foundation (Life Sciences Research Foundation Fellowship)

  • Hoong Chuin Lim

Research Corporation for Science Advancement (Cottrell College Science Award 22525)

  • Benjamin M Swarts

National Institutes of Health (U01CA221230)

  • M Sloan Siegrist

National Institutes of Health (Training Grant T32 GM008515)

  • Emily S Melzer

The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.

Copyright

© 2018, García-Heredia et al.

This article is distributed under the terms of the Creative Commons Attribution License permitting unrestricted use and redistribution provided that the original author and source are credited.

Metrics

  • 6,154
    views
  • 839
    downloads
  • 105
    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)

  1. Alam García-Heredia
  2. Amol A Pohane
  3. Emily S Melzer
  4. Caleb R Carr
  5. Taylor J Fiolek
  6. Sarah R Rundell
  7. Hoong Chuin Lim
  8. Jeffrey C Wagner
  9. Yasu S Morita
  10. Benjamin M Swarts
  11. M Sloan Siegrist
(2018)
Peptidoglycan precursor synthesis along the sidewall of pole-growing mycobacteria
eLife 7:e37243.
https://doi.org/10.7554/eLife.37243

Share this article

https://doi.org/10.7554/eLife.37243

Further reading

    1. Epidemiology and Global Health
    2. Microbiology and Infectious Disease
    Moagi Tube Shaku, Bavesh Davandra Kana
    Insight

    A better understanding of the mechanisms underpinning the growth of mycobacteria could help identify targets for new antibiotics.

    1. Biochemistry and Chemical Biology
    2. Genetics and Genomics
    Federico A Vignale, Andrea Hernandez Garcia ... Adrian G Turjanski
    Research Article

    Yerba mate (YM, Ilex paraguariensis) is an economically important crop marketed for the elaboration of mate, the third-most widely consumed caffeine-containing infusion worldwide. Here, we report the first genome assembly of this species, which has a total length of 1.06 Gb and contains 53,390 protein-coding genes. Comparative analyses revealed that the large YM genome size is partly due to a whole-genome duplication (Ip-α) during the early evolutionary history of Ilex, in addition to the hexaploidization event (γ) shared by core eudicots. Characterization of the genome allowed us to clone the genes encoding methyltransferase enzymes that catalyse multiple reactions required for caffeine production. To our surprise, this species has converged upon a different biochemical pathway compared to that of coffee and tea. In order to gain insight into the structural basis for the convergent enzyme activities, we obtained a crystal structure for the terminal enzyme in the pathway that forms caffeine. The structure reveals that convergent solutions have evolved for substrate positioning because different amino acid residues facilitate a different substrate orientation such that efficient methylation occurs in the independently evolved enzymes in YM and coffee. While our results show phylogenomic constraint limits the genes coopted for convergence of caffeine biosynthesis, the X-ray diffraction data suggest structural constraints are minimal for the convergent evolution of individual reactions.