DOCSLIB.ORG

Lmarinelli ETD 080608.Pdf

Total Page：16

File Type：pdf, Size：1020Kb

Download full-text PDF Read full-text
Lmarinelli ETD 080608.Pdf CHARACTERIZATION OF THE ROLE OF THE RPF MOTIF IN MYCOBACTERIOPHAGE TAPE MEASURE PROTEINS by Laura Jane Marinelli Bachelor of Science, Youngstown State University, 2001 Submitted to the Graduate Faculty of Arts and Sciences in partial fulfillment of the requirements for the degree of Doctor of Philosophy University of Pittsburgh 2008 UNIVERSITY OF PITTSBURGH FACULTY OF ARTS AND SCIENCES This dissertation was presented by Laura J. Marinelli It was defended on May 15th, 2008 and approved by Karen Arndt, Ph.D. Associate Professor, Department of Biological Sciences Roger Hendrix, Ph.D. Professor, Department of Biological Sciences Saleem Khan, Ph.D. Professor, Department of Microbiology and Molecular Genetics Valerie Oke, Ph.D. Assistant Professor, Department of Biological Sciences Dissertation Advisor: Graham Hatfull, Ph.D. Professor, Department of Biological Sciences ii Copyright © by Laura J. Marinelli 2008 iii CHARACTERIZATION OF THE ROLE OF THE RPF MOTIF IN MYCOBACTERIOPHAGE TAPE MEASURE PROTEINS Laura J. Marinelli, Ph.D. University of Pittsburgh, 2008 In order to inject their DNA into the bacterial cytoplasm and establish infection, bacteriophages must ensure their genetic material successfully traverses both the bacterial membrane(s) and the layer of peptidoglycan surrounding the host cell. Phages accomplish this in a variety of ways, and some have virion-associated murein hydrolase enzymes that facilitate this process, particularly in conditions where the peptidoglycan is highly cross-linked. Phages that infect the mycobacteria must also contend with these barriers to infection, as well an impermeable layer of mycolic acids that decorates the cell surface; however, the mechanisms by which they do this are mostly unknown. In this regard, three small sequence motifs have been identified within mycobacteriophage tape measure proteins (TMPs) extended molecules that span the tail lumen and determine its length at least two of which have similarity to host proteins with muralytic activity. This suggests that phages may utilize regions of the TMP, which because of its location within the tail might be uniquely primed for host interaction, to facilitate localized peptidoglycan hydrolysis and DNA injection. The focus of this study is the motif found in the TMPs of mycobacteriophages Barnyard and Giles that has identity to a group of bacterial proteins known as resuscitation promoting factors (Rpfs). These Rpf proteins stimulate growth of non-growing bacteria and seem to exert their activity by cleaving inert peptidoglycan in the cell wall. Notably, the Barnyard Rpf Motif is contained within a 70 kDa C-terminal cleavage product of TMP, which appears to be cell wall- iv and/or membrane-associated during infection. Further, mycobacteria expressing TMP fragments containing this motif show aberrant behavior in culture and on solid media, and hybrid proteins in which the Rpf domain of the Micrococcus luteus Rpf protein is replaced with either of the phage motifs have muralytic activity in vivo. A recombineering-based method for generating mutations on lytically replicating mycobacteriophages has been developed and utilized to make multiple mutations in the Giles TMP motifs. Mutant phages infect host cells in late-stationary phase with a reduced efficiency, an observation that further supports a role for these motifs in cell wall hydrolysis during infection. v TABLE OF CONTENTS PREFACE.................................................................................................................................. xvii 1.0 INTRODUCTION........................................................................................................ 1 1.1 BACTERIOPHAGE TAPE MEASURE PROTEINS...................................... 3 1.1.1 The Tape Measure of Bacteriophage λ ....................................................... 4 1.1.2 Tape Measures in Other Bacteriophages.................................................... 6 1.2 THE CELL WALL.............................................................................................. 9 1.2.1 The Gram-Negative Cell Wall ................................................................... 12 1.2.2 The Gram-Positive Cell Wall..................................................................... 13 1.2.3 Mycobacteria and the Mycobacterial Cell Wall ...................................... 16 1.2.4 Cell Wall Modifying Enzymes ................................................................... 21 1.2.5 The Cell Wall as a Barrier ......................................................................... 25 1.3 BACTERIOPHAGE-ASSOCIATED HYDROLASE PROTEINS............... 26 1.3.1 Phages of Gram-Negative Hosts ................................................................ 