Browsing by Subject "S105"
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Item Biochemical and Genetic Characterization of Bacteriophage Holins(2013-11-06) To, Kam HoBacteriophages infect and kill bacterial cells. During the infection cycle, a phage attaches to the host cell surface, then ejects its DNA into the cytoplasm, where its progenies are subsequently assembled. The final step of the infection cycle is host cell lysis, which allows the progeny virions to escape into the environment. However, the timing of lysis, and thus the length of the infection cycle, is independent of endolysin biosynthesis and rather depends on the function of a second class of lysis proteins, the holins. Holins are small integral membrane proteins that accumulate harmlessly in the membrane during the infection cycle, until they suddenly form lethal lesions in the membrane at an allele-specific time. This membrane damage allows the endolysin to attack the cell wall. This dissertation focuses on several aspects of the structural and functional aspect of holins. First, Y is the putative holin gene of the paradigm coliphage P2. Although Y is not related to the S holin of phage lambda according to its primary structure, its characterization might prove useful in discerning the essential traits for holin function. In this instance, physiological and genetic approaches are utilized to show that Y exhibits the essential holin functional criteria, namely, allele-specific delayed-onset lethality and sensitivity to the energization of the membrane. These results suggest that class I holins share a set of unique features that are needed for their remarkable ability to program the end of the phage infection cycle with precise timing. Nevertheless, I report studies involving phenotypic analysis of a systematic library of clustered site-directed mutants of S105, and then conclude with experiments designed to probe the structure of the mature ?S-hole? in the membrane of the cell using chemical probes. Furthermore, I address whether the Y holin and the S21 pinholin of phage 21 effect membrane depolarization with the same all-or-nothing fashion as S while using the same tethered- cell assay previously employed for studying S. Finally, the holin and antiholin in Mu, one of the few paradigm coliphage, were identified and characterized. The introductory chapter is intended to serve as an update to the last major review on holin function in 2000.Item Characterization of the Bacteriophage Lambda Holin and Its Membrane Lesion(2011-10-21) Dewey, Jill SayesBacteriophage holins are a diverse group of proteins that are responsible for the spontaneous and specifically-timed triggering of host cell lysis. The best-studied holin, S105 of phage lambda, is known to form lesions, or ?holes?, in the inner membrane of E. coli which are large enough to allow the endolysin through to the periplasm. S105 has been studied extensively by both genetic and biochemical approaches; however, little is known about the mechanism of hole formation or the structure of the lambda holin and its inner membrane lesion. An in vitro system for reconstituting hole formation by S105 was developed in which liposomes containing a self-quenched fluorophore served as artificial cell membranes (1-2). Upon delivery of solubilized S105 to the liposomes, an increase in fluorescence was observed, indicating that the fluorophore within the liposomes had escaped into the surrounding media via an S105-mediated hole in the membrane. This in vitro system, which has been optimized in this work, has been a valuable biochemical tool for analysis and reconstitution of the pathway to S105 hole formation in the cell membrane. Due to the difficulty associated with over-expression and purification of toxic membrane proteins, there are no solved structures of bacteriophage holins. Sample preparation and experimental conditions for NMR spectroscopy were optimized and structural information about a lambda holin mutant protein was obtained. Specifically, micellar contacts of transmembrane domain regions versus water contacts of the C-terminal region, secondary structure, and backbone dynamics were determined. Cryo-electron microscopy was used to visualize the inner membrane lesions formed by phage holins [lambda] S105, P2 Y, and T4 T. Therefore, the large holes initially seen in cells expressing S105 are not specific to the lambda holin, nor to class I holins. The S105 holes average ~340 nm (3), and are the largest membrane lesions ever observed in biology. They are stable at their original size, and are not localized to a specific region of the membrane. In addition, missense mutants of S105 were used to correlate hole size, protein accumulation, and lysis timing in a current model for the S105 hole formation pathway.Item What makes the lysis clock tick? A study of the bacteriophage holin(2009-05-15) White, Rebecca LynnThe timing of host lysis is the only decision made in the bacteriophage lytic cycle. To optimize timing, double-stranded DNA phages use a 2-component lysis system consisting of a muralytic enzyme, the endolysin, and a small membrane protein, the holin, which controls the timing of lysis. The best characterized holin gene to date is the S gene of bacteriophage ?. One unusual feature of the S gene is that it produces two proteins of opposing function: the holin, S105, and the antiholin, S107. Raab et al isolated and characterized a number of S mutants, but all of them expressed both the holin and the antiholin; it is possible, then, that the true extent of the holin-holin interactions were masked by interactions with the antiholin. Thus, a large number of S105 mutants were created, and their phenotypes characterized in the absence of the antiholin. The interaction between those mutants and the wild-type were examined in an attempt to better understand what determines the timing of hole formation by S105. S105 and S107 differ only by two amino acids at the N-terminus; S107 has an additional Met-Lys sequence. Previous studies have shown that S107 may have a different topology to S105, where the N-terminus of S107 is located in the cytoplasm and is cannot flip through the membrane because of the extra cationic side chain. This study investigates the role of the N-terminal transmembrane domain of the S proteins in terms of hole formation and its role in the antiholin character of S107. Previous results suggest that S105 forms hole via a large oligomeric structure termed the ?death raft?. The death raft model states that after S105 is inserted into the membrane, it forms ?rafts?, which grow in size until a spontaneous channel forms leading to depolarization of the membrane and hole formation. This study investigates the pathway of hole formation at the single-cell level, using a C-terminal fusion of S105 and green fluorescent protein, and attempts to address several of the predictions posed by the death raft model.