MOLECULAR CHARACTERISATION OF MEMBRANE TRANSPORTERS ASSOCIATED WITH SAXITOXIN BIOSYNTHESIS IN CYANOBACTERIA A DISSERTATION SUBMITTED BY JASPER JOHN LOBL PENGELLY B. SC. BIOTECHNOLOGY (HONS.) UNSW IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE AWARD OF DOCTOR OF PHILOSOPHY (PH.D.) IN THE SCHOOL OF BIOTECHNOLOGY AND BIOMOLECULAR SCIENCES THE UNIVERSITY OF NEW SOUTH WALES SYDNEY, AUSTRALIA MARCH 2008 i ii MOLECULAR CHARACTERISATION OF MEMBRANE TRANSPORTERS ASSOCIATED WITH SAXITOXIN BIOSYNTHESIS IN CYANOBACTERIA A DISSERTATION SUBMITTED BY JASPER JOHN LOBL PENGELLY B. SC. BIOTECHNOLOGY (HONS.) UNSW IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE AWARD OF DOCTOR OF PHILOSOPHY (PH.D.) IN THE SCHOOL OF BIOTECHNOLOGY AND BIOMOLECULAR SCIENCES THE UNIVERSITY OF NEW SOUTH WALES SYDNEY, AUSTRALIA SUPERVISOR PROFESSOR BRETT A. NEILAN SCHOOL OF BIOTECHNOLOGY AND BIOMOLECULAR SCIENCES THE UNIVERSITY OF NEW SOUTH WALES SYDNEY, AUSTRALIA iii iv 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………………………………………………………………………………… Date………………… 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………………… 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………………… v vi Acknowledgements I would like to acknowledge and express my appreciation for the help and support of my supervisor Brett Neilan and many of the past and present members of the BGGM laboratory. In no particular order: Thank-you to the members of the prestigious ARS group, Troco M, Rosi C, Ralf K, Young-Jae J and Julia M for making toxin research always so much fun – may we all get published eventually; Thank-you to my elder and greater scientific teachers, Ken-lee N, Janine C, Leanne P, Francesco P, Brendan B, Michelle G, Anne J and Tim S for all the endless questions and showing me the way to the other-side of the long dark tunnel; Thank-you to my many comrades in the struggle for freedom, Falicia G, Hannah, R, Kristin M, Ralitza A, Will C, Reut A, Jamal T, Alper Y, Stefan L, Aiden K, Linda Ly, other BGGM groovers, honours students, summer students, visiting students and other BABS teachers and students for bringing a smile to my face every day. Thank-you my friends. Thank-you so very much to Alexandra Anne Roberts, my scientific mentor, uni-buddy, sister-in-crime and the greatest scientist in the world, without whom I would have gone a-lot madder a-lot sooner and not had nearly as much fun throughout this journey A special mention for the readers and editors of this thesis document, especially Brett Neilan, Michelle Gehringer and Hannah Root, for all your hard work and effort, it is much appreciated. Thank-you to the school of Biotechnology and Biomolecular Sciences, CRC for Water quality and Treatment and Graduate Research School for your financial support, which allowed me to complete this doctorate, travel to national and international conferences, and attend regular research meetings at the Royal hotel… Thank-you as well to all of you who have gifted me with strains/plasmids/DNA from various organisms, given me experimental advice, and physically helped during my studies – none of it would have been possible without you. vii Finally, thank-you to my family, friends and my gorgeous partner Jasmine, for all of your support and love, which cannot adequately be described on paper, yet deserves a description larger than the following document… viii Abstract Cyanobacteria produce a diverse range of secondary metabolites, many of which are toxic to mammals. It is important to understand the factors affecting the release of toxins from aquatic cyanobacteria so as to develop management strategies that minimise contamination of potable and recreational water systems. Release of toxins from cyanobacterial cells is thought to occur primarily after cell lysis, yet recent evidence also suggests active toxin export. The cyanobacterium Cylindrospermopsis raciborskii T3, contains a gene cluster, sxt, believed to be responsible for the production of the neurotoxic alkaloid saxitoxin (STX) and its derivatives. This gene cluster also includes two genes putatively encoding members of the multi-drug and toxic compound extrusion (MATE) transporter family, sxtF and sxtM. In addition, the cyanobacterium A.circinalis genome contains a putative sodium-dependant transporter gene, naDt, shown to be associated with STX-producing strains. These transporter proteins were predicted to be involved in the export of STX from cyanobacterial cells. The main aim of this project was to characterise the transporters associated with STX biosynthesis, by investigation of their genetic prevalence, functional substrates and specific regulation, through an approach involving bioinformatic analysis, protein expression, mutagenesis and transcriptional assessment. An sxtM homologue was discovered in A. circinalis 131C, as part of the sxt cluster, and found to be uniquely associated with STX-producing strains. Membrane topology analysis predicted 12 membrane-spanning regions for the sxt transporter genes, characteristic of members of the MATE family. Putative MATE transporter genes were also identified in strains of Nostoc and Nodularia, yet were not found to be linked to toxic compound production. Bioinformatic and phylogenetic analysis showed that the translated sxt transporters clustered with the NorM prokaryotic MATE sub- family, whereas the Nostoc and Nodularia MATEs clustered with the DinF sub-family. The genetic loci surrounding naDt in A. circinalis 131C were sequenced and found not to include an sxt gene cluster. To characterise the functional substrates of the putative STX-transporters, naDt and sxtM from A.circinalis 131C and sxtF and sxtM from C.raciborskii T3, were heterologously expressed in the antibiotic-sensitive E. coli strain KAM32. Expression ix of the sxt MATES complemented host sensitivity to the cationic fluroquinolone antibiotics ciprofloxacin and ofloxacin. The functional substrate range of these transporters was also investigated in the non-toxic model cyanobacterium Synechocystis sp. PCC6803. Disruption of gene homologues of naDt and the sxt MATE genes in this cyanobacterium yielded mutant strains with increased sensitivity to the toxic organic cations, methyl viologen and acriflavine. Transcript analyses of mutants growing under sub-lethal acriflavine stress showed increased expression of these transporters, supporting their hypothesised role in resistance to toxic compounds via efflux. Increased transcription of other putative transporter genes, from the ABC and RND multi-drug resistance families, in mutant strains indicated the likelihood of a complementary system of exporters contributing to drug efflux in Synechocystis sp. PCC6803. Transcription of the putative STX transporters, naDt and sxtM from A.circinalis 131C and sxtF and sxtM from C.raciborskii T3, and the putative STX biosynthesis gene sxtA present in both strains, was studied under alkali and Na+ stress at two stages of growth. As the Na+ levels, and alkalinity of the growth media of C.raciborskii T3 have been previously shown to influence STX levels, intra- and extracellular STX was measured using HPLC. Alkali stress (pH 9) decreased total STX levels in A. circinalis 131C, and correlated with down-regulation of the putative transport and biosynthetic genes. In C.raciborskii T3, alkali stress induced higher extracellular STX levels and lower intracellular levels than in control cultures, which correlated with large increases in transcription of the putative STX transport genes. No observable effects were seen in response to Na+ stress. Overall, these experiments have provided evidence that the sxt MATEs present exclusively in STX-producing