27 1.3.2 Phages of Gram-Positive Hosts.................................................................. 32 1.4 MYCOBACTERIOPHAGE TAPE MEASURE MOTIFS............................ 34 1.5 BACTERIAL RPF PROTEINS ....................................................................... 39 1.5.1 The Micrococcus luteus Rpf Protein.......................................................... 40 1.5.2 Rpf Proteins in the Mycobacteria.............................................................. 41 vi 1.5.3 Rpf Mechanism of Action........................................................................... 47 1.5.4 Evidence for Rpf-Interacting Proteins...................................................... 49 1.5.5 Distribution of Rpf-Like Proteins.............................................................. 50 1.6 SUMMARY........................................................................................................ 54 2.0 MATERIALS AND METHODS .............................................................................. 56 2.1 BACTERIAL STRAINS AND MEDIA........................................................... 56 2.2 PLASMIDS USED IN THIS STUDY .............................................................. 57 2.2.1 Commercially Available Plasmids............................................................. 57 2.2.2 Plasmids Constructed by Others ............................................................... 58 2.2.3 pLAM Plasmid Constructs......................................................................... 59 2.3 GENERAL CLONING AND DNA MANIPULATION................................. 72 2.3.1 Oligonucleotides and Primers Used in this Study.................................... 72 2.3.2 Transformation and Electroporation........................................................ 76 2.3.3 Sequencing................................................................................................... 77 2.3.4 Site-Directed Mutagenesis.......................................................................... 77 2.3.5 Cosmid Packaging Reactions..................................................................... 77 2.4 PROTEIN EXPRESSION, PURIFICATION AND ANAYLSIS.................. 77 2.4.1 Expression and Purification of the Barnyard TMP Rpf-Like Fragment .. ....................................................................................................................... 77 2.4.2 Expression and Purification of the Barnyard TMP 70 kDa C-Terminal Fragment..................................................................................................................... 79 2.4.3 Production of Polyclonal Antibodies to the Barnyard Rpf Motif........... 79 2.4.4 Immunoblot Analysis.................................................................................. 80 vii 2.4.5 M. smegmatis Cell Fractionation ............................................................... 81 2.5 BACTERIOPHAGE PREPARATION AND ANALYSIS............................. 82 2.5.1 Preparation of Bacteriophage Stocks........................................................ 82 2.5.2 Bacteriophage DNA Isolation .................................................................... 84 2.5.3 Phage Protein Analysis............................................................................... 84 2.5.4 N-Terminal Sequencing.............................................................................. 85 2.5.5 Mass Spectrometry ..................................................................................... 85 2.5.6 Mycobacteriophage Barnyard Liquid Infections..................................... 86 2.5.7 RNA Isolation.............................................................................................. 86 2.5.8 RT-PCR ....................................................................................................... 87 2.6 MICROBIOLOGY............................................................................................ 87 2.6.1 Induction of pBAD/gIII Derivative Constructs in E. coli........................ 87 2.6.2 Induction of pLAM12 Derivative Constructs in M. smegmatis .............. 88 2.7 RECOMBINEERING IN M. SMEGMATIS TO GENERATE BACTERIOPHAGE GILES TMP MUTANTS .............................................................. 89 2.7.1 Mycobacteriophage Mutant Construction and PCR Screening............. 89 2.7.2 Infectivity Assay with Mutant Phages ...................................................... 90 2.8 BUFFERS AND REAGENTS .......................................................................... 92 3.0 GLOBAL ANALYSIS OF MYCOBACTERIOPHAGE TAPE MEASURES AND TAPE MEASURE MOTIFS ...................................................................................................... 95 3.1 INTRODUCTION ............................................................................................
