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Browsing by Author "Alattas, Hibah"

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    Advancing Understanding of the Bacteriophage Lambda T4RII Exclusion (REX) Phenotype
    (University of Waterloo, 2025-10-20) Alattas, Hibah
    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
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    Isolation and Characterization of Host Mutations that Suppress the Bacteriophage Lambda (λ) Rex Phenotype
    (University of Waterloo, 2015-09-01) Alattas, Hibah
    The Bacteriophage lambda (λ) T4rII exclusion (Rex) phenotype is defined as the inability of T4rII mutant bacteriophage to form plaques on a lawn of E. coli lysogenized by bacteriophage λ. More than six decades have passed following the discovery of Rex by Seymour Benzer in 1955, yet the mechanism behind this elusive exclusion system remains a mystery. The Rex system is encoded by two genes of λ (rexA, and rexB), the expression of which, is primarily regulated by the repressor gene cI from the PM promoter. The onset of the Rex phenotype, somehow triggered by T4rII infection of a Rex+ lysogen, results in rapid membrane depolarization and a harsh cellular environment that in many ways resembles the stationary phase in metabolism and morphology. In addition, the disruption of the RexA:RexB balance, particularly the over expression of rexA to rexB, can lead to same manifestations without infection, indicating that stoichiometry of RexA:RexB is important. Despite some cell killing, infected lysogens can to some extent recover from Rex activation. The phenotype may thus be a mutualistic protection mechanism that protects both itself and the host cell from external super-infection. In this study, I have designed a system for the rapid one-step isolation of host mutations that attenuate this phenotype, in order to identify the host genes involved in Rex and elucidate the mechanism of this enigmatic exclusion system. In so doing, I was able to isolate 13 host mutations of E. coli K-12 that abrogate the Rex phenotype while simultaneously identifying for the first time several outer membrane protein genes including: ompA, ompF, ompW, and ompX that are essential in imparting Rex activity. In analyzing these omp mutants it was noted that several of these identified omp candidates play a critical role in cellular osmotic balance and the onset of stationary phase. This work substantiates a model whereby the onset of Rex shunts cells into a temporary stationary phase-like state that inhibits T4rII phage growth.