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Identification of High-Copy-Number Inhibitors IDENTIFICATION OF HIGH-COPY-NUMBER INHIBITORS OF Pl PLASMID STABILITY Natalie Erdmann A thesis submitted in conformity with the requirements for the degree of Master of Science Graduate Department of Molecular and Medical Genetics University of Toronto O Copyright by Natalie Erdmann (1998) National ïibrary Bibliothèque nationale du Canada Acquisitions and Acquisitions et Bibliographie Services seMces bibliographiques 395 Wellington Street 395, rue wermgtori ûüaw%ON K1AûN4 CRhwaON KlAW canada canada The author has granted a non- L'auteur a accordé une licence non exclusive licence allowing the exclusive permettant à la National Lïbrary of Canada to Bibliothèque nationale du Canada de reproduce, loan, distribute or sell reproduire, prêter, dimiuer ou copies of this thesis in microform, vendre des copies de cette thèse sous paper or electronic formats. la forme de microfiche/film, de reproduction sur papier ou sur format électronique. The author retains ownership of the L'auteur conserve la propriété du copyright in this thesis. Neither the droit d'auteur qui protège cette thèse. thesis nor substantial extracts hmit Ni la thèse ni des extraits substantiels may be printed or otherwise de celle-ci ne doivent être imprimés reproduced without the author's ou autrement reproduits sans son permission. autorisation. Identification of High-Copy-Number Inhibitors of Pl Plasmid Stability by Natalie Erdmann Master of Science, Graduate Department of Molecular and Medical Genetics, University of Toronto, 1998 ABSTRACT Pl is a low-copy-number plasmid that requires an active partition system for stable maintenance in Escherichia coli. The Pl par operon contains two cotranscribed genes, parA and parB, and a downstrearn cis-acting site, parS. ParA and ParB are both essential for partition, but overproduction of either protein disturbs partition and destabilizes P 1. Partition must position Pl plasmids so that each daughter cell receives one copy. Bacterial host factors are believed to participate in the positionhg process. A genetic screen using a hi&-copy library of E. coli genes was performed to identiQ host factors, as it was anticipated that overproduction of key host proteins would disrupt the partition process and destabilize P 1. 1 identified ten gene products that destabilize Pl when overproduced, but are not directly required for partition. Subsequent tests were performed to elucidate the functions of these gene products, but their roles in Pl partition remain unknown. ACKNOWLEDGEMENTS 1 have to thank my supe~sor,Dr. Barbara E. Funnell, for her continued guidance and support, and for making my graduate school experience one of intellectual and personal growth. I am also grateful to my supervisory committee members, Dr. Andrew Spence and Dr. Johanna Rommens, for their advice and expertise. 1 am indebted to my lab-mates - Jennifer Surtees, Megan Davey, and Jean Yves Bouet - for their boundless help and generosity in answering my (seemingiy) endless questions. Thanks also to my Med Gen fiiend, fellow shopaholic, and confidante, Beaûice Seguin. To Cathy, Jenn, Blake, and Kerry - thank-you for your friendship and for providing me with what linle social life 1 had ... and to Michael, for distracting me when 1 should have working. Finally, 1 would like to express my deepest gratitude to my family - Mom,Dad, and Mark. 1 owe any success 1 have to their examples of hard work and dedication. and their constant encouragemenf support, and pride. iii TABLE OF CONTENTS Abstract Acknowledgements Table of Contents List of Figures and Tabtes Chapter 1 General Introduction - Escherichia coli chromosome partition and ce11 division - Plasmid stability models - The P 1 partition system - Involvement of host factors Chapter 2 Materiah and Methods Chapter 3 Results -Chamterization of library inserts - Examining the roies of the interference genes in Pl partition - Effects on E. coli gross morphology - Effects on P 1 copy number - Effects on Par protein expression - Interactions of library plasmid gene products Chapter 4 Discussion -Interference genes -Essential par components -Future directions Figures Tables Re ferences List of Figures and Tables Figure 1. Models for active plasmid partition Figure 2. The sop/par family of partition operons for plasrnids P 1, P7, and F Figure 3. The porS site of P 1 Figure 4. A model of Pl plasmid partition Figure 5. Potential roles for host factors in Pl plasmid partition Figure 6. Vectors Figure 7. The test plasrnid pBEF2 18 Figure 8. Replication Test plasrnids Figure 9. Bacterid DNA inserts of final library candidates Figure 10. Characterization of potential partition inhibitors Figure 11. Determination of copy nurnbers of Replication Test plasmids Figure 12. Effect of library plasrnids