SODIUM HOMEOSTASIS AND THE
PRODUCTION OF SAXITOXIN IN
CYANOBACTERIA
FRANCESCO POMATI
A Thesis Submitted in Partial Fulfilment of the Requirements for the Degree of Doctor of Philosophy (Ph.D.)
SCHOOL OF BIOTECHNOLOGY AND BIOMOLECULAR SCIENCES
THE UNIVERSITY OF NEW SOUTH WALES
SYDNEY, AUSTRALIA
December, 2003 Certificate of Originality
I hereby declare that this submission is my own work and to the best of my knowledge it contains no material previously published or written by another person, nor material which to a substantial extent has 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.
CERTIFICATE OF OIUGINALITY
I hereby_ declare that this submission is my own work and to the best of my knowledge it contams no matenals previously published or written by another person nor material wh_ich to a substantial extent has been accepted for the award of any ~!her 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.
Francesco Pomati 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.Jru:,pro~£!'s design and conception •• "''" •=-·.. ~-"''?
11 a Valeria, Donate/la, Giancarlo e Matteo
vincer potero dentro a me l'ardore ch'i' ebbi a divenir del mondo esperto, e de li vizi umani e del valore; ma misi me per l'alto mare aperto (Dante Alighieri, Inferno, Canto XXVI)
111 TABLE OF CONTENTS
Abstract Xll
Acknowledgments Xlll List of publications xv List of abbreviations xvn
Preface, by RalfKellmann XIX
CHAPTER! INTRODUCTION 1 1.1 Overview 1 1.1.1 Harmful algal blooms 1 1.1.2 Cyanobacterial blooms 2 1.2 Cyanobacteria 6 1.2.1 Order Nostocales: genera Anabaena and Cylindrospermopsis 9 l .2.2 Anabaena circinalis l 0 l.2.3 Cylindrospermopsis raciborskii 12 1.3 Cyanobacterial bioactive metabolites 14 1.3.1 Cyanotoxins 15 1.3.2 LPS 15 1.3.3 Cyclic peptides 16 1.3.4 Alkaloids 19 1.3.5 PSP toxins 22 1.3.5.1 Chemistry of PSP toxins 25 1.3.5.2 Pharmacology and toxicology of PSP toxins 28 1.3.5.3 Detection methods for PSP toxins 33 1.3.5.4 Ecology and physiology of PSP toxins 39 1.3.5.5 Biosynthesis and Biodegradation of PSP toxins 42 1.3.5.6 Genetics of PSP toxins 46
IV 1.4 Objectives and scopes 48 1.4.1 Aims 48 1.4.2 Hypothesis 48
CHAPTER2 MATERIALS AND METHODS 49 2.1 Bacterial strains and culturing 49 2.1.1 Cyanobacteria 49 2.1.2 Escherichia coli 50 2.2 Analysis of cyanobacterial growth 51 2.2.1 Measurement of growth 51 2.2.2 Total protein assay 51 2.3 Toxin detection and quantification 51 2.3.1 HPLC analysis 51 2.3.2 Protein phosphatase inhibition assay 52 2.3.3 Toxins standards 52 2.4 Flame photometry analysis 53 2.5 Statistical analyses 53 2.6 Nucleic acid extraction 53 2.6.1 Genomic DNA extraction 53 2.6.2 Total RNA extraction 54 2.7 Suppression subtractive hybridisation (SSH) 55 2. 7 .1 Overview 55 2. 7 .2 Driver and tester DNA preparation 58 2.7.3 Subtractive hybridisation 58 2.7.4 PCR amplification 59 2.8 DNA cloning and automated sequencing 59 2.9 DNA microarray design and production 60 2.10 Nucleic acid labelling 61 2.10.1 Preparation of DNA 61 2.10.2 Preparation of total RNA 61 2.10.3 Labelling of genomic DNA and cDNA 61
V 2.11 Microarray hybridisation 62 2.12 Microarray scanning, data acquisition and statistical analysis 62
CHAPTER3 ENHANCEMENT OF INTRACELLULAR SAXITOXIN ACCUMULATION IN Cylindrospermopsis raciborskii T3 BY LIDOCAINE HYDROCHLORIDE 64 3.1 Background 65 3.2 Experimental procedures 66 3.2.1 Reagents 66 3.2.2 Growth conditions and cyanobacterial cultures 66 3.2.3 Extraction for HPLC analysis 67 3.3 Results 67 3 .3 .1 Effect of lidocaine hydrochloride on growth 67 3.3.2 Effect oflidocaine hydrochloride on STX accumulation 71 3.3.3 Time course oflidocaine hydrochloride effect on STX intracellular concentration 72 3.3.4 Effect of pH and Na+ concentration on the lidocaine hydrochloride- induced STX accumulation 73 3 .4 Discussion 75 3.5 Conclusions 77 3.6 Summary 78
CHAPTER4 INTERACTIONS BETWEEN INTRACELLULAR Na+ LEVELS AND SAXITOXIN PRODUCTION IN Cylindrospermopsis raciborskii T3 79 4.1 Background 80 4.2 Experimental procedures 80 4.2.1 Reagents 80 4.2.2 Growth conditions and cyanobacterial cultures 81 4.2.3 Flame photometry analysis 81 4.2.4 Extraction and HPLC analysis 82
VI 4.2.5 Total protein content 82 4.3 Results 83 4.3.1 Effects of pH on Na+-K+ levels and STX content 83 4.3.2 Effect ofNaCl on growth, Na+-K+ levels and STX accumulation 84 4.3.3 Effects of channel-blockers on Na+ _K+ levels and STX accumulation 87 4.4 Discussion 90 4.5 Conclusions 93 4.6 Summary 94
CHAPTERS EFFECTS OF SAXITOXIN AND VERATRIDINE ON BACTERIAL NA+-K+ FLUXES: A PROKARYOTIC-BASED STX BIOASSAY 95 5.1 Background 96 5.2 Experimental procedures 97 5.2.1 Reagents 97 5.2.2 Cyanobacterial strains and culture conditions 97 5.2.3 Total cellular Na+ and K+ content and flame photometry 98 5.2.4 Cyanobacterial cells lysis test 98 5.2.5 Cell titre assay for metabolic activity 99 5.2.6 Luminescent bacteria test 100 5.3 Results 101 5.3.1 Effect of Na+ stress, VTD and STX on total cellular Na+-K+ levels 101 5.3.2 Lysis test with toxic and non-toxic cyanobacteria 102 5.3.3 Effect ofVTD and STX on cyanobacterial metabolic activity 104 5 .3 .4 Effect on bioluminescence by Vi brio fischeri I 05 5 .4 Discussion 107 5.5 Conclusions 110 5.6 Summary 110
vu CHAPTER6 EVIDENCE FOR DIFFERENCES IN THE METABOLISM OF STX AND C1+2 TOXINS IN Cylindrospermopsis raciborskii T3 112 6.1 Background 113 6.2 Experimental procedures 114 6.2.1 Reagents 114 6.2.2 Growth conditions and cyanobacterial cultures 114 6.2.3 In viva experiments 114 6.2.4 In vitro experiments 115 6.2.5 HPLC analysis 116 6.2.6 Protein phosphatase inhibition assay 116 6.2.7 Total protein content 116 6.3 Results 117 6.3.1 Effect of CAM on cyanotoxin accumulation 117 6.3.2 Time course of CAM influence on MCYST and PSP toxin production 118 6.3.3 Effect of substrates on PSP toxins levels 120 6.3.4 In vitro synthesis of PSP toxins under CAM stress and arginine supplementation 123 6.4 Discussion 124 6.5 Conclusions 126 6.6 Summary 127
CHAPTER 7 IDENTIFICATION OF A Na+ DEPENDENT TRANSPORTER ASSOCIATED WITH SAXITOXIN PRODUCING STRAINS OF Anabaena circinalis 128 7 .1 Background 129 7 .2 Experimental procedures 130 7.2.1 Cyanobacteria 130 7 .2.2 DNA extraction 130 7.2.3 PCR amplifications and DNA sequencing 131
Vlll 7.2.4 SSH 132 7.2.5 Microarray design and production 133 7.2.6 Genomic DNA labelling and hybridisation 133 7.2.7 Microarray scanning, data acquisition and statistical analyses 133 7.2.8 Nucleotide sequence accession numbers 134 7.3 Results 134 7.3.1 HIPl genomic polymorphism 134 7.3.2 SSH ofHIPl genomic libraries 137 7.3.3 Microarray hybridisation 141 7.3.4 Amplification of genes encoding Na+ dependent transporters 141 7.3.5 Phylogeny of Na+ dependent transporter proteins 142 7.3.6 Screening for STX-producing A. circinalis by multiplex PCR 143 7.4 Discussion 14 7 7.5 Conclusions 150 7.6 Summary 151
CHAPTERS PCR-BASED POSITIVE HYBRIDISATION TO DETECT GENOMIC DIVERSITY ASSOCIATED WITH BACTERIAL SECONDARY METABOLISM 152 8.1 Background 153 8.2 Experimental procedures 155 8.2.1 Cyanobacterial strains and growth conditions 155 8.2.2 DNA extraction 155 8.2.3 SSH 155 8.2.4 PPH 157 8.2.5 PCR amplifications 159 8.2.6 Microarray design and production 160 8.2.7 Labelling of genomic DNA 160 8.2.8 Microarray hybridisation 160 8.2.9 Microarray scanning, data acquisition and statistical analyses 160 8.2.10 Nucleotide sequence accession numbers 161
lX 8.3 Results 161 8.3.1 Suppression subtractive hybridisation 161 8.3.2 PCR-based positive hybridisation 168 8.3.3 DNA microarray analysis of SSH and PPH libraries 168 8.3.4 PCR amplification of putative unsubtracted PPH toxic-specific sequences 169 8.4 Discussion 170 8.5 Conclusions 172 8.6 Summary 173
CHAPTER9 TRANSCRIPTIONAL ANALYSIS IN Anabaena circinalis USING THE BGGM1 DNA-MICROARRAY: A COMPARATIVE STUDY OF TOXIC AND NON-TOXIC STRAINS AND THE EFFECTS OF LIDOCAINE HYDROCHLORIDE 174 9.1 Background 175 9.2 Experimental procedures 176 9.2.1 Cyanobacterial strains and growth conditions 176 9.2.2 Total RNA extraction 176 9.2.3 Labelling of total RNA 176 9.2.4 Microarray hybridisation 177 9.2.5 Microarray scanning, data acquisition and statistical analyses 177 9.3 Results 177 9.3.1 Comparative analysis of gene expression in toxic and non-toxic strains 177 9.3.2 Effect of lidocaine on gene expression using BGGM1 DNA microarray 178 9 .4 Discussion 185 9.5 Conclusions 188 9.6 Summary 189
X CHAPTERl0 DISCUSSION 190 10.1 General discussion 191 10.2 The physiology of STX production in cyanobacteria 193 10.3 The molecular biology of STX production in cyanobacteria 194 10.4 STX "genes" and STX "enzymes" 195 10.5 Conclusions and significance 198 10.6 Future directions 199
APPENDIX A CYANOBACTERIAL STRAINS USED IN THIS STUDY 202
APPENDIXB GROWTH MEDIA PREPARATION 203
APPENDIXC C.1) PCR-BASED GENOMIC SUBTRACTION ADAPTOR AND PRIMER SEQUENCES 207 C.2) PCR PRIMERS USED IN THIS STUDY 208
APPENDIXD BGGM1 DNA MICROARRAY GENE LIST 209
APPENDIXE EXAMINATION OF COMBINED MICROARRAY DATA SETS 219
APPENDIXF NUCLEOTIDE SEQUENCE GENBANK ACCESSION NUMBERS 221
REFERENCES 223
Xl ABSTRACT
Saxitoxin (STX) is the most potent representative among the paralytic shellfish poisoning (PSP) toxins, that are lethal natural Na+ channel-blocking alkaloids. In this study the production of STX in cyanobacteria was investigated by means of combined physiological and molecular biology approaches. The aim was to identify the ecological and molecular basis of STX biosynthesis in these photosynthetic prokaryotes. Ecophysiological investigations demonstrated that alkaline pH and Na+ stress enhanced STX production by Cylindrospermopsis raciborskii T3, and that STX biosynthesis can be modulated by affecting cyanobacterial Na+ fluxes. This evidence suggested a possible interaction between STX and cyanobacterial Na+ uptake. This hypothesis was verified by developing a bacterial bioassay for STX based on the antagonism of this toxin with the Na+ channel activator veratridine. The novel STX bioassay was employed in a standardised toxicity test for the detection of PSP toxins in the environment. Molecular biology methods were applied with the aim of obtaining candidate genes involved in STX production. PCR-based techniques, such as suppression subtractive hybridisation and highly iterated palindrome genotyping, were used to identify genomic differences between STX-producing and non-toxic strains of Anabaena circinalis. A novel method was also developed to recover possible genes laterally exchanged among toxic A. circinalis isolates. Putative toxic-strain specific fragments were employed to prepare a DNA microarray, comprising genes implicated in cyanobacterial toxigenicity and ecophysiology. Candidate genes were assayed for their toxic-strain specificity by microarray analysis. A Na+ dependent transporter, putatively involved in cyanobacterial pH and Na+ homeostasis, was detected among the toxic-strain specific genes and successfully applied as a probe for the environmental screening of STX-producing A. circinalis. The DNA microarray was also used to study the expression profiles of toxic and non-toxic A. circinalis strains. Additionally, by providing the conditions previously found to modulate STX production, genes that correlated with the expected increase in STX biosynthesis were studied in toxic strains. The results shown here suggest that PSP toxin production may represent, for the producing cyanobacteria, a potential evolutionary advantage for adapting to drastic environmental conditions. The genetic differences between STX-producing and non-toxic strains also indicated a distinctive evolutionary adaptation to specific environmental characteristics.
Xll ACKNOWLEDGEMENTS
As someone once said, science is the truth inside the lie. The truth of this work is simple enough. Most of the studies here presented, and most of the three years of life behind them, would have not been successful without the assistance and the contribution of a number of wonderful people, in Australia and across the seven seas. In particular I would like to acknowledge:
Brett Neilan, for the great opportunity he gave me to live and work in Australia, for the friendly and stimulating environment I found when I came here, for believing in me, and for having been such an example for me in science and in life. Since the first time we met (I nearly ended up in jail in USA), he's been making a little-big difference in my life. Thanks Brett.
I wish to thank every single member of Brett's lab, the Blue Green Groove Machine, including all the guests that came and went. I am grateful for all the help and support offered by you guys along the way. Tim, Michelle Moffy, Leanne, Melanie, Harvey, Brendan, Ralf, Janine, Michelle G., Michelle A., Falicia, Toby, Kenlee, Anne, Rosy and Sensei Sugiura San. Each of you has made it a privilege to work in the lab.
In particular, special thanks to Michelle Moffitt, Ralf Kellmann and Tim Salmon. Thank you for all the things you taught me. Many of the results here presented have been influenced by your assistance and advice. I am also grateful to Harvey Fernandez and Bronwyn Robertson for their technical support during production and utilisation of the DNA microarray. Thanks to Andy Netting, Kevin Barrow and Lyndon Llewellyn for their collaboration and advice, and to all the Danish students that contributed to this project in some extent.
Many special thanks to Brendan Bums, for having let me share with him the famous Office of Love (OOL). Without your jokes, your friendship and your help I'm not sure if I could have made it. Thanks also for your everyday life wisdom and poetry. One more for the man
Xlll that officially baptised the OOL by putting a sign on its door, Steve Leach. Thanks buddy for the pub-talks, the surfing trips and all the rest.
Here is to Anne Marie and Iain Couperwhite, for all their friendship and support. Thank you Anne for being a patient swimming coach and thank you Iain for reviewing this thesis, and also for helping me in developing my passion for Scotch Whisky.
All this work has been made possible by the University of New South Wales and the School of Biotechnology and Biomolecular Sciences (formerly Microbiology and Immunology), by kindly granting an International Post-graduate Research Scholarship (IPRS), a University Post-graduate Research Scholarship (UPRS), and extra money for living expenses. An Adrian Lee travel scholarship is also acknowledge from the School of Microbiology and Immunology.
Now let's go overseas.
There are a number of individuals that actively contributed to this work from the University of Insubria, Varese, Italy. First, thanks to Carlo Rossetti for granting me all the lab-space and support I desired in the months I spent back in my home town, and for always being there when I needed an advise. Thanks to Gianluca Manarolla, Alessadro Martinengo (Egy) and Monica Molteni for the experimental assistance. Last, but not least, thanks to Davide Calamari for investing in my strange ideas and for coming here to visit his old student with a bottle of Campari.
Thanks to all my friends that have been so close to me even if we've been distant for a long time. We're scattered all around the world, but some things will never change. In particular, the music of the Jimmy Island Band always kept my heart beating.
Finally, thanks to Valeria for all the love, patience and strength demonstrated during the last three years. Thank you for believing in the dream that will soon become true. I am grateful to my family, Donatella, Giancarlo and Matteo, that encouraged and supported my choices, my ideas, my studies and my aspirations since as far as I can remember. It's simple as that: without your love and sustain, I wouldn't be here.
XIV LIST OF PUBLICATIONS
Peer reviewed articles in journals
- Pomati F., Neilan B. A., Manarolla G., Suzuki T. and Rossetti C. 2003. Enhancement of intracellular saxitoxin accumulation by lidocaine hydrochloride in the cyanobacterium Cylindrospermopsis raciborskii T3 (Nostocales). Journal of Phycology 39, 535-542. - Pomati F., Rossetti C., Calamari D. and Neilan B. A. 2003. Effects of Saxitoxin and Veratridine on Bacterial Na+ -K+ Fluxes: a Prokaryotic-based STX Bioassay. Applied and Environmental Microbiology 69, in press. - Pomati F., Rossetti C., Manarolla G., Bums B. P. and Neilan B. A. 2003. Combined variations Intracellular Na+ Levels and Saxitoxin Production in Cylindrospermopsis raciborskii T3. Microbiology, in press. - Pomati F. and Neilan B. A. PCR-based Positive Hybridisations to Detect Genomic Diversity Associated with Bacterial Secondary Metabolism. 2003. Nucleic Acids Research, in press. - Pomati F., Netting A. G., Calamari D. and Neilan B. A. Effects of erythromycin, tetracycline and ibuprofen on the growth of Synechocystis sp. and Lemna minor. Aquatic Toxicology, submitted. - Pomati F., Bums B. P. and Neilan B. A. Identification of a Na+ Dependent Transporter Associated with Saxitoxin Production in the Cyanobacterium Anabaena circinalis. Applied and Environmental Microbiology, submitted. - Suzuki T., Nakasato K., Shapiro S., Pomati F. and Neilan B. A. 2003. Effects of synthetic local anaesthetics on the growth of the cyanobacterium Synechococcus leopoliensis. Journal of Applied Phycology, in press. - Bums B. P., Goh F., Pomati F. and Neilan B. A. Big genes, small molecules - the molecular basis for toxicity and biosynthesis of natural products in cyanobacteria. Marine Biotechnology, submitted.
xv Pomati F., Moffitt M. C., Cavaliere R. and Neilan B. A. Evidence for Differences in the Metabolism of Saxitoxin and Cl +2 Toxins in the freshwater cyanobacterium Cylindrospermopsis raciborskii T3 (Nostocales). FEBS Letters, submitted. Sugiura N., Bums B. P., Kameyama K., Itayama T., Pomati F., Neilan B. A. and Inamori Y. Adsorption and biodegradation of musty odorous compounds, 2- methyisobomeol and geosmin. Manuscript in preparation. Pomati F. and Neilan B. A. Comparative Expression Study of Toxic and Non Toxic Anabaena circinalis Strains and Effects of Lidocaine Hydrochloride. Manuscript in preparation.
Contributions to academic conferences
- Pomati F., Neilan B. A., Suzuki T., Manarolla G. and Rossetti C. Lidocaine promotes growth and saxitoxin accumulation in Cylindrospermopsis raciborskii. Poster presentation, The Fifth International Conference on Toxic Cyanobacteria, Noosa, Queensland, Australia, July 15 - 20, 2001. - Pomati F., Rossetti C., Manarolla G., Kellmann R. and Neilan B. A. Na+ cycle and saxitoxin regulation in the freshwater cyanobacterium Cylindrospermopsis raciborskii T3. Poster presentation, Tenth International Conference on Harmful Algae, St. Petersburg, Florida, 21-25 October, 2002. - Pomati F. and Neilan B. A. Na+ cycle and saxitoxin regulation in the freshwater cyanobacterium Cylindrospermopsis raciborskii T3. Biotechnology and Biomolecular Sciences Symposium, The University of New South Wales, Sydney, Australia, 8th November, 2002. Pomati F. and Neilan B. A .. PCR-based positive hybridisation to detect genomic diversity associated with bacterial secondary metabolism. Biotechnology and Biomolecular Sciences Symposium, The University of New South Wales, Sydney, Australia, ?1h November, 2003.
XVI LIST OF ABBREVIATIONS
AT amidinotransferase AWQC Australian Water Quality Centre AWT Australian Water Technology BGGM Blue Green Groove Machine BLAST basic local alignment search tool bp base pairs bw bodyweight CAM chloramphenicol DNA deoxyribonucleic acid ECF extracytoplasmic function ELISA enzyme-linked immunosorbent assays GR growth rate GTX gonyautoxin GYP gas vesicle protein HAB harmful algal blooms HIP highly iterated octameric palindrome HPLC high performance liquid chromatography 1.p. intraperitoneal IAA indole-3-acetic acid kb kilobase (103 bases) LPS lipolysaccharides LSD least significant difference MCYST microcystins MU mouse units MW molecular weight NaDT Na+ dependent transporter
XVll NODLN nodularin NRPS non-ribosomal peptide synthetase OD optical density ORF open reading frame PCR polymerase chain reaction PKS polyketide synthase PM plasma membrane pp protein phosphatase PPH PCR-based positive hybridisation PSP paralytic shellfish poisoning PTT phosphopantetheinyl transferase rDNA ribosomal RNA (rRNA) RNA ribonucleic acid SSH suppression subtractive hybridisation STX saxitoxin TTX tetrodotoxin UV ultraviolet VAN o-vanadate VGSC voltage-gated sodium channel VTD veratridine
xvm PREFACE
by RalfKellmann
HISTORICAL ASPECTS OF SAXITOXIN
Harmful algal blooms have been recorded since the earliest time of written history, such as in the Bible in Exodus 7: 20-21: "And all the fish that were in the river turned into blood. The fish that were in the river died, the river stank, and the Egyptians could not drink the water of the river. So there was blood throughout all the land of Egypt." This account probably describes a massive dinoflagellate bloom, commonly referred to as "red tide", which discoloured the water and turned it anoxic, but did not appear to produce any toxins. Ancient Greek authors actually applied the name "Red Sea" as a result of the frequent red water blooms in that region [1]. A small proportion of less than 2% out of 4000 known species of micro-algae are capable of producing a diverse range of potent phycotoxins. Despite the small number of toxic species, they have a global impact, due to their world-wide distribution. The PhD thesis introduced here deals with saxitoxin and its analogues, which are the most potent among the phycotoxins and are historically associated with red tides. These toxins cause, within minutes upon ingestion, numbness and tingling of the lips and extremities. In severe cases, the usually fully conscious victim experiences an ascending paralysis of the whole body, which may lead to death through cardio-pulmonary arrest. Since shellfish are a common vector of these toxins to humans, this syndrome is commonly referred to as paralytic shellfish poisoning (PSP). Research on saxitoxin has been historically characterised by slow progress. The oldest medical account, describing the specific symptoms of PSP, dates back to 1689 [2], but the reason for the sporadic and unpredictable toxicity of shellfish remained unknown for almost another 250 years. In the 1930s, a Californian research group investigated the cause of the shellfish toxicity, speculations of which included stagnant water, putrefactive processes, and phytoplankton [3]. Interestingly, native Indian tribes in America already knew about the link between algal blooms and shellfish toxicity long before Europeans arrived in their country, and they provided the first public quarantine. The Indians observed the sea at night for bioluminescence of the sea water, which was caused by non-toxic Noctiluca sp. that is frequently associated with toxic Gonyaulax catenella. When
XIX bioluminescence was observed, they posted sentries to warn the unwary inland traveller not acquainted with the ways of the sea that shellfish must not be eaten during this period [4]. Notable historic accounts of PSP can be found in the log book of Captain George Vancouver [5]. He and his crew were surveying an area near Fitzhugh Sound in 1793, now called Poison Cove, in honour of a man who died within 5 hours of eating local mussels, while several others recovered within a few days. Another interesting account describes Aleutian hunters in Alaska, who stopped at Peril Way in 1799, and consumed a dinner of mussels, after which more than 100 men died within 2 hours [6, 7]. Due to the potency of saxitoxin, the American CIA was interested in using it as a chemical agent or weapon, and collected a huge supply of tens of grams for experimental purposes during the 1950s. Allegedly, saxitoxin was trailed in suicide capsules, but stocks were later destroyed or supplied to scientists via the National Institutes of Health for research purposes. Saxitoxin is currently still classified as a Schedule 1 warfare agent by the United Nations. Nearly 40 years after the link between dinoflagellate blooms and shellfish toxicity was made, the molecular structure of saxitoxin was determined, while the biosynthetic pathway was elucidated another ten years later. Since then, no significant progress has been achieved on the molecular biology of saxitoxin, however, the present PhD thesis by this inspiring young man, Francesco Pomati, has given us valuable new insight into the molecular biology of saxitoxin.
RalfKellmann, December 2003
1. Brongersma-Sanders, M., Mass mortality in the sea, in Treatise on Marine Ecology and Paleoecology, J.W. Hedgpeth, Editor. 1957, Waverly Press: Baltimore. p. 941-1010. 2. Chevalier, A. and E.A. Duchesne, Memoire sur /es empoisonnements par /es huitres, les moules, /es crabes, et par certain poissons de mer et de riviere. Annales d'Hygiene Publique (Paris), 1851. 45: p. 387-437. 3. Sommer, H. and K.F. Meyer, Paralytic shellfish poisoning. AMA Archieves of Pathology, 1937. 24: p. 560-598. 4. Carson, R.L., The changing year, in The sea around us, R.L. Carsons, Editor. 1951, Oxford University Press: New York. p. 33. 5. Vancouver, G., in A Voyage ofDiscovery to the North Pacific Ocean and around the World. 1801, John Shockdale: London. p. 44-47. 6. Petroff, I., Report on the population, industries, and resources ofAlaska., in U.S. Census Reports, 10th Census. 1884. p. 106. 7. Dall, W.H., Alaska and its resources. 1870, Boston: NSA. 319.
XX Chapter 1 Introduction
CHAPTERl
INTRODUCTION
1.1 OVERVIEW
1.1.1 Harmful algal blooms
Harmful algal blooms (HABs) are caused by microalgae and have a negative impact on human activities. Certain algae produce potent toxins that impact upon human health in a range of different ways. As well as causing human intoxication, harmful algae may indirectly impinge upon aquatic resources, including wild and cultivated fish, animals or marine invertebrates. HABs can also affect the functional components of aquatic ecosystems (Sivonen and Jones 1999, Rossetti et al. 2000). Beside the production of toxins, the degradation of bloom biomass can deplete or exhaust oxygen supplies, thus killing commercially important organisms unable to leave the anoxic area. Algal blooms may also cause intense discoloration of waters, which earned some of them the name of "red-tides" (Fig. 1.1) . Even if non-toxic, HABs are often Figure 1.1 A red-tide in the Canadian Atlantic coast characterised by the presence of malodorous scums and mucilage that can lead to severe degradation of the environment, preventing its recreational use. Over the past three decades, the frequency and global distribution of toxic algal blooms appears to have increased, and human intoxications from novel algal sources have occurred (Van Dolah 2000). This increase parallels recent evidence of large-scale Chapter 1 Introduction
ecologic disturbances that coincide with trends in global warming. The extent to which human activities and eutrophication have contributed to their increase is still in question.
1.1.2 Cyanobacterial blooms
In marme environments the list of microalgal species that are potentially involved in HABs comprises mainly diatoms and dinoflagellates (Zingone and Enevoldsen 2000). In freshwaters, however, problems with toxin-producing phytoplankton are almost exclusively associated with cyanobacteria. Cyanobacterial blooms (Fig. 1.2) occur world-wide in eutrophied coastal areas, m freshwaters as well as in drinking-water reservoirs. Poisonous blooms of cyanobacteria in freshwater and brackish lakes represent a worldwide concern, not only for the health of domestic animals and wildlife, but also for the human population that depend on the water for drinking or recreation.
Figure 1.2 Cyanobacterial blooms in Mission Lake, Saskatchewan
Numerous cases of animal poisoning ( often lethal) substantiate the concern of health hazards for humans exposed to cyanobacteria. Historically, the first report of cyanobacterial poisoning was of the deaths of cattle, sheep, dogs, horses and pigs after drinking a scum of Nodularia spumigena in Lake Alexandrina, Australia (Francis 1878). Since that time there have been frequent instances of farm animal poisonings from cyanobacterial water blooms. Apart from pet dogs, other affected animals include ducks, coots and other waterfowl, skunks and mink, and even rhinoceros (Carmichael Chapter 1 Intrrxluction
1992). An additional source of intoxication for terrestrial animals can also be the bioaccumulation of cyanotoxins in the food chain (Ressom et al. 1994). One of the most convincing mammalian poisonings has been the documented death of sheep drinking from a farm dam contaminated with the neurotoxic Anabaena circinalis in Australia (Negri et al. 1995). The authors recovered high concentrations of saxitoxins from cyanobacteria in the farm dam and from the ruminal fluid of the dead sheep. Beside Australia, animal deaths from cyanobacterial toxicity have been reported from North and South America, Europe and Africa. The major injury reported is hepatotoxicosis. The causative cyanobacteria have been Microcystis aeruginosa, Nodularia spumigena and Oscillatoria (Planktothrix) agardhii. In the most recent cases, post mortem examination has shown evidence of cyanobacterial ingestion as well as characteristic tissue injury in the liver. The other main cause of livestock and pet deaths due to cyanobacterial toxins has been from acute neurotoxicity leading to respiratory failure, with no post mortem indications of organ injury. In one case (Gunn et al. 1992) the neurotoxin, anatoxin-a, was isolated from the stomach contents of a dog. Henriksen et al. (1997) demonstrated lethality in wild ducks due to anatoxin-a(s). The cyanobacteria associated with deaths from neurotoxicity are Anabaena jlos-aquae, Anabaena circinalis, Aphanizomenon jlos aquae and Oscillatoria sp. The toxins anatoxin-a, anatoxin-a(s) and saxitoxins have all been implicated in different cases. Some of the toxic bloom-forming cyanobacteria that cause animal death may also adversely affect human health in many ways. Evidence for effects of cyanotoxins on human health derives from epidemiological evidence including human and animal poisonings, as well as from toxicological investigations. Epidemiological evidence mainly results from studies of human populations that have shown symptoms of poisoning or injury attributed to the presence of cyanotoxins in drinking water or other sources of water. Reports of gastro-enteritis after the appearance of cyanobacterial blooms in drinking water come from North and South America, Africa and Europe. Most of these cases of human injury have been studied retrospectively, and complete epidemiological data, especially regarding exposure (number of organisms, type and concentration of cyanotoxins), are rarely available.
3 Chapter 1
Nevertheless, this information is of special importance in directly demonstrating the link between toxin exposures and human health issues, which otherwise cannot be derived directly from animal experiments. The recorded cases of gastrointestinal and hepatic illness that can be reliably attributed to cyanobacterial toxins in water supplies have all been coincident with either the breakdown of a natural cyanobacterial bloom or with the artificial lysis of a bloom by application of copper sulphate. Both mechanisms lead to cyanotoxin release from decomposing cells. Whereas treatment procedures might have removed cyanotoxins bound in intact cells, they were not effective in removing the cyanotoxins dissolved in water. The earliest reported cases of gastroenteritis from cyanobacteria were in the population of a series of towns along the Ohio River in 1931 (Tisdale 1931 ). In Harare, Zimbabwe, children living in an area of the city supplied from a particular water reservoir, developed gastroenteritis each year at the time when a natural bloom of Microcystis was decaying in the reservoir (Zilberg 1966). The most lethal outbreak attributed to cyanobacterial toxins in drinking water occurred in Brazil, when a newly flooded dam developed an immense cyanobacterial bloom. Eighty-eight deaths, mostly children, were reported to have occurred (Teixera et al. 1993). Examples of illness following the use of copper sulphate to destroy a cyanobacterial bloom in a water storage reservoir have been also described in the USA and in Australia. A severe outbreak of cyanobacterial toxicity in a human population occurred on an island off the north-eastern coast of Australia. Due to complaints of bad taste and odour in the water supply, which were attributed to a cyanobacterial bloom, the authorities treated the reservoir with copper sulphate. Within a week numerous children developed severe hepatoenteritis, and a total of 140 children and 10 adults required hospital treatment (Byth 1980). Cylindrospermopsis raciborskii was identified as the cyanobacterium responsible for this episode. Within human populations, for a variety of reasons, there will be individuals who are at a much greater risk of injury from cyanotoxins than the population as a whole. Children are the most obvious example. However, kidney dialysis patients, if exposed to microcystins in the water used for dialysis, are especially vulnerable because treatment exposes them intravenously to large volumes of water. In a disastrous incident in Caruaru, Brazil, 117 patients developed cholestatic liver disease and at least 47
4 Chapter 1 Intrrxluctinn
deaths were attributed to dialysis with water containing cyanobacterial toxins. Examination of the carbon filter from the dialysis unit demonstrated microcystin-LR, as did the blood and liver tissue of deceased patients (Jochimsen et al. 1998). While acute toxicity is the most obvious problem in cyanobacterial poisoning, a chronic risk may also be present. Short-term exposures to toxins may result in long-term injury, and chronic low-level exposure may cause adverse health effects. Animal experiments have shown chronic liver injury from continuing oral exposure to microcystins. In particular the possibility of carcinogenesis and tumour growth promotion need careful evaluation, because both have been shown in animal experimentation. The incidence of human hepatocellular carcinoma in China is one of the highest in the world, and studies have explored whether cyanobacterial toxins play a part in the incidence of this disease. Cyanobacteria are abundant in surface waters in south-east China where the incidence of hepatocellular carcinoma is highest, and it has been proposed that microcystins in the drinking water are responsible for the higher incidences of cancer among drinkers of pond and ditch water (Yu 1989, 1995). On the other hand, there have been repeated descriptions of adverse health consequences for swimmers exposed to cyanobacterial blooms. Even minor contact with cyanobacteria in bathing water can lead to skin irritation and increased likelihood of gastrointestinal symptoms (Pilotto et al. 1997). Some severe skin reactions have been reported, particularly from contact with the marine cyanobacterium Lyngbya majuscula, which causes deep blistering when trapped under the bathing suit of swimmers (Grauer 1961 ). In this case the organism contains a powerful dermal toxin. Illnesses can also derive from accidental swallowing of cyanobacteria during swimming.
5 Chapter 1
1.2 CYANOBACTERIA
Cyanobacteria are oxygenic phototrophic prokaryotes containing chlorophyll-a and accessory pigments and are among the most ancient life forms on earth. This phylum contains about 150 genera and circa 2000 species. Cyanobacteria have a cosmopolitan distribution and are common in all kinds of habitats, including soils, thermal springs and Antarctic lakes. Cyanobacteria may be unicellular, colonial or filamentous (Fig. 1.3) and they may live as symbionts with plants and fungi or in the benthos, but they are commonly known as planktonic members of the water column in marine and freshwater ecosystems.
Figure 1.3 Different morphotypes of colonial cyanobacteria. In clockwise order from top-left: Anabaena circinalis, Aphanizomenon f/os-aquae, Cylindrospermopsis raciborskii, Lyngbya sp., Spirulina sp., Oscillatoria princeps, Nostoc sp., and Microcystis aeruginosa.
The ability to withstand high salinity allows cyanobacteria to dominate many hypersaline environments such as marine lagoons and inland saline lakes (Stal 1995). Many of these organisms, however, grow better at salinities lower than that found in their most common habitats (Garcia-Pichel et al. 1998). Many isolates from coastal habitats showed to be halotolerants instead to be halophytic (Reed et al. 1984). In contrast to eukaryotic microalgae, cyanobacteria do not possess membrane bound sub-cellular organelles, and their photosynthetic pigments are located in thylakoids that lie free in the cytoplasm near the cell periphery. Cell colour vary from
6 Chapter 1 Intrrxluaim blue-green to violet-red. The green of chlorophyll-a is usually masked by carotenoids (e.g. beta-carotene) and accessory pigments such as phycocyanin, allophycocyanin and phycoerythrin (phycobiliproteins). The pigments are embodied in phycobilisomes, which are found in rows on the outer surface of the thylakoids. All cyanobacteria contain chlorophyll-a and phycocyanin. The basic features of photosynthesis in cyanobacteria have been well described (Ormerod 1992). Cyanobacteria possess two kinds ofreaction centres, PS I and PS II, in their photosynthetic apparatus. With the accessory pigments mentioned above, they are able to use effectively that region of the light spectrum between the absorption peaks of chlorophyll-a and the carotenoids. The ability for continuous photosynthetic growth in the presence of oxygen, together with having water as their electron donor for C02 reduction, enables cyanobacteria to colonise a wide range of ecological niches (Whitton 1992). Phycobiliprotein synthesis is particularly susceptible to environmental influences, especially light quality. Chromatic adaptation is largely attributable to a change in the ratio between phycocyanin and phycoerythrin in the phycobilisomes. Cyanobacteria have a remarkable ability to store essential nutrients and metabolites within their cytoplasm. Prominent cytoplasmic inclusions for this purpose can be seen with the electron microscope (e.g. glycogen granules, lipid globules, cyanophycin granules, polyphosphate bodies, carboxysomes) (Fay and Van Baalen 1987). Reserve products are accumulated under conditions of an excess supply of particular nutrients. During their long history of evolution, beside light harvesting pigments, cyanobacteria have gained special environmental adaptations such as nitrogen fixation, buoyancy regulation, circadian clocks, and differentiated cell types for reproduction or resting. Nitrogen fixation is a fundamental metabolic process of cyanobacteria, giving them the simplest nutritional requirements of all living organisms. By using the enzyme nitrogenase, they convert N2 directly into ammonium (NH4) using solar energy to drive their metabolic and biosynthetic machinery. Since nitrogen-fixing processes require an anoxic environment, some filamentous cyanobacteria ( e.g. Anabaena, Nostoc, Cylindrospermopsis) have evolved specialised cells for this purpose called heterocysts (Stewart 1973). There are, however, several well-documented examples of nitrogen fixation in cyanobacteria which do not form heterocysts (e.g. Trichodesmium)
7 Cliapter 1
(Carpenter et al. 1992). Under predominantly nitrogen limited conditions, but when other nutrients are available, nitrogen fixing cyanobacteria may be favoured and gain growth and reproductive success. Mass developments (blooms) of such species m freshwaters and marine environments are common phenomena worldwide. Many species of cyanobacteria possess gas vesicles. These are cytoplasmic inclusions that enable buoyancy regulation and are gas-filled, cylindrical structures. Their function is to give planktonic species an ecologically important mechanism enabling them to adjust their vertical position in the water column (Walsby 1987). To optimise their position, and thus to find a suitable niche for survival and growth, cyanobacteria use different environmental stimuli ( e.g. photic, gravitational, chemical, thermal) as clues. Gas vesicles become more abundant when light is reduced and the growth rate slows down. Cyanobacteria can, by such buoyancy regulation, poise themselves within vertical gradients of physical and chemical factors. Other ecologically significant mechanisms of movement shown by some cyanobacteria are photomovement by slime secretion or surface undulations of cells (Hader 1987, Paerl 1988). Cyanobacteria have a number of special properties which determine their relative importance in phytoplankton communities. However, the behaviour of different cyanobacterial taxa in nature is not homogeneous because their ecophysiological properties differ widely. The optimum temperature for growth of many cyanobacteria is higher by at least several degrees than for most of the eukaryotic algae. This characteristic may play an important role in the noted summertime dominance of cyanobacteria in temperate latitudes. Some species tolerate even higher temperatures, inhabiting hot springs up to 74°C (Castenholz 1984), terrestrial rock surfaces and hot desert soils (Garcia-Piche! and Belnap 1996). Certain cyanobacteria can be, in fact, desiccation-resistant, hence the prevalence of extensive cyanobacterial mats in ephemeral hypersaline bodies of water, as epilithic crusts, and in terrestrial and sub aerial habitats in numerous tropical and subtropical regions (Potts, 1994). Cyanobacteria maintain effective structure and function of the cells with low energetic supply and a higher growth rate in comparison with other phytoplanktonic organisms when the light intensity is particularly low (Van Li ere et al. 1979). This capability allows them to have an advantage over many competitors. Although
8 Chapter 1 Irttraluaian cyanobacteria are generally considered resistant and adaptive organisms, they show very high sensitivity to rapid environmental changes. Species of the genera Aphanizomenon, Anabaena and Microcystis can grow at high PAR (photosynthetic active radiation) and temperature but a rapid change in temperature of even 5°C can lead to the decay of the population in few days (Pearl 1985). The same has been reported for other ecological parameters such as particular ions, osmolarity, pH. For these reasons, cyanobacteria generally dominate calm and stable environments in which bloom formation is a result of synergistic conditions of nutrient availability, high temperature and pH, and low light intensity.
1.2.1 Order Nostocales: genera Anabaena and Cylindrospermopsis
The two genera of freshwater filamentous cyanobacteria Anabaena and Cylindrospermopsis belong to the order Nostocales, which is the largest within the class of Cyanobacteria. In the new taxonomic system by Komarek and Anagnostidis (1989, Anagnostidis and Komarek 1988, 1990), the non-filamentous species are classified into one single order while the filamentous forms are classified in three orders, Oscillatoriales, Nostocales and Stigonematales. Order Nostocales compnses filamentous forms with sometimes false-branching but always with differentiated cells named heterocysts, specialised in nitrogen fixation. Another common feature of Nostocales is the reproduction ofhormogonia, in which the filaments divide in only one plane by random trichome breakage. In this process, short pieces of trichome become detached from the parent filament and move away by gliding, eventually developing into a separate filament. Some germinate from akinetes (perennial structures filled with endospores ), but generally they are indistinguishable from mature vegetative trichomes. Both heterocysts and akinetes occur in the genera Anabaena and Cylindrospermopsis but with different polarity: in Cylindrospermopsis akinetes are adjacent to heterocysts that are exclusively terminal and form at both ends of the trichomes, while in Anabaena heterocysts can be intercalary or terminal and the position of akinetes is extremely variable (Fig. 1.4). The two genera are also very different in the morphology of vegetative cells, which are isodiametric or cylindrical in Cylindrospermopsis and
9 Chapter 1
generally spherical or ovoid m Anabaena species. Both Anabaena and Cylindrospermopsis have similar growth requirements and characteristics. They are all adapted to low light intensity (less then 10 µE), alkaline pH conditions and they can grow with or without the presence of inorganic nitrogen in the culture medium. Media supplemented with nitrogen sources is always associated with the disappearance of heterocysts and, generally, the optimum growth temperature ranges from 25 to 27 °C.
B) 10 microns
K \ !
Figure 1.4 Photomicrographs of Anabaena circinalis (A) and Cylindrospermopsis raciborskii (B). Heterocysts (H) and akinetes (K) are visible.
1.2.2 Anabaena circinalis
Anabaena circinalis (Fig. 1.4-A) is a bloom-forming cyanobacterium common in temperate and sub-tropical rivers, lakes, reservoirs and dams. This species has been identified in phytoplankton communities from Europe, North America, Asia, South Africa, Japan, New Zealand, and Australia. A. circinalis blooms are a major worldwide problem due to their production of a range of toxins, in particular the neurotoxins anatoxin-a and paralytic shellfish poisoning (PSP) toxins. The first neurotoxin to be identified in A. circinalis was anatoxin-a, a tropane-related alkaloid that acts as a powerful depolarising neuromuscular blocking agent (Stevens and Krieger 1991 ). In common with other cyanobacteria, A. circinalis blooms can impart tastes and odours to drinking and recreational waters (Sklenar and Home 1999).
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A. circinalis has been recognized as an important health risk especially in Australia, were it was often reported as neurotoxic to animals. Since 1973 (May and McBarron), A. circinalis has been widely blamed for sheep and cattle losses (Mc Barron et al. 1975, Runnegar et al. 1988), with blooms regularly forcing the closure of water bodies for recreation and domestic supplies. This cyanobacterium is very resistant to environmental stresses and during the drought of 1991, a bloom of neurotoxic A. circinalis stretched for nearly 1000 km on the Darling River in New South Wales (Bowling and Baker 1996). Incidentally, the neurotoxins of A. circinalis remained unidentified until recently, when Humpage et al. (1994) identified PSP toxins in several Australian samples. Toxin profiles detected in A. circinalis were always dominated by the N-sulfocarbamoyl-11-hydroxysulfate C toxins, Cl and C2, with the remainder consisting of gonyautoxins (GTX) -2, -3, and -5, decarbamoyl gonyautoxins (dcGTX) - 2 and -3, saxitoxin (STX), and decarbamoyl saxitoxin (dcSTX). N-1-hydroxy PSP toxins, such as neoSTX, GTXl and GTX4 were not detected in any field sample or cultured strain, suggesting that A. circinalis lacks the enzyme responsible for N-1- hydroxylation (Negri et al. 1997, Velzeboer et al. 2000). Among different isolates of this species, often having similar toxin profiles, toxin production varied extremely (Llewellyn et al. 2001). Although A. circinalis is found worldwide, there is a geographical segregation of neurotoxin production among strains. American and European isolates of this species produce only anatoxin-a, while Australian isolates exclusively produce PSP toxins. The reason for this geographical segregation of neurotoxin production by A. circinalis has been recently explored by molecular techniques. Beltran and Neilan (2000) determined the phylogenetic structure of A. circinalis by analysing 16S rRNA gene sequences. A. circinalis was found to form a monophyletic group of international distribution. However, the PSP- and non-PSP toxin-producing A. circinalis formed two distinct 16S rRNA gene clusters. The geographical segregation of neurotoxin production therefore demonstrated that it was a result of genetic heterogeneity within isolates of A. circinalis, and not mere morphological misclassification. The genetic difference between toxic and non-toxic Australian A. circinalis strains may be the result of adaptation to different microhabitats or specific environmental pressures.
11 Chapter 1 Intrrxluction
1.2.3 Cylindrospermopsis raciborskii
Cylindrospermopsis raciborskii (Fig. 1.4-B) is one of the major components of phytoplankton communities in tropical and sub-tropical freshwater ecosystems. In Australia and Brazil this cyanobacterium is known to be a prominent constituent of the blue-green algal flora of lakes, water supply reservoirs, slow-flowing rivers, dams and aquaculture ponds. The presence of C. raciborskii is of particular concern in water bodies used for human and stock consumption because of its potential for biosynthesis of potent toxins. Australian isolates and environmental samples of C. raciborskii showed the presence of cylindrospermopsin (CYLN). CYLN is a hepatotoxic alkaloid and was implicated in the worst Australian case of human poisoning by cyanobacteria: during 1979 139 children and 10 adults were affected by CYLN in Palm Island (Byth, 1980). Beside outbreaks of human sickness, C. raciborskii has been implicated also in cattle mortality (Saker et al. 2003). On the other hand, the same species in Brazil was found to be responsible for toxic blooms in which CYLN was detected more rarely. Recently, during a screening of freshwater cyanobacterial blooms in Brazil, three C. raciborskii strains isolated from the State of Sao Paulo were found to be characterized by the presence of PSP toxins (Lagos et al. 1999). The presence of STX, neoSTX, GTX2 and 3 and C-toxins in the isolates, named TI, T2 and T3, was proven by high performance liquid chromatography (HPLC) and liquid chromatography-mass spectrometry (LC-MS) (Lagos et al. 1999, Bernardo and Azevedo pers. Comm.). Toxin concentrations detected in C. raciborskii were in the order of magnitude of nmol / mg of dry weight, comparable to those previously found in A. circinalis (Humpage et al. 1994). C. raciborskii is also becoming one of the most notorious freshwater cyanobacteria. Beside its potential toxicity and its tendency to form dense blooms, it is tending to colonise more temperate regions, such as North America and Europe (Chapman and Schelske 1997, Coute et al. 1997, Saker at al. 2003). This new distribution of C. raciborskii has been noted only in the last 10 years, when it has been detected in many previously unaffected water bodies (Saker and Griffiths 2001). Reasons of the global increase of incidents involving C. raciborskii include the general trend in global warming and the high competitiveness and adaptiveness of this species
12 diapter 1 Intrrxluction
in nutrient utilisation and resistance to environmental stresses (Padisak, 1997; Pearl pers. comm.). Padisak (1998) proposed dispersal of C. raciborskii akinetes (resting spores) with migratory birds or in the vegetative form with the importation of tropical fish as the most likely dispersal mechanisms. More recently, Neilan et al. (2003) investigated the genetic variation between strains of C. raciborskii isolated from freshwater rivers and reservoirs in Australia, Brazil, Germany, Hungary, Portugal and the USA. Strains were characterised by both analysis of their 16S rRNA gene nucleotide sequences and cyanobacterium-specific short tandem repeat sequence technique, HIP 1. Greater genetic similarity was found between European and Australian strains, suggesting the possibility that a transfer from the Australasian region to Europe could have provided the source of this cyanobacterium which is now invading many previously unaffected European water bodies.
13 Chapter 1 IrtlrrXiuction
1.3 CYANOBACTERIAL BIOACTIVE METABOLITES
Cyanobacterial metabolites, beside having attracted attention because they can be the causative agents of toxic algal blooms, also represent a potentially rich source of new drugs. In the recent years, there has been an increasing interest in cyanobacterial products as a source of potentially new medicines and antibiotics (Shimizu 1996, Longley et al. 1991; Zhang et al. 1997, Chen et al. 2003 ). Many of these compounds have chemical structures unprecedented in other aquatic or terrestrial organisms, and display new and interesting biological activity. Some cyanobacterial genera, such as Microcystis, Anabaena, Nostoc, Oscillatoria and Lyngbya are comparable to actinomycetes in their capability of producing a variety of biologically active molecules (Shimizu 1993). Cyanobacteria can produce antitumour, antiviral, antibiotic and antifungal compounds. Of the cyanobacterial extracts screened by an Hawaiian research group, 0.8 per cent showed solid tumour selective cytotoxicity (Moore et al., 1996). Depsipeptides (peptides with an ester linkage) and an indolizine derivative (named ACEMOC) isolated from Nostoc sp. and Anabaena cylindrica strains are promising candidates for a anticancer drugs {Trimurtulu et al., 1995, Suzuki et al. 1999 and 2003). The cyanobacterial metabolite calothrixin-A can effectively kill tumour cells by inducing apoptosis (Chen et al. 2003). Several new cyclic or linear peptides and depsipeptides from cyanobacteria have been characterised as protease inhibitors (Namikoshi and Rinehart, 1996). Cyanobacteria can also synthesise several other bioactive compounds, some of which are toxic to other cyanobacteria, bacteria, algae and zooplankton. Severe intoxication of fish and frog embryos by crude cyanobacterial extracts have been also reported (Oberemm et al. 1999, Prati et al. 2002). However, research on cyanobacterial metabolites has a very short history. Only limited knowledge is available about the chemotaxonomical position and biosynthetic mechanisms of such compounds. The understanding of genetic systems and the regulation of cyanobacterial toxin synthesis can have a substantial industrial application in pharmaceutical treatments (Suzuki et al. 1999, Radau 2000) or agrochemical industries (Iwasaki and Shirai 2000).
14 Chapter 1 Intrrxluctian
1.3.1 Cyanotoxins
The presently known toxigenic cyanobacteria are represented by approximately 50 species. The principal mechanisms of cyanobacterial toxicity are diverse and range from hepatotoxic, neurotoxic and dermatotoxic effects to general inhibition of protein synthesis. The harmful toxins produced by cyanobacteria can be classified according to their chemical structure as lipolysaccharides (LPS), cyclic peptides (microcystin and nodularin) and alkaloids (cylindrospermopsin, aplysiatoxins, lyngbyatoxins, anatoxin-a, anatoxin-a(s) and saxitoxins). Substantial data are available about the toxicology and chemistry of cyanotoxins, and few molecules have also been studied according to their genetic basis and molecular biology. Environmental factors affecting toxin content in cyanobacteria showed that the production of toxin by a single strain seems to be constant and changes fluctuate only within a range of less than an order of magnitude. The majority of studies indicated that cyanobacteria produce toxins under conditions which are the most favourable for their growth.
1.3.2 LPS
LPS produced by some cyanobacteria are irritant toxins. The first isolation of cyanobacterial LPS from Anacystis nidulans occurred in the late 1960s (Weise et al. 1970), and numerous reports of endotoxins in cyanobacteria have followed. Lipopolysaccarides are generally found in the outer membrane of the cell wall of Gram negative bacteria, including cyanobacteria, where they form complexes with proteins and phospholipids. They are commonly pyrogenic and toxic (Weckesser and Drews 1979). LPS, as the name implies, are condensed products of a sugar and a lipid (C14- C18 fatty acid). The many structural variants of LPS are generally phylogenetically conserved, i.e. particular orders, genera and occasionally species, have identical or similar fatty acid and sugar components contained in their cell wall LPS (Kerr et al., 1995).
15 Chapter 1
In general it is the fatty acid component of the LPS molecule that elicits an irritant of allergenic responses in human and animal tissues that come in contact with the compound. Although comparatively poorly studied, cell wall components, particularly LPS endotoxins from cyanobacteria may contribute to human health problems associated with exposure to mass occurrences of cyanobacteria. The chemical stability of cyanobacterial LPS in surface waters is unknown. The few results available indicate that cyanobacterial LPS is less toxic than the LPS of other bacteria, such as Salmonella (Raziuddin et al. 1983), and that they may have interesting and novel effects on the immune system cells (Prati et al. 2001, Rossetti unpublished data).
1.3.3 Cyclic peptides
The cyclic peptides are the most common toxins detected in cyanobacterial blooms and they are considered hepatotoxins in terms of their toxicity to animals. They are commonly found in both fresh and brackish water blooms, and in particular the peptide-toxins microcystin and nodularin (Fig. 1.5) pose a major challenge for the production of safe drinking water in many countries.
Figure 1.5
A) General structure of microcystins. X and Z represent variable L-amino acids. R1 and R2 can be either H or CH3.
B) General structure of nodularin. Z can be either L-arginine, L-valine or homoarginine.
16 Chapter 1 Intrrxluctinn
Cyclic peptides are comparatively large natural products, with a molecular weight (MW) of - 800-1, 100 Da, although small compared with many other cell oligopeptides and polypeptides (MW > 10,000). Microcystins have been found in over 60 different chemical isoforms in strains of Microcystis, Anabaena, Oscillatoria, Nostoc and Anabaenopsis (Sivonen and Jones 1999), while nodularin is to date produced only by some strains of Nodularia spumigena in four derivatives (Namikoshi et al. 1994). These toxins are cyclic hepta- (microcystins) and penta- (nodularin) peptides, with the two terminal amino acids of the linear peptide being condensed to form a cyclic compound (Fig. 1.5). They are water soluble and, except for a few more hydrophobic microcystins, they are unable to penetrate directly the lipid membranes of animal, plant or bacterial cells, therefore, to elicit their toxic effect, uptake into cells occurs through membrane transporters. Microcystins and nodularin target liver cells in animals due to their binding to an organic anion carrier (bile acid transporter) on the hepatocytes membrane. In mammalian cells, they inhibit the key cellular enzymes called protein phosphatases (PP) -1 and -2A leading to cytoskeletal damage and liver failure. Long term exposure to low concentrations of these toxins has been also associated with tumour promotion (Yu 1989, 1995). Most of the structural analogues of microcystin and nodularin are highly toxic within a comparatively narrow range of acute dose: intra-peritoneal (i.p.) mouse toxicities vary from 50 to 300 µg kg- 1 (Sivonen and Jones 1999). Only a few non-toxic variants of these molecules, characterised by structural modifications to the Adda glutamate region of the toxins, have been identified (Harada et al. 1990, Rinehart et al. 1994). Microcystin and nodularin are virtually the only cyanotoxins for which the biosynthetic pathways and the gene clusters have been elucidated and documented. By means of gene disruption and mutant analysis, the microcystin synthase gene cluster (55 kb large) has been identified in M aeruginosa (Dittmann et al. 1997, Tillett et al. 2000). This gene cluster is composed of 10 bidirectionally transcribed open reading frames (ORFs), arranged in two putative operons (mcyA-C and mcyD-J). The presence and expression of these ORFs has been correlated with microcystin production in several cyanobacterial strains (Neilan et al. 1999). Of the 48 sequential catalytic reactions involved in microcystin synthesis, 45 have been assigned to catalytic domains within
17 Chapter 1
six large multienzyme synthases/synthetases (McyA-E, G), belonging to the family of non-ribosomal peptide synthetase (NRPS) and polyketide synthase (PKS). These multienzyme complexes incorporate the precursors phenylacetate, malonyl-CoA, S adenosyl-methionine, glutamate, serine, alanine, leucine, methyl-isoaspartate, and arginine in the final cyclic peptide microcystin (Tillett et al. 2000). The additional four monofunctional proteins are putatively involved in O-methylation (McyJ), epimerization (McyF), dehydration (Mcyl), and transport (McyH) of the toxin molecule. Similarly, it has recently been demonstrated that the other hepatotoxic cyclic peptide nodularin is biosynthesised in Nodularia spumigena NSORl O by a 48 kb gene cluster consisting of nine ORFs ndaA-1, transcribed into two polycistronic mRNA from a bi-directional regulatory promoter region. The structure of the putative nodularin synthetase (ndaS) gene cluster is similar to the microcystin synthetase (mcyS) gene cluster (Moffitt and Neilan 2001, Moffitt 2003). Additional evidence suggests a common progenitor for the two cyclic peptide-synthesising gene clusters (Moffitt 2003). In the natural aquatic environment, microcystins and nodularin usually remain contained within the cyanobacterial cells, and are released in substantial amounts along with increasing cell lysis. Recent studies however, demonstrated an increase in microcystin NRPS and PKS genes transcription as a result of high light intensities (Kaebemick et al. 2000). It was shown that light quality (in the red light spectrum) was responsible for this effect on genes involved in microcystin biosynthesis and these variations in gene expression were not reflected by any observed change in intracellular toxin bioactivity (Kaebemick et al. 2000). It was found that microcystins can be exported in the medium by M aeruginosa cells under certain conditions of light intensity (Dittmann, unpublished results). Active release of microcystins from the cell is supposedly operated by an ABC transporter whose gene (mcyH) is located upstream the hybrid NRPS/PKS gene mcyDE in the microcystin synthase gene cluster (Pearson and Neilan, unpublished). A similar effect of light on nodularin genes regulation and nodularin accumulation has been described in N. spumigena NSORI 0 (Moffitt 2003). The production of both microcystin and nodularin are known to be affected also by nutrient availability and iron limitation (Utkilen and Gjolme 1995, Rapala et al. 1997, Moffitt 2003). Microcystin has been shown to bind Iron (111) and other cations,
18 Chapter 1 Intrrxluainn suggesting that microcystin can function as a chelator of cations, particularly Iron (111) (Humble et al. 1994). In some instances, microcystin intracellular content was shown to increase proportionally to the concentration of iron (111) in the media (Utkilen and Gjolme 1995), while conflicting results have shown the production of microcystin increasing in response to low levels of iron (111) (Bickel et al. 2000). Recent data indicated that nodularin may also act as siderophore to scavenge available iron (Ill) from the environment (Moffitt 2003). These studies may indicate microcystin and nodularin syntheses are complex responses to light and iron (Ill), associated with other external influences.
1.3.4 Alkaloids
The alkaloids are, in general, a broad family of heterocyclic nitrogenous compounds usually characterised by low to moderate molecular weights (< 1000 Da). Like cyclic peptide toxins, these kind of molecules are considered secondary metabolites, which means compounds that are "apparently not absolutely essential to the life and growth of the producing organism" (Bentley 1999). They are produced, in particular, by higher plants and by some microorganisms (algae and bacteria), and are invariably bioactive and commonly toxic. Normally, alkaloids have varying chemical stabilities, often undergoing spontaneous transformations to by-products which may have higher or lower potencies than the parent toxin. Some are also susceptible to direct photolytic degradation. Cyanobacterial alkaloids can be classified, according to their biological toxicity, as dermatotoxins, cytotoxins and neurotoxins. The category of cyanobacterial dermatotoxins is represented by aplysiatoxins, debromoaplysiatoxins and lyngbyatoxins (Fig. 1.6), and produced primarily by benthic marine strains of Lyngbya, Oscillatoria and Schizothrix. Figure 1.6 Lyngbyatoxin These compounds can be either protein kinase C activators or inhibitors of microtubulin assembly (Shimizu 1996), resulting in
19 diapter 1
inflammatory activity that may produce severe dermatitis among swimmers and fishermen. These toxins have also been reported to be potential tumour promoters (Sivonen and Jones 1999). The best known cytotoxic cyanobacterial OH H alkaloid is cylindrospermopsin (Fig. 1. 7), 0 a tricyclic guanidine with a MW of 415 Da, which suppress glutathione and Me protein synthesis. Recently, this toxin was demonstrated to inhibit also the Figure 1.7 Cylindrospermopsin activity of the cytochrome P450 in mammalian cells (Froscio et al. 2003). It has also been suggested to have carcinogenic activity (Humpage et al. 2000). The toxicology of cylindrospermopsin, however, is complex and still not well understood. It is known to affect the liver as the principal target, although it induces pathological symptoms in the kidneys, heart and thymus (Sivonen and Jones 1999). The cyclic guanidine structure of cylindrospermopsin is believed to be biosynthesised by multimodular NRPS and PKS enzymes similarly to microcystins (Burgoyne et al. 2000). This hypothesis has been recently supported by published evidence of an amidinotrasferase, NRPS and PKS specific to cylindrospermopsin producing cyanobacterial strains (Schembri et al. 2001, Shalev Alon et al. 2002, Fergusson and Saint 2003). Cylindrospermopsin is produced in a couple of structural variants (Senogles et al. 2000) by strains of Cylindrospermopsis raciborskii, Umezakia natans and Aphanizomenon ovalisporum (Harada et al. 1994, Hawkins et al. 1997, Banker et al. 1997). The biosynthesis of cylindrospermopsin in C. raciborskii has been found to be strongly promoted by nitrogen limitation and low temperature (Saker and Neilan 2001). Among the cyanobacterial neurotoxins, two classes have bee studied in detail: the anatoxins and the saxitoxins. Saxitoxin and derivative compounds, also known as PSP toxins, are purine-like alkaloids that block the voltage-gated sodium channels in excitable cells, and are described exhaustively in paragraph 1.3.5. Anatoxins, on the other hand, are potent neurotoxic alkaloids that block the neural transmission at the synapses level. Anatoxin-a (Fig. 1.8-A) is the most common representative of the family and is a secondary amine, structurally related to the
20 Chapter 1 Irmx:luaion
tropane-alkaloids produced by higher plants (Moore A) 1999). Anatoxin-a(s) (Fig. 1.8-B), on the contrary, is a unique methyl phosphate ester of a cyclic N hydroxyguanidine and the only naturally occurring 8 organophosphate known (Matsunga et al. 1989).
