Regulation and Release of VSH-1, a Prophage and Gene Transfer Agent of Brachyspira Hyodysenteriae Eric Glenn Matson Iowa State University
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Iowa State University Capstones, Theses and Retrospective Theses and Dissertations Dissertations 2004 Regulation and release of VSH-1, a prophage and gene transfer agent of Brachyspira hyodysenteriae Eric Glenn Matson Iowa State University Follow this and additional works at: https://lib.dr.iastate.edu/rtd Part of the Microbiology Commons Recommended Citation Matson, Eric Glenn, "Regulation and release of VSH-1, a prophage and gene transfer agent of Brachyspira hyodysenteriae " (2004). Retrospective Theses and Dissertations. 1183. https://lib.dr.iastate.edu/rtd/1183 This Dissertation is brought to you for free and open access by the Iowa State University Capstones, Theses and Dissertations at Iowa State University Digital Repository. It has been accepted for inclusion in Retrospective Theses and Dissertations by an authorized administrator of Iowa State University Digital Repository. For more information, please contact [email protected]. NOTE TO USERS This reproduction is the best copy available. ® UMI Regulation and release of VSH-1, a prophage and gene transfer agent of Brachyspira hyodysenteriae by Eric Glenn Matson A dissertation submitted to the graduate faculty in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Major: Microbiology Program of Study Committee: Thaddeus B. Stanton, Co-major Professor Gregory J. Phillips, Co-major Professor Gwyn A. Beattie F. Chris Minion Vijay Sharma Richard L. Zuerner Iowa State University Ames, Iowa 2004 UMI Number: 3158357 INFORMATION TO USERS The quality of this reproduction is dependent upon the quality of the copy submitted. Broken or indistinct print, colored or poor quality illustrations and photographs, print bleed-through, substandard margins, and improper alignment can adversely affect reproduction. In the unlikely event that the author did not send a complete manuscript and there are missing pages, these will be noted. Also, if unauthorized copyright material had to be removed, a note will indicate the deletion. UMI UMI Microform 3158357 Copyright 2005 by ProQuest Information and Learning Company. All rights reserved. This microform edition is protected against unauthorized copying under Title 17, United States Code. ProQuest Information and Learning Company 300 North Zeeb Road P.O. Box 1346 Ann Arbor, Ml 48106-1346 ii Graduate College Iowa State University This is to certify that the doctoral dissertation of Eric G. Matson has met the requirements of Iowa State University Signature was redacted for privacy. ^Co-major Profess Signature was redacted for privacy. Co-m Professor Signature was redacted for privacy. For the Major Program iii To my parents, Charles and Faye and my wife, Theresa for their enduring support and encouragement iv TABLE OF CONTENTS LIST OF FIGURES vi LIST OF TABLES viii CHAPTER 1. GENERAL INTRODUCTION 1 Dissertation Organization 2 Literature Review 2 References 27 CHAPTER 2. IDENTIFICATION OF GENES OF VSH-1, A DEFECTIVE BACTERIOPHAGE AND GENE TRANSFER AGENT OF BRACHYSPIRA HYOD YSENTERIAE 43 Abstract 43 Introduction 44 Materials and Methods 45 Results 49 Discussion 51 References 61 CHAPTER 3. CHARACTERIZATION OF AN ENDOLYSIN OF VSH-1, A DEFECTIVE BACTERIOPHAGE AND GENE TRANSFER AGENT OF BRACHYSPIRA HYODYSENTERIAE 67 Abstract 67 Introduction 68 Materials and Methods 69 Results 75 Discussion 78 References 93 CHAPTER 4. INDUCTION AND CHARACTERIZATION OF VSH-1 TRANSCRIPTION IN BRACHYSPIRA HYODYSENTERIAE 97 Abstract 97 Introduction 98 Materials and Methods 100 Results 108 Discussion 113 References 129 V CHAPTER 5. GENERAL CONCLUSIONS AND RECOMMENDATIONS FOR FUTURE RESEARCH 133 General Conclusions 133 Recommendations for Future Research 135 ACKNOWLEDGEMENTS 137 vi LIST OF FIGURES CHAPTER 1. Figure 1. B. hyodysenteriae cell 7 Figure 2. VSH-1 particles 17 CHAPTER 2. Figure 1. PGR amplicons pattern for the svplOl region 59 Figure 2. Genetic map of VSH-1 and adjacent ORFs 60 CHAPTER 3. Figure 1. Basic structure of a peptidoglycan oligosaccharide chain shown with a peptide cross-link 87 Figure 2. L-arabinose induction of E. coli clones 88 Figure 3. Purification of recombinant LysV 89 Figure 4. Lysis of E. coli cells by purified LysV 90 Figure 5. Activity of LysV on purified B. hyodysenteriae peptidoglycan 91 Figure 6. Reducing sugar assay for peptidoglycan hydrolysis 92 CHAPTER 4. Figure 1. Northern slot blot of B. hyodysenteriae RNA 120 Figure 2. Transcript levels of VSH-1 genes in B. hyodysenteriae cells treated with mitomycin C 121 Figure 3. Transcript levels offlaAl in B. hyodysenteriae cells treated with mitomycin C 122 Figure 4. Intergenic RT-PCR amplification of VSH-1 genes 123 Figure 5. Northern blots of B. hyodysenteriae RNA 124 Figure 