on par gene expression Table 1. List of abbreviations Table 2. Bacterial strains Table 3. Categorization of blue phenotypes produced by librq plasmids Table 4. Summary of library inserts CHAPTER 1 GENERAL INTRODUCTION The bacterial chromosome encodes al1 of the essentiai information for the bacterial ceIl and therefore requires a highly accurate segregation system to ensure that it is stably maintained fiom generation to generation. The process of segregation in bacteria, called partition, in which daughter chromosomes are separated and positioned prior to septum formation and ce11 division, involves mechanisms that are largely unknown. Segregation factors resembling those involved in mitosis in eukaryotes have not yet been identified in bacteria. Like the bacterial chromosome, low-copy-number plasmids require an active partition system to guarantee that each daughter ce11 receives at least one copy of the plasmid. Plasmids are extrachromosomal, usuaily circular DNA duplexes that are not essential for the host, but cm be beneficial for the ce11 by providing antibiotic resistance genes and virulence factors. For example, the pINV family of plasmids allow Shigellaflemeri and enteroinvasive species of Escherichia coli to enter epithelial cells and cause intestinal disease (Sasakawa et uZ., 1986; Makino et al., 1988). Plasmids are also usually much smaller and less complex than the chromosome, making them easier to manipulate and analyze. These characteristics make plasmids convenient models for the midy of the partition process. A variety of low-copy-number plasmids possess homologous partition systems, and I have used the Pl plasmid to investigate an important aspect of partition: the identification of potential host factors that position the newly replicated DNA molecules on each side of the division plane. Such factors are believed to be essential cornponents for partition, and 1 have performed a genetic screen in Escherichia coli to idena them. Because plasmids often use bacterially encoded components for their own survival, it seems likely that the partition systems of plasrnids and bacterial chromosomes may share some components adorsteps. If not, it would at lest be expected that there are comparable components that perform equivalent functions in segregation. For this reason, examination of 2 the partition mechanisms in a variety of systems is important to gain an understanding of dlof the required elements for active segregation. Escheridia coli chromosome partition and ceil division Separation of catenanes and resolution of dimers after DNA replication. DNA replication is initiated at oriC by a multienzyme complex including the DnaA initiator protein, RNA polymerase, DNA gyrase, DNA Polymerase III holoenzyme, single-stranded binduig protein (SSB), histone-like protein (HU), DnaB helicase, DnaC protein, and DnaG primase. and proceeds bidirectionally (Kornberg and Baker, 1992). Termination of replication occurs when the two replication forks meet at the temiinus region. Mer DNA replication is completed the daughter chromosomes are often present as catenanes or as circular dimers. These structures result fiom incomplete topological uniinking durhg replication or fiom homologous recornbination, respectively. They must be completely unlinked and resolved into single, circuiar chromosomes to allow partition to be completed. Decatenation of catenanes is carried out primarily by the type 2 topoisomerase, topoisomerase IV (topo IV), encoded by the parc and parE genes. The Parc subunit of topo IV appears to be associated with the cytoplasmic membrane, implying that the decatenating machinery is localized at the membrane (Kato et al., 1992). Overexpression of the other known bacterial type 2 topoisomerase, DNA gyrase, can partially suppress the growth defect observed in topo IV mutants. This observation indicates that DNA gyrase and topo N may have functional overlap, although DNA gyrase7smain role is to catalyze the ATP-dependent negative supercoiling of DNA to relieve ovenvinding during replication. Zechiedrich and Conarelli (1995) demonstrated that DNA gyrase is approximately 100-fold less efficient than topo N in unlinking replicated daughter plasmids in vivo, indicating that topo N is the critical decatenase in Escherichia coli Chromosornal dimers are resolved into monomers by a site-specific recombination event at the replication terrninus region. Resolution is catalyzed by the bacterial XerC-XerD recombinase at the terminal dif (deletion-induced- -filamentation) site (Clerget, 199 1; Blakely et al., 199 1; Kuempel et al., 1991). Strains containing mutations in xerC, xerD, and Mifform filamentous cells and are defective in chromosome segregation. This defect is suppressed by recA mutations, illustrating that dimerization occurs as
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