Bacteriophage ë has been used as a model replicon system for forty years. While the basic ë replication initiation scheme has been elucidated for several decades, many aspects of the mechanisms are unclear. I wished to study two unanswered issues in ë replication initiation. </p><p>Replication initiation of E. coli and ë each depend upon a protein generally called a licensing factor, which brings the DnaB helicase protein to the origin site to begin DNA synthesis. The licensing factors are the products of host gene dnaC and ë gene P. The synthesis of P from ë DNA in an E. coli cell can competitively interfere with DnaC activity needed for E. coli replication initiation. I wished to learn more about what happens to a host cell when exposed to extended P expression. Previous studies in this laboratory suggested that i) the continuous expression of P was tolerated by a subset of exposed cells and that ii) host defects mapping to dnaB could suppress the effect of extended P expression (P-lethality). I used DNA sequencing to determine if these suppressor mutations were within dnaB. I screened known host mutations for their influence on P-lethality. In summary: E. coli strains with GrpD55 and GrpA80 defects were found to each have two point mutations within their dnaB genes. I was unable to isolate mutations within P that suppressed P-lethality and instead obtained regulatory mutations preventing wild type P expression. Two of these sequenced mutations showed that a cI[Ts] lambda repressor was reverted to cI wild type, blocking P expression at all assay temperatures. P-lethality was reversible in cells exposed to P for up to five hours, causing me to suggest that P-Interference be used in place of the term P-lethality. A non-inducible allele of lexA prevented P-mediated cellular filamentation and enhanced P-Interference. This suggests that induction of the SOS response helps cells to tolerate extended P expression. A host strain containing a defective ClpXP protease significantly enhanced cellular sensitivity to P-Interference. This suggests an important role for the ClpXP chaperone-protease complex in degradation of P and cellular resistance to P expression. I present models to explain the P-Interference Phenotype.</p><p>Recent reports have re-opened the possibility that the tO-oop-pO element influences ë DNA replication initiation. I have also been investigating this possibility. I found that a plasmid with tO-oop-pO (the terminator, nucleotide sequence and promoter for OOP RNA) and orië DNA sequence was inhibitory to the development of repë phages, and designated this the Inhibition Phenotype (IP). In pursuing the mechanism for this inhibition, I mutated the tO-oop-pO and orië elements. I found that the expression of the 77nt OOP RNA transcript and the presence of four 18 base pair repeats (iterons) within orië were required for the IP. I isolated spontaneous phage mutants, resistant to the IP. I determined that singly infected cells were sensitive to the IP but that multiply infected cells escaped the IP. I propose that the IP to repë phage development is directed to the initial or theta mode of ë replication initiation. I found that the theta-mode of ë replication initiation can be bypassed, likely via recombination between multiple phage genomes within a singe cell. I propose models to explain the IP and also suggest a role for OOP RNA in the regulation of ë DNA replication.

The purpose of the work described in this dissertation is to better understand the role of Rz and Rz1 function with respect to phage lysis. We determined using both a genetic and biochemical approach that the Rz protein is an inner membrane protein containing a single N-terminal transmembrane domain (TMD) with an Nin/Cout topology. Consistent with previous work on Rz1, the Rz1 lipoprotein was found to be localized to the outer membrane (OM). Following localization, both Rz and Rz1 form homodimers in vivo due to intermolecular disulfide formation. Despite being localized to apposing membranes, the two proteins form a complex. A small number of phages encode a potential single protein equivalent of Rz-Rz1. This protein, termed a spanin, is predicted to tether the inner and outer membranes by a single polypeptide chain. Based on complementation, it was concluded that gp11 from the phage T1 is a functional equivalent of Rz-Rz1. Gp11, and by analogy the Rz-Rz1 two-component spanin complex, threads the meshwork of the PG layer. The presence of an Rz-Rz1 complex, which forms in the presence of peptidoglycan (PG), is supported by in vivo results. The soluble periplasmic domains of Rz and Rz1, which are dimeric and monomeric respectively, were purified. Circular dichroism analysis indicates that Rz is structured, with significant α-helical content, whereas Rz1, in which 10 out 39 residues are proline, is unstructured. Mixing the proteins results in the formation of a complex with significant new α-helical content. Negative-stain images reveal ~ 25 nm x ~ 4 nm rod-shaped structures. Holin independent activity of Rz and Rz1 is found to disrupt whole cells. Furthermore, time lapse microscopy of λ and λRzam lysis allows us to conclude that Rz and Rz1 are essential for lysis. These results suggest a model for Rz-Rz1 function which begins with Rz and Rz1 forming a complex through direct interaction prior to holin and endolysin function. Holin-mediated hole formation allows the endolysin to degrade PG which sterically hinders Rz-Rz1 activity. Removal of PG by endolysin degradation thus triggers Rz-Rz1 OM disruption via fusion of the inner and outer membranes.

The bacteriophage lambda tail protein gpK is required for tail assembly. The activity of the protein can be found at the assembling tail tip and is believed to be localized to this structure. GpK is a 27 kDa protein that has sequence identity to two families of proteins: the Mov34 family of peptidases and the NlpC/P60 family of peptidoglycan endopeptidases. Point substitutions and complementation data confirm that gpK possesses each of these domains and that they can function in trans. When the Mov34 domain is inactivated tail assembly is disrupted whereas when the NlpC/P60 domain is inactivated tails assemble but are inactive. Evidence is presented here that the C-terminal domain possesses lytic activity in isolation but not when part of the full-length protein.

The bacteriophage lambda tail protein gpK is required for tail assembly. The activity of the protein can be found at the assembling tail tip and is believed to be localized to this structure. GpK is a 27 kDa protein that has sequence identity to two families of proteins: the Mov34 family of peptidases and the NlpC/P60 family of peptidoglycan endopeptidases. Point substitutions and complementation data confirm that gpK possesses each of these domains and that they can function in trans. When the Mov34 domain is inactivated tail assembly is disrupted whereas when the NlpC/P60 domain is inactivated tails assemble but are inactive. Evidence is presented here that the C-terminal domain possesses lytic activity in isolation but not when part of the full-length protein.

Thesis (Ph. D.)--University of Oregon, 2004. / Typescript. Includes vita and abstract. Includes bibliographical references (leaves 163-172). Also available for download via the World Wide Web; free to University of Oregon users.

Errata sheet pasted opposite Table of Contents. / Includes bibliographies. / 142, [171] leaves : ill. ; 30 cm. / Title page, contents and abstract only. The complete thesis in print form is available from the University Library. / This thesis investigates the UV-induced inhibition of oriC initiation function and the nature of the phenotype of the mutant origin of replication of Escherichia coli, oriCc. The specific aims of this study are to delineate the source of the UV-induced trans-acting inhibition of oriC function, utilizing the phage vector [lambda]poriCc, and to demonstrate the oriCc allele as an enhanced DNA replication initiator from oriC. / Thesis (Ph.D.)--University of Adelaide, Dept. of Biochemistry, 1997?

Thesis (Ph. D.)--Ohio State University, 2007. / Title from first page of PDF file. Includes bibliographical references (p. 152-161).

Thesis--Carleton University. / Also available in electronic format on the Internet.

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