Load more

Recommended publications
  • Contents More Information
    Cambridge University Press 978-0-521-86297-4 - Horizontal Gene Transfer in the Evolution of Pathogenesis Edited by Michael Hensel and Herbert Schmidt Table of Contents More information Contents ix Preface page xi Contributors xv PART I Theoretical Considerations on the Evolution of Bacterial Pathogens 1 Genomes in Motion: Gene Transfer as a Catalyst for Genome Change 3 Jeffrey G. Lawrence and Heather Hendrickson 2 Bacterial Recombination in vivo 23 Xavier Didelot and Daniel Falush PART II Mobile Genetic Elements in Bacterial Evolution 3 Phage-bacterium Co-evolution and Its Implication for Bacterial Pathogenesis 49 Harald Brussow¨ 4 The Role of Bacteriophages in the Generation and Spread of Bacterial Pathogens 79 Roger W. Hendrix and Sherwood R. Casjens 5 Genomic Islands in the Bacterial Chromosome – Paradigms of Evolution in Quantum Leaps 113 Tobias Olschl¨ ager¨ and Jorg¨ Hacker PART III Paradigms of Bacterial Evolution 6 Genomic Islands in Plant-pathogenic Bacteria 137 Dawn L. Arnold and Robert W. Jackson © Cambridge University Press www.cambridge.org Cambridge University Press 978-0-521-86297-4 - Horizontal Gene Transfer in the Evolution of Pathogenesis Edited by Michael Hensel and Herbert Schmidt Table of Contents More information 7 Prophage Contribution to Salmonella Virulence and Diversity 159 Sebastien´ Lemire, Nara Figueroa-Bossi, and Lionello Bossi 8 Pathogenic Yersinia: Stepwise Gain of Virulence due to Sequential Acquisition of Mobile Genetic Elements 193 Elisabeth Carniel 9 Genomic or Pathogenicity Islands in Streptococcus pneumoniae 217 Barbara Albiger, Christel Blomberg, Jessica Dagerhamn, Staffan Normark, and Birgitta Henriques-Normark 10 The Mobile Genetic Elements of Staphylococcus aureus 237 Richard P.
    [Show full text]
  • Dynamics and Genetic Diversification of Escherichia Coli During Experimental Adaptation to an Anaerobic Environment
    Dynamics and genetic diversification of Escherichia coli during experimental adaptation to an anaerobic environment Thomas J. Finn1,2,3, Sonal Shewaramani1,2,4, Sinead C. Leahy1, Peter H. Janssen1 and Christina D. Moon1 1 Grasslands Research Centre, AgResearch Ltd, Palmerston North, New Zealand 2 New Zealand Institute for Advanced Study, Massey University, Auckland, New Zealand 3 Department of Biochemistry, University of Otago, Dunedin, New Zealand 4 Department of Molecular and Cell Biology, University of Connecticut, Storrs, CT, United States of America ABSTRACT Background. Many bacteria are facultative anaerobes, and can proliferate in both anoxic and oxic environments. Under anaerobic conditions, fermentation is the primary means of energy generation in contrast to respiration. Furthermore, the rates and spectra of spontaneous mutations that arise during anaerobic growth differ to those under aerobic growth. A long-term selection experiment was undertaken to investigate the genetic changes that underpin how the facultative anaerobe, Escherichia coli, adapts to anaerobic environments. Methods. Twenty-one populations of E. coli REL4536, an aerobically evolved 10,000th generation descendent of the E. coli B strain, REL606, were established from a clonal ancestral culture. These were serially sub-cultured for 2,000 generations in a defined minimal glucose medium in strict aerobic and strict anaerobic environments, as well as in a treatment that fluctuated between the two environments. The competitive fitness of the evolving lineages was assessed at approximately 0, 1,000 and 2,000 generations, in both the environment of selection and the alternative environment. Whole genome re-sequencing was performed on random colonies from all lineages after 2,000-generations.
