Host-associated microbe PCR (hamPCR) enables convenient measurement of both microbial load and community composition

  1. Derek S Lundberg  Is a corresponding author
  2. Pratchaya Pramoj Na Ayutthaya
  3. Annett Strauß
  4. Gautam Shirsekar
  5. Wen-Sui Lo
  6. Thomas Lahaye
  7. Detlef Weigel  Is a corresponding author
  1. Max Planck Institute for Developmental Biology, Germany
  2. University of Tübingen, Germany

Abstract

The ratio of microbial population size relative to the amount of host tissue, or 'microbial load', is a fundamental metric of colonization and infection, but it cannot be directly deduced from microbial amplicon data such as 16S rRNA gene counts. Because existing methods to determine load, such as serial dilution plating, quantitative PCR, and whole metagenome sequencing, add substantial cost and/or experimental burden, they are only rarely paired with amplicon sequencing. We introduce host-associated microbe PCR (hamPCR), a robust strategy to both quantify microbial load and describe interkingdom microbial community composition in a single amplicon library. We demonstrate its accuracy across multiple study systems, including nematodes and major crops, and further present a cost-saving technique to reduce host overrepresentation in the library prior to sequencing. Because hamPCR provides an accessible experimental solution to the well-known limitations and statistical challenges of compositional data, it has far-reaching potential in culture-independent microbiology.

Data availability

All data in this manuscript have been deposited in the European Nucleotide Archive (ENA) under the project number PRJEB38287. At https://www.ebi.ac.uk/ena.

The following data sets were generated

Article and author information

Author details

  1. Derek S Lundberg

    Department of Molecular Biology, Max Planck Institute for Developmental Biology, Tübingen, Germany
    For correspondence
    derek.lundberg@gmail.com
    Competing interests
    No competing interests declared.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0001-7970-1595
  2. Pratchaya Pramoj Na Ayutthaya

    Department of Molecular Biology, Max Planck Institute for Developmental Biology, Tübingen, Germany
    Competing interests
    No competing interests declared.
  3. Annett Strauß

    ZMBP-General Genetics, University of Tübingen, Tübingen, Germany
    Competing interests
    No competing interests declared.
  4. Gautam Shirsekar

    Department of Molecular Biology, Max Planck Institute for Developmental Biology, Tübingen, Germany
    Competing interests
    No competing interests declared.
  5. Wen-Sui Lo

    Department of Evolutionary Biology, Max Planck Institute for Developmental Biology, Tübingen, Germany
    Competing interests
    No competing interests declared.
  6. Thomas Lahaye

    ZMBP-General Genetics, University of Tübingen, Tübingen, Germany
    Competing interests
    No competing interests declared.
  7. Detlef Weigel

    Department of Molecular Biology, Max Planck Institute for Developmental Biology, Tübingen, Germany
    For correspondence
    weigel.elife@gmail.com
    Competing interests
    Detlef Weigel, Senior editor, eLife.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-2114-7963

Funding

Deutsche Forschungsgemeinschaft (SPP 2125 DECRyPT)

  • Derek S Lundberg
  • Pratchaya Pramoj Na Ayutthaya
  • Detlef Weigel

Human Frontiers Science Program Long-term Fellowship (LT000565/2015-L)

  • Derek S Lundberg

Max Planck Society

  • Derek S Lundberg
  • Pratchaya Pramoj Na Ayutthaya
  • Gautam Shirsekar
  • Wen-Sui Lo
  • Detlef Weigel

Cluster of Excellence EXC2124 Controlling Microbes to Fight Infection (390838134)

  • Derek S Lundberg
  • Pratchaya Pramoj Na Ayutthaya
  • Gautam Shirsekar
  • Wen-Sui Lo
  • Detlef Weigel

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

Reviewing Editor

  1. Rebecca Bart, The Donald Danforth Plant Science Center, United States

Publication history

  1. Received: December 31, 2020
  2. Accepted: July 19, 2021
  3. Accepted Manuscript published: July 22, 2021 (version 1)
  4. Version of Record published: August 25, 2021 (version 2)

Copyright

© 2021, Lundberg 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

  • 2,849
    Page views
  • 434
    Downloads
  • 3
    Citations

Article citation count generated by polling the highest count across the following sources: Crossref, PubMed Central, Scopus.

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. Derek S Lundberg
  2. Pratchaya Pramoj Na Ayutthaya
  3. Annett Strauß
  4. Gautam Shirsekar
  5. Wen-Sui Lo
  6. Thomas Lahaye
  7. Detlef Weigel
(2021)
Host-associated microbe PCR (hamPCR) enables convenient measurement of both microbial load and community composition
eLife 10:e66186.
https://doi.org/10.7554/eLife.66186
  1. Further reading

Further reading

    1. Microbiology and Infectious Disease
    Josué Flores-Kim et al.
    Research Article

    Penicillin and related antibiotics disrupt cell wall synthesis in bacteria causing the downstream misactivation of cell wall hydrolases called autolysins to induce cell lysis. Despite the clinical importance of this phenomenon, little is known about the factors that control autolysins and how penicillins subvert this regulation to kill cells. In the pathogen Streptococcus pneumoniae (Sp), LytA is the major autolysin responsible for penicillin-induced bacteriolysis. We recently discovered that penicillin treatment of Sp causes a dramatic shift in surface polymer biogenesis in which cell wall-anchored teichoic acids (WTAs) increase in abundance at the expense of lipid-linked teichoic acids (LTAs). Because LytA binds to both species of teichoic acids, this change recruits the enzyme to its substrate where it cleaves the cell wall and elicits lysis. In this report, we identify WhyD (SPD_0880) as a new factor that controls the level of WTAs in Sp cells to prevent LytA misactivation during exponential growth and premature lysis. We show that WhyD is a WTA hydrolase that restricts the WTA content of the wall to areas adjacent to active PG synthesis. Our results support a model in which the WTA tailoring activity of WhyD during exponential growth directs PG remodeling activity required for proper cell elongation in addition to preventing autolysis by LytA.

    1. Evolutionary Biology
    2. Microbiology and Infectious Disease
    Maliheh Safari et al.
    Research Article Updated

    Overlapping coding regions balance selective forces between multiple genes. One possible division of nucleotide sequence is that the predominant selective force on a particular nucleotide can be attributed to just one gene. While this arrangement has been observed in regions in which one gene is structured and the other is disordered, we sought to explore how overlapping genes balance constraints when both protein products are structured over the same sequence. We use a combination of sequence analysis, functional assays, and selection experiments to examine an overlapped region in HIV-1 that encodes helical regions in both Env and Rev. We find that functional segregation occurs even in this overlap, with each protein spacing its functional residues in a manner that allows a mutable non-binding face of one helix to encode important functional residues on a charged face in the other helix. Additionally, our experiments reveal novel and critical functional residues in Env and have implications for the therapeutic targeting of HIV-1.