Combining Comprehensive Analysis of Off-Site Lambda Phage Integration with a CRISPR-Based Means of Characterizing Downstream Physiology.

Yu Tanouchi,Markus W Covert,Sang Yup Lee

doi:10.1128/mbio.01038-17

Abstract

ABSTRACTDuring its lysogenic life cycle, the phage genome is integrated into the host chromosome by site-specific recombination. In this report, we analyze lambda phage integration into noncanonical sites using next-generation sequencing and show that it generates significant genetic diversity by targeting over 300 unique sites in the host Escherichia coli genome. Moreover, these integration events can have important phenotypic consequences for the host, including changes in cell motility and increased antibiotic resistance. Importantly, the new technologies that we developed to enable this study—sequencing secondary sites using next-generation sequencing and then selecting relevant lysogens using clustered regularly interspaced short palindromic repeat (CRISPR)/Cas9-based selection—are broadly applicable to other phage-bacterium systems.

Highlights

During its lysogenic life cycle, the phage genome is integrated into the host chromosome by site-specific recombination
IMPORTANCE Bacteriophages play an important role in bacterial evolution through lysogeny, where the phage genome is integrated into the host chromosome
We further developed an assay to examine the phenotypic consequence of such diverse integration events and found that phage integration can cause changes in evolutionarily relevant traits such as bacterial motility and increases in antibiotic resistance

Summary

Introduction

During its lysogenic life cycle, the phage genome is integrated into the host chromosome by site-specific recombination. Phage integration into secondary sites has been shown to disrupt surrounding host genes and cause phenotypic changes such as auxotrophy [8, 9]. Using phage lambda and its host Escherichia coli, arguably the best-studied phagebacterium infection model, we sought to characterize the genomic landscape of lambda secondary integration sites.

Results

Conclusion