Characterisation of prophages from Wolbachia symbionts maintained in insect cell culture systems.

Dyer, Philip (2025) Characterisation of prophages from Wolbachia symbionts maintained in insect cell culture systems. PhD thesis, University of Liverpool.

Text 200912226_Feb2025.pdf - Author Accepted Manuscript Access to this file is embargoed until 1 February 2026. Download (9MB)

Abstract

Wolbachia is a bacterial endosymbiont with a profound impact on the ecology of many medically or economically significant insect species. As invasive arthropods and their associated epidemiology become increasingly important, the genetic manipulation of Wolbachia could have huge implications for pest and disease control. To date, conventional approaches such as CRISPR-based techniques have not proven successful, chiefly due to Wolbachia’s obligate endosymbiosis. Additionally, Wolbachia’s abundance of mobile and repetitive elements complicate sequencing and whole-genome assembly. This study investigates the associated bacteriophage WO as an alternate route for transformation. Long-read metagenomic sequencing by Oxford Nanopore Technologies’ MinION, corrected by short-read high-accuracy Illumina sequencing, was used to generate circularised single-contig genome assemblies for the supergroup B Wolbachia strains wStri (1.75 Mb) and wBol-1b (1.47 Mb), cultured in transinfected SF9 and C6/36 host cells. Both represent the most complete genome of each strain to date. Prophage regions were identified and characterised with a variety of homology-based approaches. Transcription from within these elements was confirmed by RNA-Seq performed with reference to the new assemblies, and differential expression from prophage regions analysed between hosts. Prophage regions were found to be less transcriptionally active overall than the rest of the genome, but more likely to be differentially expressed, suggesting context-dependent regulation. Phage structural proteins were highly expressed in wStri, absent critical tail components. This may indicate production of non-infectious virions that nonetheless may have effector roles. A variety of gene editing and transgenic methodologies were investigated, with a focus on wStri as the strain with the most diverse and complete prophage. The most ambitious approach was Transformation Associated Recombination (TAR) cloning of sections of the Wolbachia genome. While unsuccessful, the attempt established a methodology and identified quality of genomic DNA extraction as a limiting factor. An alternative strategy for transformation was also investigated, inoculating Wolbachia cultures with Escherichia coli carrying conjugative plasmids, but without success. Additionally, wStri prophage genes encoding RuvC resolvases were transformed into E. coli for functional characterisation, identifying a discriminating cleavage action unlike typical viral resolvases. This study resulted in the improved genomic assembly and characterisation of the wStri and wBol1-b strains of Wolbachia, and the first transcriptomic analysis of supergroup B Wolbachia. It will support future exploration of phage and prophage in parasitic strains and lays groundwork for gene editing in Wolbachia.

Item Type:	Thesis (PhD)
Uncontrolled Keywords:	Wolbachia, Bacteriophage
Divisions:	Faculty of Health and Life Sciences Faculty of Health and Life Sciences > Institute of Infection, Veterinary and Ecological Sciences
Depositing User:	Symplectic Admin
Date Deposited:	21 Aug 2025 08:23
Last Modified:	21 Aug 2025 08:24
DOI:	10.17638/03190595
Supervisors:	Makepeace, Benjamin Darby, Alistair
URI:	https://livrepository.liverpool.ac.uk/id/eprint/3190595