7 Both these toxins interfere with neural transmission 4 at the muscular junction by blocking the nicotinic receptors (anatoxin-a) or by inhibiting acetylcholinesterase activity (anatoxin-a(s)) (Devlin et al. 1977, Matsunga et al. 1989). Anatoxin-a (MW = 165) is produced by strains of the Anabaena flos-aquae-lemmermannii group, by Anabaena planktonica, Oscillatoria, Figure 1.8 Aphanizomenon and Cylindrospermum (Sivonen A) anatoxin-a. B) anatoxin-a(s) and Jones 1999). Homoanatoxin-a (MW = 179) is the only anatoxin-a homologue, and was isolated from an Oscillatoria formosa strain
(Skulberg et al., 1992). The LD50 (lethal dose resulting in 50 per cent deaths) of anatoxin-a and homoanatoxin-a are 200 - 250 µg ki1 of body weight (bw) (Carmichael et al. l 990, Skulberg et al. 1992). The biosynthesis of anatoxin-a in A. flos-aquae and its homologue homoanatoxin-a in 0. formosa was investigated by Hemscheidt and coworkers (1995). Based on feeding experiments with 13C-labeled acetate and (S) glutamate, the pyrrolidine ring of these toxins was found to be produced in a different manner compared to that found in structurally related tropane alkaloids of higher plants, and glutamic semialdehyde was postulated as the primer unit for the triketide fragment of anatoxin and homoanatoxin. Anatoxin-a(S) (MW = 252) is produced by Anabaena flos-aquae strain NRC525-17 (Matsunaga et al. 1989), while it has more recently been identified in blooms and isolated strains of Anabaena lemmermannii (Henriksen et al. 1997,
Onodera et al. 1997a). The LD50 of anatoxin-a(S) is 20 µg kg- 1 bw (i.p. mouse) (Carmichael et al. 1990). Structural variants of anatoxin-a(S) have not been detected. Feeding experiments with stable and radiolabelled precursors established that all of the carbons of the triaminopropane backbone and the guanidino unit of anatoxin-a(S) are
21 Chapter 1 IrTtr 1.3.5 PSP toxins Paralytic shellfish poisoning (PSP) is a life-threatening affliction that results from consumption of seafood contaminated with paralytic shellfish toxins, that is, saxitoxin (STX) and analogous compounds (Kao 1993). This syndrome has been associated with both animal poisoning and deaths in humans (Anderson 1994). In some areas affected by PSP, historical reports reveal that coastal populations have been well aware for centuries of the risks associated with eating seafood at certain periods of the year, or under particular conditions such as discoloration or phosphorescence of sea water (Zingone and Enevoldsen 2000). The worldwide incidence of PSP (Kao 1993) appears to have increased markedly since the early 1970s (Fig. 1.9), with poisoning emerging in regions of the world where previously it was not known. Some possible reasons to explain this phenomenon include increased scientific awareness of toxic species (Yentsch 1984), increased utilisation of coastal waters Figure 1.9 World distribution of outbreaks of PSP (from Halstead and Schantz 1984) for aquaculture, global warming, and increased pollution due to higher nutrient loads resulting in more intense algal blooms. Currently, about 2000 cases of human poisoning due to PSP are reported each year (Zingone and Enevoldsen 2000). This is considered an underestimation due to common misdiagnosis and the lack of standardised reports from developing countries. Fatal episodes are estimated to exceed several hundred cases annually. PSP is one of the more common lethal forms of seafood poisoning and at present there is no antidote to 22 Chapter 1 Introduction treat the poisoning, the only effective control measure being the closure of the source of intoxication. Heat treatment only partially destroys saxitoxins and freezing and other forms of processing or preservation of food are ineffective in removing or destroying the toxins (Halstead and Schantz 1984). Beside the economic and environmental impacts of PSP toxin-producing algal blooms, the serious nature of PSP makes it an important public health issue. Estimates of acute toxic dose levels have been made at times of algal blooms and, for acute intoxication, the available data suggest that symptoms of toxicity can occur at intake levels of 120 µg STX for an adult human being. This can correspond to only 150 g of intoxicated shellfish or few glasses of contaminated water, and this dose can be lethal for children. STX (Fig. 1.10) is per se one of the most potent natural venoms known (Baden and Trainer 1993). With its specific pharmacological effect targeting neural transmission, saxitoxin represents both a useful physiological tool to study ion channels Figure 1.10 Chemical structure of STX in medicine and, at the same time, a possible chemical weapon and bioterrorist threat (Rose 2002). In the natural environment, PSP toxins are generally associated with red-tides. STX and derivatives, in fact, were first characterised from marine dinoflagellates of the genera Alexandrium (Fig. 1.11), Gymnodinium, and Pyrodinium (Shimizu 1977, Harada et al. 1982, Oshima et al. 1987). Some cyanobacteria and heterotrophic bacteria are also able to biosynthesise PSP toxins. Bacterial isolates from toxic dinoflagellate cultures have been shown to produce low levels of STX (Kodama et al. 1988, 1996). This controversial involvement of bacteria m the production of PSP Figure 1.11 Electron toxins, either autonomously or in conjunction with microscopy photograph of Alexandrium sp. dinoflagellates, has been reviewed by Gallacher and Smith (1999). More recently, an endosymbiotic Pseudomonas diminuta strain has been Chapter 1 isolated from toxic A. catenella and confirmed to produce STX by immunological and chemical methods (Cordova et al. 2002). Additionally, another investigation identified two strains of Enterobacter and a Klebsiella pneumoniae as the causative agents of a bovine paraplegic syndrome in Venezuela (Sevcik et al. 2003). Cultures of the three enterobacterial isolates were found to produce high levels of STX using two distinct HPLC methods. However, the unequivocal evidence of autonomous bacterial production of PSP toxins remains to be achieved. Accumulating data suggests that at least some strains play a role in the biosynthesis or the catabolism of these toxins in natural conditions, although the precise mechanisms are still unclear. PSP toxins can be produced by some filamentous cyanobacterial species, and the occurrence of neurotoxic cyanobacterial blooms and saxitoxins in freshwaters are increasing more and more every year (Kaas and Henriksen, 2000). Until the 1990's, a few strains of Aphanizomenon flos-aquae from North America were thought to be the only cyanobacteria capable of producing PSP toxins (Jackim and Gentile 1968, Sawyer et al. 1968, Alam et al. 1973). Lately, however, other cyanobacterial species have been recognised as PSP toxin-producers, including Anabaena circinalis from southern Australia (Humpage et al. 1994, Onodera et al. 1996, Negri et al. 1997), Lyngbya wollei from the USA (Carmichael et al. 1997, Yin et al. 1997, Onodera et al. 1997b), Cylindrospermopsis raciborskii from Brazil (Lagos et al. 1999), Planktothrix sp. FPl from Italy (Pomati et al. 2000), strains of Aphanizomenon from Portugal (Pereira et al. 2000, Ferreira et al. 2001), and marine strains of the genus Trichodesmium (Carmichael pers. comm.). These microorganisms produce over 20 known toxins of the PSP family and although individual species do not contain all the STX analogues, they generally contain specific suites of toxins depending on the isolate (Sivonen and Jones 1999). The chemical structure and toxicity of PSP toxins have been well described. In contrast, little is known about all the other aspects of these unusual microbial alkaloids (Cembella 1998). Being produced by dinoflagellates, photosynthetic and heterotrophic bacteria, PSP toxins are unique due to their polyphyletic taxonomic distribution, as bioactive secondary metabolites of both prokaryotes and eukaryotes. 24 Chapter 1 Intrrxluawn 1.3.5.1 - Chemistry of PSP toxins PSP toxins are a broad group of natural toxins, with a similar perhydropurine structure (Fig. 1.12) and different substitutions that alter their toxicity. The 7, 8, 9 guanidinium moiety, and not the 1, 2, 3 one, is essential for the blocking action of STX (Baden and Trainer 1993). The molecular formula of STX is C10H17N70 4 as the free base, with MW of 299. More than 20 different saxitoxin analogues and derivatives are known to date (Sivonen and Jones, 1999) and can be divided in 4 categories according to their substitutions. These carbamate alkaloids can be non-sulphated, like STX and neoSTX (NEO), singly sulphated (GTXs) or doubly sulphated (C-toxins). Except for PSP toxins, no other natural compounds containing an N-sulfocarbamoyl group have been reported (Shimizu 1993). In addition, decarbamoyl variants (dcSTX, dcNEO, dcGTXs) and several new toxins form Lyngbya wollei, characterised by acetate and carbinol as a substitution (Onodera et al. 1997b ), have been identified (Table 1. 1). Recently, other decarbamoyl variants and several new toxins have been detected in some species, such as the novel hydroxybenzoate saxitoxin analogues described in the dinoflagellate Gymnodinium catenatum by Negri and coworkers (2003). H N 7 ., ., I'.,'.\ \\±, + a .----····· NH2 g ,'/ 15 •• N H ••••••• QH 14 Rs Figure 1.12 The general chemical structure of PSP toxins. R = variable chemical groups (see Table 1.1 ). PSP toxins are a white hygroscopic solid very soluble in water, partly soluble in methanol and ethanol, but insoluble in most non-polar solvents such as ethyl and petroleum ethers. They have no absorption in the ultraviolet light range, and STX has two pKa values at 8.2 and 11.5, with an optical rotation of about +130. 25 Chapter 1 Intrrxluctim Table 1.1 Known toxins belonging to the PSP family (for the chemical structure refer to Fig . 1.12). LWTX = Lyngbya wo//eitoxin. VARIABLE CHEMICAL GROUPS TOXIN R1 R2 R3 Ri Rs STX H H H CONH2 OH GTX2 H H OsO3- CONH2 OH GTX3 H OsO3- H CONH2 OH GTX5 H H H CONHSO3- OH C1 H H OsO3- CONHSO3- OH C2 H OSO3- H CONHSO3- OH NEO OH H H CONH2 OH GTX1 OH H OsO3- CONH2 OH GTX4 OH OsO3- H CONH2 OH GTX6 OH H H CONHSO3- OH dcSTX H H H H OH dcGTX2 H H OsO3- H OH dcGTX3 H OsO3- H H OH LWTX1 H OsO3- H COCH3 H LWTX2 H OsO3- H COCH3 OH LWTX3 H H OsO3- COCH3 OH LWTX4 H H H H H LWTX5 H H H COCH3 OH LWTX6 H H H COCH3 H Under laboratory conditions, PSP toxins are stable for a long time at acidic pH, low temperature and in the dark, although these molecules are easily susceptible to alkaline hydrolysis. PSP toxins are in general very sensitive to alkaline pH values, and several reports demonstrated that PSP toxins can remain stable at high temperature with low pH, but can undergo quick degradation by heating at neutral to high pH, both in solution and in shellfish tissue homogenates (Indrasena and Gill 1999, Baker et al. 2003). With the addition of 0.1 N HCI, the stability of these toxins has been proven in the laboratory even after heating at 100°C for 10 min, while addition of 0.2 N NaOH 26 Chapter 1 Intrrxluctinn and heating at 37°C for 3 h completely degrades PSP toxins (Boyer et al. 1986). For these reasons, PSP toxins are generally extracted in acidified media including acetic acid, hydrochloric acid, acidified methanol or acidic water (Fernandez and Cembella 1995). The interconversion of various saxitoxin congeners has also been observed in the laboratory and animal tissues in consequence to chemical or enzymatically-mediated reactions (Sullivan et al. 1983, Indrasena and Gill 1999). The conversion of sulfocarbamate toxins to highly toxic carbamate derivatives has been shown to occur upon heating in the presence of acid. In the laboratory, it is also possible to obtain STX and neoSTX from GTX and C-toxins in alkaline solutions of dithiothreitol (Laycock et al. 1995). In more natural chemical conditions, saxitoxins undergo a senes of slow reductions and hydrolysis reactions. Reduction of GTX and neoSTX can form STX, while C-toxins can lose the N-sulphocarbamoyl group to give dcGTX. STX, GTX and dcGTX slowly degrade to yet unidentified non-toxic products. The half lives of PSP toxins, necessary for the breakdown reactions in neutral conditions, are in the order of 1-10 weeks, with more than three months often being required for a degradation greater than 90% (Jones and Negri, 1997). Transformation reactions may occur in living cyanobacterial cells, along with the agmg of cultures or natural blooms. Under these conditions, reduction and hydrolysis of carbamate and N-sulphocarbamoyl toxins can also yield more toxic products (such as STX and GTX) than their original precursors. A solution of PSP toxins can increase in toxicity over a period of up to three weeks before toxicity begins to abate during the succeeding 2-3 months. This has been shown for water bodies containing a natural mixture of C-toxins and GTXs from the lysis of an Australian bloom of A. circinalis (Jones and Negri 1997). No detailed studies have been carried out on the breakdown of PSP toxins in sunlight, either with or without the presence of pigments. However, the scarce absorption of UV radiation by these molecules reduces the possibility of any relevant photodegradation in natural conditions. On the other hand, it has been suggested that the photocatalytic formation of free oxygen radicals in the water could increase the natural oxidative breakdown of PSP toxins (Jones and Negri, 1997). 27 Chapter 1 Introduction 1.3.5.2 - Pharmacology and toxicology of PSP toxins All systemic actions of PSP toxins can be explained by their pharmacological effect on nerve axon membranes. This involves the specific blockage of voltage-gated sodium channels (VGSC) in a dose-dependent manner, thereby affecting (partially or completely) impulse generation in peripheral nerves and skeletal muscles, with no effect on resting membrane potential (Catterall 1980, Strichartz 1981 ). In mammals, these effects induce paralysis, respiratory depression and respiratory failure. Respiratory muscles, especially in the diaphragm, are particularly vulnerable to the paralysing action of the poison. Direct cardiac effects are usually minimal (Kao 1993). Of the various PSP toxins, only the pharmacology of STX has been studied in detail, partly because saxitoxin is the most potent representative of the PSP family and partly because the other toxins are usually not available in sufficient quantities for such studies. STX binds to nerve membrane in a 1: 1 stoichiometry with high affinity ( constant of dissociation Kd 2 nM) and in a saturable manner (Catteral et al. 1979). The dose-response curve obtained with STX is consistent with the idea that it is the combination of a single toxin molecule with a single Na+ channel that results in the blocking action. Binding is specifically associated to the VGSC and requires both a. and ~ 1 subunits. VGSC are proteins of 250 kDa, present in all animal excitable cells. For its highly selective activity, STX is considered an important medical and pharmacological tool. STX binds equally well to resting, active or inactive Na+ channels. A receptor site, common also to the other algal channel-blocker, tetrodotoxin (TTX), has been experimentally (S4). Voltage sensor determined and named as site 1 (Cestele and Catterall 2000). Receptor site 1 on sodium channels is occupied by the so called "pore-blocking" toxins. These toxins act from the extracellular side of the plasma membrane by occluding entry Figure 1.13 Three-dimensional model of a VGSC. Chapter 1 Introduction to the sodium channel pore (Fig 1.13). The positively charged guanidinium group of STX and TTX interacts with negatively charged carboxyl groups at the mouth of channel. The STX/TTX receptor site is formed by two rings of acidic amino acids residues localised in segment SS2 on the N-terminal side of the S6 transmembrane segment in each of the four domains of the VGSC (Cestele and Catterall 2000) (Fig. 1.14). All saxitoxin derivatives are presumed to interact with the same binding site. II Ill IV Site 1 {TTX, $TXI } TSIDE Pore Voltage-EenBOr 1 ·sm . • lnacli'l/atlon gata Figure 1.14 Transmembrane arrangement with functional mapping and location of STX receptor sites in the a-subunit of VGSC, as proposed by Cestele and Catterall (2000). The individual binding efficacy of PSP toxins changes in consequence to the different substitutions of the molecule and is correlated with potency, in a manner very similar to the ion flux reported in Figure 1.15 (Frace et al. 1986). The derived dissociation constants for these toxins based on potency are (in nM): STX, 5.5; GTX3, 11.4; GTX5, 14.5; C2, 16.2; GTX2, 31.5; Cl, 223.9. Chapter 1 Introduction 1.0·0-.------, 0 .75 0 50 0.25 -:- 0·.00------t~------+------~ 1 10 100 1000 Concentration of toxin (nM) Figure 1.15 Effects of PSP toxins on Na+ current in giant squid axons: • = STX, .A = GTX3, • =BI, T = C2, • = GTX2, D = Cl. Data points represent a single measurement at the given concentration, curves represent the best non-linear regression (Frace et al. 1986). Studies of sodium channels inhibition at different pH values showed that PSP toxins are more effective at neutral pH when their hydroxyl groups are protonated (Hille 1968). In fact, the key structural features of PSP toxins that are involved in the blockage of Na+ conductance through nerve membranes are the guanidinium and hydroxyl moieties. Modifications of these groups affect channel recognition by the toxins and result in the loss of biological activity. Recently, and contradictory to previous studies (Catterall 1980, Strichartz 1981), saxitoxin was also found to modify the gating properties of myocardial HERG K+ channels (Wang et al. 2003) and to inhibit L- type Ca2+ currents in mouse ventricular myocytes (Su et al. 2003). Saxitoxin-bound K+ channels are altered in their voltage inactivation and voltage-activation processes, so that STX does not simply block the ion conduction pore. Information on the effects of PSP toxins on organism physiology is mainly restricted to acute toxicity in mammals. The carbamate toxins are the most toxic (STX, neoSTX, GTXl-4) and are 10-100 times more toxic than the N-sulfocarbamoyl derivatives (the Band C toxins) (Wright 1995). In mouse bioassays, the relative toxicity Chapter 1 of the carbamate and sulfocarbamoyl toxins in descending order of toxicity is: neoSTX, STX, GTX3, GTX2, C2, B2, Bl, Cl. The toxins Bl, B2 and C2 are virtually equipotent despite their differences in structure and net charge. The potency of Cl is greatly attenuated, but it does have some intrinsic activity (Hall et al. 1990). The mouse LDso toxicity values of STX by intravenous, intraperitoneal and oral routes are 2.4 µg ki1, 10 µg kg- 1 and 263 µg ki1, respectively, for adult 20 g male white mice (Halstead and Schantz 1984). No long-term studies have been performed on mammals with PSP toxins, but they are generally considered non-carcinogenic compounds. The research on the pharmacokinetics of PSP toxins has been complicated due to the relative low sensitivity and non-specificity of the methods used to detect STX in biological fluids and tissues. Hines and co-workers (Hines et al. 1993) made the first effort to study the STX distribution in whole animal, using [3H]-saxitoxinol, a tritiated reduced saxitoxin. In rats, this STX analogue was eliminated mainly by urine, no radioactivity was found in faeces, and this compound was not metabolised during the experimental period. However, saxitoxinol is a hundred times less toxic than saxitoxin and therefore does not reproduce the intoxication effects of STX (Shimizu et al. 1981 ). More recently, Andriolo et al. (1999) used adult anaesthetised cats coupled to artificial ventilation, intravenously injected with low (2.7 µg of STX kg- 1) and high doses (10 µg of STX kg- 1) of toxin. Cardiovascular parameters were recorded, urine and blood samples were collected and STX was quantified by post-column derivatisation HPLC method. Tissue samples from brain, liver, spleen and medulla oblongata were also extracted to measure the amount of STX. Low doses of STX made no difference in haemodynamic parameters. In contrast, high doses drastically reduced blood pressure, produced myocardial failure and finally cardiac arrest. Besides STX, any other PSP toxin due to possible metabolic interconversion was not detected in any of the analysed samples, indicating that mammals cannot metabolise this molecule. STX was mainly excreted via renal clearance but was also found in the central nervous system, showing that this compound can cross the blood-brain barrier. Medical records of PSP cases among humans confirmed that saxitoxins can be readily absorbed through the gastrointestinal mucosa after ingestion, and they are mainly cleared from serum via urine excretion (Gessener et al. 1997). Death results 31 Chapter 1 from paralysis of the muscles of the diaphragm with ensuing thorax and respiratory failure, which can occur within 2-24 h. In an extensive review on the occurrence of PSP in Europe, van Egmond et al. (1993) reported illness at oral doses of 144-1,660 µg STX per person, and fatalities at 300-12,400 µg STX per person. Shumway (1995), in reviewing the findings of several other authors, concluded that as little as 120-180 µg STX can induce moderate symptoms in adults and as little as 400-1,060 µg of STX may cause death in adults. Applying a safety factor of 10 to the lowest known dose causing illness in an adult human, a "tolerable" dose would be as little as 12 µg STX. In the natural environment, direct poisoning of animals often occurs by consumption of toxic algal cells from the water or indirectly through consumption of other animals that have themselves fed on algae and accumulated these toxins. As in marine habitats, PSP toxins very commonly bioaccumulate in freshwater vertebrates and invertebrates, including fish and bivalves (Giovannardi et al. 1999). The consequent toxic effects have considerable potential of being magnified in the food chain. It is generally difficult, however, to unequivocally ascribe the deaths of natural populations of aquatic organisms, especially fish, to PSP toxins. In freshwater environments, the collapse of a large cyanobacterial bloom can lead to very low concentrations of oxygen in the water column, which can also be deleterious to animal life. Although fish and amphibians, especially their embryonic growth stage, are known to be very susceptible to cyanotoxins, few studies have been performed on the effects of PSP toxins on aquatic animals using standard laboratory tests (Oberemm et al. 1999, Prati et al. 2002). In those conditions, saxitoxins showed only acute toxicity without any carcinogenic or teratogenic effect on the aquatic animals and larvae. Similarly, it is well recognised how other cyanotoxins have many adverse effects on zooplankton, both on viability of the individuals and their reproductive capability. No detailed studies have been undertaken with regards to cyanobacterial PSP toxins and zooplankton. Saxitoxins may also impact in the growth of some species of aquatic bacteria. A recent study showed how STX is able to inhibit chemotaxis and motility (ED5o 10 µM and 1 mM, respectively) in E. coli by affecting ion fluxes through the plasma membrane (Tisa et al. 2000). No other evidence is reported in the literature on the effects of PSP toxins on prokaryotes. 32 Chapter 1 Intrrxluctian 1.3.5.3 - Detection methods for PSP toxins The methods used to detect, identify and characterise PSP toxins in water and cyanobacterial cells can vary greatly due to their degree of sophistication and the information they provide. They can be relatively simple low cost methods, appropriate to evaluate rapidly the potential hazard or, in contrast, highly sophisticated analytical techniques can be employed to determine precisely the identity and quantity of toxins. Such detection methods can be based on the chemical properties of PSP toxins, their reactivity with other chemicals or organic molecules, or they can be designed to target biological activities in cells and organisms. Chemical detection methods. Among the chemical detection methods employed to analyse the presence of PSP toxins, thin layer chromatography {TLC) played the most important role in the early PSP research (Sullivan 1993). Nowadays, the most commonly used detection methods are based on HPLC. This analytical technique uses the physicochemical properties of saxitoxins such as molecular weight, chromophores and reactivity of the functional groups to detect these molecules. HPLC methods have been primarily developed for the analysis of PSP toxins in shellfish samples, however, they have been found to be equally suitable for water and cyanobacterial studies. All HPLC methods for PSP toxins employ oxidation of the toxins with hydrogen peroxide or sodium periodate in slightly alkaline conditions to yield highly fluorescent products. Oxidation products can be visualised with a fluorescence detector at A 330 nm excitation and A 400 nm emission. The oxidation reaction can be prechromatographic (also known as pre-column) followed by liquid chromatography (Lawrence et al. 1995, 1996), or post-column with chromatographic separation prior to oxidation and fluorescence detection (Oshima 1995). The manual pre-column technique of Lawrence et al. (1995, 1996), in which the oxidation products are separated by HPLC and detected by fluorescence, can effectively distinguish among the main groups of STX derivatives, including sulphocarbamoyl, N-hydroxy and decarbamoyl toxins. The use of either peroxide or periodate results in minor changes in the efficiency of this method. This procedure is very sensitive, does not require particular hardware for post-column reactions, and utilises only one mobile phase. Different hydroxylated analogues (e.g., 33 Chapter 1 B2 and neoSTX) and isomer pairs, however, such as GTX and C toxins, yield the same oxidation products. These products cannot be separated by the pre-column HPLC conditions, resulting in poor resolution for PSP toxin screening routines. On the other hand, with automated on-line post-column oxidation and fluorescence detection it is possible to resolve all the PSP toxins known with higher sensitivity and selectivity (Oshima 1995). Although Oshima's method is considered to be the most satisfactory to date, it requires two/three different mobile phase systems (Onodera et al. 1997b) to allow analysis of all the toxins, analytically divided into decarbamoyl, sulphated and non-sulphated toxins. Furthermore, the HPLC apparatus has to be coupled with complex hardware such as the on-line post-column reaction system (PCRS). A range of mass spectrometry techniques has been described for PSP toxins, including fast atom bombardment (Mirocha et al. 1992), thermospray interface (Wils and Hulst 1993), electrospray (Hines 1993) or ion-spray ionisation (Quilliam et al. 1989). Liquid chromatography/mass spectrometry (LC/MS) has also been applied to PSP toxin detection, although this method does not allow analysis of all PSP derivatives within one LC/MS run. On the other hand, capillary electrophoresis (CE) combined with ion-spray ionisation has been successfully employed for saxitoxin analysis (Locke and Thibault 1994), though at present CE lacks satisfactory detection limits due to very small injection volumes (< 10 nL). Nuclear magnetic resonance (NMR) spectroscopy has been extensively utilised for the structural elucidation of unknown PSP toxins (Onodera et al. 1997b, Negri et al. 2003). NMR usually requires relatively large amounts of sample (mg quantities) and completely purified toxins, therefore this technique is not suitable for routine monitoring. Although excellent correlation has been demonstrated between chromatographic methods and mouse bioassay data (Sullivan 1993), all analytical techniques suffer from a global shortage of reference standard material for individual PSP toxins analysis. Recently, fluorescence chemosensors for STX have been proposed (Gawley et al. 2002). Anthracylmethyl crown ethers have been shown to bind STX with high specificity and a 1: 1 stoichiometry. The resulting enhancement of sensors fluorescence is selective for STX and can effectively detect the toxin at concentrations in the order of 100 µM. 34 Chapter 1 Animal bioassays. The mouse bioassay is the classical method for analysis of saxitoxins. It is a standardised procedure in which the strain, size and condition of the mice are all controlled. Groups of mice are challenged by injection of toxin extracts, and their responses are compared with those of mice injected with known toxin at different concentrations (AOAC 1984). The Association of Official Analytical Chemists (AOAC) method is still the main method used for detecting paralytic shellfish toxins. Acute toxicity studies have been conducted in several species with male Swiss Albino mice as the most used animals for toxicity testing for saxitoxins. Toxicity is tested by intraperitoneal injection (i.p.) of0.1-1.0 mL of a cell purified lysate or an environmental sample which has been sterilised by membrane ultra-filtration. Samples can be suspended in water or physiological saline solution if the volume to be injected is 0.5 mL or greater. Mice are observed for 24 hand then sacrificed. Toxicity is expressed in mouse units (MU) per µmol of toxin. By mouse bioassay, STX is the most potent compound among the PSP family (2.5 MU per µmol) while C toxins are the least toxic (15-143 MU per µmol) (Oshima 1995). Other organisms have also been investigated for their use in routine PSP toxins bioassays. In particular, two invertebrate assays have been documented. Adult house flies can be injected with purified toxins and natural samples (such as shellfish extracts), and toxicity results showed good correlation with mouse bioassay data (Ross et al. 1985). Flies are difficult to handle, however, and they require the problematic application of microinjections (1.5 µL). More recently, a locust bioassay was successfully validated for the detection of PSP toxins from cyanobacterial, shellfish and environmental samples (McElhiney et al. 1998). Locusts are more easy to control, samples are administered by 10 µL injections and results can be obtained within 90 minutes. In the locust assay, the LD50 for pure saxitoxin was 8 µg i 1 bw. Cell and tissue bioassays. In vitro cell bioassays were originally developed for monitoring PSP toxins in shellfish extracts in a similar fashion to the chemical detection methods. A neuroreceptor binding assay has been developed and subsequently refined (Doucette et al. 1994). This test uses radiolabelled saxitoxin and works on the basis of competitive displacement, giving good data correlation with the mouse bioassay (Cembella et al. 1995). 