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Thesisl.Knecht-Upload.Pdf Research Collection Doctoral Thesis Identification of phage receptors and enzymes essential for phage infection in Erwinia amylovora Author(s): Knecht, Leandra Publication Date: 2019-05-10 Permanent Link: https://doi.org/10.3929/ethz-b-000360576 Rights / License: In Copyright - Non-Commercial Use Permitted This page was generated automatically upon download from the ETH Zurich Research Collection. For more information please consult the Terms of use. ETH Library Diss. ETH No. 26009 Identification of phage receptors and enzymes essential for phage infection in Erwinia amylovora A thesis submitted to attain the degree of DOCTOR OF SCIENCES of ETH ZURICH (Dr. sc. ETH Zurich) presented by LEANDRA EVA KNECHT MSc ETH Zurich born on 03.10.1988 citizen of Windisch AG, Switzerland accepted on the recommendation of Prof. Dr. Martin J. Loessner, examiner Prof. Dr. Lars Fieseler, co-examiner Prof. Dr. Julia Vorholt-Zambelli, co-examiner 2019 Table of Contents Abbreviations .......................................................................................... 1 Summary ................................................................................................ 3 Zusammenfassung ................................................................................. 6 1. Introduction .......................................................................... 11 1.1. Fire blight ..................................................................................................... 11 1.1.1. Erwinia amylovora ......................................................................................... 11 1.1.1. Virulence factors............................................................................................ 11 1.1.2. The disease ................................................................................................... 12 1.1.3. Treatment options ......................................................................................... 14 1.1.4. Fire blight in Switzerland ............................................................................... 15 1.2. Bacteriophages ........................................................................................... 16 1.2.1. History ........................................................................................................... 16 1.2.2. Phage classification ....................................................................................... 17 1.2.3. Life cycle ....................................................................................................... 17 1.2.4. Phage resistance ........................................................................................... 19 1.2.5. Phage biocontrol ........................................................................................... 20 1.2.6. Phage biocontrol of E. amylovora .................................................................. 21 1.2.7. Phages of E. amylovora ................................................................................ 22 1.3. Aim of the study .......................................................................................... 23 1.4. References ................................................................................................... 24 2. Manuscripts ......................................................................... 37 2.1. Manuscript I: Receptor identification for phage cocktail composition .... 39 2.2. Manuscript II: Phage infection of E. amylovora requires cellulose ......... 65 2.3. Manuscript III: The role of topB1, rfaE and pgm in phage resistance ...... 95 2.4. Manuscript IV: Y2 resistance affects phage infectivity ........................... 123 3. Conclusions and Outlook .................................................... 149 4. Acknowledgements ............................................................. 155 5. Curriculum Vitae ................................................................. 157 I Abbreviations Abbreviations CFBP Collection Française de Bactéries associées aux Plantes CFU Colony forming units CHF Swiss francs CPC Cetylpyridinium chloride Da Dalton ddH2O Double distilled water DNA Deoxyribonucleic acid ds Double stranded EOP Efficiency of plating EPS Extracellular polysaccharides bp Base pair LB Lysogeny broth LPS Lipopolysaccharide M Molarity min Minutes mM Milimolar MOI Multiplicity of infection NCBI National Centre for Biotechnology Information OD Optical density PAGE Polyacrylamide gel electrophoresis PBS Phosphate-buffered saline PCR Polymerase chain reaction PFU Plaque forming unit RBS Ribosomal binding site RT Room temperature SD Standard deviation SOC Super optimal broth with catabolite repression spp. Species pluralis T3SS Type three secretion system wt Wildtype 1 Summary Summary The Gram-negative bacterium Erwinia amylovora is the causative agent of fire blight. This plant disease was classified as one of the ten most devastating plant diseases and affects members of the Rosaceae family. Fire blight first appeared in 1780 in Northern America from where it developed into an almost global threat for apple and pear farms. Under favourable conditions, the highly contagious pathogen is able to infect an entire orchard within a single season. Disease treatments are time and cost intensive since susceptible plants must be monitored and sanitized regularly. Antibiotics such as streptomycin are efficient in controlling the plant infection. However, the rise of antibiotic resistant strains and increasing public health concerns prompted increasing numbers of countries to ban streptomycin for agricultural purposes. Alternatives to antibiotics are therefore urgently needed. Over recent years, bacteriophages have been emerging as possible alternatives to conventional antibiotic treatments. These bacterial viruses specifically target and destroy host cells by recognizing receptors on their surface and have several advantages over antibiotics. Phages pose the most abundant entity on earth and outnumber bacteria by ten fold. This abundance ensures almost unlimited possibilities in combining different phages. Phages are highly specific and can only target host bacteria, leaving potentially beneficial bacteria unharmed. Since phages require their host metabolism for reproduction, phage numbers will increase in the presence of their hosts but start to decay in their absence. Finally, phages are considered as non toxic and environmentally safe. Although phages can adapt to modifying hosts, the abundance of estimated 1025 phage infections occurring per second, forces bacteria to adapt to the phages and develop phage resistance. To ensure that phage biocontrol is an efficient and especially long-lasting treatment option against pathogenic bacteria, the risk of resistance development must be minimized. The targeted receptor on the host surface can be modified, thereby ensuring phage resistance. Combinations of phages targeting different receptors could be applied to circumvent resistance development, since the bacterium would have to mutate several receptors simultaneously. To identify host receptors targeted by six well-characterized, highly E. amylovora specific phages, a transposon mutagenesis library was screened for phage resistant mutants. Transposon insertions in Bue1 and Y2 resistant mutants could mostly be linked to LPS biosynthesis and LPS export. It is likely that both phages require LPS structure of E. amylovora for host identification and successful infection. The phages L1 and S2 were unable to lyse mutants with disrupted ams genes. The ams operon is responsible for encoding the amylovoran synthesis apparatus, which generates one of the major EPS components secreted by E. amylovora and functions as virulence factor in fire blight pathogenicity. These findings and the fact that both phages harbour depolymerases, which are able to degrade 3 Summary amylovoran suggests that amylovoran is targeted by L1 and S2 as receptor. M7 and S6 were unable to infect mutants with transposon insertions in the bcs operon. This operon encodes the bacterial cellulose synthase, which is required for bacterial cellulose production and secretion. This carbohydrate polymer is required for stable biofilm production. Deletion of the entire operon as well as deletion of the key genes bcsA encoding the catalytically active subunit, or bcsC, which encodes the outer membrane protein BcsC demonstrated M7 and S6 resistance. Experiments with the cellulose binding dye Congo Red also protected bacteria from phage infection, suggesting that both the cellulose synthase complex and cellulose are required for M7 or S6 infection. In addition, a collection of genes in the M7 and S6 genomes were identified that could encode enzymes with cellulase or endoglucanase activity. The incubation of entire phages with cellulose verified cellulolytic activity for S6 phages. These findings support the hypothesis that M7 and S6 specifically target bacterial cellulose secreted by the host bacterium. These enzymes could have potential to target biofilm-forming bacteria such as E. amylovora. The fact that phages encode cellulase and are able to target bacterial cellulose or the cellulase synthase complex as host receptors is, to our knowledge a novelty and should be further investigated. The results generated from the screen were used to test different phage combinations in vitro and on blossoms for their potential as biocontrol agent. M7 or S6 alone were the most potent single treatment in vitro. Combinations of phages belonging to different phage receptor groups
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