Tasmanian Institute of Agriculture A study of key meat spoilage bacteria By Tamsyn Stanborough Master of Science Submitted in fulfilment of the requirements for the Doctor of Philosophy (Agriculture) February 2018 Declaration of originality This thesis contains no material which has been accepted for a degree or diploma by the University or any other institution, except by way of background information and duly acknowledged in the thesis, and to the best of my knowledge and belief no material previously published or written by another person except where due acknowledgement is made in the text of the thesis, nor does the thesis contain any material that infringes copyright. (Tamsyn Stanborough) University of Tasmania February 2018 Statement on authority of access This thesis may be made available for loan and limited copying and communication in accordance with the Copyright Act 1968. (Tamsyn Stanborough) University of Tasmania February 2018 i Statement regarding published work contained in this thesis The publishers of the papers comprising Chapters 2, 4 and 5 hold the copyright for that content and access to the material should be sought from the respective journals. The remaining non published content of the thesis may be made available for loan and limited copying and communication in accordance with the Copyright Act 1968. (Tamsyn Stanborough) University of Tasmania February 2018 Statement of ethical conduct The research associated with this thesis abides by the international and Australian codes on human and animal experimentation, the guidelines by the Australian Government's Office of the Gene Technology Regulator and the rulings of the Safety, Ethics and Institutional Biosafety Committees of the University. (Tamsyn Stanborough) University of Tasmania February 2018 ii Co-authorship statements The following people and institutions contributed to the publication of work undertaken as part of this thesis: Author 1: Tamsyn Stanborough, Tasmanian Institute of Agriculture, University of Tasmania (Candidate) Author 2: Shane M. Powell, Tasmanian Institute of Agriculture, University of Tasmania (Primary supervisor) Author 3: Mark Tamplin, Tasmanian Institute of Agriculture, University of Tasmania (Co- supervisor) Author 4: P. Scott Chandry, Agriculture and Food, CSIRO (Co-supervisor) Author 5: Narelle Fegan, Agriculture and Food, CSIRO (Co-supervisor) Author 6: Tanoj Singh, Agriculture and Food, CSIRO (external collaborator) Author 7: Randy Suryadinata, Manufacturing, CSIRO (external collaborator) Author 8: Stewart D. Nuttall, Manufacturing, CSIRO (external collaborator) DETAILS OF THE AUTHOR ROLES Chapter 2: Insight into the genome of Brochothrix thermosphacta, a problematic meat spoilage bacterium. Published as: Stanborough T., Fegan N., Powell S.M., Tamplin M. and Chandry P.S., 2017. Insight into the genome of Brochothrix thermosphacta, a problematic meat spoilage bacterium. Applied and Environmental Microbiology, 83, e02786-16. Author 1 contributed 70% (conceptualised the study, conducted experimental work, analysed data and wrote the manuscript), authors 5, 2 and 3 each contributed 5% (assisted iii with study development and commented on the manuscript) and author 4 contributed 15% (guided experimental design and study development, assisted with data analysis and commented on the manuscript). Chapter 3: Characterisation of a putative Brochothrix thermosphacta sortase A enzyme. Submitted for publication as: Stanborough T., Suryadinata R., Fegan N., Powell S.M., Tamplin M., Nuttall S.D. and Chandry P.S. Characterisation of a putative Brochothrix thermosphacta sortase A enzyme. Author 1 contributed 70% (conceptualised the study, conducted all experimental work, analysed data and wrote the manuscript), author 7 contributed 8% (assisted with experimental design and commented on the manuscript), authors 5, 2 and 3 each contributed 3% (commented on the manuscript), author 8 contributed 8% (guided experimental design and study development and commented on the manuscript) and author 4 contributed 5% (contributed to study development and commented on the manuscript). Chapter 4: Genomic and metabolic characterisation of spoilage-associated Pseudomonas species. Published as: Stanborough T., Fegan N., Powell S.M., Singh T., Tamplin M., and Chandry P.S., 2018. Genomic and metabolic characterisation of spoilage-associated Pseudomonas species. International Journal of Food Microbiology, 268: 61-72. Author 1 contributed 70% (conceptualised the study, conducted all experimental work, analysed data and wrote the majority of the manuscript), authors 5, 2 and 3 each iv contributed 5% (assisted with the interpretation of results and commented on the manuscript), author 6 contributed 8% (assisted with experimental design, data analysis and writing of the methods section of the headspace analysis, and commented on the manuscript) and author 4 contributed 7% (assisted with experimental design and commented on the manuscript). Chapter 5: Vibrioferrin production by the food spoilage bacterium Pseudomonas fragi. Published as: Stanborough T., Fegan N., Powell S.M., Tamplin M., and Chandry P.S., 2018. Vibrioferrin production by the food spoilage bacterium Pseudomonas fragi. FEMS Microbiology Letters, 365(6), fnx279. Author 1 contributed 75% (conceptualised the study, conducted all experimental work, analysed data and wrote the manuscript), authors 5, 2 and 3 each contributed 5% (assisted with the interpretation of results and commented on the manuscript) and author 4 contributed 10% (guided experimental design and commented on the manuscript). We the undersigned agree with the above stated “proportion of work undertaken” for each of the above published (or submitted) peer-reviewed manuscripts contributing to this thesis: Signed: (Shane M. Powell) (Prof. Holger Meinke) Primary supervisor Head of School School of Land and Food School of Land and Food University of Tasmania University of Tasmania Date: February 2, 2018 v Publications and communications arising from this research Journal publications Stanborough T., Fegan N., Powell S.M., Tamplin M. and Chandry P.S., 2017. Insight into the genome of Brochothrix thermosphacta, a problematic meat spoilage bacterium. Applied and Environmental Microbiology, 83, e02786-16. Stanborough T., Fegan N., Powell S.M., Singh T., Tamplin M., and Chandry P.S., 2018. Genomic and metabolic characterisation of spoilage-associated Pseudomonas species. International Journal of Food Microbiology, 268: 61-72. Stanborough T., Fegan N., Powell S.M., Tamplin M., and Chandry P.S., 2018. Vibrioferrin production by the food spoilage bacterium Pseudomonas fragi. FEMS Microbiology Letters, 365(6), fnx279.. Conference presentations (poster format) Stanborough T., Fegan N., Powell S.M., Tamplin M. and Chandry P.S., 2016. Comparative genomics of the meat spoilage bacterium, Brochothrix thermosphacta. Australian Society of Microbiology 2016 Annual Scientific Meeting, Perth, Australia. Stanborough T., Suryadinata R., Fegan N., Powell S.M., Tamplin M., Nuttall S.D. and Chandry P.S., 2016. Characterisation of a putative Brochothrix thermosphacta sortase enzyme. Australian Society of Microbiology 2016 Victoria/Tasmania Bi-state Conference, Launceston, Tasmania. Stanborough T., Chandry P.S., Powell S.M., Tamplin M. and Fegan N., 2017. A putative siderophore locus of Pseudomonas fragi: Solving an iron problem. International Association of Food Protection 2017 Annual Meeting, Tampa, USA. vi Acknowledgements First and foremost, I would like to acknowledge the funding support from the Australian Meat Processor Corporation and CSIRO, without which this research would not have been possible. I would like to express my sincere gratitude to all of my supervisors, Scott Chandry, Narelle Fegan, Shane Powell and Mark Tamplin. Without their outstanding knowledge and leadership, invaluable suggestions and constructive criticism, this work would not have been possible. I am also truly grateful for the encouragement I received from all of my supervisors, which helped me to stay positive and confident when times were tough. My sincere appreciation goes to Stewart Nuttall for giving me the opportunity to conduct the research from chapter 3 under his guidance at CSIRO Manufacturing, and I would also like to thank Randy Suryadinata for his help and advice for this section. I am very grateful to David Ratkowsky for the statistical help he provided for the work in chapter 6 and I would like to thank Steve Petrovski for providing strains, plasmids and advice for the work in chapter 5. I am also incredibly grateful to Ed Fox and Tanoj Singh for their help, advice and insight, which were invaluable for improving the work in this thesis. I have been very lucky to have been based for my research at CSIRO Agriculture and Food in Werribee. The support and kindness I received from all of the staff members was amazing and made my time thoroughly enjoyable. Last but not least, I would like to thank my partner Barbara Koch for putting up with my whinging and worrying, for her constant encouragement and advice, and for being the best listener. vii Table of contents Declaration of originality ........................................................................................................... i Statement on authority of access .............................................................................................. i Statement regarding published work contained in this thesis ............................................... ii Statement of ethical conduct