    [Show full text]
  • Amplification–Mutagenesis: Evidence That ‘‘Directed’’ Adaptive Mutation and General Hypermutability Result from Growth with a Selected Gene Amplification
    Amplification–mutagenesis: Evidence that ‘‘directed’’ adaptive mutation and general hypermutability result from growth with a selected gene amplification Heather Hendrickson*†, E. Susan Slechta*, Ulfar Bergthorsson*, Dan I. Andersson‡, and John R. Roth*§ *Department of Biology, University of Utah, Salt Lake City, UT 84112; and ‡Swedish Institute for Infectious Disease Control, S 17182 Solna, Sweden Communicated by Nancy Kleckner, Harvard University, Cambridge, MA, December 18, 2001 (received for review September 9, 2001) When a particular lac mutant of Escherichia coli starves in the presence We have suggested (9) that apparently directed mutation of lactose, nongrowing cells appear to direct mutations preferentially could be explained if the leaky lac allele used in this experiment to sites that allow growth (adaptive mutation). This observation were amplified so as to allow slow growth of a clone on lactose. suggested that growth limitation stimulates mutability. Evidence is Reversion would then occur when the growing clone has accu- provided here that this behavior is actually caused by a standard mulated enough mutant lac copies. What appears to be a Darwinian process in which natural selection acts in three sequential directed single step mutation in a nongrowing cell can result steps. First, growth limitation favors growth of a subpopulation with from selection acting on growing cells within a colony. The ؉ an amplification of the mutant lac gene; next, it favors cells with a lac required growth will not be evident in the lawn between colonies, revertant allele within the amplified array. Finally, it favors loss but can be revealed by appropriate analysis of developing ؉ of mutant copies until a stable haploid lac revertant arises and colonies (9).
    [Show full text]
  • Title 1 Ribosome Provisioning Activates a Bistable Switch
    bioRxiv preprint doi: https://doi.org/10.1101/244129; this version posted April 24, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. 1 Title 2 Ribosome provisioning activates a bistable switch coupled to fast exit from stationary 3 phase 4 5 Authors: 6 P. Remigi1*, G.C. Ferguson2, S. De Monte3,4 and P.B. Rainey1,5,6* 7 1 New Zealand Institute For Advanced Study, Massey University, AucKland 0745, New 8 Zealand 9 2 Institute oF Natural and Mathematical Sciences, Massey University, AucKland 0745, New 10 Zealand 3 11 Institut de biologie de l’Ecole normale supérieure (IBENS), Ecole normale supérieure, CNRS, 12 INSERM, PSL Université Paris 75005 Paris, France 13 4 Department oF Evolutionary Theory, Max PlancK Institute For Evolutionary Biology, Plön 14 24306, Germany 15 5 Department oF Microbial Population Biology, Max PlancK Institute For Evolutionary Biology, 16 Plön 24306, Germany 6 17 Ecole Superieure de Physique et de Chimie Industrielles de la Ville de Paris (ESPCI Paris 18 Tech), CNRS UMR 8231, PSL Research University, 75231 Paris, France 19 20 * Corresponding authors: 21 Philippe Remigi ([email protected]) and Paul B. Rainey ([email protected]) 22 bioRxiv preprint doi: https://doi.org/10.1101/244129; this version posted April 24, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. 23 Abstract: 24 Observations oF bacteria at the single-cell level have revealed many instances oF phenotypic 25 heterogeneity within otherwise clonal populations, but the selective causes, molecular bases 26 and broader ecological relevance remain poorly understood.
    [Show full text]
  • Recombineering in Mycobacteria Using Mycobacteriophage Proteins
    RECOMBINEERING IN MYCOBACTERIA USING MYCOBACTERIOPHAGE PROTEINS by Julia Catherine van Kessel B.S. Biology, Utica College of Syracuse University, 2003 Submitted to the Graduate Faculty of Arts and Sciences in partial fulfillment of the requirements for the degree of Doctor of Philosophy University of Pittsburgh 2008 UNIVERSITY OF PITTSBURGH SCHOOL OF ARTS AND SCIENCES This dissertation was presented by Julia Catherine van Kessel It was defended on July 24, 2008 and approved by Roger W. Hendrix, Ph.D., Biological Sciences, University of Pittsburgh William R. Jacobs, Jr., Ph.D., Albert Einstein College of Medicine Jeffrey G. Lawrence, Ph.D., Biological Sciences, University of Pittsburgh Valerie Oke, Ph.D., Biological Sciences, University of Pittsburgh Dissertation Advisor: Graham F. Hatfull, Ph.D., Biological Sciences, University of Pittsburgh ii Copyright © by Julia Catherine van Kessel 2008 iii RECOMBINEERING IN MYCOBACTERIA USING MYCOBACTERIOPHAGE PROTEINS Julia Catherine van Kessel, Ph.D. University of Pittsburgh, 2008 Genetic manipulations of Mycobacterium tuberculosis are complicated by its slow growth, inefficient DNA uptake, and relatively high levels of illegitimate recombination. Most methods for construction of gene replacement mutants are lengthy and complicated, and the lack of generalized transducing phages that infect M. tuberculosis prevents simple construction of isogenic mutant strains. Characterization and genomic analysis of mycobacteriophages has provided numerous molecular and genetic tools for the mycobacteria. Recently, genes encoding homologues of the Escherichia coli Rac prophage RecET proteins were revealed in the genome of mycobacteriophage Chec9c. RecE and RecT are functional analogues of the phage λ Red recombination proteins, Exo (exonuclease) and Beta (recombinase), respectively. These recombination enzymes act coordinately to promote high levels of recombination in vivo in E.
