Metagenomics of Cyanobacterial Blooms Author Pope, Phillip Byron Published 2007 Thesis Type Thesis (PhD Doctorate) School School of Biomolecular and Biomedical Sciences DOI https://doi.org/10.25904/1912/1772 Copyright Statement The author owns the copyright in this thesis, unless stated otherwise. Downloaded from http://hdl.handle.net/10072/368095 Griffith Research Online https://research-repository.griffith.edu.au METAGENOMICS OF CYANOBACTERIAL BLOOMS A Thesis submitted in fulfilment of the requirements for the degree of Doctor of Philosophy, Griffith University by Phillip Byron Pope (BSc. Hons) School of Biomedical and Biomolecular Science, Faculty of Science, Griffith University, Nathan Campus, Queensland, Australia January 2007 DECLARATION I declare that this work has not been previously submitted for a degree or diploma in any university. To the best of my knowledge and belief, the thesis contains no material previously published or written by another person except where due reference is made in the thesis itself. Phillip B Pope ii ACKNOWLEDGEMENTS Upmost and foremost I would like to thank Professor Bharat KC Patel for the opportunity to work with him. His continuing advice, support and assurance gave me the confidence and enthusiasm needed to make my PhD candidature as productive and enjoyable as possible. Many thanks are forwarded to Dr Glenn Shaw and Dr Anthony Greene for their additional support and guidance throughout my candidature. I appreciate the advice, assistance and friendship of Mr Peter Bain, Mr Jamie Nourse, Ms Janet Li Zhang, Mr Matthew Walker-Brown, Dr Kerry Inder, Dr Renee Stirling, Mr Frederich Huynh, Dr Marwan Abu-Halaweh and Dr Lyle McMillen. The visitors from Nanyang Polytechnology, Singapore, Tok Zheny Rony (Alex), Daphne Lim and Wee Yang Hoa are also mentioned for their contribution to this project. Special thanks must also go out to the entire CRC for Water Quality and Treatment “team”, particularly Professor Dennis Mulcahy who has always been there ready to provide words of wisdom and lend a helping hand. Carolyn Bellamy and George Turelli are also mentioned for their solid support through out my candidature especially for assistance regarding conference travel. I gratefully acknowledge the financial support from The Australian Research Council Project Grant and the CRC for Water Quality and Treatment for my PhD program as well as funding provided by Griffith University for conference travel. Finally I thank my family and friends for their continued interest and enduring support in all that’s been done and what still lies ahead. iii PUBLICATIONS AND CONFERENCE PROCEEDINGS Publications in preparation: 1. Phillip B Pope and Bharat KC Patel. Metagenomics of a Aphanizomenon and Cylindrospermopsis Dominated Toxic Cyanobacterial Bloom. Applied and Environmental Microbiology. In preparation. 2. Phillip B Pope and Bharat KC Patel. Community Composition associated with Cyanobacterial blooms: Analysis of Communities in Lake Samsonvale and Lake Ainsworth. Microbial Ecology. In preparation. Conference proceedings: 1. Phillip B Pope and Bharat KC Patel (2006). Metagenomic Analysis of a Toxic Cyanobacterial Bloom. 11th International Symposium on Microbial Ecology, 20-25 August, Vienna, Austria.(oral) 2. Phillip B Pope and Bharat KC Patel (2006). Comparative Genomic Analysis of DNA Fragments from a Cyanobacterial Bloom. 12th International Conference on Harmful Algal Blooms, 4-8 September, Copenhagen, Denmark. 3. Phillip B Pope (2006). The Quest for Treasure in a Toxic Green World. Cooperative Research Centre’s Association National Conference, 17-19 May, Brisbane. – National Finalist for ‘Showcasing CRC PhD’s’. (oral) 4. Phillip B Pope (2006). Cyanobacterial Diversity and the Expression of Secondary Metabolites in Environmental Blooms. 5th Biennial Postgraduate Student Conference of the CRC for Water Quality and Treatment, 10-13 July, Melbourne Australia. – Runner up best presentation. (oral) 5. Phillip B Pope and Bharat KC Patel (2005). Metagenomics of Cyanobacterial Blooms. GRC conference for Applied and Environmental Microbiology, 24-29 July, New London, Connecticut, USA. iv ABSTRACT Cyanobacteria are a diverse and widely distributed group of organisms common in soil and in both marine and freshwater. Under favorable conditions they can reproduce explosively, forming dense concentrations called blooms. Fresh water cyanobacterial blooms in particular are commonly associated with toxin production in drinking water supplies and are increasingly becoming a risk to human health. Beyond toxin production these extremely complex, constantly interacting and changing microbial communities have vast impacts on their surrounding ecosystem. The triggers that initiate bloom formation and/or toxin production