35 Chapter 1 Several types of Na+ channel blocking assays are, instead, described in the literature, which work on the principle that saxitoxins bind to Na+ channels in nerve cell membranes and disrupt normal depolarisation. A mouse neuroblastoma cell bioassay kit for PSP toxins has also been developed (Jellett et al. 1992). However, this test suffers from the limited shelf-life of these cell lines (1-3 weeks) and false positive results due to interfering substances. Manger et al. (1997) described a mouse neuroblastoma cell bioassay for the detection and quantification of PSP toxins based on enzymatic colorimetric end-point assessment of cell viability with tetrazolium salts. The assay has been modified to allow detection of either Na+ channel blockers or enhancers. Low-end detection was 0.1 ng, proving to be considerably more sensitive than the mouse bioassay (Hungerford and Weckell 1992). Based on the work of Manger and colleagues, a haemolysis assay has been subsequently made available for the detection of sodium channel specific toxins using fish red blood cells (RBC) (Shimojo and Iwaoka 2000). This test relies on the use of veratridine, a Na+ channel activator, and ouabain, an inhibitor of sodium ATPases, to affect the permeability of the RBC membrane. The presence of STX or analogue channel blockers prevents RBC lysis or changes in the cells morphology. The haemolysis assay of Shimojo and Iwaoka (2000) was approximately the same sensitivity and detection limits of the standard mouse bioassay. Very recently, a fluorimetric microplate assay has been developed for the detection and quantitation of PSP toxins using human neuroblastoma cells (Louzao et al. 2003). This test is based on a fluorescent dye, bis-oxonol, measuring direct changes in membrane potential. Fluorescence intensity increases due to cell depolarisation, induced with veratridine, and dose-dependent repolarisation can be detected as an increase in fluorescence in the presence of PSP toxins. Cheun et al. (1998) instead reported the development of a tissue biosensor, consisting of a Na+ electrode covered with a frog bladder membrane, all integrated within a flow cell. This tissue sensor measures the direction of Na+ transfer across the bladder membrane in the presence of channel-blocking toxins. Bacterial bioassays. For practical, ethical and economic reasons alternative tests to animals and animal-cell bioassays have been investigated in PSP toxin screening procedures. Several bacterial bioassays have been studied to determine whether they 36 Chapter 1 Intrrxluawn could provide simple routine methods for PSP toxin detection. Among the common standardised tests, the Microtox® and similar bioluminescence assays have been the most promising. These bioassays indicate toxicity by a reduction in the light emitted by bioluminescent bacteria (Photobacterium phosphoreum or Vibrio fischieri). Although it has been suggested that this test may be suitable for detection of some cyanotoxins (Lawton et al. 1990), the assay can give false positives due to the presence in natural samples of unknown components rather than cyanobacterial toxins (Campbell et al. 1994). A second bacterial bioassay, utilising the inhibition of pigment (prodigiosin) formation in Serratia marcescens as an indication of toxicity, has been proposed by Dierstein et al. (1989). This assay was also investigated for saxitoxin detection, however, poor correlation between actual content of toxin and inhibition of pigment formation was shown (Lawton et al. 1994). Immunodetection. Immunodiagnostic systems for the detection of PSP toxins have primarily been developed to replace the mouse bioassay for routine testing of shellfish samples. Both radioimmunoassays and enzyme-linked immunosorbent assays (ELISA) have been employed, involving rabbit serum antibody preparations and monoclonal antibodies (Chu and Fan 1985, Usleber et al. 1991, 1995, 1997). The presence of cross-reactions with lower binding specificity and the lack of response for all toxins within the saxitoxin family limits the use of these systems. Antibodies have been principally raised to saxitoxin mainly because it has been the most extensively studied, hence methods reliably detect this variant but fail to cross-react with neosaxitoxin which is of similar toxicity. More successful methods have recently been developed and may provide a suitable routine monitoring system in the future. Micheli and colleagues (2002) reported the production of saxitoxin-specific polyclonal antibodies, obtained from rabbits immunized with saxitoxin-keyhole limpet haemocyanin (STX-KLH). The optimisation of a 96-well microplate test was coupled by a comparison of two competitive ELISA formats (direct and indirect) for the detection of STX. A goat anti-rabbit IgG peroxidase conjugate was used to enable detection. This study showed saxitoxin detection limits of 3 and 10 pg mL- 1 for direct and indirect ELISA formats, respectively. 37 Chapter 1 Intrrxluctim On the other hand, Kawatsu and coworkers (2002) developed a qualitative enzyme immunoassay based on a GTX 2+ 3-specific monoclonal antibody and a saxitoxin-horseradish peroxidase conjugate (STX-HRP). The resulting direct competitive enzyme immunoassay (GTX-EIA) showed that all of the GTX components examined and STX are detectable at concentrations lower than the regulatory limit of 80 µg 100 i 1 of shellfish tissue (Association of Official Analytical Chemists). Similar immunological detection methods have been successfully applied to the screening for STX production in bacteria isolated from toxic dinoflagellate samples (Cordova et al. 2002), demonstrating the efficacy and specificity of these techniques. Saxiphilin binding assay. According to Negri and Llewellyn (1998), bioassays for STX may be compromised by cross reactivity to tetrodotoxin in natural samples. These authors have developed an alternative binding assay based on a novel protein called saxiphilin. Saxiphilin is a hydrophilic protein with a high affinity and specificity for PSP toxins and is found in the circulatory fluid of many invertebrates and ectothermic vertebrates. Saxiphilin has been first found in bullfrogs and it is closely related to the iron binding transferrins (Morabito and Moczydlowski 1994), a group of proteins that range in molecular weight between 70 and 90 kDa. One saxiphilin isoform, isolated from the Australian centipede Ethmostigmus rubripes, has been used to develop a microtitre plate assay for PSP toxins which relies upon detection of radiolabelled [3H]STX. Saxiphilin binds STX with high affinity (<1 pM: the half-life for toxin dissociation is almost 2 days) and has similar affinities for dcSTX, neoSTX and Bl, but differs from the Na+ channel in not having any affinity for tetrodotoxin, however, it binds C-toxins with a much lower affinity (Llewellyn et al. 1997). Toxic extracts are evaluated by their inhibition of the [3H]STX binding to saxiphilin. Using the conditions reported by Llewellyn and Doyle (2001 ), this assay can be used to reliably detect 1.3 µg STX eq 100 g- 1 shellfish tissue. 38 Chapter 1 Intraluctwn 1.3.5.4 - Ecology and physiology of PSP toxins The stimuli inducing or repressing saxitoxin production in cyanobacteria are currently unknown, as is the metabolic role of PSP toxins within the producing microorganisms. Factors influencing the biosynthesis of saxitoxin and related compounds in cyanobacteria have been poorly studied, both in the laboratory and in the environment. One of the most detailed reports in the literature, underlies the physico-chemical conditions associated with one of the largest PSP toxin producing blooms ever witnessed (Bowling and Baker 1996). The cyanobacterial bloom, dominated by Anabaena circinalis up to half a million cells per mL, affected 1000 km of river waters in the Barwon-Darling basin, New South Wales, Australia. Stock deaths were reported during this occurrence, and the neurotoxicity of A. circinalis samples was demonstrated by mouse bioassay. This cyanobacterial bloom has been attributed to low river flow, caused by an intense drought period. In such environmental conditions nutrient concentrations, especially phosphorus, were found to be very high. Alkaline pH (> 8.5) and very high ammonia values, in some instances more than 1 mg L- 1, also characterised the majority of sampling sites along the river. Additionally, most of the neurotoxic A. circinalis water samples were collected from localities typified by high electrical conductivity (Bowling and Baker 1996). The bloom decline was attributed to the increased river flow following heavy catchment rainfalls. Analogous environmental parameters, in particular elevated pH and high conductivity, have also been described in Australia for another potentially toxic bloom of A. circinalis in Lake Cargelligo, New South Wales, during 1991 (Bowling 1994). In the laboratory, studies with cyanobacterial isolates documented that some environmental factors can induce changes in toxicity or in toxin concentration, although such variations ranged from two to four fold. For PSP toxins, most of the research in the field of the environmental effects on toxin production comes from studies on toxic dinoflagellates. Among the environmental parameters tested on growth and PSP toxin accumulation are culture age, temperature, light, principal nutrients, salinity, pH and micronutrient concentrations. The few studies undertaken, as outlined below, indicate that cyanobacteria apparently produce saxitoxin under conditions which are most 39 Chapter 1 Intrrxluctinn favourable for their growth. This has also been reported for other cyanobacterial secondary metabolites (Sivonen and Jones 1999). Some dinoflagellate species conversely were shown to be responsive to some critical factors in their production of PSP toxins. Cyanobacteria produce more PSP toxins during their late exponential growth phase, and it has been reported that STX biosynthesis in Aphanizomenon flos-aquae NH-5 was inversely proportional to the growth rate (Gomaa 1990, cited by Yin et al. 1997). Comparable results were also described for L. wollei (Yin et al. 1997). Dinoflagellate toxin content in nutrient-supplied batch cultures has been found conversely to peak during early exponential phase, rapidly declining prior to the onset of the plateau phase (Usup et al. 1994). Moreover, a study using synchronised cultures of Alexandrium Jundyense documented that STX is accumulated during a discrete period of time localised in the G1 phase of the eukaryotic cell cycle {Taroncher Oldemburg et al. 1997). Studies on light intensity documented a reduction of toxicity in Lyngbya wollei cultures under high levels of light irradiation, coupled by an increase in biomass (Yin et al. 1997). Although the authors hypothesised possible photodegradation of PSP toxins with increased light intensity, several other studies on cyanobacteria and dinoflagellates showed no relevant effect of this condition on saxitoxin quota per unit of biomass (Anderson 1990, Usup et al. 1994, Negri et al. 1997, Hwang and Lu 2000). Light is nevertheless the principal stimulus inducing the onset of the G1 phase of the dinoflagellate cell cycle, associated with STX biosynthesis {Taroncher-Oldemburg et al. 1997). On the other hand, temperature seems to have a marked effect on PSP toxins production. Dinoflagellates tend to have a higher toxin concentration (up to 3-fold) when grown at lower temperatures (Ogata et al. 1987, Anderson 1990, Usup et al. 1994, Hwang and Lu 2000). In cyanobacteria, an analogous effect of temperature has been described for the production of the alkaloid cyanotoxin cylindrospermopsin in C. raciborskii (Saker and Griffitths 2000). Cylindrospermopsin increased and decreased proportionally along with the reduction or rise of temperature, respectively. Yin and colleagues (1997) documented a net decrease in PSP toxin content of L. wollei cultures with increasing media temperature. Similarly, A. circinalis was found to produce more 40 Chapter 1 Intrrxluction STX and GTX 2+3 per cell at low temperatures (15°C) compared to values above 30°C (Rossetti and Pomati, unpublished data). These documented effects have been explained by either less stability or higher biodegradation of saxitoxins at high temperature, as well as by changes in the rate of cell division (Anderson 1990, Yin et al. 1997, Saker and Griffiths 2000). In the environment however, toxic blooms of A. circinalis have been associated with water temperatures ranging from 25 to 30°C (Bowling and Baker 1996). To date, the consequence of pH variation on PSP toxin production in cyanobacteria have not been investigated, but in toxic dinoflagellates this environmental parameter has not been correlated to any significant change in toxin content (Ogata et al. 1987, Anderson 1990, Hwang and Lu 2000). Alkalinity of water is a main feature characterising toxic cyanobacterial blooms, in particular those dominated by PSP producing species such as A. circinalis (Bowling and Baker 1996). Nutrients, and in particular nitrogen and phosphorus, are essential for cyanobacterial growth. Phosphorus is usually the limiting factor for autotrophic growth in freshwaters, and hence small changes in this nutrient's concentrations may influence toxin production merely as a result of influencing growth. In L. wollei, optimum growth levels of PO4-P correspond to the best conditions for PSP toxins production (Yin et al 1997). On the contrary, toxic dinoflagellates produce more saxitoxins when phosphorus is deficient, probably because these conditions reduce cell duplication while toxin production remains constant (Ogata et al. 1987, Anderson 1990, Hwang and Lu 2000). A large portion of microbial secondary metabolites are molecules very rich in nitrogen. Therefore, non-nitrogen fixing organisms, including toxic dinoflagellates, produce more toxins under nitrogen-rich conditions (Ogata et al. 1987, Anderson 1990). Nitrogen fixing species, such as PSP toxin producing cyanobacteria, are not dependent on nitrogen in the media for their toxin production (Rapala et al. 1993, Lehtimaki et al. 1997), although very high concentrations of this element were found to inhibit PSP toxin accumulation in cultures of L. wollei (Yin et al. 1997). Rising levels of calcium have been shown to increase PSP toxin synthesis in L. wol/ei (Yin et al. 1997), while no other metal or trace element has been investigated for their effect on the production of STX in cyanobacteria. In dinoflagellates, there is evidence to suggest that there is no specific effect of trace elements on PSP toxin 41 Chapter 1 Intraluctian accumulation, the optimum concentrations for growth being optimal for toxin production (Anderson 1990, Hwang and Lu 2000). Several papers described that dinoflagellates increased toxicity under conditions of high salinity (Proctor et al. 1975, Boyer et al. 1987, Ogata et al. 1987, Hwang and Lu 2000). In some studies the cellular toxin levels increased under salt stress, while in other instances it was the composition of toxin profiles that changed under these circumstances. In Alexadrium minutum, low salinity stimulated the cells to produce higher amounts of GTX 1, with high salinity triggering the production of peak levels of the more toxic GTX 2+ 3 (Hwang and Lu 2000). Although environmental conditions of increased salinity of fresh and brackish waters are common during drought summer periods in sub-tropical and temperate regions, the effects of salt stress on cyanobacterial PSP toxin production haven't been yet investigated. High salinity has been documented as increased conductivity in correlation with neurotoxic blooms of A. circinalis in Australia (Bowling 1994, Bowling and Baker 1996). Another parameter that has been found to affect STX production in dinoflagellates was the presence of contaminating heterotrophic bacteria (Uribe and Espejo 2003). Without their typical bacterial flora some dinoflagellate species, such as A. catenella, appeared to strongly diminish the production of PSP toxins. In cyanobacteria, there is little evidence to suggest any correlation between toxicity or toxin profile and the amount or type of associated bacteria (Yin et al. 1997, Beltran and Neilan unpublished data). Unlike dinoflagellates, no bacterial strains capable of producing PSP toxins have been isolated from cyanobacterial cultures, to date. 1.3.5.5 - Biosynthesis and Biodegradation of PSP toxins One of the most striking aspects of microbial metabolites is the unforeseen way in which some of these compounds are biosynthesised. Almost 30 years after the correct structure of STX was elucidated by X-ray crystallography (Schanz et al. 1975), there have been many speculations as to how this peculiar alkaloid is synthesised, although very few investigations substantially contributed to the biosynthetic knowledge of PSP toxin production. 42 Chapter 1 Intraluction In the early 1980s, Shimizu and colleagues documented a fundamental study concerning the biosynthesis of the STX carbon perhydropurine-skeleton (Shimizu et al. 1984, Shimizu 1986a, 1986b ). According to feeding experiments carried out with 13C and 2H-labled precursors in the dinoflagellate Alexandrium tamarense and the cyanobacterium Aphanizomenon flos-aquae, the molecule of STX was found to be built from arginine and acetate via a Claisen-type condensation between C2 of arginine (or omithine) and C 1 of acetate (Fig. 1.16). This kind of reaction is rather uncommon, although it characterises the first step of porphyrin biosynthesis with the condensation of succinate and glycine to form aminolevulinic acid (Shimizu 1996). Further, the amino group of arginine is transformed into a guanido group by transfer of an amidino moiety from another molecule of arginine. The guanido carbon of arginine is also incorporated in the two guanidine groups in the nucleus of the side-chain carbamate. The side-chain C13 comes from the S-adenosylmethionine (SAM) methyl group. In total, to synthesise one STX molecule three arginine, one acetate and one methionine molecule are required. ' 'H;N'-';;,.¾.NH NH;; ··-··-• STX Figure 1.16 Biosynthetic pathway of STX in cyanobacteria and dinoflagellates as speculated from various feeding studies (Shimizu 1993, 1996) 43 Chapter 1 Given this sequence of biochemical reactions, some candidate enzymes in STX biosynthesis have been hypothesised. As previously mentioned, the first Claisen-type condensation between the a-carbon of arginine and Cl of acetate can be catalysed by aminolevulinate synthase (involved in the metabolism of chlorophyll and heme synthesis) or by a biosynthetic arginine decarboxylase (implicated in polyamine metabolism) (Ralf Kellmann, pers. comm.). Alternatively, this reaction could be operated by a mixed NRPS/PKS enzyme with an acyl adenylation, 13-ketoacyl synthase and thioesterase domains. The transfer of an amidino group from a molecule of arginine to the incomplete skeleton of STX can be catalysed by an amidinotransferase, followed by the cyclisation of the molecule possibly directed by a thioesterase. A C methyltransferase, or a S-adenosylmethionine-dependent methyltransferase, could be involved in the methylation of the STX backbone and a cytochrome P450 may oxidise this new methyl group (Ralf Kellmann, pers. comm.). A carbamoyl transferase and a dioxygenase could also be implicated in the last steps of STX biosynthesis, while sulfotransferases are likely to be involved in the modification of the STX basic structure to give the sulphated derivatives. In a recent study, three more candidate enzymes in STX production have been identified. Using differential display, Taroncher-Oldenburg and Anderson (2000) characterised the differential gene expression in the toxic dinoflagellate A. fundyense during the early G1 phase of the cell cycle, which coincides with the onset of toxin production in this orgamsm (Taroncher-Oldemburg et al. 1997). An S adenosylhomocysteine hydrolase, a methionine aminopeptidase, and a histone-like protein were isolated, although none of these genes showed direct correlation with the hypothesised STX biosynthetic pathway. On the other hand, Sako and coworkers (2001) purified and characterised a N sulfotransferase from the toxic dinoflagellate G. catenatum that was uniquely and specifically capable of transferring a sulphate residue from 3 '-phosphoadenosine 5 ' - phosphosulfate (PAPS) to the N-21 carbamoyl group present in STX and GTX2+3. This enzyme, a monomeric 60 kDa protein, was functional only with three substrates and each yielded a distinct product: STX ~ GTX5, GTX2 ~ Cl, and GTX3 ~ C2. Based on the activity of this N-21 sulfotransferase, Sako et al. (2001) suggested a 44 diapter 1 possible pathway for STX synthesis in dinoflagellates in which STX represents the basic structure for the production of all other PSP derivatives. The hypothesised sequence of conversions, STX~ GTX2+3 ~ C-toxins, is the reverse of a previous speculative pathway proposed by Taroncher-Oldenburg et al. (1997) based on the time-dependent accumulation of various STX analogues during the synchronised growth of Alexandrium fundyense. During the cell cycle of this toxic dinoflagellate, the accumulation of C2 preceded GTX2+3, which preceded STX. In the case of the N-sulfotransferase characterised by Sako et al. (2001), it is possible that the enzyme does not normally play a role in STX biosynthesis. This N-21 sulfotransferase has not been purified in a sufficient quantity for protein sequencing, therefore it was not possible to verify the toxic-specificity of the corresponding gene in dinoflagellate isolates. Furthermore, earlier evidence documented the detection of N-sulfotransferase activity in a non-toxic strain of G. catenatum, although not in a toxic A. tamarense (Oshima 1995). Little work has been undertaken on the biodegradation of saxitoxins. Jones and Negri (1997) observed no bacterially-mediated degradation of PSP toxin in a range of surface water samples. More recently, initial evidence suggested a possible degradation pathway for PSP toxins in the cyanobacterium Planktothrix sp. FP 1, related to the nitrogen micro-cycles in water environments (Pomati et al. 2001). It is common for cyanobacteria to use traces of purines and ureides as nitrogen source for growing, and the relationship between PSP toxins accumulation and the activation of the purine degradation pathway was thus investigated. The activity of allantoicase, an inducible enzyme of that catabolism, has been used as a tool for assaying the activation of purine metabolism in Planktothrix sp. Although induction of the degradative pathway by allantoic acid (the direct substrate of allantoicase) resulted in a net increased accumulation of toxins compared to the controls, cultures fed with adenine, urea and E. coli crude extracts showed low PSP toxin levels. Thus, as proposed for purine alkaloids in higher plants (Ashihara et al. 1998), it was suggested that saxitoxin and derivatives could be broken down in cyanobacteria via the degradative pathway of purine compounds (Pomati et al. 2001). 45 Chapter 1 1.3.5.6 - Genetics of PSP toxins As mentioned before, PSP toxins represent a unique example among microbial secondary metabolites, being produced by organisms distributed in two of the three kingdoms of life and across phylogenetically very distant genera. The biosynthetic capability of PSP toxin production is not, however, universal to a species, but is exceptional and limited to certain strains. Of the various attempts to explain the nature of this scattered toxigenicity the most accredited theory, although still controversial, hypothesises the lateral acquisition of STX biosynthetic genes by dinoflagellates, cyanobacteria and bacteria from an original prokaryotic source. According to the biosynthetic pathway hypothesised in chapter 1.3.5.5 (Fig. 1.16), the production of saxitoxins requires circa a dozen enzymes (Shimizu 1996). To be transferred horizontally between two or more organisms, these biosynthetic genes have to be all clustered in a small region, and then exchanged by means of plasmid, transposon or phage. However, until the eventual discovery of the so-called STX gene cluster, the origin and the evolution of PSP toxin production remains a mystery. To date, very little information has been obtained from studies focusing on the genetic basis of saxitoxin synthesis, especially in cyanobacteria. Thus far, investigations have been undertaken to address predominately dinoflagellate toxin synthesis. In Alexandrium sp., PSP toxin production was found to be inherited according to Mendelian rule and to be chromosome dependent (Sako et al. 1992, Ishida et al. 1993, Sako et al. 1995). In the experiments performed, when gametes from two Alexandrium clones with different toxin profiles were conjugated, the FI clone cultures inherited each parental toxin profile in a 1: 1 ratio. The F2 clones showed the same toxin profiles as the Fl and profile inheritance was not related to cell sexuality, suggesting that the STX biosynthetic genes are not mapping on a mating type locus in the dinoflagellate genome. More recently, STX production in A. fundyense was established to be active only during a short period of the dinoflagellate cell cycle, localised in the G1 phase of DNA synthesis (Taroncher-Oldenburg et al. 1997). This discontinuous toxigenesis, combined with the hypothesised chromosomal integration of STX genes, suggested that 46 Chapter 1 Intrrxiuawn STX biosynthesis could be regulated at the transcriptional level with a cyclic pattern. A recent preliminary report also indicates that STX production in bacteria could be regulated by chaperonins or heat-shock proteins (Cordova et al. 2002). In cyanobacteria, the toxigenicity of A. circinalis has been studied recently by a molecular phylogeny approach (Beltran and Neilan, 2000). PSP toxins producing and non-toxic strains of A. circinalis from geographically diverse locations have been analysed by 16S rRNA gene sequencing and the phylogenetic structure of this species determined. Interestingly, PSP toxin producing and non-toxic strains formed two distinct 16S rRNA gene clusters, with few exceptions. These results suggested the existence of a common ancestor of the neurotoxic Anabaena strains, with the current PSP toxin producers being a relatively recent divergent population (Beltran and Neilan, 2000). Data were also consistent with the possible horizontal acquisition of the PSP toxin biosynthetic genes by Australian A. circinalis. 47 Cl.tapter 1 1.4 OBJECTIVES AND SCOPES 1.4.1 AIMS The objectives of this research project were: • Investigate the physiological and environmental conditions leading to the production and regulation of PSP toxins in cyanobacteria. • Isolate and characterise candidate genes involved in the biosynthesis or regulation of PSP toxins. • Study the candidate genes identified according to their differential expression under the above mentioned physiological conditions. The three objectives are interrelated. The physiological experiments will provide the necessary basic information for the gene expression studies. On the other hand, the genetic determinations will help in elucidating the function of PSP toxins and their role in the cyanobacterial physiological response to environmental stresses. The ultimate goal of this research is to acquire a better understanding of why and how some cyanobacterial strains produce PSP toxins. 1.4.2 HYPOTHESES 1. PSP toxins are metabolites involved in the maintenance of the cell homeostasis. 2. PSP toxin-producing cyanobacteria have a potential advantage over other non-toxic strains under conditions of critical Na+ levels. 3. PSP toxins can be modulators of cation channel activity in prokaryotes. 4. There are genes or groups of genes whose expression patterns are related to PSP toxin production. 48 Chapter2 Mat:erials and Methals CHAPTER2 MATERIALS AND METHODS 2.1 BACTERIAL STRAINS AND CULTURING 2.1.1 Cyanobacteria The cyanobacterial strains used in this study are listed in Appendix A. Specific growth conditions are mentioned in each of the following chapters. Reported here are the sources and main characteristics of the cyanobacterial strains utilised. The filamentous freshwater cyanobacterium Cylindrospermopsis raciborskii T3 was kindly provided by Sandra Azevedo (Federal University of Rio de Janeiro, Brazil). This strain was isolated in Brazil from a cyanobacterial bloom in the State of Sao Paulo and found to produce the neurotoxic alkaloids of the PSP-toxins family Cl +2 toxins and STX (Lagos et al. 1999). The cyanobacterium was cultivated in MLA medium (CSIRO Marine Laboratories, Hobart, Tasmania, Australia), modified MLA medium (MLA-E), or ASM-1 medium (Gorham et al. 1964) (Appendix B). PSP-toxin producing and non-toxic strains of Anabaena circinalis were obtained from the Australian Water Quality Centre (AWQC, Adelaide, South Australia) and maintained in Jaworski's Medium (JM) (Humpage et al. 1994) (Appendix B). Strains were originally isolated from cyanobacterial blooms samples collected in New South Wales, Victoria, Queensland and South Australia (Llewellyn et al. 2001). Cultures were grown at a constant temperature of 26° C, under continuous irradiance of cool white light at an intensity of 15 µmol photon m-2 s- 1• Cylindrospermopsis raciborskii A WT205 was obtained from Peter R. Hawkins (Australia Water Technologies, EnSight, West Ryde, NSW, Australia). The strain was grown in ASM-1 medium at 26°C, under continuous irradiance of cool white light at an intensity of 15 µmol photon m-2 s- 1• 49 Chapter 2 Materials ard Methals Nodularia spumigena strain NSORl0 was obtained from S. Blackburn, CSIRO Marine Laboratories (Hobart, Tasmania, Australia). Batch cultures were maintained in ASM-1 medium at 25°C and light (12 h)/dark (12 h) cycling between 16 and 0 µmol p h otons m -2 s . -1 Microcystis aeruginosa strain PCC7806 was obtained from the Pasteur Culture Collection (PCC), Paris, and cultured in BG-11 freshwater medium (Rippka et al. 1979) (Appendix B).Cells were grown at 25°C and light (12 h)/dark (12 h) cycling between 16 and 0 µmol photons m-2 s- 1• All the cyanobacterial cultures employed in this investigation were unialgal but not axenic. 2.1.2 Escherichia coli E. co/i cultures (Table 2.1) were grown at 37°C in Luria-Bertani (LB) broth (10 g L- 1 tryptone, 5 g L- 1 yeast extract, 10 g L- 1 NaCl) (Oxoid Pty Ltd, Hampshire, England) in conical flasks with constant shaking for 15-18 hours. For growth on solid media, LB supplemented with 15 g L- 1 of bacteriological agar (Oxoid Pty Ltd) was used. For selection of E. coli that were harbouring plasmids containing antibiotic resistance genes, E.coli was grown in the presence of 100 µg mL- 1 ampicillin. Table 2.1 Strains of E.coli used in this study and their genotypes E. coli strain Genotype Reference supE, Mac (~80 /acZL\M15), hsdR, recA, DH5a (Sambrook 2001) endA, gyrA, thi, re/A F' mcrA L\(mrr-hsdRMS-mrcBC) ~80 /acZL\M15, MacX74 recAl deoR araD139 (Invitrogen, Carlsbad, TOPl0F' L\(ara-leu)7697 ga/U rpsL (StrR) endAl CA) nupG 50 Chapter 2 Materials am Methals 2.2 ANALYSIS OF CYANOBACTERIAL GROWTH 2.2.1 Measurement of growth Cyanobacterial cultures were monitored spectrophotometrically by recording the optical density at 750 nm (OD1so) with a Lambda 10 UVNS spectrometer (Perkin Elmer, Inc., Shalton, CT), and microscopically by viewing total cells under an Olympus EHA (Tokyo, Japan) phase contrast microscope. 2.2.2 Total protein assay Total protein concentration in cyanobacterial cultures and media was determined by means of the method of Bradford (1976), using bovine serum albumin as a standard. 2.3 TOXIN DETECTION AND QUANTIFICATION 2.3.1 HPLC analysis Depending on the different experiments, PSP toxins were extracted from cyanobacterial cells with either Milli-Q water or acetic acid 0.5 M (see also Section 1.3.5.1). Screening for PSP toxins was performed by prechromatographic oxidation with H20 2 followed by HPLC separation. Chromatography was carried out according to the method of Lawrence et al. (1996) (see also Section 1.3.5.3). Briefly, chemical analyses were performed on a Waters 600 HPLC apparatus coupled with a Waters 470 fluorescence detector (Millipore Corp., Bedford, MA). The column used was a Supelcosil LC-18 (150 x 4.6 mm, id 5 µm) (Supelco, Bellefonte, PA). The samples and standard mixture were oxidized as previously described (Lawrence et al., 1996) using hydrogen peroxide prior to injection. Oxidation products (25 µL injection volume) were eluted under isocratic conditions with 1% acetonitrile (v/v) in 0.1 M ammonium formate, pH 6.0, at a flow rate of 1.0 mL min- 1• 51 Chapter 2 Materials am Methals Concentrations of PSP toxins in the samples were calculated by comparing the peak area corresponding to the toxins in the cyanobacterial extracts to that of the standard solutions. When necessary, hydrogen peroxide was removed from the oxidant solution to verify the oxidation dependence of HPLC peaks. 2.3.2 Protein phosphatase inhibition assay The concentration of microcystins (MCYST) or nodularin (NODLN) in a sample was analysed using the colorimetric protein phosphatase 2A (PP2A) inhibition assay described by An and Carmichael (1994). Ten microliters of standard or sample, appropriately diluted in Milli-Q water, were placed in duplicate in a 96-well plate followed by the addition of 0.04 units of PP2A (Promega, Madison, WI) diluted in enzyme diluent buffer (0.05 M Tris-HCl [pH 7.0], 2 mM DTT, 2 mM MnCh, 1 mg mL- 1 BSA). The sample was incubated with the enzyme at 37°C for 5 minutes to allow inhibition of the enzyme to occur. One hundred microlitres of reaction buffer (0.2 M Tris-HCl [pH 8.1], 0.4 mM MnCh, 84 mM MgCh, 2 mg mL- 1 BSA, 4 mM DTT) containing 20 mM of the enzyme substrate, P-nitrophenol phosphate (P-NPP), was mixed with each sample. The reaction was incubated at 3 7°C for 80 minutes and the absorbance read at 405 nm using a Benchmark™ Microplate Reader (Bio-Rad, Hercules, CA). 2.3.3 Toxins standards Certified standard calibration solutions for analysis of paralytic shellfish poisoning toxins (PSP-lC and STXdiHCl-C) were obtained from the Institute of Marine Bioscience (1MB), National Research Council of Canada (Halifax, NS, Canada). Protein phosphatase inhibition assay standard, nodularin, was HPLC purified from cyanobacterial cell extracts (supplied by Harri Kaankaanpaa). 52 Chapter 2 Materials am MethaJs 2.4 FLAME PHOTOMETRY ANALYSIS In this study, flame photometry was used to assay total cellular Na+ and K+ levels in cyanobacteria. Two milliliter aliquots of cyanobacterial cultures were collected by centrifugation in 2 mL plastic tubes and sampled pellets resuspended in 0.5 mL of diluent flame solution (3 mM Li in Milli-Q water). Analysis of total Na+ - K+ cellular content was performed with a FLM3 Flame Photometer (Radiometer, Copenhagen, Denmark). 2.5 STATISTICAL ANALYSES All graphical and descriptive statistical analyses were performed using the software for PC Origin 5.0 (Microcal Software, Inc., Northampton, MA). ANOVA and post-hoe analysis of means using the least significant difference (LSD) test were carried out with Statistica software for Windows, release 4.3 (Osiris Technology Systems, Sunderland, UK). For all tests, the statistical significance level used was P < 0.05. 2.6 NUCLEIC ACID EXTRACTION 2.6.1 Genomic DNA extraction Cyanobacterial cultures were filtered through a 3.0 µm pore size filter (Millipore, Billerica, MA), and cells washed twice with sterile water. This method has been previously demonstrated to be effective in the removal of possible contaminating heterotrophic bacteria (Beltran and Neilan 2000, Beltran 2001, Kaebernick 2001 ). Genomic DNA was then extracted from filtered and washed cyanobacterial cells using the XS procedure as described previously (Tillett and Neilan 2000). Briefly, cell cultures were harvested by centrifugation {5,000 x g, 10 min) and the cell pellets resuspended in 50 µL ofTER (10 mM Tris-HCl, pH 7.4; 1 mM EDTA, pH 8; 100 µg of RNase A per mL) and 750 µL of freshly made XS buffer (1 % potassium ethyl 53 Cbapter 2 Materials am MethaJs xanthogenate; 100 mM Tris-HCl, pH 7.4; 20 mM EDTA, pH 8; 1% sodium dodecyl sulfate; 800 mM ammonium acetate). The tubes were incubated at 70°C for 40 min, vortexed and placed on ice for 30 min. Cell debris was removed by centrifugation at 12,000 x g for 10 min, supematants transferred to fresh 1.5 mL tubes, nucleic acid precipitated with isopropanol, and centrifuged for 10 min at 12,000 x g. The DNA pellets were washed with 70% ethanol, air dried, and resuspended in TE (10 mM Tris HCl, pH 7.4; 1 mMEDTA, pH 8). 2.6.2 Total RNA extraction For total RNA extraction, cyanobacteria were collected by filtration through 3.0 µm pore size filters (Millipore), washed twice with sterile water and immediately frozen in liquid nitrogen. Cells and filters were crushed in liquid nitrogen with a pre-cooled mortar and pestle and the cellular extract recovered with 2 mL of TRizol reagent (Invitrogen). Crude cell extracts were added to 0.4 mL of chloroform and incubated at room temperature for 5 min. The mixture was then vortexed and centrifuged at 4 °C and 6,000 x g for 15 min. The upper aqueous layer was transferred to fresh 1.5 mL tubes, nucleic acid precipitated with isopropanol, and centrifuged for 10 min at 12,000 x g. The RNA pellets were washed with 75% ethanol in RNAse-free water, air dried, and resuspended in 50 µL ofRNAse-free water. 54 Chapter 2 Materials am Methals 2.7 SUPPRESSION SUBTRACTIVE HYBRIDISATION (SSH) 2. 7.1 Overview Although there are several different methods, the basic theory behind DNA subtraction implies that the genomic-DNA sample containing the sequences of interest is named "tester," and the reference sample is named "driver." Tester and driver DNAs are hybridised, and the hybrid sequences are then removed. Consequently, the remaining unhybridised DNAs represent tester-specific sequences. Traditional subtractive hybridisation methods involve several rounds of hybridisation and require large amounts of DNA. In contrast, the method developed by Akopyants et al. (1998) overcomes these and other technical limitations of traditional subtraction procedures. The suppression subtractive hybridisation (SSH) method (Diatchenko et al. 1996, Akopyants et al. 1998) requires only 1-2 µg of genomic DNA, takes only 2-3 days, and does not involve the physical separation of single stranded and double stranded molecules. Furthermore, suppression PCR (Diatchenko et al. 1996) prevents undesirable amplification while enrichment of target molecules proceeds. The molecular basis of the PCR-based SSH is shown in Figure 2.1 First, genomic DNA is isolated from the two strains of bacteria being compared (1-2 µg of DNA required per subtraction). These tester and driver DNAs are digested with the appropriate four-base-cutting restriction enzyme. The tester DNA is then subdivided into two portions, each of which is ligated with a different adapter. The ends of the adapters are unphosphorylated, so only one strand of each adapter attaches to the 5' ends of the DNA. The two adapters have stretches of identical sequence, which allows annealing of the same PCR primer to both ends once the recessed ends have been filled in by Taq polymerase (see Appendix C).Two hybridisations are then performed. In the first, an excess of driver is added to each adapter-ligated tester sample. The samples are then heat denatured and allowed to anneal, generating the type a, b, c, and d molecules in each sample (Figure 2.1 ). The single stranded type a molecules are significantly enriched for tester-specific sequences, as DNA fragments that are not tester specific form type c molecules with the driver. At the same time, the concentration of fragments, 55 Cliapter 2 Materials am Methals present in single and multiple copies in the bacterial genome, is equalized among the type a molecules. This is because reannealing is faster for the more abundant molecules due to the second-order kinetics of hybridisation. During the second hybridisation, the two primary hybridisation samples are mixed together without denaturing. This ensures that only the remaining equalized and subtracted single stranded tester DNAs can reassociate and form new type e hybrids. These new hybrids are double stranded tester molecules with different ends; the ends correspond to the sequences of Adapters 1 and 2R (Appendix C, Section C. l ). Freshly denatured driver DNA is added (again, without denaturing the subtraction mix) to further enrich fraction e for tester-specific sequences. After DNA polymerase has filled in the ends, the type e molecules - the fraction that is greatly enriched for tester specific sequences - have different primer annealing sites on their 5' and 3' ends. The entire population of molecules is then subjected to PCR to amplify the tester-specific sequences. Type a and d molecules are missing primer-annealing sites, and thus cannot be amplified. Due to the suppression PCR effect, most type b molecules form a panhandle-like structure that prevents their exponential amplification. Type c molecules have only one primer annealing site and can only be amplified linearly. Only type e molecules, which have two different adapters, can be amplified exponentially. These molecules are equalised and enriched for tester-specific sequences. Next, a secondary PCR amplification is performed using nested primers (Appendix C, Section C.1) to further reduce any background PCR products and enrich for tester-specific sequences. The subtracted DNA can be directly inserted into a cloning vector and then tester specific fragments can be identified by sequence and hybridisation analysis. 56 Cliapter 2 Materials and MethaJs Tester DNA with Adaptor 1 Driver DNA lin excess) Tester DNA with Adaptor 2R == - ==----c::a a == == b cm C - == d{ Second hybridization: mix samples, add fresh denatured driver, and anneal a, b, c, d + e ..-=====a• cm i Fill in the ends am= M::;;-== b - M C -== --== e M en'.;·- - I Add primer ._ • Amplify by PCR a, d no amplification ~ b-+b' no amplification c linear amplification ._ e exponential amplification 5'M cm 3' (Although there is a primer binding sequence on both and ends of the type e molecules, the shorter overall 3· aec:i-----cm s· homology at the two ends practically negates the ., suppression PCR effect-except for very short molecules.) Figure 2.1 Schematic diagram of SSH (Akopyants et al. 1998). Type e molecules are formed only if the sequence is present in the tester DNA, but absent in the driver DNA. Solid lines represent the Rsa I-digested DNA. Solid boxes represent the outer part of the Adapter 1 and 2R longer strands and corresponding PCR Primer 1 sequence. Clear boxes represent the inner part of Adapter 1 and the corresponding Nested Primer 1 sequence; shaded boxes represent the inner part of Adapter 2R and the corresponding Nested Primer 2R sequence (Appendix C, Section C.1 ). 57 Chapter 2 Materials ard Methals 2.7.2 Driver and tester DNA preparation One microgram of tester and of driver DNA was each digested to completion with 15 units of Rsal (New England Biolabs) for 16 h at 37°C in 50 µL reaction volumes, extracted with phenol and precipitated with ethanol according to standard protocols (Sambrook et al. 1989), and resuspended in 6.5 µL sterile Milli-Q water at a final concentration of 200 ng µL- 1• Two aliquots of tester DNA (120 ng each) were ligated separately to 2 µL of the two adapters, each in a total volume of 10 µL (2mM final concentration). Unsubtracted tester control DNA was prepared by mixing 1.5 µL of each aliquot of tester DNA and adapter preparations, to obtain tester DNA ligated to both the terminal adapters (see Fig. 8.1). Reactions were incubated at 16°C overnight, using 1 µL of T4 DNA ligase (5 units, Promega, Madison, WI) and 1 µL of T4 RNA ligase (Promega, 1 unit) in the buffer supplied by the manufacturer. After ligation, 1 µL of 0.2 M EDTA was added, and the samples were heated at 70°C for 5 min to inactivate the ligase and then stored at -20°C. Unsubtracted tester control DNA (3 µL) was diluted in 1 mL sterile Milli-Q water before storage (unsubtracted tester DNA is used as positive control in subsequent PCR amplifications). 2.7.3 Subtractive hybridisation Two microliters of driver DNA (600 ng) were added to 1 µL (12 ng) of each of the adapter-ligated tester DNAs (50:1 ratio). One microliter of 4x hybridisation buffer (2 M NaCl; 200 mM Hepes, pH 8.3; 0.8 mM EDTA) was added to each tube, the solutions were overlaid with mineral oil, DNA denatured (2 min, 98°C) and allowed to anneal for 90 min at 63°C. After this first hybridisation, the two samples (the first with adapter 1, the second with adapter 2) were combined without intermediate denaturation, 300 ng more of heat-denatured driver DNA was added in 1 µL of 2x hybridisation buffer, and the sample was allowed to hybridise at 63°C overnight. This final 13 µL reaction was diluted in 200 µL with dilution buffer (50 mM NaCl; 5 mM Hepes, pH 8.3; 0.2 mM EDTA), heated at 63°C for 10 min to eliminate non-specific hybridisation, and stored at -20°C until use in PCR. 58 Cliapter 2 Mat:erials and Methals 2. 7.4 PCR amplification Two sequential PCRs were carried out. The first PCR contained 1 µL of subtracted and unsubtracted genomic DNA prepared as described, 2.5 µL of PCR primer Pl (10 µM), in 25 µL reactions containing 200 µM dNTP, 2.5 mM MgCh, Taq polymerase Buffer, and 1 U Taq FI polymerase (Promega). This first PCR was incubated at 72°C, 5 min followed by 94°C, 25 sec and then subjected to 25 cycles of 94°C, 10 sec; 66°C, 30 sec; 72°C, 1.5 min. The amplified products were then diluted 20-fold in sterile Milli-Q water, and 2 µL of each diluted sample was used in the second PCR with 2.5 µL of nested PCR primers NPl and NP2 (10 µMeach) in 25 µL reactions containing 200 µM dNTP, 2.5 mM MgCh, Taq polymerase Buffer, and 1 U Taq FI polymerase. PCRs were cycled using a temperature profile of 94 °C, 25 sec followed by 25 cycles of 94°C, 10 sec; 68°C, 30 sec; and 72°C, 1.5 min, and concluded with one cycle of 72°C for 5 min. PCRs were carried out in a Perkin-Elmer (Shalton, CT) GeneAmp system 2400. 2.8 DNA CLONING AND AUTOMATED SEQUENCING PCR products were inserted into the pGEM-TE vector (Promega) following the instructions provided by the manufacturer. Ligated DNAs were transformed into E. coli strains DH5a or TOPlOF' (Section 2.1.2) by heat shock following standard protocols (Sambrook 2001). Clones were selected for ampicillin resistance and amplified using the pGEM-TE vector-specific primers (mpF and mpR, Appendix C, Section C.2). DNA was sequenced by PRISM Automated BigDye terminator sequencing and an ABI 373 sequencer (PE Applied Biosystems, Foster City, CA), with reactions performed using 3 µL (-150 ng) of each PCR product and 10 pmol of each appropriate primer in a half-scale reaction as specified by the manufacturer. 59 Chapter2 Materials and Methals 2.9 DNA-MICROARRAY DESIGN AND PRODUCTION The PCR clones obtained in this and other studies (Moffitt 2003) were organised, together with more than 200 other DNA fragments including 16S rDNA of the investigated strains as housekeeping genes, in the BGGM1 DNA-microarray. The complete BGGM1 array list of genes is reported in Appendix D and accessible via web through the BGGM web-server (http:l/149.171.168.73/microarray/arraylist.html). PCR products and clones were amplified, purified using 96 well multi-screening membrane plates (Millipore) and resuspended in 70 µL sterile Milli-Q water. Purified DNA was diluted to reach a final concentration of -200 ng µL- 1 in 150 mM NaP04 (pH 8.0). Six microliters of each sample was then transferred to a 384 well microplate for printing. Slide preparation and printing were performed at the Ramaciotti Centre for Gene Function Analysis (UNSW, Sydney, Australia). DNA samples were arrayed with four pins (SMP-3 Stealth, Arraylt, Sunny-vale, CA) at a spacing distance of 180 µm on polysine-coated 25 by 75 mm glass slides (Menzel-Glaser, Braunscheig, Germany) using a Chip WriterPro robotic printer (BioRad, Herne! Hempstead, UK) under conditions of 65% relative humidity. Each gene probe was spotted in duplicate at a volume of 600 pL (spot diameter of 100 µm), and each glass slide contained two copies of the BGGM1 array. Following printing, slides were allowed to age for 3 days before post-processing. DNA microarrays were then baked at 80°C for 30 min and kept in the dark for long term storage. Before use, gene arrays were rehydrated over a 40°C water bath for 15 s and dried on a heating block at 90°C for 5 seconds. DNA on the microarrays was fixed by UV cross-linking at 60 mJ in a Hoefer UVC UV Crosslinker 115 VAC (Amersham Pharmacia Biotech, Piscataway, NJ), washed once in 0.2% SDS and twice with water. The glass slides were then treated with 6 g succinic anhydride (Sigma) dissolved in 350 mL of l-methyl-2-pyrrolidinone (Aldrich) and 15 mL of 1 M boric acid (pH 8.0) (Aldrich). Immediately following blocking, the DNA was denatured by immersing the slides in MilliQ water at 95°C for 2 min. The microarrays were then rinsed briefly in 95% ethanol at 4 °C for 1 min, spun dry at 500 x g for 5 min, and stored dry in the dark. 60 Cli.apter2 Materials and M ethals 2.10 NUCLEIC ACIDS LABELLING Fluorescently labelled nucleic acids were prepared indirectly by incorporating amino-ally! dUTP followed by coupling with the fluorescent dyes. 2.10.1 Preparation of DNA One microgram aliquots of genomic A. circinalis DNA were prepared by digestion with Rsal as described in Section 2.7.2. DNA was then extracted with phenol and precipitated with ethanol following standard protocols (Sambrook et al. 1989). 2.10.2 Preparation of total RNA Extracted and purified total RNA was quantified by UV spectroscopy (DU® 640 UV Spectrophotometer, Beckman, Fullerton, CA), with 10 µg of RNA retro-transcribed for each microarray hybridisation using the following protocol. RNA was diluted in 11.5 µL of water, combined with 1 µL of 50 µM random nonamers (Sigma) and heated at 65°C for 10 min. For each reaction, 4 µL 5x RT buffer (Invitrogen), 2 µL 0.1 M DTT, 0.5 µL 10 mM dNTPs and 1 µL (200 U) Superscript enzyme (Invitrogen) were combined on ice in a final volume of20 µL. Reactions were incubated at 42°C for 2.5 h, RNA hydrolysed by adding 1 µL of 1 M NaOH per mixture and then incubated at 65°C for 10 min. Solutions were buffered with 2.5 µL of 2 M Tris-HCI (pH 7.5) and the cDNA purified using the QIAquick PCR purification Kit (QIAGEN, Germantown, MD). 2.10.3 Labelling of genomic DNA and cDNA Amino-ally! (aa) dUTP labelling of cyanobacterial genomic DNA or cDNA was achieved as follows. DNA was diluted in 38 µL of water and denatured at 99°C for 10 min and chilled on ice. Five µL of l0x NEB labelling buffer (New England Biolabs), 3 µL of amino-ally! dNTP mix (3 mM dGTP, dATP, dCTP, 1.8 mM aa-dUTP, 1.2 mM 61 Chapter 2 Materials and MethaJs dTTP) and 2 µL (10 U) of the large Klenow fragment of DNA polymerase I (Promega) were combined in a final reaction volume of 50 µL. The reaction mixture was incubated at 37°C for 2 h and unincorporated amines removed by QIAquick purification (QIAGEN). DNA samples were dried in a speed-vac, resuspended in 9 µL of 0.1 M NaHCO3, pH 9.0, and added to 2 µL of prepared Cy3 or Cy5 dye aliquots (Amersham Pharmacia Biotech). Reactions were incubated for 60 min at room temperature in the dark and purified (QIAquick PCR Kit). Labelled DNA samples were dried to -20 µL, combined according to the different experiments, and evaporated to dryness. 2.11 MICROARRAY HYBRIDISATION For each single hybridisation, fluorescently labelled DNA was resuspended in 20 µL of hybridisation solution, containing DIG Easy buffer (Roche Applied Science, Penzberg, Germany) and 500 µg of yeast tRNA (Sigma), denatured for 2 min at 99°C, cooled to ambient temperature and applied to the microarray slide. Glass 22 x 22 mm coverslips were placed over the solution and hybridisation was performed overnight at 37°C in a water-tight humidified hybridisation chamber. Array slides were washed in two stages: three washes at 50°C for 15 min with pre-heated 1 x SSC buffer 0.1 % SDS followed by three rinses at room temperature with decreasing concentrations of SSC buffer (0.5 x, 0.25 x, 0.1 x in Milli-Q water). Slides were spun dry at 500 x g for 5 min, and kept in the dark prior to scanning. 2.12 MICROARRAY SCANNING, DATA ACQUISITION AND STATISTICAL ANALYSIS Clean slides were scanned with the ArrayWorx "e" Microarray Scanner from Applied Precision, Inc. (Issaquah, WA). Scanned slide images were generated automatically with ArrayWorx software provided with the ArrayWorx Scanner. Images were quantified using the GenePix Pro software from Axon Instruments (Foster City, CA). Erroneous spots were manually flagged and removed from the final data set. All microarray data were filtered to remove any spots in which less than 60% of the signal 62 Cliapter 2 Mat:erials and Methals pixels exceeded the local background value for both lasers (595 and 685 run). The median Cy5/Cy3 ratio automatically generated by the GenePix Pro software for the filtered data for each spot was used for subsequent normalization and analysis. Normalised microarray data sets were assembled and analysed by hierarchical clustering analysis with the software GeneSpring 5.0.2 (Silicon Genetics, Redwood City, CA, USA). Pearson and Spearman correlation coefficients, as well as Euclidean distances, were used as a measure of similarity for the analysis of both gene and experiment data sets. 63 Chapter 3 Ejfects ifL ida:aine an STX prrxluctinn CHAPTER3 ENHANCEMENT OF INTRACELLULAR SAXITOXIN ACCUMULATION IN CYLINDROSPERMOPSIS RACIBORSKII T3 BY LIDOCAINE HYDROCHLORIDE Seven deadly sins Seven ways to win Seven holy paths to hell And your trip begins (A. Smith, B. Dickinson) 64 Chapter 3 Ejfects cfL ida:aine an STX pmductian 3.1 BACKGROUND Lidocaine (2-( diethylamino )-N-(2,6-dimethylphenyl)acetamide) is a synthetic compound widely used in medical and veterinarian practice as a local anaesthetic and intravenous antiarrhythmic. Its physiological effect is mainly related to the blockage of sodium channels in excitable cells. Lidocaine, in animals, binds to voltage-gated sodium channels and inhibits the activation of the inward Na+ ion current (Postma and Catteral 1984) and its effects have been compared to those of natural channel-blocking toxins (Narahashi et al. 1994, Sunami et al. 2001). This local anaesthetic has also been shown to have affinity for other receptors on the plasma membrane (PM), such as the Na+ /H+ exchanger (Shibamoto et al. 1990, Ohsuka et al. 1994, Bidani and Heming 1997) and a protein kinase (Kanai et al. 2001 ). The mechanism of action of lidocaine on the sodium cycle of plants, algae and prokaryotes has still to be fully understood, although there are several reports confirming that this agent has effects on membrane ion fluxes in a broad spectrum of organisms. Lidocaine has been used to study membrane properties of algal, microalgal and bacterial cells (Scholuebbers et al. 1984, Bloodgood and Salomonsky 1990, Nosaka et al. 1992, Tisa et al. 2000), in which this compound was able to affect gliding or motility, chemotaxis, and certain other PM related functions (Todo and Yonei 1983, Scholuebbers et al. 1984). Depolarization and imbalance of the PM potential in Gram positive and Gram-negative bacteria has also been associated with the antibacterial activity of lidocaine (Ohsuka et al. 1994). More recently, the activity of lidocaine hydrochloride and similar Na+/K+ channel-blockers has been assayed in Synechococcus sp., a freshwater unicellular cyanobacterium, and an effect on Na+ fluxes by these compounds has been proposed (Allakhverdiev et al. 2000). Lidocaine hydrochloride and related compounds, on the other hand, have been shown to promote the growth of plants and microorganisms (Cachita-Cosma and Ardelean 1996). In particular, recent studies reported the stimulatory effect of local anaesthetics on the growth rate of some cyanobacterial species belonging to the genera Anabaena and Synechococcus (Suzuki et al. 2000, Suzuki et al. submitted). The aim of this study was to examine the effects of lidocaine hydrochloride on the neurotoxic cyanobacterium C. raciborskii T3. Physiological responses to the local 65 Chapter 3 E jfects ifL ida:aine an STX prrxluctwn, anaesthetic were recorded as both growth rate (GR) and intracellular STX concentration. Although strain T3 is known to produce Cl/C2 toxins, the focus of the present investigation addressed only STX content, which is the most important and harmful toxin of the PSP-toxins family. Moreover, the roles of Na+ concentration and pH on the activity of lidocaine hydrochloride were evaluated. The promoting effect of this compound on intracellular STX accumulation is discussed with regards to the possible connection between STX regulation and cyanobacterial homeostasis. To date, this study represents the first report of a specific factor promoting the accumulation of STX. 3.2 EXPERIMENTAL PROCEDURES 3.2.1 Reagents All reagents were purchased from Sigma-Aldrich (Dorset, UK). A Lidocaine hydrochloride solution (100 µM) was prepared fresh in Milli-Q water prior to each experiment and diluted in the culture medium to reach final test concentrations. 3.2.2 Growth conditions and cyanobacterial cultures C. raciborskii strain T3 was maintained in MLA medium (Appendix B) modified by the addition of NaHCO3 to a final concentration of 2 mM (MLA-E) and adjusted to pH 9.5, as well as in standard MLA (0.2 mM NaHCO3) adjusted to pH 7, 8.5 or 9.5. Cultures were grown in glass 250 mL flasks in a thermostatically controlled bath at a constant temperature of 26° C and under continuous irradiance of cool white light at an intensity of 15 µmol photon·m-2-s- 1. Cylindrospermopsis cultures were monitored for growth by recording the optical density at 0D150 (Section 2.2.1) in 1 cm disposable cuvettes. Culture in late-logarithmic growth phase was used as inocula for new batch cultures (150 mL) exposed to lidocaine hydrochloride at O (control), 0.01, 0.1 and 1 µM. Culture densities were monitored by means of OD1so measured once every 24 h. The 66 Chapter 3 E jf«ts cfL ida:aine on STX praluctim, initial cell density was set to approximately 0.1 and then measured during the active growth phase (15 days). The growth rates (GR), expressed as increments in OD750 · daf 1, were calculated as the slope ± standard error of the linear fit applied to the linear growth phase (8 days), while the final growth yield was calculated from the Boltzman functions asymptote applied to total growth curves. The time course experiment was carried out for 144 h in 1 L flasks with 600 mL of culture, while the evaluation of pH effect on lidocaine hydrochloride activity was performed on 150 mL cultures induced for 2 h. In both studies C. raciborskii was exposed to 1 µM of lidocaine hydrochloride. All experiments were performed in triplicate. 3.2.3 Extraction for HPLC analysis Cyanobacterial cells (100 mL of culture) in the late-logarithmic growth phase were harvested by centrifugation (15 min, 2,000 g) in plastic tubes and lysed by sonication (3 min, 100 W) in 3 mL Milli-Q water. The cellular extract was prepared by centrifugation (10 min, 10,000 g) to remove cell debris and stored frozen at -20°C until HPLC analysis. The screening for PSP-toxins was performed by prechromatographic oxidation followed by HPLC separation as described in Section 2.3.1. 3.3 RESULTS 3.3.1 Effect of lidocaine hydrochloride on growth The ability of lidocaine hydrochloride to stimulate the growth of C. raciborskii T3 at concentrations of 0.01 to 1 µM was investigated during the active cyanobacterial growth phase ( 15 days) and the results summarized in Fig. 3 .1. Cell density was recorded daily as OD at 750 nm, with increased growth evident for lidocaine hydrochloride at 0.1 and 1 µM based on the cell density ratio of the stimulated cultures compared to the controls (Fig. 3. lA). During the course of these experiments, cultures exposed to different concentrations of lidocaine hydrochloride were examined 67 Chapter 3 Ejfeas cfL ida:aine an STX praluawn microscopically for variations in cyanobacterial morphology. No changes in the mean cell size or trichome structure, as well as pigmentation, were evident in the lidocaine hydrochloride treated samples. 1.30 A) -o-0.01 µM -e 1.25 -o-0.1 µM C: -t::.-1 µM -8 1.20 lil -Cl 1.15 0 1.10 -Q) -a. E 1.05 m g 1.00 -Cl 0 0.95 0.90 0 2 4 6 8 10 12 14 16 0.5 8) C) 0.4 0.3 0.2 0 LO r--. 0.1 0 0 0.5 E) 0.4 0.3 0.2 --Boltzman Fit 0.1 ··unear Fit 0 2 4 6 8 10121416 0 2 4 6 8 10 12 14 16 days Figure 3.1 (A) Effect of lidocaine hydrochloride on the photoautotrophic growth of C. raciborskii T3. The experiments were performed in triplicate and the points reported represent the ratio of the tested sample over the controls expressed as Mean ± SE. Growth curves of C. raciborskii T3 cultures exposed to the three different doses of lidocaine hydrochloride, 0 (B), 0.01 (C), 0.1 (D) and 1 µM (E). Dash lines represent the linear fit functions applied to the linear growth phase (8 days), while straight lines represent the Boltzman fit of the original optical density data. Error bars are ± SE. 68 Chapter 3 Ejfects ifL ida:aine an STX prrxluctwn For quantitative analysis of this growth stimulation, data points were analysed by means of sigmoidal (Boltzman fit) and linear fit functions (Fig. 3.1, B-E). The Boltzman asymptotes applied to total growth curves (Table 3.1) of C. raciborskii cultures only showed an increase in the final growth yield for exposure to lidocaine hydrochloride at 0.1 and 1 µM (Fig 3.2A), which were calculated respectively as 18.2 ± 5.6 and 17. 7 ± 3.1 % relative to the controls. The lowest dose of lidocaine hydrochloride, 0.01 µM, showed negative growth yield (-5.1 ± 2.2 %) which was, however, still within the standard error range (± 6.1 %) of the measurements of control cultures densities. Table 3.1 Principal parameters of the Boltzman fit functions applied to the growth curves of C. raciborskii T3 cultures exposed to lidocaine hydrochloride at 0 (control}, 0.01, 0.1 and 1 µM. The growth yield was calculated as (0D750) ± SE. Growth ±SE x,2 yield Control 0.351 0.0215 4.876 · 10-5 0.01 µM 0.333 0.0072 5.324 · 10-5 0.1 µM 0.415 0.0233 5.108 · 10-5 lµM 0.413 0.0130 2.924 · 10-5 Cyanobacterial growth rates (OD75o · daf1) were calculated as the slope of the linear fit function (Table 3.2) applied to the linear growth phase (first 8 days, Fig. 3.1, B-E) of C. raciborskii cultures exposed to lidocaine hydrochloride at 0.01, 0.1 and 1 µM. The GR was stimulated in a dose-dependent manner, as shown in Fig. 3.2 (B).The highest incremental change in the rate of cell proliferation was recorded for 1 µM lidocaine hydrochloride (39 ± 4.8%), while 0.1 and 0.01 µM doses resulted in increases of 34.9 ± 4.7% and 9.9 ± 8%, respectively, relative to the controls. 69 Chapter 3 Efleets ifLida-aim an STX prrxluctim, Table 3.2 The GR (increment in OD750 I day) ± SE for the linear growth phase (8 days) of C. raciborskii T3 cultures exposed to lidocaine hydrochloride at 0 (control), 0.01, 0.1 and 1 µM. Intercept Slope ±SE R p Control 0.105 0.0215 8.0141 · 10·4 0.995 < 0.0001 0.01 µM 0.102 0.0231 8.4488 · 10-4 0.996 < 0.0001 0.1 µM 0.092 0.026 1.02. 10·3 0.995 < 0.0001 lµM 0.923 0.027 7.3445 · 10·4 0.997 < 0.0001 25 A) "'C Q) 20 ·5,. T ..c 15 1 10 !i.;;. C) 5 -;:R. 0 0 -5 T I 1 I Control 0.01 µM 0.1 µM 1 µM 0.0275 B) 2 ~ 0.0250 ..c l (!)e 0.0225 0.02004-.-,-,.-~~~~--~~-~~~~ Control 0.01 µM 0.1 µM 1 µM Figure 3.2 The growth yield (% over control ± SE) (A) and GR (OD750 · day°1 ± SE) (B), calculated from the Boltzman asymptotes and the slope of the linear fit functions applied to the C. raciborskii T3 cultures exposed to lidocaine hydrochloride. The curve in (B) represents the sigmoidal fit of data. 70 Chapter 3 Ejfects ifL id«aine an STX pra/uctinn 3.3.2 Effect of lidocaine hydrochloride on STX accumulation The cyanobacterial cultures were extracted at the end of the growth phase and analysed for intracellular STX concentration by means ofHPLC. The chromatograms in Fig. 3.3 show the HPLC profile of the C. raciborskii T3 cell extract (B) in comparison with the standard mixture of PSP-toxins comprising gonyautoxin 2/3 (GTX2/3) and STX (A). The cyanobacterium was confirmed to produce STX. 5 A) 4 GTX2/3 STX 3 2 1 i 0 ~ -1 C: 5 B) i 4 0 ::::, 3 [I 2 1 0 2 4 6 8 10 12 14 16 18 20 22 24 Elution time (min) Figure 3.3 HPLC chromatograms of the STX and GTX2/3 standard solutions (A) and a control culture of C. raciborskii T3 (B). STX concentrations in the extracted cultures were calculated by comparing the peak areas of STX standard solution (201 µg / L) to those of the analysed samples. The dosing of cultures grown in MLA-E medium, pH 9.5, with the three different concentrations of lidocaine hydrochloride stimulated STX accumulation in C. raciborskii T3 cells in comparison to the controls (Fig. 3.4). Concentrations were calculated by comparing the peak area corresponding to STX in the culture extracts to that of the STX standard solution and normalising for cell number by the optical density at 750 nm of the culture sample. As with GR, the cyanobacterial intracellular concentration of STX increased in a dose-dependent manner in response to the presence oflidocaine hydrochloride in the culture medium (Fig. 3.4), giving a 14 ± 8.4% increase 71 Chapter 3 E jfects ifL ida:aine an STX prrxluction over the untreated cultures for the 0.01 µM dose, 49 ± 27.8% for 0.1 µMand a 114 ± 12.9% increase in STX after the addition of 1 µM lidocaine hydrochloride. Exposure to lidocaine hydrochloride did not affect the chromatographic profile of toxins produced by C. raciborskii T3 as indicated by the HPLC chromatograms for the various stimulated cultures and controls. These results suggested that lidocaine hydrochloride promoted STX accumulation in C. raciborskii T3 without affecting the toxin profile of the cyanobacterium. ..... 550 ' ~ 500 8 450 C: 0 400 +:i ~ 350 +-' C: 300 ~ C: 250 8 200 150 (/)~ 100 Control 0.01 µM 0.1 µM 1 µM Figure 3.4 The intracellular STX concentrations ([STX] = µg / L ± SE) detected in C. raciborskii T3 cultures exposed to lidocaine hydrochloride. All data represent the mean from three independent cultures and were normalised by the 0D750 of the sample. The curve represents the sigmoidal fit of data. 3.3.3 Time course of lidocaine hydrochloride effect on STX intracellular concentration The time course of intracellular STX concentrations was analysed in cultures of C. raciborskii T3 exposed to lidocaine hydrochloride at 1 µMin MLA-E medium. The mean STX concentrations at 0, 1, 2, 8, 24, 48 and 144 hours are shown in Fig. 3.5. Toxin levels appeared to have the greatest incremented change during the first two 72 Chapter 3 Ejfects ifL ida:aine an STX prrx/uction hours after initial stimulation (298.1 ± 6.4% increase over the sample at O h), decreased slightly from 8 to 48 h, and achieved the maximum concentration after 144 h (6 days), corresponding to a 305.7 ± 14% increase. In the short term, the intracellular concentration of STX showed the highest rate of change between the first and the second hour of exposure to 1 µM lidocaine hydrochloride and then increased only slightly over the next 6 h. Over the following 15 days the STX concentration decreased, reaching a value of the same magnitude as those reported above for cultures at the end of the active growth phase (data not shown). 800 " 700 ~ 8 600 C .Q 500 1§ c 400 ~ C 300 8 ~ 200 I- 100 0 1 2 3 4 5 6 7 8 20 40 60 80 100 120 140 160 Time (h) Figure 3.5 Time course of the intracellular STX concentration in C. raciborskii T3 cultures exposed to 1 µM lidocaine. Values represent the average from three independent cultures and are expressed as mean STX concentration ([STX] = µg / L) ± SE normalised by the 0D750 of the culture. 3.3.4 Effect of pH and Na+ concentration on the lidocaine hydrochloride -induced STX accumulation In order to evaluate whether the effect of lidocaine hydrochloride on STX intracellular accumulation in C. raciborskii T3 was due to its channel-blocking properties, we assayed STX levels in cyanobacterial cultures exposed to 1 µM lidocaine 73 Chapter 3 Ejfects ifLida:aine an STX prrxlucti,an, hydrochloride for two hours in standard MLA medium (0.2 mM NaHCO3), at pH 7, 8.5 and 9.5. The results are summarised in Fig. 3.6. The lidocaine hydrochloride-induced STX accumulation in C. raciborskii T3 was dependent on alkaline pH. Moreover, at neutral pH the local anaesthetic seemed to reduce STX intracellular content, rather than having no effect. The percentage variation relative to the controls were -14 ± 11.6, 8.2 ± 8.2 and 35.2 ± 6.2 % for pH 7, 8.5 and 9.5, respectively. 50 pH9.5 e 40 C: 30 -8 pH8.5 .... 20 ~ 10 pH? C: 0 :;::::. 0 ·;::m m -10 > -20 0~ -30 -40 Figure 3.6 The effect of 2 h lidocaine hydrochloride 1 µM exposure at pH 7, 8.5 and 9.5, on intracellular STX accumulation in C. raciborskii T3. The experiment was performed in triplicate, in MLA medium 0.2 mM NaHC03• Values are expressed in percentile, mean from three independent experiments, as increment over the control levels ± SE. Average control value = 186.49 µg (STX) I L · 0D150. Comparing lidocaine hydrochloride-induced STX accumulation in C. raciborskii T3 cultures exposed to the local anaesthetic in MLA-E medium with 2 mM NaHCO3 or in MLA with 0.2 mM NaHCO3, an order of magnitude difference in the stimulation effect was noted. Within two hours after the induction, the STX intracellular concentration increased 300% in the presence of 2 mM Na+ (Fig. 3.5) and 35% with 0.2 mM sodium ion (Fig. 3.6). These results suggested that the effect of lidocaine hydrochloride on STX accumulation may have been mediated by the same cellular processes involved in the maintenance of pH and Na+ homeostasis in the cyanobacterial cells. 74 Chapter 3 Ejfects cfLidcx:aim on STX prrx/uction, 3.4 DISCUSSION The local anaesthetic lidocaine hydrochloride stimulated the growth and, at the same time, promoted intracellular saxitoxin accumulation in cultures of the cyanobacterium C. raciborskii T3. The growth stimulation effects on the cyanobacterial cultures were similar to those observed for lidocaine hydrochloride and related compounds in previous studies on both freshwater filamentous and unicellular cyanobacteria (Suzuki et al. 2000, Suzuki et al. unpublished results). Local anaesthetics have been shown to promote the photoautotrophic growth of Anabaena cylindrica, Anabaena variabilis and Synechococcus leopoliensis within the same range of concentrations tested in the present study. Lidocaine hydrochloride, in the range of 0.1-1.0 µM, has been shown to be the most effective synthetic local anaesthetic for enhancing growth rate, with an increase of 80-100% over control cultures (Suzuki et al. submitted). C. raciborskii T3 was less sensitive to the same concentrations of lidocaine hydrochloride than these previously tested cyanobacteria, achieving a GR increase of 35-39% over control levels, similar to the response observed for procaine supplementation of Anabaena and Synechococcus cultures (Suzuki et al. 2000). The growth rate enhancing properties of lidocaine hydrochloride also increased the final growth yield in C. raciborskii T3. The promotion of cyanobacterial growth rate induced by lidocaine hydrochloride is well established, however few tentative studies have been made to elucidate the mechanism by which this agent and related compounds could increase the rate of cell division. Recently, the effect of local anaesthetics has been compared to the one exerted by certain phytohormones, such as indole-3-acetic acid (IAA). This auxin and lidocaine hydrochloride have similar growth-rate promoting properties and homologous chemical and structural characteristics (Suzuki et al. unpublished results). In this model, the channel-blocking activity of local anaesthetics may suggest a possible growth enhancement pathway mediated by changes in cellular ion fluxes, as has previously been described for the effect of auxins on plants (Kim et al. 2001). Recently, the ability of cyanobacteria to produce IAA has also been demonstrated (Sergeeva et al. 2002). In C. raciborskii T3, lidocaine hydrochloride at 1 µM markedly stimulated STX 75 Chapter 3 Ejfects ifL idcraine an STX praluction intracellular accumulation. The dose-response increases induced by lidocaine hydrochloride on GR and STX concentration (Fig 3.2B, Fig. 3.4) showed different trends, suggesting independent effects on growth and intracellular STX levels. This phenomenon was confirmed during the time course studies. The highest rate of incremental change in STX accumulation was detected within the first and the second hour after induction. Subsequently, cellular STX concentrations remained almost constant for the following week, decreasing only slightly as the culture aged. This increase in STX intracellular concentration could be explained therefore by the upregulation of toxin biosynthesis, repression of STX catabolism, or changes in extracellular transport in C. raciborskii T3. Little data is available, however, regarding possible degradation pathways of STX and related compounds. Recently it has been suggested that this degradation might follow the same catabolic metabolism of purines (Pomati et al. 2001). In addition, there is no evidence in the literature regarding inhibition of this catabolism by lidocaine hydrochloride and related compounds. Moreover, these local anaesthetics are structurally very different from PSP-toxins and it is unlikely that these synthetic molecules might be involved in the cyanobacterial purine degradation pathway. The timing of STX intracellular accumulation indicated by this study, on the other hand, suggests a direct response to lidocaine hydrochloride through transcriptional or post-transcriptional activation of STX synthesis. Though the eventual increase in STX biosynthesis in C. raciborskii T3 could be simply due to metabolic stimulation, we hypothesize that STX is regulated in consequence to variations in cell homeostasis, which is perturbed by the activity of lidocaine hydrochloride. During the course of the present study, the effect of lidocaine hydrochloride was found to be dependent on pH and Na+ concentration of the culture media. In the presence of lidocaine hydrochloride, an order of magnitude decrease in Na+ ion concentration (from 2 mM to 0.2 mM) corresponded to an order of magnitude decrease in intracellular STX levels (from ea. 300% to 35%). The alkaline pH dependence of lidocaine hydrochloride-induced STX accumulation in C. raciborskii T3 could be the result of two distinct factors. First, lidocaine hydrochloride can exert its channel blocking properties only when the net charge of the molecule is equal to 0, which is achieved when pH ~ 8.5 (Hille 1997). Second, alkaline pH represents a stress for 76 Chapter 3 Ejfects ifL ida:airx? an STX prrxluawn, cyanobacterial cells which utilise channels and Na+ /H+ exchangers to maintain cytoplasmic pH neutrality. Under this condition, a H+ gradient is created by photosynthesis (Brown et al. 1990), and Na+ is extruded from the cytosol during the process. Together with the evidence that, in C. raciborskii T3, lidocaine hydrochloride induced STX accumulation was dependent on the NaHCO3 concentration of the culture medium, suggests a possible interference of lidocaine hydrochloride with sodium fluxes in cyanobacterial cells. Such evidence may also indicate a potential regulation of STX biosynthesis based on variations of intracellular concentration of Na+ ion or pH. This hypothesis, together with the role of channels and membrane transporters regulating homeostasis and/or the intracellular concentration of STX, are currently under investigation. For example, PSP toxins are considered to be strictly endotoxins (released into the environment only after cell lysis) (Negri et al. 1997). It has recently been discovered that other cyanobacterial toxins, such as microcystin, may be subject to active transport outside the cyanobacterial cell by means of ABC transporters (Dittmann et al. 2001, Pearson et al. 2001). 3.5 CONCLUSIONS The present study provides initial evidence of a specific factor promoting STX accumulation in a cyanobacterium and demonstrates the usefulness of well defined pharmaceuticals for studying physiology and toxin production in cyanobacteria. Traditionally, this type of study would have been approached using the predicted effects of known ecological parameters. Lidocaine hydrochloride and related agents are potential tools to further investigate the metabolism and regulation of saxitoxin, its derivatives, and other alkaloid biotoxins. The results revealed here indicate an association between cellular alkaline pH/Na+ homeostasis and STX production in Cylindrospermopsis raciborskii T3. This association may be present in various other species of cyanobacteria, bacteria and algae also. 77 Chapter 3 Ejfects ifLidocaine an STX prrxluctim, 3.6SUMMARY The metabolic effects of three different concentrations of lidocaine hydrochloride (0.01, 0.1, 1 µM) on growth and saxitoxin (STX) production by the freshwater cyanobacterium Cylindrospermopsis raciborskii T3 were analysed. Lidocaine hydrochloride increased both the growth rate and the final growth yield, in the toxic cyanobacterium, with a maximum of 25% and 18% for a 1 µM dose, respectively. Moreover, C. raciborskii T3 samples harvested at the end of the growth phase and analysed for STX content by high performance liquid chromatography showed an increase in STX intracellular concentration of 14.3 and 49.3% after exposure to 0.01 µM and 0.1 µM lidocaine hydrochloride, respectively, while 1 µM lidocaine hydrochloride resulted in a 113.8% incremental change in STX content. The time course of the 1 µM lidocaine hydrochloride effect showed the highest rate of increase in mean STX intracellular concentration (298.1 %) within the first 2 hours after induction. The increase in STX content induced by lidocaine hydrochloride in C. raciborskii T3 was dependent on the concentration of Na+ ions in the culture medium and alkaline pH. The results suggest an influence of lidocaine hydrochloride on membrane ion fluxes and a potential linkage between cyanobacterial homeostasis and STX regulation is hypothesised. 78 Chapter4 STX M etabdismand Salium C')de CHAPTER4 INTERACTIONS BETWEEN INTRACELLULAR Na+ LEVELS AND SAXITOXIN PRODUCTION IN CYLINDROSPERMOPSIS RACIBORSKII T3 Remembering games, and daisy chains and laughs, got to keep the loonies on the path. (R. Waters) 79 Chapter4 STX Metahdismand Salium Cyle 4.1 BACKGROUND Although much is known about the pharmacology and chemistry of PSP toxins, the metabolism and physiology of saxitoxin-producing cyanobacteria have been studied rarely. The stimuli inducing or repressing STX production in cyanobacteria are currently unknown, as well as the metabolic role of PSP toxins within the producing microorganisms. Laboratory studies documented that cyanobacterial isolates preferentially produce PSP toxin under conditions which are most favourable for their growth. In PSP toxin-producing dinoflagellates however, high salinity has been found to increase cell toxicity (see Section 1.3.5.4). The effects of salt stress and pH variations on cyanobacterial PSP toxins production have yet to be investigated. Alkalinity of water is a main feature characterising toxic cyanobacterial blooms. This parameter, combined with high salinity, has been reported in correlation with blooms of PSP toxins producing species such as Anabaena circinalis in Australia (Bowling and Baker 1996). In this study the effects of pH, salt and of two channel-blockers ( amiloride and lidocaine) on total Na+-K+ cellular content and STX accumulation were investigated in the cyanobacterium C. raciborskii T3. The results presented suggest that in C. raciborskii T3, STX production is responsive to changes in intracellular sodium levels. The present study also reports essential elements for future research on saxitoxin biosynthesis and indicates a possible correlation between STX metabolism and cyanobacterial homeostasis. 4.2 EXPERIMENTAL PROCEDURES 4.2.1 Reagents All reagents were purchased from Sigma-Aldrich (Dorset, UK). Lidocaine hydrochloride and amiloride solutions (100 µM and 100 mM, respectively) were prepared freshly in Milli-Q water prior to each experiment and, diluted in culture medium to obtain the final concentrations required. 80 Chapter4 STX Metabdismand Salium Cyk 4.2.2 Growth conditions and cyanobacterial cultures Cylindrospermopsis raciborskii strain T3 was maintained in ASM-1 medium (Gorham et al. 1964) adjusted to pH 9.5. Cultures were grown in glass 250 mL flasks in a cabinet at a constant temperature of 26° C and under continuous irradiance of cool white light at an intensity of 15 µmol photon m-2 s- 1. Cylindrospermopsis cultures were monitored spectrophotometrically by recording the 0D750 and microscopically by viewing cells under a phase contrast microscope as reported in Section 2.2.1. For the growth experiments, a culture in late-logarithmic growth phase was used as inocula for new batch cultures exposed to 0 (control), 1 and 10 mM of NaCl. The initial cell density was adjusted to approximately 0.1 0D750 and then measured for 7 days once every 24 h. Short term experiments were carried out for 2 h or 4 h with cultures in standard growth conditions at pH 9.5 or at pH 7.5, when required. For all experiments, cultures in late logarithmic growth phase were exposed to NaCl (1, 5, 10 mM), lidocaine at 1 µMand amiloride 1 mM. These concentrations of lidocaine and amiloride were chosen based on previous studies on cyanobacteria (Chapter 3) and general physiological investigations on animal sodium homeostasis (Kim and Smith 1986). To evaluate the effect of pH on total Na+- K+ cellular levels and STX accumulation, aliquots of the same culture were adjusted to different pH values and analysed after 2 h. All experiments were performed in triplicate or quadruplicate. 4.2.3 Flame photometry analysis Total cellular Na+ and K+ levels m cyanobacteria were assayed by flame photometry. Two millilitre aliquots of C. raciborskii T3 cultures were collected by centrifugation in 2 mL plastic tubes at 11000 g for 15 min. Samples were harvested immediately after exposure (0 min) and at 30, 60 and 120 min. The control culture (unexposed) was monitored for an additional sample at 90 min. In the lidocaine and amiloride experiments, culture aliquots were withdrawn prior to exposure (-5 min) and immediately after the addition of the agents (0 min). All sampled pellets were 81 Oiapter4 STX Metabdismand Salium Cyle resuspended in 0.5 mL of diluent flame solution and analysed for total Na+ - K+ cellular content as described in Section 2.4. 4.2.4 Extraction and HPLC analysis Cyanobacteria (100 mL culture) were harvested after 0, 30, 60, 120 or 240 min by centrifugation (15 min, 4000 g), cells resuspended for extraction in 3 mL Milli-Q water and lysed by sonication (3 min, 100 W). The supernatant (growth medium) was either discarded or filter-sterilised (0.2 µm membrane), freeze-dried and analysed after resuspension in 2 mL MilliQ-water. Cellular aqueous extracts were prepared by centrifugation (10 min, 13 000 rpm) to remove cell debris and stored frozen at -20°C until HPLC analysis. Screening for PSP toxins was performed by prechromatographic oxidation as reported in Section 2.3.1. In this study, no autofluorescent peaks with the same retention time of STX standard solution were detected. STX data were normalised by the optical density at 750 nm of the culture sample to account for differences in cell numbers of the experimental replicates. 4.2.5 Total protein content Protein concentrations in the C. raciborskii T3 culture medium were determined as described in Section 2.2.2. 82 Chapter4 STX Metabdismand Salium Cyle 4.3 RESULTS 4.3.1 Effects of pH on Na+_K+ levels and STX content Total cellular Na+ and K+ levels were monitored in C. raciborskii T3 by means of flame photometry, and the results summarised in Fig. 4.IA. 0.6 0.050 A) 0.045 0.5 0.040 0.035 + 0.4 ro ·:::.:::: 0.030 z ::iE ::iE E E 0.3 0.025 ,,,,'l 0.020 0.2 0.015 :0: 0.010 0.1 -~---- 0.005 0.0 0.000 2 4 6 8 10 12 1000 8) • 800 I •I -;- I I lil I I gaoo I I :.I ~400 ' ~ 200 ,'A,'. --- 0 •--- ·------• 7.5 8.0 8.5 9.0 9.5 10.0 pH Figure 4.1 A) Total cellular Na•(•) and K+ (O) concentrations measured by flame photometry (mM ± Standard Error in the final cells suspension) in C. raciborskii T3 cell suspensions adjusted to different pH and analysed after 2 h. B) Intracellular STX concentrations (.A., [STX] = µg L-1) in cultures adjusted to different pH and analysed by HPLC after 2 h. Data were normalised by the 0D750 of the corresponding culture sample. Solid line: Gauss fit, dash lines: exponential fit. 83 Chapter4 STX Metabdismand Salium Cyle At low pH (3 .1 ), both total cellular Na+ and K+ contents were at their lowest concentration. In response to rising alkalinity, K+ levels reached their maximum for optimal growth pH (7 to 10.5), but decreased for pH values higher than 11. On the other hand, cellular Na+ content increased exponentially with the rising alkalinity of media, achieving the highest concentration at pH 12. In addition, STX intracellular concentrations, as measured by HPLC, increased exponentially in response to the rising pH of the culture medium (Fig. 4.1B), with the highest rate for pH> 9. The maximum cyanobacterial STX content was observed at the highest pH tested (10.0), a value 69- fold greater than the amount recorded at pH 7.5. 4.3.2 Effect of NaCl on growth, Na+-K+ levels and STX accumulation The effect of NaCl at 1 and 10 mM on the growth of C. raciborskii T3 was investigated for 7 days (Fig. 4.2A). A decreased growth rate was evident at day 6 for 10 mM NaCl based on the cell density ratio of the experimental cultures compared to the controls. During the course of the following experiments, cultures of C. raciborskii T3 exposed to the different agents and conditions were also monitored spectrophotometrically and microscopically for short term variations in cyanobacterial cells density and morphology. In this study, no significant changes in OD75o, mean cell size or trichome structure were evident after either 2 h or 4 h of treatment. To investigate the ability of Na+ ions to promote intracellular STX accumulation in C. raciborskii T3, cyanobacterial cultures grown at pH 9.5 were exposed to either NaCl (1, 5 and 10 mM), sorbitol at 20 mM (osmotic stress control), or MgCh at 5 mM (ionic stress control) for 2 h (Fig. 4.2B). Treatment of cultures with increasing concentrations of NaCl stimulated STX accumulation in a dose dependent manner in comparison to the unexposed and the osmotic/ionic stress controls. MgCh at 5 mM and sorbitol at 20 mM administered to cyanobacterial cells showed no statistically significant difference in STX accumulation compared with untreated controls. By ANOV A analysis and post-hoe comparison using the LSD test (Section 2.5), the mean STX concentration corresponding to cultures dosed with NaCl at 10 mM was demonstrated to be significantly distinct from average control levels. STX values 84 Chapter4 STX Metabdismand Salium Cyle recorded in the 5 and 10 mM NaCl experiments were instead statistically different from the mean concentration recorded after exposing cells to MgCh at 5 mM (P :S: 0.05). Intracellular STX levels, normalised against the hyperosmotic effect induced by sorbitol on cyanobacterial cells, increased by 24.4 ± 5.6% for 5 mM and 29 ± 6.6% after the addition of 10 mM Na+. 1.35 "7 A) 1.30 .....e C 1.25 I 8 1.20 1.15 J 1.10 0 Q) 1.05 c. 1.00 E 0.95 rn ':Q; 00g o.oo 0 0.85 ·--1--- •. 0 0.80 1 2 3 4 5 6 7 days 450 B) 400 0 I.O cf 0 350 -~ en...... 300 250 Control 1 mM 5mM 10mM 20mM 5mM NaCl NaCl NaCl Sorbitol MgCl2 Figure 4.2 A) Effect of NaCl at 1 (D) and 10 mM (O} on the photoautotrophic growth of C. raciborskii T3. Data points represent the 0D750 ratio of the test sample over the control (0 mM NaCl}, expressed as Average ± Standard Error. B) Comparison of the intracellular STX concentrations ([STX] = µg L"1 ± Standard Error) of cultures exposed to O (control}, 1, 5, 1O mM NaCl, 20 mM sorbitol and 5 mM MgCl2 for 2 h at pH 9.5. All data were normalised by the 0D750 of the corresponding culture sample. 85 d:iapter 4 STX Metabdismand Salium C'){ie This result suggested that STX accumulation in the cyanobacterial cells was promoted by Na+ ion stress. The variations induced by 10 mM NaCl on total cellular Na+ -K+ levels and STX accumulation were thus investigated (Fig. 4.3). 50 A) 40 30 20 10 0 - 4~! -f=!--- -10 C E -20 !--~I o -30 L.. -..,...... ~~~~~~~~~~~~~~ Q) > 0 20 40 60 80 100 120 0 60 30 '#- B) 50 25'7 C 40 Q) 20 e 30 ~ a. 15 ro +-' 20 0 I- +-' 10 10 ,...... , > Figure 4.3 A) Time course of total cellular Na+ - K+ levels in C. raciborskii T3 cultures exposed to 10 mM NaCl for 2 h (Na+=•. K+ =•),in comparison with untreated samples (Na+= D, K+ = o). B) Time course of the intracellular (•) and extracellular (t.) STX concentrations in cultures exposed to 10 mM NaCl for 4 h. Intracellular values were normalised by the 0D750 of the corresponding culture sample while extracellular concentrations were normalised by the total-protein content (mg ml-1) of the corresponding filter-sterilized culture medium. All values are exoressed as averaae oercentile variation over the samole at O min ± Standard Error. 86 Chapter4 STX Metabdismand Soium Cyle In comparison with untreated cyanobacteria (Fig. 4.3A), exposure to NaCl at 10 mM resulted in an increase of total Na+ levels coupled with a corresponding decrease in cellular K+. The highest and lowest values reached for the two ions over the samples at 0 min were 36 ± 10.6% and - 25 ± 4.6% for Na+ and K+, respectively. In C. raciborskii T3, intracellular STX accumulation (Fig. 4.3B) also increased in consequence to Na+ stress, with the greatest increment of change during the first two hours after initial exposure (45.8 ± 4.9% over time 0). These levels decreased slightly between 2 and 4 h (30.1 ± 1.9% ). In order to evaluate the possible involvement of extracellular transport in the observed trend of STX intracellular accumulation under Na+ stress, the time course of extracellular STX was also studied. Total protein concentrations were quantified in the filter-sterilised culture medium, as an indication of cell lysis. The extracellular concentration of STX represented 25 to 30% of the total STX content of the cultures (which ranged from 179 to 247 µg / L). Extracellular levels, after normalizing for the total protein concentration in the cell-free culture medium, were found to remain constant over time (Fig. 4.3B). These results suggested that no active extracellular transport of STX was involved in the effect seen for NaCl in C. raciborskii T3. 4.3.3 Effects of channel-blockers on Na+-~ levels and STX accumulation To confirm the observed correlation between changes in Na+ cellular levels and STX concentrations, we utilised amiloride and lidocaine to alter Na+ and K+ fluxes in C. raciborskii T3 over 2 h of exposure. The two sodium channel blockers elicited a rapid response by ion cycles, as seen by comparison of Na+ and K+ values at -5 min and 0 min (i. e., immediately after addition of the blockers). Amiloride at 1 mM (Fig. 4.4A) induced a decrease in total cellular Na+ and K+ levels. However, Na+ values diminished by only 21 ± 3.1 % after 2 h, while K+ content decreased to -76 ± 1% over samples at -5 min. Lidocaine hydrochloride at 1 µM, in contrast, promoted the cellular increase of both Na+ and K+ values within 30 minutes (Fig. 4.4B). Potassium ions eventually decreased to control levels after 60 minutes. The highest and lowest values reached for Na+ and K+, compared with samples at -5 min, were 42 ± 11 % and -15 ± 1.6% at 60 and 120 minutes, respectively. 87 Chapter4 STX Metabdismand Salium C')(ie -10 -20 -30 -40 -50 .EC -60 -70 LO I -80 ~ +:i 60 L... B) Q) 50 ~ 40 0~ 30 20 -rI~ 10 0 ----- ~ ------10 -20 1-:o: -30 0 20 40 60 80 100 120 minutes Figure 4.4 Effects of amiloride at 1 mM (A) and lidocaine hydrochloride at 1 µM (B) on the total cellular Na+ (•) and K+ {O) levels in C. raciborskii T3 cultures at pH 9.5. Values are expressed as average percentile variation over the sample at -5 min ± Standard Error. In terms of the STX intracellular concentration, at pH 7.5, amiloride only slightly inhibited the accumulation of STX by C. raciborskii T3 after 4 h exposure (Fig. 4.5A). The effect detected at pH 9.5, however, was markedly unambiguous. At alkaline pH, amiloride significantly reduced STX intracellular levels (-33.5 ± 4.3%) after 2 h exposure and remained constant during the next 2 h (-34.1 ± 4.3%). Lidocaine at 1 µM and pH 9.5, promoted STX intracellular accumulation by C. raciborskii T3 over the 4 h time course. STX concentrations (Fig. 4.5B) increased linearly with time, reaching levels 43 ± 14% greater than the sample at O min. 88 Chapter4 STX Mettlhdismand Salium Cyk 30 A) 20 10 0 -I~!------10 -20 -30 .EC: -40 0 L.. 60 ~ B) 0~ 50 40 30 1 20 10 0 -10 ?::>i~------ -20 • I -30 0 50 100 150 200 250 minutes Figure 4.5 A) Comparison of the effect of amiloride 1 mM at pH 7.5 (0) and 9.5 (•) on the time course of intracellular STX accumulation in C. raciborskii T3. B) Time course of the intracellular STX concentration in C. raciborskii T3 cultures exposed to 1 µM lidocaine hydrochloride (ti) and lidocaine 1 µM + amiloride 1 mM ('Y). All values were normalized by the 0D750 of the corresponding culture sample and expressed as the percentile average increase over the sample at O min ± Standard Error. Lidocaine-induced STX accumulation was, however, suppressed by the addition of 1 mM amiloride (Fig. 4.5B), and displayed the same temporal pattern as seen in Fig. 4.5A for pH 9.5. The positive effect of lidocaine on STX production prevailed during the first hour (18 ± 1.5% and 14.5 ± 0.7%, after 30 and 60 minutes, respectively), while at 2 and 4 h STX levels dropped compared to control values (- 21 ± 1.5%) similarly to the trend observed in Fig. 4.5A. 89 Chapter4 STX Metabdismand Salium Cyle 4.4 DISCUSSION In a previous study (Chapter 3), the effect of lidocaine hydrochloride, a synthetic Na+ channel-blocker (Suzuki et al. 2000), has been investigated on growth and STX intracellular accumulation in the freshwater cyanobacterium Cylindrospermopsis raciborskii T3. Focusing on the metabolism of saxitoxin, it was previously found that lidocaine stimulated STX intracellular accumulation, and that the increase in STX content induced by this agent was dependent both on the concentration of Na+ ions in the culture medium and on alkaline pH (Chapter 3). In the present study, the reliance on alkaline pH for lidocaine's effects was coupled by a similar dependence in the inhibition of STX accumulation by amiloride (Fig. 4.5A). Amiloride is used in human medicine as a weak diuretic with potassium sparing properties. In animals, amiloride acts on the distal renal tubule of the nephron to inhibit sodium-potassium ion exchange. This Na+ channel-blocker has been associated with the inhibition of mechano-gated epithelial sodium channels (Hamill and McBride 1996) and similar proteins utilised in the maintenance of pH and sodium homeostasis in eukaryotes (Schaefer et al. 2001). Eukaryotic channels interested by the effect of amiloride are, however, structurally unrelated to the voltage-gated sodium channels acted on by STX. Additionally, amiloride has been found to interfere with Na+ channels in prokaryotes (Rowbury et al. 1994), including the cyanobacterium Synechocystis PCC 7120 (Maestri and Joset 2000). Together with the pH dependence of the effects induced by lidocaine and amiloride, here it was documented that the accumulation of STX itself is dependent upon alkaline pH (Fig. 4.18). The majority of freshwater cyanobacteria, including C. raciborskii T3, are alkaliphilic microorganisms, growing naturally and preferentially at pH>8. In alkaliphilic bacteria, the principal active process employed for the maintenance of cytoplasmic pH neutrality involves the cycle of ions (mainly Na+ and K+) across cell membranes (for review, see Horikoshi 1991, Krulwich et al. 2001). In this study, the predicted imbalance of total cellular Na+-K+ induced by applied pH and sodium stresses was verified (Fig. 4.1-4.3). In cyanobacteria however, K+ is thought to play a minor role and intracellular pH neutrality is achieved by net H+ accumulation coupled to Na+ efflux as mediated by the Na+/H+ antiporter (Lengeler et al. 1999, Maestri and Joset 2000, 90 Chapter4 STX Metabdismand Salium Cyf,e Waditee et al. 2001 ). This process is energised by an imposed proton motive force (Apte and Thomas 1986, Sonoda et al. 1998), with uptake of Na+ required in alkaline conditions. Na+ uptake can be achieved by general sodium/solute symporters, cation channels (Miller et al. 1984, Krulwich et al. 2001) or pH-gated Na+ channels (Lengeler et al. 1999). A schematic diagram summarising proteins involved the sodium cycle is shown in Fig. 4.6. Na+ Na+ Na+ Figure 4.6 Primary elements which have defined roles in the Na+ cycle and alkaline pH homeostasis of facultatively alkaliphilic microorganisms. Na+ ions can be removed from the cytosol either by H+ exchangers or by ATP-dependent ABC type sodium transporters. Na+ uptake can be achieved by sodium/solute symporters and cation channels, such as NtpJ-, KtrB-, MotAB/PomAB-, and HKT- 1-like proteins (Miller et al. 1984, Krulwich et al. 2001, Shibata et al. 2002). Homologues of these molecules are present in cyanobacteria (Kaneko et al. 1996, Waditee et al. 2001, Shibata et al. 2002, Kaneko et al. 2001 ). The existence of a pH-gated sodium channel (pH-Na) is also hypothesised (Lengeler et al. 1999). Therefore, to study in detail the interaction between cellular Na+ levels and STX accumulation, for subsequent experiments we chose the strain optimal conditions for growth and STX production, corresponding to pH 9.5, which are also associated with active Na+ homeostasis. 91 Chapter4 STX Metabdismand Sodium Cyk The possibility of an extracellular transport of STX cannot be refuted or supported by the data presented in this study (Fig. 4.3B). However, based on the time course of extracellular concentration of the toxin under salt stress, no evidence was found that indicated this process was involved in the changes of STX content shown by C. raciborskii T3. This consideration is consistent with previously published data indicating no export of PSP toxins by the cyanobacterium Anabaena circinalis (Negri et al. 1997). Given the promoting effect of NaCl on STX intracellular accumulation (Fig. 4.2) and the strong correlation seen in total cellular Na+ and intracellular STX over time (Fig. 4.3), our results suggest that STX metabolism can somehow be regulated by Na+ levels within the cyanobacterial cell. To verify this hypothesis, amiloride and lidocaine hydrochloride were employed to modulate cellular Na+ and K+ levels. According to the model of alkaline pH homeostasis previously discussed, the inhibition ofNa+/H+ antiporters would result in a net Na+ intracellular accumulation, while the blockage of sodium uptake would lead to a concomitant cytoplasmic decrease in this ion. Here we confirmed that amiloride and lidocaine can interfere with cyanobacterial Na+ uptake and Na+ export mechanisms, respectively, affecting in opposite ways the total cellular sodium concentrations (Fig. 4.4). In addition, such variations in total cellular Na+ levels were observed to be coupled with corresponding changes in intracellular STX, as verified by simultaneous exposure of cyanobacterial cells to both the compounds (Fig. 4.5). Curiously, Na+ stress, lidocaine and amiloride displayed similar values for their effect on STX accumulation ( a variation at between 30 and 40% compared to the controls). This indicated, at a given pH, that threshold levels of intracellular STX may exist. Exposure to salts and channel-blocking agents did not affect the HPLC chromatographic profile of toxins produced by C. raciborskii T3, compared to otherwise stimulated or control cultures. This also indicated that no detectable toxin transformations were involved in the observed variations in STX levels. The time course of STX production, under the different conditions studied, however, resembled regulation of a metabolic pathway. Strong changes in ion fluxes, as shown here, are known to cause up- or down-regulation of genes involved in the maintenance of cell homeostasis (Maestri and Joset 2000). 92 Chapter4 STX Metabdismand Salium Cyle The possibility of a regulation of STX metabolism in C. raciborskii T3 in response to cellular Na+ levels, may suggest either that STX biosynthesis is influenced by certain processes involved in cyanobacterial homeostasis, or that the toxin itself could play a role in the maintenance of cell functions. In response to Na+ stress, cyanobacteria are known to activate the production of amine osmolites, such as proline, that are synthesised from arginine via the urea cycle (Quintero et al. 2000). Arginine is also the principal biosynthetic precursor of the STX perhydropurine skeleton (Shimizu 1996). Enhanced production of osmolites could, through increased availability of arginine, favour STX biosynthesis. This process has already been documented in plants, where amine-derived alkaloids increase under salt stress (Ali 2000). Alternatively, STX may interact with membrane ion fluxes in C. raciborskii T3, preventing the deleterious effect of intracellular Na+ increase in this freshwater cyanobacterium. The blockage of Na+ uptake by STX has been demonstrated in cyanobacterial strains of C. raciborskii and A. circinalis (Chapter 5). This inhibition of sodium uptake by STX raises questions regarding the potential advantage that PSP toxin-producing cyanobacteria could have over other non-toxic species under conditions of high pH or salt stress. Similar circumstances may prevail in sub-tropical and temperate regions during natural cycles of flood and drought periods, or as a consequence of human exploitation. During 1991 high pH and elevated water conductivity were associated with the most extensive STX producing bloom of A. circinalis in Australia (Bowling and Baker 1996). The same circumstances correlated with the dominance of a neurotoxic strain of C. raciborskii in Brazilian freshwaters (S. Azevedo, personal communication). 4.5 CONCLUSIONS The present study demonstrates a strong correlation between variations m cellular Na+ levels and STX production in the cyanobacterium C. raciborskii T3. The evidence reported suggests that either STX metabolism or the toxin itself could be linked to the maintenance of cyanobacterial homeostasis under alkaline pH or Na+ stressed conditions. The model proposed could also apply to other PSP toxin-producing bacteria, cyanobacteria or dinoflagellates, and may represent an important element in 93 diapter4 STX Metabdismand Sali,um Oyle understanding the ecology of PSP toxin-producing microorganisms. In addition, the results reported here will be important for further physiological, biochemical or gene expression studies of saxitoxin and related compounds in cyanobacteria and other . . m1croorgamsms. 4.6SUMMARY The effects of pH, salt, amiloride and lidocaine hydrochloride on total cellular levels of Na+ and K+ ions and STX accumulation in cultures of the cyanobacterium Cylindrospermopsis raciborskii T3 are described here. Both Na+ levels and STX intracellular concentrations increased exponentially in response to rising alkalinity. NaCl inhibited cyanobacterial growth at a concentration of 10 mM. In comparison with osmotic stressed controls however, NaCl promoted STX accumulation in a dose dependent manner. A correlation was seen in the time course of both total cellular Na+ levels and intracellular STX for NaCl, amiloride and lidocaine exposure. The increase in cellular Na+ induced by NaCl at 10 mM was coupled by a proportional accumulation of STX. The two sodium channel-blocking agents amiloride and lidocaine had opposing effects on both cellular Na+ levels and STX accumulation. Amiloride at 1 mM reduced ion and toxin concentrations, while lidocaine at 1 µM increased the total cellular Na+ and STX levels. The effects of the channel-blockers were antagonistic and dependent on an alkaline pH. The results presented suggest that, in C. raciborskii T3, STX is responsive to cellular Na+ levels. This may indicate that either STX metabolism or the toxin itself could be linked to the maintenance of cyanobacterial homeostasis. 94 Chapter 5 STX Blaks Bacteria/, Na+ -K+ Uptake CHAPTERS EFFECTS OF SAXITOXIN AND VERATRIDINE ON BACTERIAL NA+- K+ FLUXES: A PROKARYOTIC-BASED STX BIOASSAY And if you listen very hard The tune will come to you at last. When all are one and one is all To be a rock and not to roll. (J. Page, R. Plant) 95 Chapter 5 5.1 BACKGROUND All non-analytical detection systems for PSP toxins rely on bioassays based on whole animals or animal cell-lines (Harada et al. 1999, Manger et al. 1993), since much is known regarding the pharmacological effect of STX on eukaryotes. On the other hand, the effects of PSP-toxins on prokaryotic cells have not been comprehensively studied (Tisa et al. 2000). For both economic, practical and ethical reasons, alternatives to the standard animal biological tests are desired. Here the first investigation of the feasibility of a prokaryotic-based bioassay for saxitoxin and its analogues is described. In a previous study it was reported that, in the STX-producing cyanobacterium C. raciborskii T3, STX accumulation directly correlated to variations in intracellular Na+ levels (Chapter 4). Those results suggested a possible role of this toxin in the maintenance of cyanobacterial homeostasis under Na+ stress. Questions regarding whether STX-producing cyanobacteria have a potential advantage over other non neurotoxic strains under conditions of critical Na+ levels, or whether STX interferes with bacterial Na+ fluxes as it does in eukaryotic cells arose from that study. In this study the effect of STX and veratridine (VTD), a sodium channel activator that increases Na+ permeability in eukaryotic cells (Catteral and Nirenberg 1973), was first investigated on the cyanobacterial total Na+-K+ cellular contents measured by flame photometry. C. raciborskii A WT205, a cyanobacterium known not to produce any neurotoxin including STX (Hawkins et al. 1997), was exposed to STX and VTD. Further, by using VTD to induce intracellular Na+ stress, we assayed PSP toxins producing and nontoxic strains of C. raciborskii and A. circinalis in a "cyanolytic" test similar to those employed with eukaryotic cells (Manger et al. 1993, Shimojo and Iwaoka 2000). In the assay developed here cyanobacteria were stressed with VTD and o-vanadate (VAN), an inhibitor of bacterial ion pumps (ion translocating P-type ATPases) (Hafer et al 1989, Eddy and Jablonski 2000). The detection of cell lysis was used as the endpoint. Two non-neurotoxic strains ( C. raciborskii A WT205 and A. circinalis 271C) were then employed in the same test to assay the presence of channel-blockers such as lidocaine, amiloride, STX and neoSTX. In order to investigate whether the demonstrated sensitivity of C. raciborskii A WT205 to VTD and STX was also related to changes in the cell's metabolic activity, the test was applied to a cell titre 96 Chapter 5 cytotoxicity assay. Subsequently, the method based on VTD-induced Na+ stress was employed for assaying the presence of STX with the commercially available and standardised toxicity test LUMIStox using the bioluminescent bacterium Vibrio fischeri. 5.2 EXPERIMENTAL PROCEDURES 5.2.1 Reagents All reagents and chemicals were obtained from Sigma-Aldrich (Sigma-Aldrich Co., Dorset, UK). Lidocaine hydrochloride, amiloride, and a-vanadate solutions (100 µM, 100 mM and 10 mM, respectively) were prepared in Milli-Q water, stored at 4°C protected from light and diluted into the culture medium to obtain the desired concentration. Veratridine was dissolved to a final concentration 10 mM in acidic Milli Q water (pH 2) and stored at -20°C. Certified standard solutions of paralytic shellfish poisoning toxins (PSP-1 C and STXdiHCl-C) were stored at -20°C with the stock solutions diluted into the culture medium to obtain the final test concentrations. 5.2.2 Cyanobacterial strains and culture conditions Cylindrospermopsis raciborskii AWT205, a non PSP-toxin producer (Hawkins et al. 1997) and C. raciborskii T3, STX producer (Lagos et al. 1999) were maintained in ASM-1 medium (Gorham et al. 1964). PSP-toxin producing strains of Anabaena circinalis, 344B, 134C, 150A, 131C, and the non-toxic strains 271C, 306A, and 332H (see also Appendix A) were maintained in JM medium (Humpage et al. 1994). All cyanobacterial cultures were grown in glass 250 mL flasks at a constant temperature of 26° C, under continuous irradiance of cool white light at an intensity of 15 µmol photon m-2 s-1. Cyanobacterial growth was monitored by recording the OD750 as described in Section 2.2.1. Mid-exponential phase cultures were chosen for the following tests. 97 Chapter 5 5.2.3 Total cellular Na+ and IC content and flame photometry To evaluate the effect of STX at 1 µM and VTD at 100 µM on total Na+-K+ cellular levels, aliquots of the same culture (20 mL) of C. raciborskii A WT205 were adjusted to pH 8.1 by adding HEPES buffer to a final concentration of 10 mM. Samples (2 mL) were harvested before exposure (-5 min), immediately after exposure (0 min), and at 10, 30, and 60 min post exposure. Experimental replicates included negative controls (unexposed culture sample) and positive controls (10 mM NaCl). Aliquots of challenged A WT205 cultures were collected by centrifugation in 2 mL plastic tubes at 11,000 g for 15 min. All sampled pellets were resuspended in 0.5 mL of diluent flame solution (3 mM lithium in MQ water) and analysed for total Na+ -K+ cellular content as reported in Section 2.4. All experiments were performed in quadruplicate. Control traces were subtracted from the tested samples for threshold correction and the data normalised by expressing values as the percentile variation over samples at -5 min. 5.2.4 Cyanobacterial cells lysis test The assay was based on the same principles as the animal neuroblastoma and red blood cell culture assays reported elsewhere (Manger et al. 1993, Shimojo and Iwaoka 2000), with the exception of the use of VAN to inhibit Na+-K+ ATPase activity instead of ouabain since this compound is known to be ineffective against algal Na+ pumps (Glimmer 2000). Briefly, 96-well microtitre plates were used for the cyanolytic assay, in which 100 µL of the cyanobacterial cultures were inoculated and exposed to the agents. Controls consisted of untreated aliquots of cyanobacterial cultures and samples with 4 µL of either 10 mM VTD or 4 µL of 10 mM VAN. Toxic and nontoxic cyanobacteria were also assayed after the addition of a combination of 4 µL of 10 mM VTD + 4 µL of 10 mM VAN. The final concentration ofVTD and VAN, corresponding to 400 µM, was chosen based on previous studies (Hafer et al. 1989, Shimojo and Iwaoka 2000). For the treatment of C. raciborskii AWT205 and A. circinalis 271C with STX and neoSTX at 1 µM, the PSP-toxins were added to the tested wells 30 min prior to the addition ofVTD and VAN, to simulate natural the conditions of toxic cultures. In 98 Chapter 5 these experiments, the positive controls consisted of samples exposed to VTD+VAN added to lidocaine hydrochloride at 1 µM. Replicates dosed with amiloride at 10 mM were used as negative controls. In a previous study, lidocaine hydrochloride was demonstrated to induce an increase in total cellular Na+, while amiloride reduced the cellular ion levels in cyanobacteria (Chapter 4). In both assays, the microtitre plates were incubated at room temperature (25°C) for 5-8h (minimum time for complete cell lysis observed in the VTD +VAN samples). Determination of cell lysis was performed by light microscopic inspection every 30-60 min from the onset of the test, and cells counted by means of a Neubauer improved counting chamber (0.1 mm deep). Complete cyanobacterial lysis in the VTD + VAN samples was utilised as the end-point of the assay. If no cyanolysis was observed in the inoculated wells the presence of a channel-blocking agent, including PSP-toxins, was indicated. Three to five trials were performed for each strain or treatment to test reproducibility of the results. 5.2.5 Cell titre assay for metabolic activity Microtitre plate cytotox1c1ty assays on cyanobacterial cells were performed usmg the CellTitre 96® Non-Radioactive Cell Proliferation Assay kit (Promega Corporation, Madison, WI, USA). This method utilises, as an indicator of a cell's metabolic activity, the cellular conversion of a tetrazolium salt into a formazan product that can be quantified with a spectrophotometer plate reader. Assays were performed essentially as suggested in the standard protocol provided with the kit. C. raciborskii cells in mid-exponential growth were centrifuged (15 min at 4000 g) and concentrated to reach approximately OD75o=l. Subsequently, 100 µL aliquots of concentrated cyanobacterial suspension were inoculated in 96-well microtitre plates and exposed to the test agents. For comparison between the metabolic responses of C. raciborskii strains AWT205 and T3, the culture samples were tested with a combination of 4 µL of 10 mM VTD + 4 µL of 10 mM VAN giving a final concentration 400 µM of each compound. Controls consisted of untreated cyanobacteria. In the evaluation of the effect of combined VTD and STX on C. raciborskii A WT205, cyanobacterial cells were exposed to 1 µM STX, incubated at room temperature for 30 min and then added to 99 diapter 5 VTD at 100 µM. Controls consisted, for each sample, of untreated cells and cyanobacteria exposed to VTD at 100 µM. The dye solution was added at different times and the stop solution after 4 h of incubation at room temperature (25°C). Plates were let to rest overnight and read recording the absorbance at 600 nm with a microplate reader Metertech L960 (Metertech Inc., Taipei, Taiwan). All experiments were performed in quadruplicate and the data expressed as an average percent variation of sample values over untreated control levels. 5.2.6 Luminescent bacteria test Inhibition ofbioluminescence in cultures of Vibrio fischeri NRRL-B-11177 was performed using the commercially available Dr. Lange standard luminescent bacteria LUMIStox test kit (Dr Bruno Lange GmbH & Co, Dusseldorf, Germany), and specific analytical equipment, including the LUMIStox 300 measuring station and Lumistherm thermostat. Reactivation of freeze-dried bacteria and preparation of samples was operated following the instructions provided. Light emission of reactivated bacteria was adjusted to approximately 1000 RI (relative intensity) by diluting with sterile 2% NaCl. For the test, 0.5 mL of luminescent bacterial suspension was combined with 0.5 mL of the test solutions. Test solutions were prepared in sterile saline medium (2% NaCl) and adjusted to pH 7 with 10 mM phosphate buffer. When needed, STX supplemented the solutions to the desired final concentration. Bacterial suspensions and samples were maintained at 15°C, combined, and monitored with the luminometer while allowing bacteria to adapt for 5-10 minutes. Subsequently, VTD at 100 µM was added and the light emission measured over time. Positive and negative controls were included for each test and consisted of bacteria exposed to only VTD and unexposed samples, respectively. The effect on bioluminescence was monitored and expressed as the percent inhibition relative to the untreated controls. Values were calculated using, as a correction factor, the changes in intensity of the controls. This was achieved by subtracting the trace negative control reading from the test samples over the duration of the experiment. 100 Chapter 5 5.3 RESULTS 5.3.1 Effect of Na+ stress, VTD and STX on total cellular Na+-K+ levels In C. raciborskii AWT205, the stress induced by 10 mM NaCl increased total cellular Na+ levels compared to the untreated controls (Fig. 5.lA). Na+ uptake by the cells was shown to be very rapid, and the total cyanobacterial sodium content remained stable over the 60 min course of the experiment. Total K+ content of cells was only slightly affected by 10 mM NaCl, indicating that the homeostasis of K+ is of marginal consequence in the overall cyanobacterial Na+ stress response. A) aool700 600 100 75 C: 50 .E 25 LO 0 l!. -25 -ea -50 -+-..--,--,--,---.---.--r--r--.--...--...--,.--.--,---.---. ~ 140 ea 120 A) > I 0 100 ~ 80 0 60 40 20 ~I 0 ------~ ><==i -20 -40 -60 -80 -t-..--,--,,...... ,---.---.--r--r--.--...--...--,.--,---..--.---, -10 0 10 20 30 40 50 60 Time (min) Figure 5.1 A) Time course of total cellular Na+ -K+ levels in C. raciborskii AWT205 cultures exposed to 10 mM NaCl (Na+=•. K+ = D) in comparison with untreated control samples (Na+ = •. K+ = 0). B) Effects of STX at 1 µM (Na+= T, K+ = V) and VTD at 100 µM (Na+= A, K+ = fl.) on total cellular Na+-K+ concentrations in C. raciborskii AWT205. All values are the mean of 4 experimental replicates and are expressed as the percentage variation over time. 101 Chapter 5 On the other hand, in C. raciborskii AWT205 both Na+ and K+ cellular levels were altered due to the effects of STX at 1 µM and VTD at 100 µM (Fig. 5.lB). The addition of VTD dramatically stimulated cyanobacterial Na+-K+ accumulation while STX markedly inhibited the cellular uptake and hence intracellular levels of both ions. These results suggest that, in cyanobacteria, Na+ flux is not the only cellular response elicited by these two compounds. 5.3.2 Lysis test with toxic and non-toxic cyanobacteria The effects of VTD and VAN each at 400 µM and in combination were monitored by microscopically counting cells of the two strains of C. raciborskii, AWT205 and T3. Cell densities were recorded over a 180 min time period (Fig. 5.2). 20 0 rn -20 Q) ::J -C: E -40 0 L.. Q) > -60 0 ~ -80 -100 f 0 20 40 60 80 100 120 140 160 180 Time (min) Figure 5.2 Effects of VTD and VAN at 400 µM and their combination on cell numbers in samples of C. raciborskii AWT205 and T3. All values represent the average of 5 experimental replicates and are expressed as percentage variation over time. C. raciborskii AWT205: • = VAN, • = VTD, 'Y = VAN + VTD; C. raciborskiiT3: D = VAN, o = VTD, V =VAN+ VTD. 102 Chapter 5 VAN had no significant effect on both strains, while culture samples of A WT205 were manifestly more sensitive to VTD and the combination of VTD and VAN compared to the STX-producing strain T3. For AWT205, the first indications of cyanobacterial lysis occurred after 60 minutes of exposure to VTD or VTD + VAN, while for both T3 and A WT205 cell lysis was evident 120 minutes after the onset of the experiment. The combination of VTD + VAN was more effective than VTD alone in both strains according to the decrease in number of intact cells (Fig. 5.2). In AWT205 cultures, 180 min exposure to VTD + VAN resulted in almost complete lysis of cells. By microscopic examination, cyanobacterial lysis occurred after evident enlargement of the cells, which subsequently burst. This indicated that lysis could have been caused by a dramatic increase in the intracellular osmotic pressure due to excessive ion uptake. PSP-toxin producing and non-neurotoxic strains of the cyanobacterial species C. raciborskii and A. circinalis have been assayed for resistance to VTD + VAN using the method shown above with the results summarized in Table 5.1. All the non-PSP toxin producing strains tested showed complete cell lysis after 3 to 8 h with no filaments recorded, while the neurotoxic strains where characterized by intact filaments and a lack of complete cell lysis even after overnight exposure to these agents. Table 5.1 Cyanobacterial strains used in this study and their relative resistance to treatment with VTD (400 µM) + VAN (400) µM. +=complete cell lysis and filaments absent; - = partial cell damage with intact filaments present. Strain PSP-toxins Lysis Time C. raciborskii A WT205 NO + 3-5 h C. raciborskii T3 YES 3-5 h A. circinalis 344B YES 3-5 h A. circinalis 134C YES 3-5 h A. circinalis 150A YES 3-5 h A. circinalis 131 C YES 3-5 h A. circinalis 271C NO + 3-5 h A. circinalis 306A NO + 3-5 h A. circinalis 332H NO + 5-8 h 103 Chapter 5 Two non-PSP toxin producing cyanobacterial strains, C. raciborskii AWT205 and A. circinalis 271C, were also chosen to evaluate the effect of STX and neoSTX at 1 µM on the stress induced by exposure to VTD + VAN. The results, summarised in Table 5.2, demonstrated that the production of PSP toxins prevented, in culture samples, the complete cells lysis caused by VTD + VAN, as seen for the positive controls and in the cyanobacteria added with lidocaine hydrochloride at 1 µM. In cyanobacteria exposed to lidocaine, cell lysis was observed at the same time as the untreated controls. On the other hand, the effects of amiloride at 1 mM, STX at 1 µM and neoSTX at 1 µM were comparable with regards to the level of inhibition of cyanobacterial lysis. Table 5.2 Results of the application of channel-blocking agents to two non-PSP toxin producing cyanobacteria over a 5 h exposure time to VTD (400 µM) + VAN (400 µM). Control refers to samples with no channel-blocker added. +=complete cell lysis and filaments absent; - = partial cell damage with intact filaments present. Cell l:ysis Strain Control Lidocaine Amiloride STX neoSTX + lµM lmM lµM lµM C. raciborskii A WT205 + + A. circinalis 271C + + 5.3.3 Effect of VTD and STX on cyanobacterial metabolic activity In order to investigate whether the increased Na+ uptake in cyanobacterial cells was coupled to a consequent measurable decrease in general metabolic activity, cultures of C. raciborskii A WT205 were treated with 400 µM VTD + 400 µM VAN, 100 µM VTD, or 100 µM VTD + 1 µM STX and monitored by the CellTitre 96® Cell Proliferation Assay. As a control, cultures of the STX-producing C. raciborskii T3 were also exposed to 400 µM VTD + 400 µM VAN and the metabolic activity measured over time. The treatments with VTD and VTD + VAN both resulted in a reduced metabolic activity of C. raciborskii strains over the 90 min exposure, however the toxic strain T3 104 diapter 5 was affected to a lesser extent compared to AWT205 (Fig. 5.3). STX at 1 µMalone did not result in any adverse effect on cyanobacterial metabolism ( data not shown). The addition of STX at 1 µM to cultures of A WT205, followed by VTD at 100 µM, induced a less dramatic decrease in metabolic activity compared to exposure to VTD 100 µM alone (Fig. 5.3). 5 -5 .EC: -10 0 '- Q) -15 > 0 -20 ~0 -25 -30 -35 -40 0 20 40 60 80 100 Time (min) Figure 5.3 Time course of metabolic activity in culture samples of C. raciborskii AWT205 treated with VTD (100 µM) {.A.), VTD (400 µM) + VAN (400 µM) (T) and VTD (100 µM) + STX (1 µM) (0). Control culture samples of C. raciborskii T3 were added with VTD (400 µM) + VAN (400 µM) (X). All values are the average of 4 experimental replicates and are expressed as the percentage variation over time. 5.3.4 Effect on bioluminescence by Vibrio fischeri In the standard toxicity test LUMIStox, no significant effect of varied concentrations of STX (from 100 nM to 1 µM) on the luminescence of Vibrio fischeri was observed. On the other hand, treatment with VTD at 100 µM reduced bacterial light emission over time, as shown in Fig. 5.4A. Addition of STX (1 µM) to the Vibrio cultures after exposure to VTD 100 mM resulted in slight recovery of bioluminescence (maximum 4.4% after 70 min) compared to samples with no STX added (Fig. 5.4A). Exposure of the bioluminescent bacteria to STX prior to the addition ofVTD (100 µM), 105 Chapter 5 gave rise to different responses over time for the test samples compared to the controls that had no STX added. Figure 5.4B shows the time course of percentage light emission for the various treatments, with values corrected by subtracting the trace levels of unexposed control samples to eliminate threshold variations in bioluminescence. 1400 A) 1350 1300 1250 1200 1150 -o- added 2% NaCl -•-added STX 1.2 µM 1100 0 25 50 75 100 125 150 175 200 225 8) vro 1ooµM 10 -15 0.0 2.5 5.0 7.5 10.0 12.5 15.0 17.5 20.0 minutes Figure 5.4 A) Time course of bioluminescence in Vibrio fischeri: VTD (100 µM) was added 52 min after the onset of the experiment. Subsequently half of the experimental replicates were supplemented with STX (1 µM) (•) while the other half were supplemented with physiological saline solution (D). B) Effects of VTD (100 µM) on light emission by samples of V. fischeri exposed to STX at 0 (•). 300 (0), 600 ("') and 1200 nM (T), expressed as the percentage variation over time and corrected for the control levels. All values are the ::ivP.r::tnP. nf 4 P.XnP.rimP.nt::il rP.nlir.::itP.i::. 106 Chapter 5 In the first two minutes after addition of VTD at 100 µM, samples not exposed to STX drastically decreased their light emission compared to bacterial solutions treated with the channel-blocking toxin (Fig. 5.4B). STX at 600 nM was the most effective treatment in preventing the VTD effect (+21 % compared to VTD controls), followed by STX at 300 nM (+19.5%) and at 1.2 µM (+18.3%). Five minutes after the addition of VTD, samples with no STX added reached a level of bioluminescence that were, on average, 6% higher than the untreated controls. Bacterial solutions with STX at 300 and 600 nM showed no significant variation over control levels. Samples with STX added to 1.2 µM did not completely recover control levels of light emission, attaining an average of -3.8% of the luminescence in unexposed bacteria. 5.4 DISCUSSION During the course of the present study, opposing effects of STX and VTD on cyanobacterial Na+-K+ ion fluxes, as measured by flame photometry analysis, have been observed. The effects detected were rapid but not Na+ specific, as predicted by the interaction of these compounds with eukaryotic cells. These results suggested either that STX and VTD have less specific effects on prokaryotic cells than those reported in the literature for eukaryotic sodium fluxes, or that the target of these two agents on cyanobacterial cells is a binding protein involved in both Na+ and K+ homeostasis. The latter hypothesis is consistent with several reports in the literature demonstrating the presence, in cyanobacterial cells, of channel proteins permeable to both sodium and potassium ions (Kaneko et al. 1996, Murata et al. 1996, Nakamura et al. 1998, Kaneko et al. 2001 ). Based on these observations, PSP-toxin producing and non-neurotoxic cyanobacterial strains have been assayed to investigate whether, in vivo, the production of STX would have prevented the excessive ion uptake and subsequent cell lysis induced by VTD stress. As noted from microscopic observation, non-PSP toxin producing cyanobacteria under VTD + VAN stress were swollen, probably due to an increase in the internal osmotic pressure, and subsequently collapsed within 5-8 h. Similar findings were reported in previous studies using animal cells subjected to the 107 Chapter 5 activity of the sodium channel activator and the ion-pump inhibitor ouabain (Shimojo and Iwaoka 2000). In contrast, PSP-toxin producing cultures exhibited lower rates of cell lysis. This differential sensitivity to VTD and VAN exposure is proposed to be due to the presence of channel-blocking compounds in the cultures, which interfere with the action of the channel-activating agents. To verify this hypothesis, and exclude the possibility of a variable intrinsic sensitivity of the toxic strains to the chemicals used, the two non-neurotoxic cyanobacteria C. raciborskii A WT205 and A. circinalis 271 C were exposed to VTD + VAN in the presence of STX and neoSTX at 1 µM. This experiment clearly revealed the acquired resistance to lysis of non-neurotoxic strains after the addition of PSP-toxins to the cultures. Therefore, in vivo, a direct antagonism of STX and VTD was demonstrated in a prokaryotic microorganism, similar to what has been noted in the eukaryotic cell-based assays for channel-blocking toxins (Manger et al. 1993). Consistent with our hypothesis that the main effect of VTD on cyanobacterial cells was due to the increased uptake of Na+/K.