Epigenetic Segregation of Microbial Genomes from Complex Samples Using Restriction Endonucleases HpaII and McrB

PLoS One. 2016 Jan 4;11(1):e0146064. doi: 10.1371/journal.pone.0146064. eCollection 2016.

Abstract

We describe continuing work to develop restriction endonucleases as tools to enrich targeted genomes of interest from diverse populations. Two approaches were developed in parallel to segregate genomic DNA based on cytosine methylation. First, the methyl-sensitive endonuclease HpaII was used to bind non-CG methylated DNA. Second, a truncated fragment of McrB was used to bind CpG methylated DNA. Enrichment levels of microbial genomes can exceed 100-fold with HpaII allowing improved genomic detection and coverage of otherwise trace microbial genomes from sputum. Additionally, we observe interesting enrichment results that correlate with the methylation states not only of bacteria, but of fungi, viruses, a protist and plants. The methods presented here offer promise for testing biological samples for pathogens and global analysis of population methylomes.

Publication types

  • Research Support, U.S. Gov't, Non-P.H.S.

MeSH terms

  • 5-Methylcytosine / analysis*
  • CpG Islands / genetics
  • DNA Methylation
  • DNA Restriction Enzymes* / isolation & purification
  • DNA Restriction Enzymes* / metabolism
  • DNA, Bacterial / genetics
  • DNA, Bacterial / isolation & purification*
  • DNA, Fungal / genetics
  • DNA, Fungal / isolation & purification*
  • DNA, Plant / genetics
  • DNA, Plant / isolation & purification*
  • DNA, Protozoan / genetics
  • DNA, Protozoan / isolation & purification*
  • DNA, Viral / genetics
  • DNA, Viral / isolation & purification*
  • Deoxyribonuclease HpaII* / isolation & purification
  • Deoxyribonuclease HpaII* / metabolism
  • Escherichia coli Proteins* / isolation & purification
  • Escherichia coli Proteins* / metabolism
  • Gene Library
  • Genetics, Microbial / methods*
  • Genomics / methods*
  • Humans
  • Metagenome*
  • Microbiota / genetics
  • Sequence Analysis, DNA
  • Sputum / microbiology
  • Substrate Specificity

Substances

  • DNA, Bacterial
  • DNA, Fungal
  • DNA, Plant
  • DNA, Protozoan
  • DNA, Viral
  • Escherichia coli Proteins
  • 5-Methylcytosine
  • DNA Restriction Enzymes
  • Deoxyribonuclease HpaII
  • mcrB protein, E coli

Grants and funding

Funding for this research was provided in full through contract HSHQDC-10-C-00019 to FLIR Systems, Inc., by the Department of Homeland Security (DHS), Science and Technology Directorate (S&T), http://www.dhs.gov/science-and-technology. DHS S&T reviewed and approved the manuscript for publication. DHS S&T had no additional roles in the study design, data collection, analysis or preparation of the manuscript. FLIR Systems, Inc., Singlera Genomics Inc. and Zova Systems, LLC, provided support in the form of salaries for authors GL, CQW, LKP, SW, HB, PK, DS, RTY and RAF, but did not have any additional role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript. The specific roles of these authors are articulated in the ‘author contributions’ section.