    [Show full text]
  • Wednesday 28Th August Thursday 29Th August
    QMB Programme Queenstown Molecular Biology Week and Webster Centre for Infectious Diseases Symposium: Of Microbes and Men – Translational Medical Microbiology in the 21st Century 29 August – 30 August, 2013 Rydges Hotel, Queenstown, New Zealand th Wednesday 28 August Time Details Location 7.00pm - Late QMB conference dinner Skyline Restaurant th Thursday 29 August Time Details Location 9.00am-9.10am Introduction and opening remarks: Clancy’s Room Professor Kurt Krause Director of the Webster Centre for Infectious Diseases, University of Otago 9.10am-10.00am Opening plenary: Chair: Professor Kurt Krause Clancy’s Room Associate Professor Bill Hanage Department of Epidemiology, Harvard School of Public Health The power of many: population genomics for epidemiology and ecology 10.00am-10.30am Morning tea Trade Area Session 1: From bench to bedside: advances in molecular diagnostics Chair: Dr Deborah Williamson 10.30am-10.45am Professor Jane Hill Clancy’s Room University of Vermont Toward using breath to diagnose lung infections 10.45am-11.10am Professor David Murdoch Clancy’s Room sponsored by Thermo Fisher Scientific University of Otago Molecular testing for respiratory pathogens: benefits and pitfalls 11.10am-11.40am Dr Steven Tong Clancy’s Room Menzies School of Health Research, Darwin How whole genome sequencing is changing the diagnostic landscape 11.40am-12.05pm Dr Sally Roberts Clancy’s Room Auckland District Health Board Advances in TB molecular diagnostics and typing methods 12.05pm-12.30pm Dr Barry Bochner Clancy’s Room BIOLOG technologies
    [Show full text]
  • Amplification–Mutagenesis: Evidence That ''Directed'' Adaptive Mutation
    Amplification–mutagenesis: Evidence that ‘‘directed’’ adaptive mutation and general hypermutability result from growth with a selected gene amplification Heather Hendrickson*†, E. Susan Slechta*, Ulfar Bergthorsson*, Dan I. Andersson‡, and John R. Roth*§ *Department of Biology, University of Utah, Salt Lake City, UT 84112; and ‡Swedish Institute for Infectious Disease Control, S 17182 Solna, Sweden Communicated by Nancy Kleckner, Harvard University, Cambridge, MA, December 18, 2001 (received for review September 9, 2001) When a particular lac mutant of Escherichia coli starves in the presence We have suggested (9) that apparently directed mutation of lactose, nongrowing cells appear to direct mutations preferentially could be explained if the leaky lac allele used in this experiment to sites that allow growth (adaptive mutation). This observation were amplified so as to allow slow growth of a clone on lactose. suggested that growth limitation stimulates mutability. Evidence is Reversion would then occur when the growing clone has accu- provided here that this behavior is actually caused by a standard mulated enough mutant lac copies. What appears to be a Darwinian process in which natural selection acts in three sequential directed single step mutation in a nongrowing cell can result steps. First, growth limitation favors growth of a subpopulation with from selection acting on growing cells within a colony. The ؉ an amplification of the mutant lac gene; next, it favors cells with a lac required growth will not be evident in the lawn between colonies, revertant allele within the amplified array. Finally, it favors loss but can be revealed by appropriate analysis of developing ؉ of mutant copies until a stable haploid lac revertant arises and colonies (9).
    [Show full text]
  • Untreatable Gonorrhoea, Are We There Yet?
