Identifying stress-tolerance genes in hyperarid desert soils using functional metagenomics by Gideon Jacobus de Jager Submitted in partial fulfilment of the requirements for the degree Magister Scientiae In the Faculty of Natural & Agricultural Sciences Department of Genetics University of Pretoria Pretoria 12 February 2015 Supervisor: Prof. Don A. Cowan Co-supervisor: Dr René Sutherland Declaration I, Gideon Jacobus de Jager declare that the thesis, which I hereby submit for the degree Magister Scientiae at the University of Pretoria, is my own work and has not previously been submitted by me for a degree at this or any other tertiary institution. Signature: DdeJager Date: 12 February 2015 ii Dedication This thesis is dedicated to my mom, dad and Mari. Without your support and belief I would not have been able to reach this point. iii Acknowledgements I would like to thank the National Research Foundation (NRF) for providing me with a bursary so I could pursue this degree. The financial assistance of the NRF towards this research is hereby acknowledged. Opinions expressed and conclusions arrived at, are those of the author and are not necessarily to be attributed to the NRF. Prof Don Cowan, thank you for your supervision and guidance during this research project and for always expecting the best from me and believing in my abilities. You are an inspiration in both science and life. Dr René Sutherland, thank you for your hands-on supervision and for your continuing support after your departure, despite having so much else on your plate! Dr Eloy Ferreras and Dr Eldie Berger, thank you for taking over from René as my go-to people when I needed advice about which experimental approaches to use and for your guidance in carrying these out. Dr Leandro Guerrero, Dr Pieter de Maayer and Dr Angel Valverde, thank you for helping me acquire valuable skills in bioinformatic and statistical analyses. I would like to thank the Sequencing Facility at the University of Pretoria and the ION PGM team for their professionalism and particularly Elritha van Zyl for her help in ensuring I had enough good quality DNA to be sequenced. My thanks go to the following people for taking the time to read various chapters of my thesis and for giving constructive commentary on these: Dr Eloy Ferreras, Dr Evelien Adriaenssens, Dr Pieter de Maayer, Marc Van Goethem and Andi Wilson. To everyone at CMEG, thank you for being such a great group of people to work and socialise with! Those wine evenings never ceased to entertain (thanks Prof Cowan for the wine). To Marc, the cricket matches, random challenges, honest trailers, tester, #LabStories and countdown beers at Oom Gert‘s kept us both sane! Thanks for that. To my friends and family, thank you for your support and for understanding (or trying to, at least) that I sometimes couldn‘t attend the numerous social gatherings, but also for twisting my arm on other occasions to join the fun and unwind in the midst of thesis writing! Andi, thank you for your support and understanding, for always believing in me and my abilities and never even letting me forget what I am capable of achieving also! iv Summary Microorganisms represent an intriguing and underexploited resource for the discovery of novel genes. Extremophiles in environments like the Namib Desert are exposed to multiple stress factors including UV radiation, desiccation, osmotic, oxidative, nutrient and temperature stress. These microorganisms are well adapted to tolerate extended exposure to stressors and rapid change in the environmental conditions. Functional metagenomics is a powerful tool for the discovery of novel genes and enzymes. As sequence based approaches are limited by sequence homologues, functional screening allows for the identification of unique genes involved in stress tolerance. In this study, novel genes involved in stress tolerance were identified by screening a Namib Desert soil metagenomic fosmid library (>1x106 clones) in Escherichia coli EPI300 under hyperosmotic (NaCl), oxidative (H2O2), heat (46°C) and UVB radiation (280 – 315 nm) stress. Increasing the sodium chloride (NaCl) concentration in growth media to 5% resulted in the identification of 12 salt-tolerant clones. All clones, except one, showed significantly increased growth compared to the host (containing an empty vector) in liquid media augmented with 5% NaCl after 48 hours (P-value <0.05). The clones contained fosmids of approximately 42 kilobases (kb) with estimated insert sizes ranging from 30 to 34 kb. Fosmid DNA of salt-tolerant clones was sequenced using the Ion Torrent PGM platform at the University of Pretoria, generating 1.4 Gb of sequence data. Sequences were assembled using MIRA and open reading frames were predicted using the RAST server. Phylogenetic analysis, using conserved