A quantitative proteomics investigation of cold adaptation in the marine bacterium, Sphingopyxis alaskensis Thesis submitted in partial fulfilment of the requirements for the Degree of Doctor of Philosophy (Ph.D.) Lily L. J. Ting School of Biotechnology and Biomolecular Sciences University of New South Wales January 2010 COPYRIGHT STATEMENT ‘I hereby grant the University of New South Wales or its agents the right to archive and to make available my thesis or dissertation in whole or part in the University libraries in all forms of media, now or here after known, subject to the provisions of the Copyright Act 1968. I retain all proprietary rights, such as patent rights. I also retain the right to use in future works (such as articles or books) all or part of this thesis or dissertation. I also authorise University Microfilms to use the 350 word abstract of my thesis in Dissertation Abstract International (this is applicable to doctoral theses only). I have either used no substantial portions of copyright material in my thesis or I have obtained permission to use copyright material; where permission has not been granted I have applied/will apply for a partial restriction of the digital copy of my thesis or dissertation.' Signed ……………………………………………........................... 21st April, 2010 Date ……………………………………………........................... AUTHENTICITY STATEMENT ‘I certify that the Library deposit digital copy is a direct equivalent of the final officially approved version of my thesis. No emendation of content has occurred and if there are any minor variations in formatting, they are the result of the conversion to digital format.’ Signed ……………………………………………........................... 21st April, 2010 Date ……………………………………………........................... Originality statement I hereby declare that this submission is my own work and to the best of my knowledge it contains no materials previously published or written by another person, or substantial proportions of material which have been accepted for the award of any other degree or diploma at UNSW or any other educational institution, except where due acknowledgement is made in the thesis. Any contribution made to the research by others, with whom I have worked at UNSW or elsewhere, is explicitly acknowledged in the thesis. I also declare that the intellectual content of this thesis is the product of my own work, except to the extent that assistance from others in the project's design and conception or in style, presentation and linguistic expression is acknowledged. Signed………………………………………………… Dated………………………………………………….21st April, 2010 L. Ting, UNSW. i Abstract The marine bacterium Sphingopyxis alaskensis was isolated as one of the most numerically abundant bacteria from cold (4–10°C) nutrient depleted waters in the North Pacific Ocean. The objective of this study was to examine cold adaptation of S. alaskensis by using proteomics to examine changes in global protein levels caused by growth at low (10ºC) and high (30ºC) temperatures. Stable isotope labelling-based quantitative proteomics was used, and a rigorous post-experimental data processing workflow adapted from microarray-based methods was developed. The approach included metabolic labelling with 14N/15N and normalisation and statistical testing of quantitative proteomics data. Approximately 400,000 tandem mass spectra were generated resulting in the confident identification of 2,135 proteins (66% genome coverage) and the quantitation of 1,172 proteins (37% genome coverage). Normalisation approaches were evaluated using cultures grown at 30ºC and labelled with 14N and 15N. For 10ºC vs. 30ºC experiments, protein quantities were normalised within each experiment using a multivariate lowess approach. Statistical significance was assessed by combining data from all experiments and applying a moderated t-test using the empirical Bayes method with the limma package in R. Proteins were ranked after calculating the B-statistic and the Storey-Tibshirani false discovery rate. 217 proteins (6% genome coverage) were determined to have significant quantitative differences. In achieving these outcomes a range of factors that impact on quantitative proteomics data quality were broadly assessed, resulting in the development of a robust approach that is generally applicable to quantitative proteomics of biological system. The significantly differentially abundant proteins from the proteomics data provided insight into molecular mechanisms of cold adaptation in S. alaskensis. Important aspects of cold adaptation included cell membrane restructuring, exopolysaccharide biosynthesis, lipid degradation, carbohydrate and amino acid metabolism, and increased capacity of transcriptional and translational processes. A number of cold adaptive responses in S. alaskensis were novel, including a specific cold-active protein folding pathway, a possible thermally-controlled stringent response, and biosynthesis of intracellular polyhydroxyalkanoate reserve material. The overall study provided important new insight into the evolution of growth strategies necessary for the effective competition of S. alaskensis in cold, oligotrophic environments. ii L. Ting, UNSW. Acknowledgements Thank you to everyone who has helped me along the way. Special thanks go to my parents for their understanding, generosity and constant support. To Rick Cavicchioli and Mark Raftery, thank you for your guidance and supervision; this PhD would have been impossible without your support. Thanks also go to the past and present members of the RC lab; especially to Maz and Maine for listening and sharing, and to Haluk and Tim for your inputs of wisdom. Thanks to Linda from BMSF for your friendship, advice and proofreading! A big thanks to Mark Cowley for a fantastic collaboration. And, a special thank you to Prof. Bill O’Sullivan for your reading of this thesis and expert opinion. This work was supported by the Australian Postgraduate Award and the Australian Research Council. Mass spectrometric analysis for the work was performed at the Bioanalytical Mass Spectrometry Facility UNSW, and was supported in part by grants from the Australian Government Systemic Infrastructure Initiative and Major National Research Facilities Program (UNSW node of the Australian Proteome Analysis Facility) and by the UNSW Capital Grants Scheme. Finally, to Lachlan, thank you for your unending belief, patience, encouragement and confidence in me. I’m looking forwards to the future and the adventures we’ll have. Lily Ting January 2010, UNSW In memory of the late Prof. Michael Guilhaus L. Ting, UNSW. iii Table of contents Originality statement .......................................................................................................... i Abstract ............................................................................................................................. ii Acknowledgements .......................................................................................................... iii Table of contents .............................................................................................................. iv List of figures ................................................................................................................. xiii List of tables ................................................................................................................... xvi Abbreviations ............................................................................................................... xviii Publications ..................................................................................................................... xx Chapter 1. General introduction ............................................... 1 1.1. Sphingopyxis alaskensis .................................................................... 1 1.2. Living in a cold environment ........................................................... 2 1.2.1. The Arrhenius equation ................................................................................... 2 1.2.2. A definition of cold shock and cold adaptation .............................................. 2 1.2.3. Cellular responses in overcoming the challenges of the cold ......................... 3 1.2.3.1. Membrane integrity and transport systems .............................................. 4 1.2.3.2. Nucleic acid replication, transcription and turnover ................................ 5 1.2.3.3. Translation and protein folding ................................................................ 6 1.2.3.4. Compatible solutes and other cryoprotectants ......................................... 8 1.2.3.5. Protein flexibility ..................................................................................... 8 1.3. Mass spectrometry and proteomics ................................................. 9 1.3.1. Protein and peptide separation ....................................................................... 9 1.3.2. Tandem mass spectrometry and protein identification ................................. 10 1.3.3. Quantitative proteomics ................................................................................ 11 1.3.3.1. 2DE-based quantitative proteomics ....................................................... 13 1.3.3.2. Label-free quantitative proteomics ........................................................ 14 1.3.3.3. Stable isotope labelling quantitative proteomics ................................... 15 iv L. Ting, UNSW. 1.3.4. Post-experimental bioinformatics: