Temporal epigenome modulation enables efficient bacteriophage engineering and functional analysis of phage DNA modifications

PLoS Genet. 2024 Sep 4;20(9):e1011384. doi: 10.1371/journal.pgen.1011384. eCollection 2024 Sep.

Abstract

Lytic bacteriophages hold substantial promise in medical and biotechnological applications. Therefore a comprehensive understanding of phage infection mechanisms is crucial. CRISPR-Cas systems offer a way to explore these mechanisms via site-specific phage mutagenesis. However, phages can resist Cas-mediated cleavage through extensive DNA modifications like cytosine glycosylation, hindering mutagenesis efficiency. Our study utilizes the eukaryotic enzyme NgTET to temporarily reduce phage DNA modifications, facilitating Cas nuclease cleavage and enhancing mutagenesis efficiency. This approach enables precise DNA targeting and seamless point mutation integration, exemplified by deactivating specific ADP-ribosyltransferases crucial for phage infection. Furthermore, by temporally removing DNA modifications, we elucidated the effects of these modifications on T4 phage infections without necessitating gene deletions. Our results present a strategy enabling the investigation of phage epigenome functions and streamlining the engineering of phages with cytosine DNA modifications. The described temporal modulation of the phage epigenome is valuable for synthetic biology and fundamental research to comprehend phage infection mechanisms through the generation of mutants.

MeSH terms

  • Bacteriophage T4 / genetics
  • Bacteriophages* / genetics
  • CRISPR-Cas Systems*
  • DNA, Viral* / genetics
  • Epigenome*
  • Escherichia coli / genetics
  • Escherichia coli / virology
  • Genetic Engineering / methods
  • Genome, Viral
  • Mutagenesis, Site-Directed / methods

Substances

  • DNA, Viral

Grants and funding

K.H. is supported by funding from the Max Planck Society and the German Research Council (DFG SPP 2330, Project number 464500427). A.A.R.R. and D.S. are supported by the Max Planck Society within the framework of the MaxGENESYS project. M.W.S. is supported by funding from the Joachim Herz Foundation and the Studienstiftung des deutschen Volkes e.V. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.