Identifying hosts of bacteriophage transduction in microbial communities with RNA-addressable modification

Bacteriophages (phages) are the most abundant life form on earth. They facilitate important ecological interactions in all microbial ecosystems by killing their host organism or contributing to the rapid evolution of bacteria via horizontal gene transfer (HGT). Because of these traits, phages are increasingly being applied to manipulate microbial communities by killing specific hosts or delivering functional genes. Central to understanding a phage’s role in a microbial ecosystem, as well as the efficacy of their application, is knowledge of which bacterial species in a microbial community a phage can infect. Either through killing or HGT, a phage’s host is the component of microbial ecosystems that phages act on to impart their changes. The objective of this thesis is to develop and deploy a novel technology to characterize phage hosts in high throughput and in natural microbial communities. Prior techniques to connect phage to host, such as the plaque assay, are low-throughput, difficult to apply in environmental contexts, and/or require a culturable host organism. RNA-addressable modification (RAM) is a ribozyme that can be encoded into mobile genetic elements (MGE) to track their hosts. Upon infection of a host by a RAM encoded MGE, RAM will splice an RNA barcode onto the host’s 16S rRNA. Sequencing of barcoded 16S rRNA can then reveal host information for a given MGE. RAM has previously been demonstrated in plasmids delivered to a community via conjugation. For the first time we demonstrate deployment of RAM in phages and start by outlining a blueprint for incorporating RAM into various phages by adding it to the genome of the well-characterized phage P1 and P1’s associated phagemids. Addition of the P1 constructs to a synthetic microbial community of human pathogens verified RAMs ability to identify phage hosts in a community context and identified a novel host of P1 phagemids in Salmonella enterica. Further exposure of the P1 constructs to a wastewater community confirmed known hosts of P1 and captured a completely new order of P1 hosts in Aeromonadales. We finally showed that RAM can investigate host-specific phage factors and captured significant differences in host range imparted by P1’s two unique tail fibers. This thesis outlines an approach for implementing RAM in phages and lays the groundwork for the application of RAM as a foundational tool for studying phage-host ecology.

Name thesis:degree_name

Doctor of Philosophy

Level thesis:degree_level

Doctoral

Discipline thesis:degree_discipline

Engineering

Grantor

Rice University

Year dc:date.issued

2025

Author dc:creator

• LaTurner, Zach W

Advisor dc:contributor.advisor

• Stadler, Lauren B

• Bacteriophage
• Phage
• Horizontal gene transfer
• Microbial community engineering
• Host range

Statement dc:rights

• Copyright is held by the author, unless otherwise indicated. Permission to reuse, publish, or reproduce the work beyond the bounds of fair use or other exemptions to copyright law must be obtained from the copyright holder.

Language dc:language.iso

eng

Handle dc:identifier.uri

https://hdl.handle.net/1911/118518

OAI identifier oai:identifier

oai:repository.rice.edu:1911/118518