Browsing by Subject "lysis"
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Item Bacteriophage ms2 l protein: genetic and biochemical characterization(2009-05-15) McIntosh, Brenley KathleenIn order to release progeny, bacteriophages must lyse the host cell by compromising the peptidoglycan layer. There are two known strategies of lysis: the holin-endolysin system and single gene lysis (SGL), which are dependent on the genome size. Large phages encode multiple proteins, including a holin and endolysin, for lysis. In contrast, small ssRNA phages (Leviviridae and Alloleviviridae) and ssDNA phages (Microviridae) do not encode a muralytic enzyme and accomplish lysis with a single gene. The cellular target of the lysis gene E from the prototypic microvirus, ?X174, and A2 from the prototypic allolevivirus, Q?, has been elucidated. In both cases, these proteins were demonstrated to inhibit specific enzymes within the peptidoglycan biosynthetic pathway and infected cells lyse as a result of septal catastrophes. The prototype Levivirus MS2 encodes L, a 75 aa polypeptide that effects lysis without inhibiting murein synthesis. The purpose of the work described in this dissertation was to characterize MS2 L using genetic and biochemical strategies. Using a genetic approach, PcnB was shown to be important to the entry of the MS2 RNA into the cytoplasm. L accumulation during infection was quantified by comparison to purified, oligohistidine-tagged L. Biochemical experiments demonstrated the L protein behaved as a periplasmic, membrane-associated protein. The morphologies of cells undergoing L-mediated lysis are significantly different from cells lysing due to A2 expression, since L-lysing cells do not show septally localized membrane protrusions.Item Bacteriophage P1: a new paradigm for control of phage lysis(Texas A&M University, 2005-11-01) Xu, MinThe N-terminal hydrophobic domain of the phage P1 endolysin Lyz was found to facilitate the export of Lyz in a sec-dependent fashion, explaining the ability of Lyz to cause lysis of E.coli in the absence of the P1 holin. The N-terminal domain of Lyz is demonstrated to be both necessary and sufficient not only for export to the membrane but also for release into the periplasm of this endolysin. We propose that this unusual N-terminal domain functions as a "signal arrest- release" (SAR) sequence, which first directs the endolysin to the periplasm in membrane-tethered form and then allows it to be released as a soluble active enzyme in the periplasm. To understand why release from the membrane is required for the physiological expression of the lytic activity of Lyz, we examined the role of its seven cysteine residues in the biogenesis of the active endolysin. The inactive, membrane-tethered and the active, soluble forms of Lyz differ in their pattern of intramolecular disulfide bonding. We conclude that the release of Lyz from the membrane leads to an intramolecular thiol-disulfide bond isomerization causing a dramatic conformational change in the Lyz protein. As a result, an active site cleft that is missing in nascent Lyz is generated in the mature form of the endolysin. Examination of the protein sequences of related bacteriophage endolysins suggests that the presence of an SAR sequence is not unique to Lyz. Studies on holin and antiholin indicated that P1 encodes two holins, LydA and LydC. The antiholin LydB inhibits LydA by binding to it directly on the membrane. All above results demonstrate a new paradigm for control of phage lysis, which is, upon depolarization of the membrane by holin function at a programmed time, endolysin is released from the bilayer leading to the immediate lysis of the host.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 Deciphering Lysis and its Regulation in Bacteriophage T4(2012-10-19) Moussa, SamirLike all phages, T4 requires a holin (T) to effect lysis. The lysis event depends on the temporally regulated action of T, which accumulates in the inner membrane (IM) until, at an allele-specific time, it triggers to form a large "hole" in the membrane. Hole formation then releases T4 lysozyme into the periplasm where it degrades the cell wall to elicit cell lysis. Unlike other phages, T4 is unique in exhibiting real-time regulation of lysis based on environmental conditions. Specifically, lysis can be delayed indefinitely in the lysis-inhibited state (LIN), where the normal temporal schedule for holin-triggering