In 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, φX174, and A2 from the prototypic allolevivirus, Qβ, 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.

In 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, φX174, and A2 from the prototypic allolevivirus, Qβ, 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.

Most scholarship concerning Plato’s Lysis focuses on the failure of Socrates’ elenchus in its endeavor to define friendship. However, this construal of the dialogue is shortsighted. If one analyzes the dialogue’s dramatic subtext then one will discover a fairly complete theory of friendship attributable to Plato. This issue is critical, for the Lysis is a significant influence on Aristotle’s ethical theory. Thus, unless one grasps the relationship between Aristotle’s ethical theory and this particular dialogue, then one could argue that one does not really understand Aristotle’s motivations regarding his usage of friendship as the defining normative force of his political community. Similarly, understanding the Lysis is paramount to understanding Kant’s theory of friendship as well, for Kant can be interpreted as a virtue ethicist. And, analogous to other virtue ethicists such as Aristotle and Plato, Kant espouses a perspective on friendship, which utilizes friendship as the social cohesion of the moral community. However, unlike Plato and Aristotle who argue that friendship exists for the sake of the other person, Kant’s theory claims that one must participate in friendships for the sake of duty. This departure raises various issues regarding his understanding of friendship, for example, are friendships genuine? For Kant, friendship enables those involved to gain a greater understanding of the moral law and nurture relationships which will facilitate that goal. In this respect, like good Aristotelians help one another attain eudaimonia, good Kantians help each other strive towards holiness. Hence, for Kant, the empirical facets of our relationships such as aspiring towards eudaimonia, are not as important as gaining a better understanding of the moral law. Thus, to whom the actions are geared does not matter; it is the actions themselves, which are important. In this respect, while the virtuous will genuinely desire to help their friend, they do not genuinely help their friend in the Ancient Greek sense, since their actions are performed for duty’s sake. Nevertheless, Kant introduces humanistic qualities to friendship, e.g. trust, respect, and self-disclosure, which advances its study into the present day.

T4 requires two proteins: holin, T (lesion formation and lysis timing) and endolysin, E (cell wall degradation) to lyse the host at the end of its life cycle. E is a cytoplasmic protein that sequestered away from its substrate, but the inner membrane lesion formed by T allows E to gain access to the cell wall. T4 exhibits lysis inhibition (LIN), a phenomenon in which a second T4 infection occurs ≤ 3 min after primary infection results a delay in lysis. Mutations that abolish LIN mapped to several genes but only rV encoding the holin, T, and rI, encoding the antiholin, RI, are required for LIN in all hosts which support T4 replication. Antiholin RI inhibits T-mediated lysis by direct interaction with the holin. T has at least one transmembrane domain with its Nterminus (TNTD) in the cytoplasm and C-terminus in the periplasm (TCTD). In contrast, the N-terminus of RI (RINTD) is predicted to function as a cleavable signal sequence allowing the secretion of the RI C-terminal domain (RICTD) into the periplasm. Most of RI mutations which abolish LIN occur in the RICTD, suggesting RI inhibits T-mediated lysis by interacting with T via RICTD. Topological analysis of RI and T showed that fusion of PhoA signal sequence (ssPhoA) to RICTD is necessary and sufficient for LIN and ssPhoAΦTCTD interferes with RI-mediated LIN, indicating T and RI interact via periplasmic C-terminal domains. In T4 infection, LIN is observed only when superinfection takes place, indicating either the antiholin or the LIN signal must be unstable. Both RI and RINTDΦPhoA are localized to both the inner membrane and the periplasm suggesting that the RINTD is a Signal-Anchor-Release (SAR) domain. Protein stability studies indicated that the SAR domain is the proteolytic determinant of RI, and DegP is the protease that is responsible for RI degradation. To date, how TNTD participates in lysis and LIN is not known. Modifications and deletion of the N-terminus of T change the lysis time, indicating this domain is involved the in timing of lysis. GFP fusion to holin T allowed microscopic visualization of fluorescent patches on the membrane at the time of lysis.