6. VSH-1 transcriptional start signals 125 vii Figure 7. Nucleotide positions of VSH-1 transcriptional start sites 126 Figure 8. Hydrogen peroxide-induced transcription of the VSH-1 svp38 gene 127 Figure 9. Chloramphenicol inhibition of VSH-1 transcription 128 viii LIST OF TABLES CHAPTER 1. Table 1. Properties of Gene Transfer Agents 19 CHAPTER 2. Table 1. Oligonucleotide primers and probes used for PCR amplification of svplOJ and screening a B. hyodysenteriae genomic DNA library 56 Table 2. Predicted VSH-1 and adjacent bacterial ORFs 58 CHAPTER 3. Table 1. Most closely matching homologs by a BLAST search of GenBank using the lysV translated amino acid sequence 84 Table 2. Osmosensitivity assay for peptidoglycan-degrading enzymes 85 Table 3. Substrate specificity of LysV 86 CHAPTER 4. Table 1. Oligonucleotide primers and probes used for comparative RT-PCR, primer extension, cloning, and hybridization of VSH-1 and B. hyodysenteriae genes 118 Table 2. Oligonucleotide primers used for intergenic RT-PCR analysis of co-transcription of VSH-1 genes 119 1 CHAPTER 1. GENERAL INTRODUCTION Brachyspira hyodysenteriae is an anaerobic spirochete and an important pathogen of swine. B. hyodysenteriae cells harbor VSH-1, a mitomycin C-inducible prophage that mediates generalized transduction between B. hyodysenteriae strains (40). VSH-1 virions package random fragments of B. hyodysenteriae chromosomal DNA rather than a viral genome, which has complicated genetic investigations (40). N-terminal amino acid sequences generated for proteins from purified VSH-1 whole virions and tailless heads facilitated the identification of VSH-1 structural genes and allowed their assignment as head or tail associated. It remained uncertain however, as to whether or not additional genes important to virion production existed within the prophage sequence. While mitomycin C- induction of the VSH-1 prophage resulted in cell lysis pointed to bacteriophage genes for lytic growth, the identity of these genes was unknown. The ability of VSH-1 virions to mediate horizontal gene transfer in B. hyodysenteriae populations coupled with the recent observation that VSH-1 was common among Brachyspira strains suggested that VSH-1 might play an important role in Brachyspira ecology (99). This dissertation is a continuation of research on VSH-1 to understand the biology of this bacteriophage. In the following studies, additional VSH-1 genes were identified including an endolysin involved in VSH-1 escape from B. hyodysenteriae cells. The organization of genes for head and tail structures and lytic functions was found to be similar to other bacteriophage opérons. Characteristics of VSH-1 transcription were investigated, leading to the identification of an alternative to mitomycin C as VSH-1 inducing agent. 2 Dissertation Organization This dissertation has 5 chapters. Chapter 1 contains a general introduction and literature review. Chapter 2 represents this author's continuation of research initiated by Thad B. Stanton, M. Greg Thompson, Samuel B. Humphrey, and Richard L. Zuerner toward a manuscript on the identification of VSH-1 genes and the development of a VSH-1 genome map. Chapter 3 and Chapter 4 are independent manuscripts. Chapter 3 reports on the identification and characterization of a VSH-1 muralytic enzyme and Chapter 4 reports on characteristics of VSH-1 transcription. Chapter 5 contains general conclusions and recommendations for future research. Literature Review Bacteriophages The simplicity of bacteriophages as biological systems initially led to research focusing on understanding bacteriophage processes as models for more complex systems in higher organisms. Moreover, bacteriophages provided many of the genetic tools that gave rise to the field of molecular biology and are still invaluable genetic tools. A shift in bacteriophage research is reflected in the theme of a recent conference on bacteriophage biology entitled "New Phage Biology" (American Society of Microbiology Conference on the New Phage Biology, Aug 2004, Key Biscayne, FL), which focused on research directed toward understanding bacteriophage biology from the standpoints of diversity, function and ecology of bacteriophages themselves. 3 As genomic sequences have become available, it is clear that bacteriophages have genomes assembled from a vast pool of nucleic acid. Hendrix and colleagues proposed the modular theory of bacteriophage evolution, suggesting that genes and gene clusters of related function are readily exchanged among bacteriophages from disparate host organisms (35-37). Horizontally acquired bacteriophage DNA that can appropriately complement the function of another bacteriophage gene allows productive recombination and leads to genetic mosaicism of bacteriophage genomes. Evidence for both homologous and non-homologous recombination exists in bacteriophage