Advancing Understanding of the Bacteriophage Lambda T4RII Exclusion (REX) Phenotype
dc.contributor.author | Alattas, Hibah | |
dc.date.accessioned | 2025-10-20T13:31:13Z | |
dc.date.available | 2025-10-20T13:31:13Z | |
dc.date.issued | 2025-10-20 | |
dc.date.submitted | 2025-10-11 | |
dc.description.abstract | The Bacteriophage Lambda (λ) T4rII exclusion (Rex) system is a finely tuned antiviral defense mechanism in Escherichia coli (E. coli) lysogens that prevents the propagation of T4rII mutant bacteriophage. This exclusion phenotype is defined as the inability of T4rII mutant bacteriophage to propagate within λ-lysogenized E. coli cells. This phenomenon is encoded by two genes of λ (rexA and rexB), whose expression is primarily regulated and driven by the λ PM promoter under the regulation of the λ cI repressor gene. The T4rII infection of Rex+ lysogen triggers the onset of the Rex phenotype. It is characterized by a harsh cascade of cellular responses, including rapid membrane depolarization and the induction of a stress environment that mimics the physiology of the stationary phase. In addition, the disruption of the RexA:RexB balance, particularly the overexpression of rexA relative to rexB, can lead to the same manifestations without infection, indicating that the stoichiometry of RexA:RexB is relevant. Rex “activation” results in significant cell death and has been proposed to serve as an altruistic cell death system to protect the species. In contrast, despite some cell killing, infected lysogens do, to an extent, recover from Rex activation. As such, the phenotype may additionally serve as a mutualistic mechanism that protects both the λ prophage and its stability, as well as its host, by limiting superinfection. For a long time, researchers have hypothesized that E. coli host proteins are involved in this pleiotropic phenotype and identifying them has been a key goal in elucidating the Rex mechanism. Notably, Rex activation depends not only on gene expression and the stoichiometry of RexA to RexB but also on an optimal monovalent cationic environment, underscoring the importance of membrane ion channels and transport systems. Identifying all host mutations that attenuate or abrogate Rex will help in understanding the role of individual components, directly or indirectly, in triggering and inducing the cellular (physiological) manifestations of the Rex phenotype. While many E. coli membrane proteins control the passage of solutes and ions through the membranes, mutations in these proteins may disrupt the ionic environment, thereby resulting in the attenuation or abrogation of Rex. This finding was observed and confirmed by the deletion of several outer membrane proteins, including ompW, ompX, ompA, and ompF (Alattas et al., 2020). Beyond these membrane-associated proteins, the ability of additional genetic and regulatory components to modulate and influence Rex and Rex escape to varying degrees was demonstrated, including tolA, mglC, and rpoS (S). Several of the identified genes play a critical role in cellular osmotic balance and are governed by the stationary phase sigma factor (S) or envelope stress sigma factor (E) as part of the stationary phase and/or envelope distress regulons, under the control of many non-coding regulatory small RNAs. The impact of mutations in these regulatory proteins on Rex activity was examined following superinfection by T4 and T4rII, and the role of the S regulatory proteins, including RssB and IraD, in the Rex and Rex escape mechanisms was reported for the first time. However, the mechanism by which this occurs remains unclear. The impact of fluorescence fusions on colony morphology and texture was investigated, showing variations in colony size among fusion sites and Rex types. These experiments suggested that the fusion site and the expression status of the partner significantly affect Rex activity, likely by interfering with proper protein folding, complex formation, or the stoichiometric balance of RexA to RexB. N-terminal fusions severely attenuated Rex activity, while dual N-terminal tagging almost entirely abolished it. In contrast, C-terminal fusions only partially affected Rex activity, with dual C-terminal fusions showing minimal impact on exclusion capability. Together, these findings highlight the Rex system as a multifactorial, tightly regulated antiviral mechanism that integrates gene stoichiometry, membrane composition, sigma factor regulation and its RNA-mediated control. This work adopts a genetic approach to enhance and refine the existing model of Rex-mediated T4rII exclusion, leading to a new model of Rex that offers new insights into its underlying molecular determinants and paves the way for further exploration of bacteriophage-host dynamics, superinfection exclusion strategies, and programmed cell death in prokaryotes. Keyword: Rex exclusion, RexA, RexB, T4rII, T4, Rex phenotype, λ lambda, sigma factors, E. coli, bacteriophage, phage | |
dc.identifier.uri | https://hdl.handle.net/10012/22595 | |
dc.language.iso | en | |
dc.pending | false | |
dc.publisher | University of Waterloo | en |
dc.title | Advancing Understanding of the Bacteriophage Lambda T4RII Exclusion (REX) Phenotype | |
dc.type | Doctoral Thesis | |
uws-etd.degree | Doctor of Philosophy | |
uws-etd.degree.department | School of Pharmacy | |
uws-etd.degree.discipline | Pharmacy | |
uws-etd.degree.grantor | University of Waterloo | en |
uws-etd.embargo.terms | 0 | |
uws.contributor.advisor | Slavcev, Roderick | |
uws.contributor.affiliation1 | Faculty of Science | |
uws.peerReviewStatus | Unreviewed | en |
uws.published.city | Waterloo | en |
uws.published.country | Canada | en |
uws.published.province | Ontario | en |
uws.scholarLevel | Graduate | en |
uws.typeOfResource | Text | en |