Browsing by Subject "Holin"
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Item Biochemical and genetic characterization of bacteriophage 21 holin: S21 as a membrane protein and beyond(2009-05-15) Park, TaehyunThe 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 ?, the holin of lambdoid phage 21 (S21) controls lysis by forming holes in the membrane. However, unlike S?, 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 S?, S2168 forms a ?pinhole?, thus macromolecules easily pass through S? 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.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.