Genomics of Phage-Bacterium-Host Interaction of Mushroom Pathogenic Pseudomonads Nathaniel Storey
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! ! School&of&Human&and&E nvironmental&Sciences! ! ! ! Genomics of phage-bacterium-host interaction of ! Disentanglimushroomng*the*mo lpathogenicecular*mec Pseudomonadshanisms*for* persistence*of*Escherichia)coli*O157:H7*in* the*plant*environbym ent* ! Nathanielby! Storey ! Glyn*Barrett* ! ! ! Submitted*inSubmitted*partial*fu linfi lpartiallment *ofulfilmentf*the*req uofi rtheem requirementent*for*the*d foregr theee* odegreef*Doct oofr *Doctor of of*PhilosophPhilosophy,y,*March*2 0January13* 2018. ! 1 Declaration I declare that this is an account of my own research and has not been submitted for a degree at any other university. The use of material from other sources has been properly and fully acknowledged, where appropriate. Nathaniel Storey 2 Acknowledgements “Tout ce qui est simple est faux, mais tout ce qui ne l'est pas est inutilisable.” ~ Paul Valéry (1871-1945) With special thanks to my father Mark Storey for his support and encouragement, without which this project would not have been possible. At the University of Reading I would especially like to thank Geraldine Mulley for her guidance and generosity in assisting me when it was most needed; with further thanks to Rob Jackson and Ben Neuman for both their support and company during my time at the University of Reading. Furthermore, I would like to thank my fiancée Lyssa Reeve for her never-ending patience and support; and my friends Natalie Tarling and Hashem Ghazi for their assistance and company throughout all the darkest hours and to celebrate the elusive moments of success. Finally, I would like to thank Saad Mutlk for all his help and humour in the laboratory. 3 Abstract The most commonly cultivated mushroom in Europe and North America is the Agaricus bisporus, also known as the button mushroom or Portobello mushroom. Bacterial diseases of Agaricus bisporus caused by Pseudomonas species are a cause of significant crop loss and downgrading of produce, resulting in considerable economic cost. Bacteriophage have long been an attractive option for biocontrol of bacterial contamination of food products, however the precise genetic interactions between phage, bacterium and host are often inadequately explored. This project aims to explore the genetic interactions between the mushroom pathogenic bacterium Pseudomonas tolaasii and Pseudomonas agarici with the newly identified bacteriophage Pseudomonas phage NV1 and Pseudomonas phage ϕNV3. Full genome sequencing has been performed on the P. tolaasii strain 2192T and P. agarici NCPPB 2472, and the genomes of both mined for potential biosynthetic clusters involved in virulence as well as genes involved in phage resistance. Within the genome of P. tolaasii 2192T, putative non- ribosomal peptide synthases have been identified which are hypothesised to be involved in the production of the tolaasin toxin involved in disease symptom appearance on mushroom surfaces. Within the genome of P. agarici NCPPB 2472, a biosynthetic cluster was identified that is hypothesised to produce the siderophore achromobactin, an important virulence factor. P. agarici NCPPB 2472 was identified as possessing a single Type I-F CRISPR/Cas system, predicted to be involved in the development of phage resistance, as well as complete operon predicted to be involved in the production of the exopolysaccharide alginate. 4 A third Pseudomonas species was identified on the surface of disease free mushrooms which was identified as a potentially new species of Pseudomonas, named Pseudomonas sp. NS1. The genome of P. sp. NS1 was likewise sequenced and mined for potential biosynthetic gene clusters which identified a cluster demonstrated to be involved in the production of White Line Inducing Principle. The Pseudomonas phages NV1 and ϕNV3 were isolated from environmental samples and identified to be narrow host range phage specific for P. tolaasii 2192T and P. agarici NCPPB 2472 respectively. Both phage NV1 and ϕNV3 were identified as new species of the Luz24likeviruses and phiKMVlikeviruses respectively. The genomes of both phages were isolated and sequenced, with phage ϕNV3 identified as containing a conserved Signal-Arrest- Release endolysin system, which was confirmed by in vitro protein expression. Likewise, the lysis cassette proteins of ϕNV3 were identified and investigated via protein complementation assay in vitro. The full growth characteristics and life cycle of phage ϕNV3 has been investigated and reported in this study and a broad-host range mutant of ϕNV3 identified which has allowed the T7-like tail protein of ϕNV3 to be identified as the host specificity determinant. Transcriptome analyses of non-infected P. agarici NCPPB 2472 and P. agarici NCPPB 2472 infected with phage ϕNV3 at a multiplicity of infection (MOI) of 1 at 40 min post infection, were performed in triplicate using RNA-seq. A reliable method has been established that will be useful in future studies, although comparative gene expression analysis revealed no significant differences in expression between the two treatments at the multiplicity of infection and time point chosen in this case a significant quantity of phage transcripts were detected, demonstrating active phage infection. 