Metagenomic and metaproteomic analysis of the microbial communities in marine sponges Michael Yizhe Liu A thesis submitted to the University of New South Wales for the degree of Doctor of Philosophy February 2012 PLEASE TYPE THE UNIVERSITY OF NEW SOUTH WALES Thesis/Dissertation Sheet Surname or Family name: LIU First name: MICHAEL YIZHE Other name/s: Abbreviation for degree as given in the University calendar: Ph.D School: BIOTECHNOLOGY AND BIOMOLECULAR SCIENCES Faculty: SCIENCE Title: METAGENOMIC AND METAPROTEOMIC ANALYSIS OF THE MICROBIAL COMMUNITIES IN MARINE SPONGES Abstract 350 words maximum: (PLEASE TYPE) Marine sponges harbour complex communities of diverse microorganisms, which have been postulated to form intimate symbiotic relationship with their host. Despite the importance of this symbiosis - from both the ecological and biotechnological perspectives - little is known about the functional properties of the bacterial symbionts and their interactions with the host. This thesis presents the characterisation of the microbial communities associated with two sponges, Cymbastela concentrica and Rhopaloeides odorabile, from the eastern coast of Australia. A functional genomic analysis of an uncultured Deltaproteobacterium in the sponge C. concentrica showed that this organism represents a novel phylogenetic clade and lives in association with a cyanobacterium within the sponge tissue. The result also provide an overview of the predicted functional and ecological properties as well as the complex interactions of this bacterium with its surrounding. An integrated metaproteogenomic approach was used to characterise the microbial community associated with C. concentrica. The result unveiled the expression of specific proteins involved in the transport of typical sponge metabolites, respiration, stress protection and molecular interactions (e.g. eukaryotic-like proteins). This analysis highlighted the requirement for the microbial community to respond to variable environmental conditions. A subsequent study investigated the genomic and functional properties of an uncultured Phyllobacteriaceae phylotype, This organism was predicted to be capable of using host-derived compounds and to carry out anaerobic respiration through nitrate reduction. Functional signatures associated with survival using defence and host-symbiont interactions were also identified. The impact of elevated water temperature on the microbial communities within R. odorabile was investigated with a metaproteogenomic approach. Changes in taxonomic composition, species richness, genomic content and expressed functional profile of sponge microbial communities were observed during thermal stress. In particular, putative symbiotic functions, such as metabolite transport, metabolism of sponge-specific substrates and maintenance of cellular structure, were identified in unstressed sponge communities, but were absent in stress samples. The decline in expression of symbiont functions in stressed sponge sample demonstrated that sponge necrosis is likely to be caused by the disturbance of interactions within the sponge holobiont, rather than as a result of pathogen infection. Declaration relating to disposition of project thesis/dissertation I hereby grant to the University of New South Wales or its agents the right to archive and to make available my thesis or dissertation in whole or in 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 property 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 Abstracts International (this is applicable to doctoral theses only). 26-06-2012 .. .... Signature Witness Date The University recognises that there may be exceptional circumstances requiring restrictions on copying or conditions on use. Requests for restriction for a period of up to 2 years must be made in writing. Requests for a longer period of restriction may be considered in exceptional circumstances and require the approval of the Dean of Graduate Research. FOR OFFICE USE ONLY Date of completion of requirements for Award: THIS SHEET IS TO BE GLUED TO THE INSIDE FRONT COVER OF THE THESIS 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 ……………………………………………........................... 26th June 2012 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 ……………………………………………........................... 26th June 2012 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 …………………………………………… 26/02/2012 Date …………………………………………… i Abstract Marine sponges harbour complex communities of diverse microorganisms, which have been postulated to form intimate symbiotic relationship with their host. Despite the importance of this symbiosis - from both the ecological and biotechnological perspectives - little is known about the functional properties of the bacterial symbionts and their interactions with the host. This thesis presents the characterisation of the microbial communities associated with two sponges, Cymbastela concentrica and Rhopaloeides odorabile, from the eastern coast of Australia. A functional genomic analysis of an uncultured Deltaproteobacterium in the sponge C. concentrica showed that this organism represents a novel phylogenetic clade and lives in association with a cyanobacterium within the sponge tissue. The result also provide an overview of the predicted functional and ecological properties as well as the complex interactions of this bacterium with its surrounding. An integrated metaproteogenomic approach was used to characterise the microbial community associated with C. concentrica. The result unveiled the expression of specific proteins involved in the transport of typical sponge metabolites, respiration, stress protection and molecular interactions (e.g. eukaryotic-like proteins). This analysis highlighted the requirement for the microbial community to respond to variable environmental conditions. A subsequent study investigated the genomic and functional properties of an uncultured Phyllobacteriaceae phylotype, This organism was predicted to be capable of using host- derived compounds and to carry out anaerobic respiration through nitrate reduction. Functional signatures associated with survival using defence and host-symbiont interactions were also identified. The impact of elevated water temperature on the microbial communities within R. odorabile was investigated with a metaproteogenomic approach. Changes in taxonomic composition, species richness, genomic content and expressed functional profile of sponge microbial communities were observed during thermal stress. In particular, putative symbiotic functions, such as metabolite transport, metabolism of sponge- ii specific substrates and maintenance of cellular structure, were identified in unstressed sponge communities, but were absent in stress samples. The decline in expression of symbiont functions in stressed sponge sample demonstrated that sponge necrosis is likely to be caused by the disturbance of interactions within the sponge holobiont, rather than as a result of pathogen infection. iii Acknowledgements My first vote of thanks goes to my primary supervisors, Staffan Kjelleberg and Torsten Thomas. I count myself fortunate to have had the opportunity to work on this stimulating and important project with you. Staffan, thank you for your guidance and support of my project. Your honest, thorough and