    Emerging Diseases Untreatable gonorrhoea, are we there yet? Collette Bromhead and Gonorrhoea is a relatively common and easily spread bacterial sexually transmitted disease that Heather Hendrickson can cause a wide spectrum of symptoms from none to severe outcomes. Through its penchant for Downloaded from http://portlandpress.com/biochemist/article-pdf/39/3/30/852085/bio039030030.pdf by guest on 30 September 2021 (Massey University, New modifying its own genetics, Neisseria gonorrhoeae has gradually evolved to become one of modern Zealand) medicine’s greatest adversaries. In most countries we have only one antibiotic left to treat it with, and even that option is rapidly running out. So how do we stop this train wreck? What do we do when the treatments run out altogether? “I am very sorry”, says your doctor, “your gonorrhoea infection cannot be treated with antibiotics. It might Priority 1: CRITICAL# clear up on its own in 3–6 months. In the meantime, take Acinetobacter baumannii, carbapenem-resistant painkillers for the discomfort and, please don’t have sex...” Pseudomonas aeruginosa, carbapenem-resistant This scenario may seem dystopian, but it is an Enterobacteriaceae*, carbapenem-resistant, almost certain future outcome for many of the 27 3rd generation cephalosporin-resistant million cases of genital, oropharyngeal, ocular or rectal N. gonorrhoeae infection each year (2012, World Health Priority 2: HIGH Organization (WHO) estimate). And this wily organism Enterococcus faecium, vancomycin-resistant isn’t even the most critical risk we face according to the Staphylococcus aureus, methicillin-resistant, WHO, which has published its first ever list of antibiotic- vancomycin intermediate and resistant resistant ‘priority pathogens’ – a catalogue of 12 families Helicobacter pylori, clarithromycin-resistant of bacteria that pose the greatest threat to human health Campylobacter, fluoroquinolone-resistant in 2017 (see Figure 1).
    [Show full text]
  • Front Matter
    Cambridge University Press 978-0-521-86297-4 - Horizontal Gene Transfer in the Evolution of Pathogenesis Edited by Michael Hensel and Herbert Schmidt Frontmatter More information Horizontal Gene Transfer in the Evolution of Pathogenesis Horizontal gene transfer is a major driving force in the evolution of many bacterial pathogens. The development of high-throughput sequencing tools and more sophisticated genomic and proteomic techniques in recent years has resulted in a better understanding of this phenomenon. Written by lead- ing experts in the field, this edited volume is aimed at graduate students and researchers and provides an overview of current knowledge relating to the evolution of microbial pathogenicity. This volume provides an overview of the mechanisms and biological consequences of the genome rearrangements resulting from horizontal gene transfer, in both prokaryotes and eukaryotes, as well as overviews of the key mobile genetic elements involved. Subse- quent chapters focus on paradigms for the evolution of important bacte- rial pathogens, including Salmonella enterica, Streptococcus pneumoniae,and Staphylococcus aureus. The influence of socioeconomic parameters in the dis- semination of transferable elements, such as antibiotic-resistant genes in bacteria, is also discussed. Michael Hensel is currently Professor of Microbiology and Immunology at the University of Erlangen in Germany. Herbert Schmidt is currently Professor of Food Microbiology at the Univer- sity of Hohenheim in Germany. © Cambridge University Press www.cambridge.org Cambridge University Press 978-0-521-86297-4 - Horizontal Gene Transfer in the Evolution of Pathogenesis Edited by Michael Hensel and Herbert Schmidt Frontmatter More information Over the past decade, the rapid development of an array of techniques in the fields of cellular and molecular biology has transformed whole areas of research across the biological sciences.
    [Show full text]
  • Genomic Characterization and Evolutionary Relationships Among Bacteriophages in the Dairy Industry and Applications to Detect Phage Contamination
    Copyright is owned by the Author of the thesis. Permission is given for a copy to be downloaded by an individual for the purpose of research and private study only. The thesis may not be reproduced elsewhere without the permission of the Author. Genomic characterization and evolutionary relationships among Bacteriophages in the dairy industry and applications to detect phage Contamination By Prianka Rajan Supervised by Dr. Heather L. Hendrickson Abstract The prevalence of bacteriophages and their pernicious effects on the Lactococcus starter culture in the dairy industry has been an ongoing problem for several decades. The main purpose of this investigation was to understand the relationship, evolutionary history and the sources of the phages that have been isolated from the different fermentative units of Fonterra. We report the genomic comparison results of 15 phages in this study that were isolated on the host bacteria, Lactococcus lactis cremoris. These phages can be grouped in two clusters namely P335 and 936, commonly encountered tailed bacteriophages in the dairy industries. The majority of the phages belong to the P335 species with just one phage clustering with the 936 species. Although phages of the P335 group display a high level of synteny with one another, we report nine different types of P335 phages in this study. A prophage integrated in the host strain has been identified. The prophage and the phages show homology to the temperate P335 phage, R1T isolated in Netherlands in 1996. The genetic makeup of these phages is suggestive of their source and evolution from other prophages in strains that may have been co-cultured with the strain that was used in this study.
