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Type: Thesis
Title: Bacteriophage 186 - Investigating the role of transcriptional regulators CI, Apl, CII and Tum at the lytic/lysogenic switch during 186 prophage induction
Author: Isabel, Alejandra
Issue Date: 2020
School/Discipline: School of Biological Sciences
Abstract: I am not a living entity, but I am very much alive. I am of tiny proportions and yet endowed with immense power. I am constantly waging war across the prokaryotic kingdom and have proven to be an aggressive, formidable and exceptionally deadly enemy of bacteria and archaea. Who am I? I am bacteriophage. Coliphage 186 is a UV-inducible, non-lambdoid temperate phage of the family Myoviridae (genus P2-likevirus). As a temperate phage, 186 has the ability to undergo two alternative modes of development - lytic development is the active, developmental default state and lysogeny is the alternate, dormant state, where the phage DNA integrates into its host’s genome. The lysogenic state is reversible and thus the lytic pathway can be resumed upon activation of the host SOS response, a phenomenon termed prophage induction. To control the entry into, and the transition between these states, 186 employs both a lytic/lysogenic transcriptional switch and an SOS inducible operon, each existing as independent modules in the 186 genome. Whilst extensive studies of 186 have provided significant insights into how the lytic and lysogenic cycles are regulated and into the process of prophage induction, there are a number of unique aspects for which our understanding remained incomplete. To progress our understanding of 186 prophage induction and how this phage makes its developmental decisions, four separate studies we undertaken to investigate the role(s) of four key transcriptional regulatory proteins (CI, Apl, CII and Tum) at the 186 switch. This knowledge was then used to re-wire the 186 modules to design and build a simple bistable memory circuit, capable of switching between alternate states in response to a chemical signal. In Chapter 2, to investigate the role of the CI immunity repressor in prophage induction, we asked, does disruption of CI negative autoregulation reduce prophage induction efficiency? Using the goa8 mutation (a 5bp deletion between the two promoters CI regulates, pR and pL) we demonstrated that when CI negative autoregulation is disrupted, this has a negative impact on prophage induction efficiency. This outcome underlined the importance of 186 being able to establish the correct lysogenic level of CI, so as to not only maintain stable lysogeny, but to remain optimally primed for prophage induction. To investigate the role of the Apl protein, we asked, why does Apl act as a weak transcriptional repressor at pR and pL during prophage induction? A series of hypothesises were framed on the idea that Apl binding at pR.pL is required to control cII, cI and/or int gene expression during prophage induction. With the experimental outcomes resulting in the rejection of all hypotheses however, this investigation contributed only to our understanding of what Apl does not do at the 186 switch. In the context of 186 prophage induction, the role of the Tum antirepressor and the host SOS response were investigated in Chapter 3. Using a series of minimal 186-like UV- and chemically-inducible, chromosomally-integrated reporter systems and a cumic acid-inducible 186 phage, we confirmed that Tum is essential and sufficient in single-copy for stable 186 lysogenic to lytic switching and that the fundamental role of host SOS activation is to induce expression of the tum gene. In Chapter 4, we asked, what is the significance of having a short-lived/protease sensitive CII protein? By replacing the short-lived, wildtype CII with a stabilised variant (CII145), we demonstrated that not only was there a significant bias towards lysogeny, but also that prophage induction efficiency was very strongly inhibited. The outcomes of this study suggested that the key purpose of having a highly active, and rapidly degraded CII is to quickly equilibrate CI levels in a lysogen to ensure the lysogen is established and ready for induction as soon after infection as possible. Lastly, in Chapter 5 we used the data collected throughout this thesis, combined with existing knowledge on 186 to engineer a bacterial whole-cell biosensor that can establish impressively stable cellular memory, with two distinct alternate, stable states. Specific features of the 186 lytic/lysogenic switch and SOS operon were isolated, remodelled and progressively optimised to engineer such a system. We are confident that with the appropriate modifications this system could potentially serve as an environmental sensor or one that can detect and diagnose (e. g. cancer) with high sensitivity and specificity.
Advisor: Shearwin, Keith
Dodd, Ian
Dissertation Note: Thesis (Ph.D.) -- University of Adelaide, School of Biological Sciences, 2020
Keywords: Bacteriophage
prophage induction
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