remain poorly understood. This stems from the fact that there is still very little known of cyanobacterial bloom population structure and their function in the real environment. A greater understanding of the interactions of different microbial populations and their functions in the blooming process leading to toxin production could come from using metagenomics to investigate the genetic and metabolic diversity of the mixed populations rather than the difficult to culture cyanobacteria. Two distinct cyanobacterial bloom communities existing in contrasting Australian freshwater lakes were selected and high molecular weight DNA extracted. PCR-amplified 16S rRNA genes were subsequently cloned and a total of 75 clones from Lake Samsonvale and 50 clones from Lake Ainsworth were examined. Sequences identified belonged to species from 6 different phyla from the Bacterial domain, including Cyanobacteria, Actinobacteria, Firmicutes, Verrucomicrobium, Bacteroidetes, and α-, β- and γ- Proteobacteria. The majority of the bacterial sequences were most closely related to sequences recovered from other freshwater clones or isolates (<80% homology), whilst few were closely related to sequences recovered from soil or v marine habitats. In particular 9 % of the total sequences were most closely related to sequences recovered from freshwater lakes that are susceptible to cyanobacterial blooms. A total of 12 novel clusters consisting of 22 sequences were noted spanning all divisions represented in the analysis. Of this, 7 were found to lack any close relatives suggesting that sequences in these clusters may be characteristic for bloom events. Preliminary results also indicate that physio-chemical differences in lake character appear to influence bacterial community composition associated with cyanobacterial blooms. Bloom communities from Lake Samsonvale demonstrated high levels of toxin- producing Cyanobacteria and uncultured Actinobacteria. These findings were used to justify its selection for further metagenomic analysis to gain insights into the genomes of these and other organisms. DNA was fractionated and used to construct a bacterial artificial chromosome library (CBNPD1) of 2,850 clones which had an average insert size of 27 kb. A PCR-based single-gene polyketide synthase library was constructed in tandem and used as an additional assurance that high quality DNA was being extracted and cloned. Phylogenetic analysis of gene sequences recovered from this library demonstrated an abundance of novel bacterial polyketide synthase genes. Sequence-based screening of library CBNPD1 was performed to identify clones of interest and provide a physiological insight within cyanobacterial blooms. A random BAC-end sequence survey generated 67 sequences (40 kb in total) from 36 randomly selected clones. G+C composition ranged from 33.33 to 72.91%. Fifteen sequence tags (22%) were found most similar to sequences affiliated to genera with no available genome. Another 17 sequence tags (25%) were most similar to sequences affiliated to genera with available genomes, vi however similarities were less than 80%. Sequence tags were also found with affiliation to proteins involved in a wide array of cell metabolism processes including amino acid metabolism (e.g. methionine synthase), carbohydrate metabolism (cellulose), inorganic ion metabolism (nitrite/sulfite reductase), and lipid metabolism (fatty acid hydroxylase). A number of genes involved in cell structures (e.g. flagella), DNA processes, energy production (photosynthetic reaction center L subunit) and defense mechanisms (nucleases) were also affiliated to sequence tags. PCR screening of CBNPD1 was used to detect clones containing 16S rDNA to establish a link between physiological and phylogenetic information of uncharacterized microorganisms in cyanobacterial blooms. Screens from 480 clones identified 2 clones containing a 16S rRNA gene. Clone 545 and 578 contained 16S rDNA affiliated to 2 different phylogenetic genera within the Proteobacteria division, Pseudomonas and Roseateles respectively. From library screens 7 BAC inserts were selected and sequenced to completion comprising 144 kb of a cyanobacterial bloom metagenome and spanning 3 phyla including Proteobacteria, Actinobacteria and Bacteroidetes. 130 genes have been identified and assigned to COG (clusters of orthologous groups of proteins) functional categories. Also identified, were many housekeeping proteins spanning the majority of the COG functional groups as well as physiologically and ecologically important proteins some of which were looked at more in depth. These include a putative phenylacetyl catabolon, a putative RTX toxin, several putative oxidoreductases and several putative bacterial transcriptional regulators that are