Comparative Genomics and Toxin Regulation in the Cyanobacterium Cylindrospermopsis Raciborskii
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Comparative genomics and toxin regulation in the cyanobacterium Cylindrospermopsis raciborskii A DISSERTATION SUBMITTED BY Rati Sinha Masters of Applied Sciences (The University of Sydney) Masters of Science Management (The University of Technology, Sydney) IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE AWARD OF DOCTOR OF PHILOSOPHY AT THE SCHOOL OF BIOTECHNOLOGY AND BIOMOLECULAR SCIENCES THE UNIVERSITY OF NEW SOUTH WALES SYDNEY, AUSTRALIA 2014 PLEASE TYPE THE UNIVERSITY OF NEW SOUTH WALES Thesis/Dissertation Sheet Surname or Family name: Sinha First name: Rati Other name/s: Abbreviation for degree as given in the University calendar: PhD School: School of Biotechnology and Biomolecular Sciences Faculty: Science Title: Comparative genomics and toxin regulation in the cyanobacterium Cylindrospermopsis raciborskii Abstract 350 words maximum: (PLEASE TYPE) Cylindrospermopsis raciborskii is an invasive, toxin producing cyanobacterium of great ecological significance. This thesis explores the genomic plasticity and adaptive strategies of C. raciborskii strains isolated from different locations worldwide. It further explores the conservation of essential primary metabolic machinery of these strains, and discusses the potential causes of gene loss in these organisms. While strains isolated from Australia exhibited over 97% genomic similarity and 100% similarity within the cylindrospermopsin (CYN) biosynthesis (cyr) gene cluster, comparison of strains from China, Australia and Brazil highlighted diversity within the species, suggesting that geographic location could possibly play important roles in shaping the C. raciborskii genome. Nucleotide sequence based analyses exhibited strain clustering as per geographic location. Toxigenicity and taxonomic identity were not found to impact strain divergence. We report for the first time a varying spatial arrangement of the cyr biosynthesis gene cluster isolated from Chinese strains, compared to Australian strains. We, therefore hypothesize, that the arrangement of this cluster is influenced by the geographic location of isolates and not by overall strain relatedness as considered earlier. Additionally, we examined the effect of nitrogen (N) and phosphorus (P) on growth and ecotype-related changes in production of cylindrospermopsins by C. raciborskii. Significantly higher growth rates were observed, only when N and P were co-supplied. In contrast, toxin cell quotas increased significantly in treatments irrespective of whether N was supplied. These increased toxin quotas correlated with an increase in the proportion of cyrA to 16S genes in the C. raciborskii population, therefore, attributing changes in ecotype dominance as the most likely factor driving changes in toxin production between treatments. Finally, studies to gauge the changes in the transcriptional regulation of the cyr gene cluster under different intensities of light, low (10 µE) and high (100 µE) and pCO2, 500 ppm (L-CO2) and 1300 ppm (H-CO2), revealed that cylindrospermopsin production is constitutive and is not affected by the physiological parameters of light intensity and pCO2. Overall, this thesis highlights the influence of environmental drivers and location, on the divergence of C. raciborskii, thus, cautions water managers against the rampant threat of this cyanobacterium to water bodies worldwide. 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). 15-04-15 …………………………………………………………… ……………………………………..……………… ……….……………………...…….… 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 SUPERVISOR PROF. BRETT A. NEILAN SCHOOL OF BIOTECHNOLOGY AND BIOMOLECULAR SCIENCES THE UNIVERSITY OF NEW SOUTH WALES SYDNEY, AUSTRALIA CO-SUPERVISOR DR. LEANNE A. PEARSON SCHOOL OF BIOTECHNOLOGY AND BIOMOLECULAR SCIENCES THE UNIVERSITY OF NEW SOUTH WALES SYDNEY, AUSTRALIA CO-SUPERVISOR DR. TIMOTHY W. DAVIS ADJUNCT RESEARCH FELLOW AUSTRALIAN RIVERS INSTITUTE GRIFFITH UNIVERSITY SYDNEY, AUSTRALIA 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.’ Dated: ……15-04-15………………… Signed: ………………….. i 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…………………………………………… Date …15-04-15………………………………… 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 ……………………………………………........................... Date …………15-04-15…………………………………................ ii Abstract Cylindrospermopsis raciborskii is an invasive, toxin producing cyanobacterium of great ecological significance. This thesis explores the genomic plasticity and adaptive strategies of C. raciborskii strains isolated from different locations worldwide. It further explores the conservation of essential primary metabolic machinery of these strains, and discusses the potential causes of gene loss in these organisms. While strains isolated from Australia exhibited over 97% genomic similarity and 100% similarity within the cylindrospermopsin (CYN) biosynthesis (cyr) gene cluster, comparison of strains from China, Australia and Brazil highlighted diversity within the species, suggesting that geographic location could possibly play important roles in shaping the C. raciborskii genome. Nucleotide sequence based analyses exhibited strain clustering as per geographic location. Toxigenicity and taxonomic identity were not found to impact strain divergence. We report for the first time a varying spatial arrangement of the cyr biosynthesis gene cluster isolated from Chinese strains, compared to Australian strains. We, therefore hypothesize, that the arrangement of this cluster is influenced by the geographic location of isolates and not by overall strain relatedness as considered earlier. Additionally, we examined the effect of nitrogen (N) and phosphorus (P) on growth and ecotype-related changes in production of cylindrospermopsins by C. raciborskii. Significantly higher growth rates were observed, only when N and P were co-supplied. In contrast, toxin cell quotas increased significantly in treatments irrespective of whether N was supplied. These increased toxin quotas correlated iii with an increase in the proportion of cyrA to 16S genes in the C. raciborskii population, therefore, attributing changes in ecotype dominance as the most likely factor driving changes in toxin production between treatments. Finally, studies to gauge the changes in the transcriptional regulation of the cyr gene cluster under different intensities of light, low (10 µE) and high (100 µE) and pCO2, 500 ppm (L-CO2) and 1300 ppm (H-CO2), revealed that cylindrospermopsin production is constitutive and is not affected by the physiological parameters of light intensity and pCO2. Overall, this thesis highlights the influence of environmental drivers and