5 List of Abbreviations and Acronyms Abs Absorbance Amp Ampicillin ANIb Average Nucleotide Identity based on BLAST+ ANIm Average Nucleotide Identity based on MUMmer bp Base pair C Celsius Cas CRISPR associated CFU Colony Forming Units CRISPR Clustered Randomly Interspersed Short Palindromic Repeats crRNA CRISPR RNA DNA Deoxyribonucleic acid DNase Deoxyribonuclease ds Double Stranded EDTA Ethylenediaminetetraacetic Acid EPS Extracellular Polysaccharide ETEC Enterotoxigenic E. coli Gp Genome position GTP Guanosine-5'-triphosphate HHP High Hydrostatic Pressure Kan Kanamycin KB Kings B medium LB Lysogeny Broth LPS Lipopolysaccharide ml Millilitres MLSA Multilocus sequence analysis MOI Multiplicity of infection mRNA Messenger ribonucleic acid MUSCLE Multiple Sequence Comparison by Log-Expectation NRPS Non-Ribosomal Peptide Synthase OD Optical Density ORF Open Reading Frame Padj P-value adjusted for multiple testing using the Benjamin-Hochberg method PBS Phosphate Buffered Saline PCA Principal Component Analysis PCP Peptidyl Carrier Protein PCR Polymerase Chain Reaction PFU Plaque Forming Units PMBV Polymyxin B nonapeptide pv. Pathovar R-M Restriction-Modification RBP Receptor Binding Protein rDNA Ribosomal Deoxyribonucleic Acid rpm Revolutions Per Minute rRNA Ribosomal Ribonucleic Acid SAR Signal-Arrest-Release SD Standard Deviation SEM Standard Error of the Mean 6 sp. Species ss Single Stranded TBE Tris/Borate/EDTA TE Thioesterase TEM Transmission Electron Microscopy U.K United Kingdom UoR University of Reading UPEC Uropathogenic E. coli UV Ultraviolet vsd variance stabilising transformed 7 Contents Chapter 1: Introduction ........................................................................................................ 12 1.1 Cultivated mushrooms and their importance ................................................................. 13 1.2 Bacterial diseases of cultivated mushrooms .................................................................. 14 1.2.1 Pseudomonas tolaasii ............................................................................................. 15 1.2.2 Pseudomonas agarici .............................................................................................. 18 1.3 Secondary metabolites, non-ribosomal peptide synthesis and bacteriocins in Pseudomonas ....................................................................................................................... 20 1.3.1 Tolaasin ................................................................................................................... 21 1.3.2 Siderophores ........................................................................................................... 23 1.3.3 Bacteriocins and phage tail-like bacteriocins ......................................................... 24 1.4 Bacteriophage treatments of Pseudomonas infections .................................................. 26 1.5 Bacteriophage background............................................................................................. 27 1.5.1 The Podoviridae, subfamily Autographivirinae and genus Luz24likeviruses ........ 28 1.6 Endolysin systems .......................................................................................................... 34 1.6.1 Types of host cell lysis systems .............................................................................. 35 1.6.2 Lysis systems and host cell wall structure .............................................................. 37 1.6.3 Endolysins in the treatments of bacterial infections ............................................... 39 1.7 Host cell receptors and phage host specificity determinants ......................................... 41 1.7.1 Lipopolysaccharide (LPS) receptors ....................................................................... 42 1.7.2 Capsule phage ......................................................................................................... 43 1.7.3 Pili phage ................................................................................................................ 44 1.7.4 Flagella phage ........................................................................................................ 45 1.7.5 Phage receptor binding proteins.............................................................................. 46 1.8 Host resistance