UNIVERSITÀ DEGLI STUDI DI PADOVA DIPARTIMENTO DI BIOMEDICINA COMPARATA ED ALIMENTAZIONE SCUOLA DI DOTTORATO IN SCIENZE VETERINARIE Curriculum Unico Ciclo XXVIII PhD Thesis INTO THE BLUE: Spoilage phenotypes of Pseudomonas fluorescens in food matrices Director of the School: Illustrious Professor Gianfranco Gabai Department of Comparative Biomedicine and Food Science Supervisor: Dr Barbara Cardazzo Department of Comparative Biomedicine and Food Science PhD Student: Andreani Nadia Andrea 1061930 Academic year 2015 To my family of origin and my family that is to be To my beloved uncle Piero Science needs freedom, and freedom presupposes responsibility… (Professor Gerhard Gottschalk, Göttingen, 30th September 2015, ProkaGENOMICS Conference) Table of Contents Table of Contents Table of Contents ..................................................................................................................... VII List of Tables............................................................................................................................. XI List of Illustrations ................................................................................................................ XIII ABSTRACT .............................................................................................................................. XV ESPOSIZIONE RIASSUNTIVA ............................................................................................ XVII ACKNOWLEDGEMENTS ..................................................................................................... XIX ABBREVIATIONS .............................................................................................................. XXIII PREFACE ........................................................................................................................... XXVII BACKGROUND .......................................................................................................................... 1 References ............................................................................................................................... 3 AIM ............................................................................................................................................. 5 CHAPTER 1: The Microbiological Quality of Food: Pseudomonas and Related Genera....... 7 1.1. Abstract ............................................................................................................................. 9 1.2. Introduction to Pseudomonas: historical cues and significance of the genus ................. 9 1.3. Identification and tracking methods of foodborne pseudomonads: from classical microbiology towards -omics ................................................................................................ 10 1.4. Genus Pseudomonas: characteristics and ecology ......................................................... 13 1.4.1. Pseudomonas fluorescens group ............................................................................. 14 1.5. Spoilage potential of the Pseudomonas strains: a focus on the P. fluorescens group ... 17 1.5.1. Pigment production ................................................................................................. 20 1.5.2. Enzyme production .................................................................................................. 21 1.5.3. Other types of spoilage ............................................................................................ 25 1.6. Related genera: Xanthomonas and Shewanella ............................................................ 26 1.6.1. Shewanella spp. ....................................................................................................... 27 1.6.2. Spoilage potential of Shewanella spp. .................................................................... 28 1.6.3. Xanthomonas spp. .................................................................................................. 31 1.6.4. Spoilage potential of Xanthomonas spp. ................................................................ 34 VII Table of Contents 1.7. Conclusion and future perspectives: biofilm formation and control of spoilage defects .............................................................................................................................................. 35 1.8. References ...................................................................................................................... 36 CHAPTER 2: Tracking the blue: A MLST approach to characterise the Pseudomonas fluorescens group ......................................................................................................................55 2.1. Abstract ........................................................................................................................... 57 2.2. Introduction ................................................................................................................... 57 2.3. Materials and methods .................................................................................................. 60 2.3.1. Bacterial strains ...................................................................................................... 60 2.3.2. Phenotypic characterisation .................................................................................... 61 2.3.3. Primer design .......................................................................................................... 67 2.3.4. DNA extraction and PCR amplification ................................................................. 67 2.3.5. MLST data treatment and phylogenetic analyses .................................................. 69 2.3.6. Clonal and population analyses .............................................................................. 70 2.3.7. Pigment production on mozzarella cheese ............................................................. 70 2.4. Results ............................................................................................................................ 71 2.4.1. Phenotypic characterisation .................................................................................... 71 2.4.2. MLST scheme, allelic diversity and the nucleotide substitution analysis of housekeeping genes ...........................................................................................................73 2.4.3. Phylogeny of P. fluorescens ..................................................................................... 75 2.4.4. Recombination analysis of the population structure .............................................. 77 2.4.5. Pigment production on mozzarella cheese ............................................................. 78 2.5. Discussion ...................................................................................................................... 79 2.6. Acknowledgements ....................................................................................................... 84 2.7. References ..................................................................................................................... 84 2.8. Supplementary data ...................................................................................................... 90 2.9. Appendix to Chapter 2 .................................................................................................. 98 CHAPTER 3: A genomic and transcriptomic approach to investigate the blue pigment phenotype in Pseudomonas fluorescens ................................................................................ 101 VIII Table of Contents 3.1. Abstract ........................................................................................................................ 103 3.2. Introduction ................................................................................................................. 103 3.3. Materials and methods ................................................................................................ 106 3.3.1. Bacterial strains ..................................................................................................... 106 3.3.2. Genome sequencing and analysis ......................................................................... 106 3.3.3. Assessment of pigment production in MBM broth ............................................... 109 3.3.4. Transcriptome sequencing and analysis ............................................................... 109 3.3.5. Assessment of indole-derivative production test ................................................... 111 3.3.6. Biochemical characterisation of the blue pigment ................................................ 111 3.3.7. Mass spectrometry ................................................................................................. 111 3.4. Results and discussion .................................................................................................. 112 3.4.1. Genome sequencing and analysis ........................................................................... 112 3.4.2. Growth curves and pigment production ................................................................ 118 3.4.3. Transcriptome sequencing and analysis ................................................................ 118 3.4.4. Analysis of trp cluster as putatively involved in the blue pigment biosynthesis .. 122 3.4.5. Biochemical characterisation of the blue pigment ................................................ 124 3.5. Conclusions