Detection, isolation and characterization of phage-host complexes using BONCAT and click chemistry.

Abstract

Phages are viruses that infect prokaryotes and can shape microbial communities by lysis, thus offering applications in various fields. However, challenges exist in sampling, isolation and accurate prediction of the host specificity of phages as well as in the identification of newly replicated virions in response to environmental challenges. A new workflow using biorthogonal non-canonical amino acid tagging (BONCAT) and click chemistry (CC) allowed the combined analysis of phages and their hosts, the identification of newly replicated virions, and the specific tagging of phages with biotin for affinity chromatography. Replication of phage λ in Escherichia coli was selected as a model for workflow development. Specific labeling of phage λ proteins with the non-canonical amino acid 4-azido-L-homoalanine (AHA) during phage development in E. coli was confirmed by LC-MS/MS. Subsequent tagging of AHA with fluorescent dyes via CC allowed the visualization of phages adsorbed to the cell surface by fluorescence microscopy. Flow cytometry enabled the automated detection of these fluorescent phage-host complexes. Alternatively, AHA-labeled phages were tagged with biotin for purification by affinity chromatography. Despite biotinylation the tagged phages could be purified and were infectious after purification. Applying this approach to environmental samples would enable host screening without cultivation. A flexible and powerful workflow for the detection and enrichment of phages and their hosts in pure cultures has been established. The developed method lays the groundwork for future workflows that could enable the isolation of phage-host complexes from diverse complex microbial communities using fluorescence-activated cell sorting or biotin purification. The ability to expand and customize the workflow through the growing range of compounds for CC offers the potential to develop a versatile toolbox in phage research. This work provides a starting point for these further studies by providing a comprehensive standard operating procedure.