    [Show full text]
  • Testing the Potential of Mycobacteriophage Endolysins Fused
    Copyright is owned by the Author of the thesis. Permission is given for a copy to be downloaded by an individual for the purpose of research and private study only. The thesis may not be reproduced elsewhere without the permission of the Author. Testing the potential of mycobacteriophage endolysins fused to biodegradable nanobeads for controlling Mycobacteria A thesis presented in partial fulfilment of the requirements for the degree of Master of Natural Sciences at Massey University, Albany, New Zealand Courtney Grace Davies 2019 Abstract 10.4 million people are diagnosed with tuberculosis, worldwide, every year, according to the World Health Organisation. Mycobacterium tuberculosis is a Gram-positive bacterial pathogen that can easily be transmitted to health care professionals and people supplying aid in these nations. Fortunately, over the last 100 years, the bacteriophage has gained traction as a suitable therapeutic, antibiotic-alternative against bacterial pathogens, such as M. tuberculosis. Herein I describe my work utilising endolysins the lytic protein that bacteriophages usually employ to burst out of the cell, but instead using these proteins to lyse “from without”. In order to develop a proof-of-principle product, we used the expansive M. smegmatis bacteriophage collection and chose eight endolysin candidates for testing against M. smegmatis. These endolysins were bioinformatically analysed for active domains before being synthesised and inserted into an expression vector to produce fused biodegradable nanobeads made of polyhydroxyalkanoate. These nanobeads were tested for activity against M. smegmatis, a safe mycobacterium closely related to pathogenic tuberculosis. Four distinct tests were carried out to test the efficiency of these beads in causing cell death in different situations (45 minutes and 5 hours, across concentrations of 10mg/ml, 20mg/ml and 80mg/ml.
    [Show full text]
  • Te Ao Mārama Conference 24 – 25 October 2018
    Te Ao Mārama Conference 24 – 25 October 2018 The Royal New Zealand Yacht Squadron, Westhaven Marina Room: RNZYS Ballroom Location on Maps: https://goo.gl/maps/eLofdbGAc5R2 Getting to the venue: Walking: approx. 30 minutes from Britomart, along waterfront, via Wynyard Quarter. Parking information: All day parking is free of charge for event guests of RNZYS. Guests are required to park in spaces with white lines and must request a parking ticket issued from the parking machine located at the RNZYS reception and displayed on the car dashboard. Additional free parking is available at the Western side of the Marina near the Marina Office. Conference: Te Ao Mārama Conference 2018 Date: 24 - 25 October - Conference Location: RNZYS Ballroom, Royal New Zealand Yacht Squadron WEDNESDAY, 24 OCTOBER - CONFERENCE, DAY 1 8:15 AM 9:00 AM Registration OPen Arrival coffee and tea served 9:00 AM Conference Day 1 Begins 9:00 AM 9:30 AM Welcome Remarks Michael Steedman, K CamPbell 9:30 AM 10:00 AM Keynote 1: Anthony Aguirre Chair: R Easther 10:00 AM 10:20 AM Contributed Talk 1: Matthew Egbert 10:20 AM 10:40 AM Contributed Talk 2: Heather Hendrickson 10:40 AM 11:10 AM Morning Tea 11:10 AM 11:40 AM Keynote 2: Daniella Scalice Chair: N Rattenbury 11:40 AM 12:00 PM Contributed Talk 3: Sam Woolley 12:00 PM 1:10 PM Lunch 1:10 PM 1:40 PM Keynote 3: E Parke Chair: M Egbert 1:40 PM 2:00 PM Contributed Talk 4: Alex PoPinga 2:00 PM 2:20 PM Contributed Talk 5: Danielle Maddock 2:20 PM 2:40 PM Contributed Talk 6: Wayne Patrick 2:40 PM 3:00 PM Contributed Talk 7: Peter
    [Show full text]