The fate of phage-infected bacteria is determined by the holin, a small membrane protein that triggers disruption of the membrane at a programmed time, allowing a lysozyme to attack the cell wall. S2168, the holin of phage 21, has two transmembrane domains (TMDs) with a predicted N-in, C-in topology. Surprisingly, TMD1 of S2168 was found to be dispensable for function, to behave as a SAR ("signal-anchor-release") domain in exiting the membrane to the periplasm, and to engage in homotypic interactions in the soluble phase. The departure of TMD1 from the bilayer coincides with the lethal triggering of the holin and is accelerated by membrane depolarization. Basic residues added at the N-terminus of S2168 prevent the escape of TMD1 to the periplasm and block hole formation by TMD2. Lysis thus depends on dynamic topology, in that removal of the inhibitory TMD1 from the bilayer frees TMD2 for programmed formation of lethal membrane lesions. Like the holin S of λ, the holin of lambdoid phage 21 (S21) controls lysis by forming holes in the membrane. However, unlike Sλ, these holes are small, serving only to depolarize the membrane facilitating the release and activation of the SAR endolysin, R21. We were able to demonstrate that, unlike Sλ, S2168 forms a “pinhole”, thus macromolecules easily pass through Sλ but not S21 holes. This result again supports our interpretation: when S21 triggers, it only needs to collapse the membrane potential, thus causing release and activation of the membrane-tethered inactive SAR endolysin, but does not form holes in the membrane large enough to allow passage of a pre-folded, active cytoplasmic endolysin. The lysis defective S2168 mutant alleles were isolated throughout the S21 gene. Although the majority of lysis defective mutations occurred in the codons for the TMD2 domain, two mutations were found in the codons for the TMD1. This result suggests that only the TMD2 domain of S2168 is likely to participate in actual hole formation. One can assume that two mutant alleles of TMD1 are involved in two different interactions: (a) TMD1-TMD1 intermolecular interaction, (b) TMD1-TMD2 intramolecular interaction. We showed that there is a specific TMD1-TMD2 interaction. In terms of TMD1-TMD2 interaction, the mutated residues of the two TMD1 mutants might prevent a departure of TMD1 from TMD2, resulting in the lysis defective phenotype. Hopefully, these findings deliver some hints about the mechanism of S2168 hole formation and further provoke more extensive work which is required to provide a definite answer to many questions regarding this matter.

Species of Arthrobacter luteus, also known as Oerskovia xanthineolytica, can utilise yeast cells as a growth substrate. This unusual ability is due to the secretion of a battery of hydrolytic enzymes which degrade the yeast cell wall and thus lyse the cells. Although many hydrolytic enzymes are important in the degradation of the yeast cell wall, the key activities are endo beta--l,3-glucanases. In order to characterise components of the yeast lytic system and the genetic organisation of this little-understood organism, a molecular cloning approach was adopted. Large clones expressing beta-1,3-glucanase were isolated from a library of A. luteus DNA constructed in the positive selection vector pKGW. By a combination of subcloning, restriction mapping and Southern analysis, it was determined that the clones contained virtually the same inserts. Additional subcloning, transposon mutagenesis, deletion mapping and nucleotide sequencing were used to identify at least one glucanase gene. The predicted protein product had a molecular weight of about 46 kD. When the gene was expressed in a number of in vivo and vitro systems including E. coli minicells and a Streptomyces coupled transcription/translation system, the protein observed had a similar molecular weight. Furthermore, when the protein was produced in E. coli and run on activity stained gels, the beta-glucanase activity co-migrated with the major glucanase of A. luteus. In addition the E. coli-produced glucanase had the ability to cause limited lysis of yeast.

Like 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.

No description available.

The 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 λ. 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.

Proefschrift--Amsterdam. / "Theses" (4 p.).