proteins, revealed that the metagenomic DNA originated from members of the phyla Deltaproteobacteria, Fibrobacteres, Cyanobacteria, Actinobacteria and Planctomyces. Six putative salt-tolerance genes were identified through functional predictions based on conserved domains present in the encoded proteins. The proteins; an ABC transporter substrate-binding domain, a RelA-SpoT-like hypothetical protein, an HD-hydrolase domain protein, a cation export system protein, a peptidase M29 and a Na+/H+ antiporter, all shared less than 60% amino acid identity with the closest homologues. The low amino acid identity of the proteins encoded on the metagenomic DNA, to known sequences, suggests that the inserts were derived from novel taxa, highlighting the untapped microbial communities present in the Namib Desert. Over-expression of the proteins in E. coli BL21(DE3) and subsequent NaCl trials revealed that cells expressing the peptidase M29 showed significantly increased salt-tolerance at 3.5% NaCl compared to the host. Future work will entail investigating the mechanism by which the peptidase M29 confers salt-tolerance to E. coli, investigating whether any cross-protection to other abiotic v stresses is conferred and determining the optimal parameters for activity and substrate specificity of the enzyme in vitro. The identification of novel stress-tolerance genes and proteins holds potential for application in industrial biotechnology, medicine and agriculture and provides insight into the mechanisms employed by extremophiles to tolerate abiotic stress. vi Conference Outputs Sutherland, R., D. de Jager and D. Cowan. Metagenomic Gene Discovery. 2nd International Congress on Functional Metagenomics, Pretoria, South Africa. 2 – 5 June 2013. Sutherland, R., D. de Jager and D. Cowan. Metagenomic Gene Discovery. 18th Biennial Congress of the South African Society for Microbiology (SASM), Bela-bela, South Africa. 24 – 27 November 2013. Sutherland, R., D. de Jager and D. Cowan. Metagenomic Gene Discovery. Genomics Research Institute Symposium, University of Pretoria, South Africa. November 2013. *de Jager, D., R. Sutherland and D. Cowan. Metagenomic Gene Discovery. 10th International Congress on Extremophiles, St Petersburg, Russia. 7 – 11 September 2014. de Jager, D., R. Sutherland and D. Cowan. Identification of novel stress-tolerance genes from a Namib Desert soil metagenomic library, through functional screening. 2nd Joint Congress of the South African Society for Bioinformatics (SASBi) and the South African Genetics Society (SAGS), Tshwane, South Africa. 23 – 26 September 2014. de Jager, D., R. Sutherland and D. Cowan. Identification of novel stress-tolerance genes from a Namib Desert soil metagenomic library, through functional screening. Genomics Research Institute Symposium, University of Pretoria, Pretoria, South Africa. 31 October 2014. de Jager, D., R. Sutherland and D. Cowan. Identification of novel stress-tolerance genes from a Namib Desert soil metagenomic library, through functional screening. Department of Genetics Post-graduate Symposium, University of Pretoria, Pretoria, South Africa. 28 November 2014. *This poster was awarded one of five poster prizes for young scientists at this international congress. vii Table of Contents Declaration .........................................................................................................................ii Dedication ......................................................................................................................... iii Acknowledgements ........................................................................................................... iv Summary ........................................................................................................................... v Conference Outputs ......................................................................................................... vii List of Tables .................................................................................................................... xii List of Figures .................................................................................................................. xiv Table of Abbreviations ..................................................................................................... xix Chapter 1: Literature Review .............................................................................................. 1 1.1 Introduction .................................................................................................................. 1 1.2 Life in Hyperarid Deserts ............................................................................................. 3 1.3 Stress Response in Bacteria .......................................................................................