is over-ridden. Recently, it was shown that the imposition of LIN was correlated with the interaction of the periplasmic domains (PD) of RI and T. These studies have been extended in this dissertation using genetic, biochemical, and structural techniques to address the molecular mechanism of the RI-T LIN system. First, the PD of RI and an RI-T complex were purified, characterized biophysically, and crystallized to yield the first atomic resolution structures of either a holin or antiholin. The RI PD is mostly alpha-helical that undergoes a conformational change, as revealed by NMR spectroscopy studies, when bound to T. The PD of T is globular with alpha-helical, beta strand, and random coil secondary structures. Additionally, the holin was genetically characterized by mutagenesis techniques, yielding new information on its role in both lysis and LIN. Lysis defective mutants in all three topological domains: cytoplasmic, transmembrane, and periplasmic, were isolated. Analysis of these mutants revealed that both the cytoplasmic and periplasmic domains are important in the oligomerization of T. During LIN, the RI PD binds the PD of T, blocking a holin oligomerization interface. Finally, the signal for the imposition of lysis inhibition has been elucidated using NMR spectroscopy and other in vitro studies. These studies have shown that the RI PD binds DNA. From these studies, new models for lysis and LIN have been constructed. Lysis occurs with the accumulation and oligomerization of T via cytoplasmic and periplasmic domain interactions. LIN is imposed when the ectopically localized DNA of a superinfecting phage interacts with RI, stabilizing it in a conformation competent in inhibiting T oligomerization and leading to lysis inhibition.Item Exploring the Pinhole: Biochemical and Genetic Studies on the Prototype Pinholin, S21(2011-08-08) Pang, TingLysis of the host by bacteriophage 21 requires two proteins: the pinholin S21 (forms pinholes in the cytoplasmic membrane and controls lysis timing) and the endolysin (degrades the cell wall). S21 has a dual-start motif, encoding a holin, S2168, and a weak antiholin, S2171. Both proteins have two transmembrane domains (TMD) and adopt an N-in, C-in topology. The topology of S2168 is dynamic because TMD1 is a signal-anchor-release (SAR) domain which, while initially integrated into the cytoplasmic membrane, is eventually released into the periplasm. TMD1 is dispensable because the truncated protein, S2168?TMD1, retains the holin function. Adding two positive charges to N-terminus of S2168 by an irs tag (RYIRS) prevents the release of TMD1. The irsS2168 protein not only has lost its holin function, but is a potent antiholin and blocks the function of S2168. In this dissertation, the structure of S2168 was suggested by incorporating electron-microscopy, biochemical, and computational approaches. The results suggest that S2168 forms a symmetric heptamer, with the hydrophilic side of TMD2 lining the channel of ~ 15 A in diameter. This model also identifies two interacting surfaces, A and B, of TMD2. A model for the pinhole formation pathway was generated from analyzing phenotypes of an extensive collection of S21 mutants. In this model, the individually folded and inserted S21 molecules first form the inactive dimer, with the membrane-inserted TMD1 inhibiting the lethal function of TMD2 both inter- and intra-molecularly. A second inactive dimer may form, with one TMD1 released. When both TMD1s are released, the activated dimer is formed, with the homotypic interfaces A:A interaction of the TMD2s. However, this interaction might not be stable, which will shift to heterotypic A:B interactions, allowing TMD2 to oligomerize. Finally, the pinhole forms, possibly driven by the hydration of lumenal hydrophilic residues. In addition, the localization of pinholes was visualized by fusing the green fluorescent protein (GFP) to the C-terminus of pinholins. The results showed that pinholins form numerous small aggregates, designated as rafts, spread all over the cell body. The antiholin irsS2168 not only inhibits the triggering of S2168GFP, but inhibits the rafts formation as well.Item SAR Endolysin Regulation in dsDNA Phage Lysis of Gram-Negative Hosts(2012-02-14) Kuty, GabrielSAR endolysins are a recently discovered class of muralytic enzymes that are regulated by dynamic membrane topology. They are synthesized as enzymatically inactive integral membrane proteins during the phage infection cycle and then are activated by conformational remodeling upon release from the membrane. This topological duality depends on N-terminal SAR (Signal-Anchor-Release) domains, which are enriched in weakly hydrophobic residues and require the proton motive force to be maintained in the bilayer. The first SAR endolysin to be characterized was P1 Lyz, of phage P1. Its activation requires a disulfide bond isomerization involving its catalytic Cys initiated by a free cysteine thiol from the newly-liberated SAR domain. A second mode of disulfide bond regulation, as typified by Lyz103 of the Erwinia Amylovora phage ERA103, has been demonstrated. In its membrane bound form, Lyz103 is inactivated by a disulfide that is formed between cysteine residues flanking a catalytic glutamate. A second class of SAR endolysins, typified by R21, the lysozyme of the lambdoid phage 21, does not require disulfide bond isomerization for activation. Rather, these proteins are dependent on the release of the SAR domain for proper folding of the catalytic cleft. Bioinformatic analysis indicates that the regulatory theme of R21 is common in the SAR endolysins of dsDNA phages. Furthermore, bioinformatic study of endolysins of dsDNA phage of Gram-negative hosts revealed two new classes of SAR endolysins that are not homologous to T4 gpe, as all SAR endolysins were once thought to be. SAR endolysins were found in nearly 25% of sequenced dsDNA phages of Gram- negative hosts including 933W, which is involved in the release of Shiga toxin from EHEC strain EDL933. An inhibitor study against the SAR endolysin of 933W, R933W, was performed using a custom compound library in a high through-put, in vivo lysis assay. Of nearly 8,000 compounds screened, one compound, designated 67-J8, inhibited lysis but not growth. In vivo and in vitro experiments show that the compound has no effect on R933W activity, accumulation, or secretion. In vivo experiments suggest that 67-J8 increases the proton motive force, thereby presumably retaining the SAR domain in the membrane.Item The phiX174 Lysis Protein E: a Protein Inhibitor of the Conserved Translocase MraY(2010-07-14) Zheng, YiMost bacteriophages release progeny virions at the end of the infection cycle by lysis of the host. Large phages with double-stranded DNA genomes use a multigene strategy based on holins, small membrane proteins, and bacteriolytic enzymes, or endolysins. Holins mediate the control of endolysin activity and thus the timing of lysis. Phages with small genomes only encode a single protein for cell lysis. There are three known unrelated single protein lysis systems: the ?X174 E protein, the MS2 L protein, and the Q? A2 protein. None of these phages encodes a cell wall degrading activity, and previous work has shown that the lytic activity of E stems from its ability to inhibit the host enzyme, MraY, which catalyzes the formation of lipid I, the first lipid intermediate in cell wall synthesis. The purpose of the work described in this dissertation was to characterize the ?X174 E-mediated inhibition of MraY using genetic and biochemical strategies. A fundamental question was why no large phages use the single gene system. This was addressed by constructing a recombinant phage, ?E, in which the holin-endolysin based lysis cassette of ? was replaced with E. ?E was compared with ? in genetic and physiological experiments, with the results indicating that the holin-endolysin system increases fitness in terms of adjusting lysis timing to environmental conditions. Using ?E, physiological experiments were conducted to characterize the interaction between E and MraY in vivo. Transmembrane domains (TMD) 5 and 9 have been identified as the potential E binding site by isolating MraY mutants resistant to E inhibition. The five Eresistant MraY mutants were found to fall into three classes, which reflect the apparent affinity of the mutant proteins for E. Finally, an assay for MraY activity employing the dansylated UDP-MurNAc-pentapeptide and phytol-P, was used to demonstrate the inhibition of MraY by purified E protein. It was determined that E is a non-competitive inhibitor for MraY in respect with both substrates. A model for E-mediated inhibition of MraY was proposed, in which E binds to TMDs 5 and 9 in MraY and thus inactivates the enzyme by inducing a conformational change.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.