+ ions, we investigated whether such stress was correlated with a decrease in the metabolic activity of the cyanobacteria. Sodium, above a certain critical concentration, represents a threat to normal cellular functions. This ion, if in excess compared to normal physiological levels, can disrupt several crucial biological functions such as photosynthetic and electron transport activities in cyanobacterial cells (Allakhverdiev et al. 2000). The cell titre toxicity assays, applied in this study with the addition of VTD + VAN, demonstrated that the antagonistic effects of STX and VTD can also be detected via the monitoring of bacterial metabolic activity. Since measurements of metabolic activity, compared to cells lysis, represent a more precise, easy to quantify and standardised means of investigation, we applied the principle of inducing ion uptake using VTD in the bioluminescent bacterium Vibrio fischeri. Such stress, resulting in decreased primary metabolism, can be measured in this microorganism as variations in light emission by a commercially standardised method. In suspensions of Vibrio fischeri, VTD exposure reduced bioluminescence. Light emission also followed a similar pattern after the post-VTD addition of STX (Fig. 5.4A). This effect could be explained by the occurrence of non-reversible cell damage caused by either the initial VTD stress or a critical increase in cytoplasmic Na+ levels 108 di.apter 5 (Allakhverdiev et al. 2000). Alternatively, these data could indicate a difference in the affinity of VTD and STX for a putative binding ·site on the bacterial cells. STX may have less specificity than VTD for the receptor molecule in Vibrio fischeri or the binding protein(s) on the bacterial cell membranes could have a completely different structure compared to the defined targets of these two agents, i. e. the eukaryotic voltage-gated Na+ channels. On the other hand, exposure to STX pnor to the addition of VTD to bioluminescent bacteria resulted in the observed difference in the time-course of cellular metabolic activity. As expected for changes in membrane ion fluxes, the effect displayed by Vibrio fischeri was rapid and dose-dependent. The concentration of STX equal to 1.2 µM was shown to have a minor level of toxicity against bioluminescent bacteria. This effect could be a result of the prolonged inhibition of basal bacterial Na+ K+ activity by such high concentrations of the channel-blocking toxin. In general, however, during the course of the present study STX alone did not have any particular effect on bacterial (Vibrio or cyanobacteria) growth or metabolism. Accordingly, exposure to STX could not be employed directly in a bacterial bioassay. The data were consistent with reports that show low to no toxicity of STX on other microorganisms (Harada et al. 1999, Tisa et al. 2000). In contrast, no investigations of the effects of STX on prokaryotic ion fluxes have been reported. Bacterial ion channels are single domain proteins (for review see Anderson and Greenberg 2001, Catteral 2001) that are claimed to be insensitive to both STX and TTX, and confirmed by recent findings regarding the voltage-gated prokaryotic Na+ channel in the halophilic bacterium Bacillus ha/odurans (Ren et al. 2001). Further comprehensive investigations of the effect of STX on microorganisms may lead to important evolutionary findings regarding an ancestral ion channel sensitive to neurotoxins. The application of the bioluminescent bacteria method described here for toxicity assays showed a detection range for STX in the order of magnitude of nM, and a limit lower than 300 nM. Preliminary data also confirmed (Shimojo and Iwaoka 2000) that varying the concentration of VTD, VAN or the number of cells in the assay can affect the sensitivity of the test. In this study, the parameters have been selected to provide the best results in a short time-scale, as required for a rapid bioassay. We predict that additional development and standardisation of this test would afford a novel 109 Chapter 5 and accurate method for the detection and quantification of PSP-toxins. All three Gram negative bacteria tested with VTD showed a lower affinity to STX compared to their potency against eukaryotic cells. The use of microorganisms, however, represents an easy, economic and ethical alternative to animal tests for screening environmental, clinical and industrial samples for neurotoxins, including PSP-toxins, tetrodotoxin, ciguatoxins and brevetoxins. 5.5 CONCLUSIONS This study represents the first evidence of the effect of the Na+ channel-blocker STX, as well as the sodium channel-activator VTD, on Na+-K+ ion fluxes and metabolism of bacterial cells. Previously, these two agents were thought to act almost exclusively on eukaryotic membrane channels. Additionally, the applicability of VTD and STX antagonism in prokaryotic cells for the development of a novel PSP-toxin bioassay has been demonstrated. 5.6SUMMARY This study describes the antagonistic effects of the Na+ channel-blocker saxitoxin (STX) and veratridine (VTD}, a Na+ channel-activator, on three Gram negative bacteria and their application to a STX bioassay. As measured by flame photometry, STX reduced the total cellular levels of both Na+ and K+, while VTD increased their concentration relative to control ion fluxes in the cyanobacterium Cylindrospermopsis raciborskii AWT205. Endogenous STX production in toxic cyanobacterial strains of C. raciborskii and Anabaena circinalis prevented cell lysis induced by VTD stress. Microscopic cell counts showed that non-STX producing cyanobacteria displayed complete cell lysis and trichome fragmentation 5-8 h after addition of VTD and vanadate (VAN), an inhibitor of sodium pumps. Addition of STX, or its analogue neoSTX, prior to treatment with VTD + VAN prevented complete lysis 110 Chapter 5 in non-STX producing cyanobacteria. VTD also affected cyanobacterial metabolism and the presence of exogenous STX in the sample also ameliorated this decrease in metabolic activity, as measured by the cellular conversion of tetrazolium into formazan. Reduced primary metabolism was also recorded as a decrease in the light emissions of Vibrio fischeri exposed to VTD. Addition of STX prior to VTD resulted in a rapid and dose-dependent response to the presence of the channel-blocker, with samples exhibiting resistance to the VTD effect. These findings demonstrate that STX and VTD influence bacterial Na+-K+ fluxes in opposite ways and these principles can be applied to the development of a prokaryotic-based STX bioassay. 111 Chapter6 CHAPTER6 EVIDENCE FOR DIFFERENCES IN THE METABOLISM OF STX AND Cl +2 TOXINS IN CYLINDROSPERMOPSIS RACIBORSKIIT3 People say that you're no good, But I wouldn't cut you loose, baby, if I could. (G. Allman) 112 Chapter6 PSP tax in-prrx/uctwn and pratei,n synt/x5is inhibitim 6.1 BACKGROUND The chemical structure and toxicity of PSP toxins have been described. In contrast, as reviewed previously (Section 1.3.5.5), little is known about the enzymatic pathway(s) leading to the biosynthesis of these peculiar alkaloids. Shimizu and colleagues (1993, 1996) demonstrated that arginine, acetate, and a methyl group (from S-adenosylmethionine) are incorporated into the tricyclic perhydropurine skeleton of STX in cyanobacteria and dinoflagellates. Given this sequence of biochemical reactions, candidate enzymes in STX biosynthesis have been hypothesised (Shimizu 1993, Taroncher-Oldenburg and Anderson 2000). In particular, the Claisen-type condensation between arginine and acetate could be catalysed by an aminolevulinate synthase (Section 1.3.5.5). Alternatively, this reaction could involve a hybrid non ribosomal peptide synthetases/polyketide synthases (NRPS/PKS) enzyme complex with an acyl adenylation, P-ketoacyl synthase and thioesterase domains (Section 1.3.5.5). Recently, Sako et al. (2001) identified a N-21 sulfotransferase in the dinoflagellate Gymnodinium catenatum, specific for three PSP toxins and each yielding a distinct product: STX~ GTX5, GTX2 ~ Cl, and GTX3 ~ C2. Here, C. raciborskii T3 cultures were supplemented with substrates potentially involved in STX synthesis, and the resulting accumulation of PSP toxins evaluated in the presence of the antibiotic chloramphenicol (CAM), as an inhibitor of ribosomal protein synthesis. In addition, the singular effect of CAM was investigated on PSP toxin accumulation in C. raciborskii T3. PSP toxin production was studied both in vivo and in vitro, and the observed changes in intracellular Cl +2 toxins and STX accumulation discussed with regards to the possible biosynthetic pathways involved in the production of these toxic alkaloids. 113 Cliapter 6 PSP taxin-prcxluctinn and prrxei,n synt}xsis inhibition 6.2 EXPERIMENTAL PROCEDURES 6.2.1 Reagents All reagents were obtained from Sigma-Aldrich (Sigma-Aldrich Co., Dorset, UK). The analytical standard solutions used in this study are reported in Section 2.3.3. 6.2.2 Growth conditions and cyanobacterial cultures C. raciborskii strain T3 was maintained in ASM-1 medium (Ghoram et al. 1964) adjusted to pH 7.5 (Section 2.1.1). Cultures were grown in glass 250 mL flasks in a thermostatically controlled cabinet at a constant temperature of 26°C and under continuous irradiance of cool white light at an intensity of 15 µmol photon·m-2-s- 1• M aeruginosa PCC7806 and N. spumigena NSORl O were grown under natural light at 23°C in BG-11 medium (Rippka et al. 1979) and ASM-1 medium, respectively (Section 2.1.1 ). Growth of cyanobacterial cultures was monitored in 1 cm disposable cuvettes by recording the OD750 as reported in Section 2.2.1. Cultures in mid-exponential growth phase were used in this study. During the course of the following experiments, cultures of C. raciborskii T3 exposed to the different reagents were monitored spectrophotometrically for short term variations in cyanobacterial cells morphology. In this study, no significant changes in mean cell dimension or trichome structure were evident after each treatment, apart from exposure to CAM at 100 µg L- 1 that slightly reduced cell size and partially damaged C. raciborskii T3 filaments. 6.2.3 In vivo experiments Cyanobacteria were exposed to CAM at 1, 10 and 100 µg L-1 for 24 hand the culture densities monitored by means of OD at 750 nm. A culture without CAM added was used as the control. Chlorophyll-a and phycocyanin content were measured as OD at 682 and 630 nm, respectively (Section 2.2.1). Feeding experiments were performed 114 Chapter6 PSP tax in-prrxluainn and protein synt/x5is inhibitinn with arginine at concentrations from 1 to 20 mM, canavanine from 1 to 10 mM, and agmatine and proline at 5 mM. Canavanine, a natural amino acid structurally related to arginine, was used as a negative experimental reference since it cannot be incorporated into arginine-derivatives (Rosenthal 2001). Agmatine is decarboxylated arginine, a putative direct substrate utilized for the biosynthesis of the carbon skeleton of PSP toxins (Shimizu 1993, Shimizu 1996). Proline is a cyanobacterial osmolite derived from arginine via the urea cycle (Quintero et al. 2000). In the feeding experiments, cyanobacterial cultures were exposed to different concentrations of the amines for 24 or 48 h in the presence ofHEPES buffer (pH 7.5) at the final concentration of 30 mM. The time course experiments were carried out for 48 or 120 h (5 days) in 500 mL flasks. Total cell density and pigment content were monitored by OD measurements whenever samples were withdrawn for PSP toxin extraction and analysis. The effect of CAM over time and the consequence of arginine supplementation under CAM stress were studied using 10 µg L- 1 of the antibiotic. Cultures were buffered with HEPES at 30 mM (pH 7 .5). All experiments were performed in triplicate. Extraction of C. raciborskii T3 cells for HPLC analysis was performed as follows. A 15 mL culture was harvested by centrifugation (15 min, 5,000 x g), the cells resuspended in 100 µL of acetic acid at 0.5 M and lysed by sonication (3 min, 100 W). The supernatant was filter-sterilised (0.2 mm membrane), freeze-dried and analysed after resuspension in 100 µL 0.5 M acetic acid. Cellular extracts were prepared by centrifugation (15 min, 10,000 x g) to remove cell debris and stored frozen at -20°C until HPLC analysis. 6.2.4 In vitro experiments Five mL aliquots of mid-exponential growth phase cultures of C. raciborskii T3 were centrifuged 15 min at 5,000 x g, the pellets resuspended in 120 µL of 30 mM HEPES buffer (pH 7.5) and cells lysed by freezing in liquid nitrogen and thawing for 10 cycles. Extracts were sonicated on ice (1 min, 100 W) and incubated with the various reagents (CAM at 10 µg L- 1 and arginine at 1, 5, 10 or 20 mM). Samples were incubated for 2 days under the standard growth conditions, and 20 µL aliquots withdrawn at 0, 2, 4, 24 and 48 h after the onset of the experiment and stored frozen at -20°C until HPLC analysis. All experiments were performed in triplicate. 115 Chapter6 PSP tax in-pra/uction and pratei,n syr,t/Jf5is inhibitim 6.2.5 HPLC analysis Screening for PSP toxins was performed by prechromatographic oxidation with H2O2 followed by HPLC separation. Chromatography was carried out according to the method described in Section 2.3.1. Concentrations of PSP toxins in the samples were calculated by comparing the peak area corresponding to the toxins in the cyanobacterial extracts to that of the standard solutions. When necessary, H2O2 was removed from the oxidant solution to verify the oxidation dependence of HPLC peaks. In this study, no auto fluorescent peaks with the same retention time of STX or Cl +2 were detected. PSP toxin data was normalised by the total protein content (µg mL- 1) of the experimental replicates. 6.2.6 Protein phosphatase inhibition assay Five mL aliquots of mid-exponential growth phase cultures of M. aeruginosa PCC7806 and N spumigena NSORlO were incubated with CAM at 1, 10 and 100 µg L- 1 for 24 hours. Aliquots of cyanobacterial cultures (1.5 mL) were subsequently lysed by sonication on ice (1 min, 100 W), centrifuged 15 min at 5,000 x g to remove cells debris and stored at -20°C until analysis. The concentration ofMCYST or NODLN in a sample was analysed using the colorimetric PP2A inhibition assay described in Section 2.3.2. 6.2. 7 Total protein content Protein concentrations in the crude cyanobacterial cell extracts and in the culture media were determined as described in Section 2.2.2. 116 Chapter6 PSP taxin-prrxluctwnand praeinsynt/Jf5is inhibidon 6.3 RESULTS 6.3.1 Effect of CAM on cyanotoxin accumulation In consequence to CAM exposure, total MCYST values in cultures of M aeruginosa PCC7806 approximated those found in controls for all concentrations tested (Fig. 6.1 A). 80 A) 60 40 20 0 en -20 Q) ii, -40 e -60 +-' C 8 -80 i... -100 80 ~ B) C 60 0 :,;:J 40 ·c:ct:l ~ 20 0~ 0 ~ -20 ------7------40 -60 ;_/1 -80 -100 0 1 10 100 CAM concentration (µg ml-1) Figure 6.1 (A) Effects of CAM (24 h exposure) on total MCYST ( •) and NODLN (0) concentrations in cultures of M. aeruginosa PCC7806 and N. spumigena NSOR10, respectively. (B) Effects of CAM (24 h exposure) on intracellular C1+2 toxins (6) and STX (T} concentrations in cultures of C. raciborskii T3. Data points represent the percentage ratio of three experimental samples over the unexposed controls, expressed as average ± SE. All data were normalised by the total protein content of the corresponding culture sample. 117 Oiapter6 PSP tax in-praluctinn and praei,n synt}xsis inhibitinn NODLN concentrations in N spumigena NSORlO decreased by 22% and 38% for 1 and 100 µg mL- 1 of CAM, respectively, and increased by 17% for 10 µg mL- 1 dose of the antibiotic (Fig. 6. lA). On the other hand, the accumulation of PSP toxins in C. raciborskii T3 was markedly affected by CAM exposure (Fig. 6.1B). Intracellular concentrations of STX and Cl +2 dropped to -72% and -33% under control levels, respectively, after addition of CAM at 1 µg mL- 1• Ten µg mL- 1 dosage of the antibiotic resulted in no effect on C1+2 toxins and a decrease of STX by -73% compared to the unexposed samples. Treatment of C. raciborskii T3 cells with CAM at 100 µg mL- 1 promoted Cl +2 toxins accumulation (41 %), and reduced STX levels (-27%) compared to the controls. Cyanobacterial cell densities were not influenced by CAM at 1, 10 or 100 µg mL- 1 over a 24 h period, although CAM slightly decreased (average -5%) the pigment content (chlorophyll-a and phycocyanin) of the cultures for the highest dose tested (data not shown). The total protein concentrations remained constant over a 24 h period of CAM exposure, though at the 100 µg mL- 1 dosage the cyanobacterial protein contents of all the three strains investigated decreased by an average of 10%. 6.3.2 Time course of CAM influence on MCYST and PSP toxin production During the 5 days of exposure to CAM at 10 µg L- 1, the mean MCYST concentrations in cultures of M aeruginosa PCC7806 increased over time with a maximum value of 246% at 120 h relative to time zero (Fig. 6.2A). Intracellular levels of Cl +2 toxins appeared to be only slightly affected by CAM during the course of the experiment, with the highest levels observed during the first hour after initial stimulation, corresponding to a 36% average increase over the sample at O h (Fig. 6.2B). Extracellular Cl +2 concentrations decreased between O and 48 h, but returned to control levels after 120 h. The intracellular concentration of STX showed the highest rate of change in the first 24 h after exposure to CAM at 10 µg mL- 1 (Fig. 6.2B). STX increased by 52% above control levels in the first 2 h after the onset of the experiment, and then decreased to -59% at 24 h. Toxin levels remained below control levels for the 118 Chapter6 rest of the exposure time, with the lowest value of -73% attained after 48 h. Extracellular STX levels diminished immediately after the addition of CAM to the cultures, with concentrations 60% lower than control values 4 h after the initial dosing. As with intracellular concentrations, extracellular levels reached the lowest value of - 75% after 48 h of CAM stress. 350 300 A) 250 150 0 50 Q) E 0 ------:.;::::; L... -50 Q) O> -100 l I I I 140 C: 0 120 :.;::::; B) ctl 100 ·c 80 ~ 60 40 20 0 - -20 -40 -60 -80 -1 00 ....__-r----.----r----.----r----.----r----1fJ,-T"". ""Tj ---r-,.I 0 20 40 60 110120130 Time (h) Figure 6.2 Time course of A) the total MCYST content in cultures of M. aeruginosa PCC7806 exposed to CAM at 10 µg mL·1; B) the intracellular and extracellular levels of PSP toxins in C. raciborskiiT3 cultures exposed to CAM at 10 µg mL·1. Intracellular C1+2 = A; extracellular C1+2 = !:::,.; intracellular STX= T; extracellular STX= V. All data were normalised as for Fig. 1. Values represent the average (± SE) of three independent cultures and are expressed as percentage variation over toxin levels at time 0. 119 Chapter6 PSP tax in-prrxluction and pra-ei,n synth6is inhibition For the cyanobacterial strains investigated, exposure to CAM at 10 µg mL- 1 resulted in no change in cell density or pigment content after 48 h, although these parameters were found to increase in the following days, achieving average values 15 to 30% higher than the control levels at the end of the experiments (120 h, data not shown). Total protein concentrations in the M aeruginosa and C. raciborskii cultures increased by 20% after 120 h, while extracellular values remained constant over time in the culture medium. 6.3.3 Effect of substrates on PSP toxins levels Arginine stimulated, in a dose-dependent manner, the intracellular accumulation of STX, which increased 476% after 24 h above control values (10 mM, Fig. 6.3A). The production ofC1+2 toxins was also enhanced by all concentrations of arginine supplied to the cultures, with a maximum increased at 63% over the control levels. Feeding of C. raciborskii cultures with canavanine resulted in significant reduction of both Cl +2 toxins and STX (Fig. 6.3A). Arginine or canavanine supplementation did not induce significant changes on cell density, pigment content or total protein of the cyanobacterial cultures after a 24 h incubation. Canavanine, however, inhibited chlorophyll-a and phycocyanin production by 8 to 10% at all concentrations tested. The combined effect of CAM and arginine on PSP toxin accumulation in C. raciborskii T3 was assayed over a 48 h time period. Little effect was seen on Cl +2 toxin production in response to all treatments (10 µg mL- 1 CAM with arginine at 5, 10 and 20 mM), including CAM alone (10 µg mL- 1), compared to unexposed controls. STX, on the other hand, decreased over time in all samples supplemented with CAM, despite the addition of arginine (Fig. 6.3B). No changes were observed in total protein, pigment or cell densities over time for all combined treatments when compared to the unexposed controls. 120 Cbapter6 PSP tax in-prrxluaian and pratei,n syntlxsis inhibition 500 ~ 450 A) E 400 0 350 s.... Q) 300 > 0 250 C 200 0 :.::; 150 ea I------± ·c 100 ea 50 > 0 0~ -50 <------I------m I g -100 0 2 4 6 8 10 80 Amino acids (mM) 0 B) Q) 60 E :.::; 40 s.... Q) 20 > 0 0 C 0 -20 :.::; ea -40 ·c ea -60 > -80 0~ -100 0 10 20 30 40 50 Time (h) Figure 6.3 {A) Effects of arginine and canavanine (24 h exposure) on intracellular C1 +2 toxins (arginine = e; canavanine = 0) and STX (arginine = •; canavanine = D) concentrations in cultures of C. raciborskii T3. Data points represent the percentage variation of three experimental samples over the unexposed controls (0 mM). (8) Time course of intracellular STX levels in untreated cultures of C. raciborskii T3 (X), as well as exposed to CAM at 10 µg mL·1 in the presence of arginine at 0 (0), 5 (•), 10 (T) and 20 ( •) mM. Values are expressed as percentage variation over time 0. The experiments were performed in triplicate and the data presented as average ± SE. All concentrations were normalised as for Fig. 1. Feeding with other amine-derivatives of arginine was also evaluated for their effect on the production of PSP toxins by C. raciborskii T3, with or without the addition of CAM at 10 µg mL-1 (Fig. 6.4). Intracellular C1+2 toxin accumulation was stimulated to the highest level by agmatine at 5 mM or pro line at 5 mM to about 100 and 50% over control values, respectively, with or without CAM present (Fig. 6.4A). On the other 121 dlapter6 PSP tax in-prrxluction and pratei,n synt/X5is inhihition hand, STX production was only promoted by arginine in the absence of CAM (Fig. 6.4B). Proline and agmatine at 5 mM had either negative or no effect on the intracellular accumulation of STX compared to controls, in the absence or presence of the antibiotic. 300 250 A) 200 150 100 0 Q) 50 E :;:::; 0 L.. Q) -50 > 0 C -100 0 300 co B) ·c- 250 co > 200 0~ 150 100 50 0 -50 -100 1 2 3 4 5 6 Treatments Figure 6.4 Effects of amine substrates (48 h exposure) on intracellular C1 +2 toxins (A) and STX (B) concentrations in cultures of C. raciborskii T3, in the absence or presence of CAM (10 µg ml-1). 1 = arginine at 5 mM; 2 = arginine at 5 mM with CAM; 3 = agmatine at 5 mM; 4 = agmatine at 5 mM with CAM; 5 = proline at 5 mM; 6 = proline at 5 mM with CAM. The experiments were performed in triplicate. Columns represent the percentage variation of the tested sample over toxin values at time 0. Data are expressed as average ± SE. All concentrations were normalised as for Fig. 1. 122 diapter6 PSP taxin-prrxluctwn and prutei,n synt}xsis inhibition 6.3.4 In vitro synthesis of PSP toxins under CAM stress and arginine supplementation Both Cl +2 toxin and STX were produced in vitro over 48 h by crude cell extracts of C. raciborskii T3 (Fig. 6.5). A net increase in PSP-toxin concentrations was, however, only detectable 24h after the onset of the experiments. 400 A) 350 300 250 200 .... -'....J 150 C) ::1. 100 -C 0 :.::; 50 ctJ i... +-' 80 C Q) (.) 70 C 0 0 60 50 40 30 20 10 0 10 20 30 40 50 Time (h) Figure 6.5 Time course of C1 +2 toxins (A) and STX (B) concentrations (µg L"1} in C. raciborskii T3 cells crude extracts (X), as well as extracts treated with CAM at 10 µg mL"1 in the presence of arginine at 0 ( • ), 1 (0), 5 (L'l), 10 (V) and 20 (D) mM. Values represent the average from three independent cultures and are expressed as mean ± SE. 123 Chapter6 PSP tax in-prrxluctinn and prutei,n synt}xsis inhilition The highest level of toxins was observed after 48 h incubation, with Cl +2 toxin increasing by 20% and STX by 142% over the initial concentrations. Addition of CAM (10 µg mL- 1) to the cyanobacterial extracts had little or no effect on the production of PSP toxins in vitro. The antibiotic, when added in combination with arginine at 1, 5, 10 or 20 mM, only slightly inhibited STX production after 48 h (Fig. 6.5B). No significant effect on Cl +2 toxin was seen with combined arginine and CAM (Fig. 6.5A). 6.4 DISCUSSION In this study the ability of PSP toxin-producing enzymes to catalyse the biosynthesis ofC1+2 and STX under CAM stress was investigated, in viva and in vitro. These experiments were designed to assay whether multifunctional enzyme complexes, such as NRPS/PKS systems, could be involved in the first step of STX biosynthesis (Shimizu 1993). Large multienzyme complexes have long cellular half-lives while, in viva, they are metabolically unstable as free subunits (Varshavsky 1997). NRPS/PKS are resistant to degradation and can remain active for extended periods (days) (Arment and Carmichael 1996). In a previous study, the biosynthesis of MCYST was demonstrated to be independent from the blockage of protein synthesis induced by CAM (Arment and Carmichael 1996). This cyclic peptide is, in fact, produced by mixed NRPS/PKS enzymes (Dittmann et al. 1997). Additionally, the production of MCYST was shown to be functional over time both in vitro and in viva under CAM exposure, as expected for products ofNRPS and PKS. Similar results were found in the present study for MCYST and the analogous cyclic peptide NODLN. Arment and Carmichael (1996) reported a three-fold increase in MCYST content in cultures exposed to CAM at 50 µg mL- 1 for 7 days, data that were consistent with the values observed here for MCYST in cultures supplemented with CAM at 10 µg mL- 1 for 5 days (Fig. 6.2A). In similar experiments, however, the PSP toxins produced by C. raciborskii T3 showed a markedly different response to that of MCYST and NODLN. Cl +2 toxins were moderately affected by CAM compared to STX, suggesting a possible difference in the metabolic regulation of these two toxins. The extracellular levels of PSP toxins measured in this study (Fig. 6.2B) indicated that the export of these molecules was not 124 Chapter6 PSP tax in-prrxluctian and prrxei,n S'Jl1ll16is inhihition likely to be responsible for the observed variations in intracellular levels. CAM had no effect on the production of both STX and Cl +2 toxins in vitro. Taken together, these results revealed that in viva, but not in vitro, STX production was dependent on efficient protein synthesis. This suggested that STX enzymes may be subjected to a precisely balanced regulation, and that they have a short tum-over time compared to other cellular proteins, including the pigments chlorophyll-a and phycocyanin. STX biosynthetic enzymes also appeared to have a shorter turnover time compared to the proteins catalysing MCYST and NODLN biosynthesis. These results further indicated that STX biosynthesis is catalysed by enzymes different in function and half-life from NRPS and PKS. After ribosomal inactivation, STX biosynthesis enzymes were not produced and may have also been rapidly degraded in viva. Additionally, STX levels decreased over time suggesting that the toxin can also be degraded in cyanobacterial cells. Under CAM stress, STX synthesis appears to be relative to protein synthesis or protein inactivation, while toxin degradation also appears to be more rapid relative to its biosynthesis. The effects of arginine and two other amines on the production of PSP toxins by C. raciborskii T3 were also evaluated, again with or without the addition of CAM. The biosynthesis of Cl +2 toxins, as opposed to STX, was stimulated less efficiently by arginine. Addition of arginine under normal growth conditions enhanced the synthesis of STX in C. raciborskii T3. In the presence of CAM, however, this effect was not observed, supporting the possible specific rapid degradation of STX and its biosynthetic enzymes. Nevertheless, in vitro PSP toxins increased over time in the presence of CAM, indicating that the process of this specific inhibition of protein synthesis and/or toxin degradation may be inactive under these conditions. The difference between the effects of arginine seen in viva and in vitro (Fig. 6.3- 6.5) also suggest that, in vitro, arginine cannot be utilised by STX enzymes. This could result from a number of altered components in the crude extract, including the concentration of ATP or other cofactors such as acetyl-CoA, NADPH, or pyridoxal phosphate. These accessory compounds are required for the incorporation of arginine into the STX backbone (Shimizu 1993). Canavanine can disrupt several reactions that rely on argmme as the main substrate (Rosenthal 2001), including the production of STX and C1+2 toxins in C. 125 Chapter6 PSP tax in-praluctwn and prutei,n syntlx5is inhibitwn raciborskii T3 (Fig. 6.3). The results here confirmed that the PSP toxin biosynthetic enzymes rely on arginine (or one of its derivatives) as the principal substrate. The distinct effects observed during CAM stress and substrate feeding, and manifest in the profiles of STX and Cl +2 accumulation, highlight possible differences in the metabolism of these two classes of toxins in C. raciborskii T3. Proline is an amine derivative of arginine produced by cyanobacteria in consequence to salt or osmotic stress (Quintero et al. 2000). From previous investigations STX production in C. raciborskii T3 has been found to increase after pH and salt stress (Chapters 3 and 4). In this study, the feeding of C. raciborskii T3 cultures with proline lowered STX levels. This is consistent with previous results which found an intracellular decrease of a STX analogue in Planktothrix sp. FPl by amines belonging to the urea cycle (Pomati et al. 2001). Conversely, proline and agmatine strongly enhanced Cl +2 toxin accumulation in either the presence or absence of CAM. The data observed in this study suggest that STX and Cl +2 toxins are biosynthesised in C. raciborskii T3 via distinct pathways. This is in disagreement with the recent model of C 1+ 2 toxin synthesis in dinoflagellates proposed by Sako et al. (2001). If Cl+2 toxins are synthesised from STX via GTX2+3 production in cyanobacteria, as for dinoflagellates, a decrease in STX accumulation over time would result in either a corresponding decrease or a proportional increase in Cl +2 toxins. Such combined changes in toxin profiles were not observed during this study. Moreover, the presence of traces of GTX2+3 would be expected in a microorganism producing C 1+2 toxins. However, GTX2+ 3 have not been detected in C. raciborskii T3 either during this or in previous investigations (Lagos et al. 1999, Azevedo pers. comm., Chapter 3 and 4). Alternatively, in addition to the possible differences in the biosynthetic mechanisms, distinctive rates of degradation could be responsible for the observed imbalance in STX and Cl +2 toxins levels. 6.5 CONCLUSIONS This investigation presents evidence for different pathways catalysing the production of STX and Cl +2 toxins in the cyanobacterium C. raciborskii T3. 126 Chapter6 PSP tax in-prrx}uction and -protei,n syntfuis inhihitim Additionally, the mechanism of PSP toxin synthesis in cyanobacteria may be distinct from that proposed for dinoflagellates. This data also supported the hypothesis that STX biosynthetic enzymes have a short half-life in cyanobacterial cells, especially when compared to the proteins synthesising the cyclic peptide toxins MCYST and NODLN. Moreover, PSP toxins were shown to accumulate in vitro in a crude cyanobacterial extract. These findings may also have substantial relevance in the management and control of PSP toxin-producing cyanobacterial blooms m the environment, and establishes a fundamental basis for future research on PSP enzymology in cyanobacteria. 6.6SUMMARY In this study the activity of saxitoxin (STX) biosynthetic enzymes was assayed after inhibiting protein synthesis with chloramphenicol (CAM) in the cyanobacterium Cylindrospermopsis raciborskii T3. The production of the paralytic shellfish poisoning (PSP) toxins, Cl +2 and STX, was more sensitive to CAM when compared to the biosynthesis of the cyclic peptide toxins microcystin and nodularin in Microcystis aeruginosa PCC7806 and Nodularia spumigena NSORl 0, respectively. In particular, both intracellular and extracellular levels of STX decreased by 70% after 24 h exposure to the antibiotic. PSP toxin production was strongly promoted by arginine supplementation, with a maximum 476% increase in intracellular STX concentrations after 24 h exposure to 10 mM of the amino acid. However, arginine had no stimulating effect on PSP toxin levels if supplemented in combination with CAM at 10 µg L-1• Addition of agmatine and pro line to C. raciborskii T3 cultures in the presence of 10 µg L-1 CAM increased Cl +2 toxins levels, while having a negative or no effect on STX accumulation. In vitro, PSP toxin levels increased naturally in cyanobacterial extracts, with CAM and arginine having no influence on either Cl +2 or STX synthesis. The evidence presented in this study suggests a possible difference between the metabolism of STX and the Cl +2 toxins and indicated a high tum-over rate of STX biosynthetic enzymes in C. raciborskii T3. 127 Chapter7 Tax ic-strain Specific Genes in A. ci:ranalis CHAPTER 7 IDENTIFICATION OF A Na+ DEPENDENT TRANSPORTER ASSOCIATED WITH SAXITOXIN PRODUCING STRAINS OF ANABAENA CIRCINALIS I knew a girl, Her name was truth, She was a horrible liar. (Ben Harper) 128 Chapter7 Taxic-strain Spocific Gent5 inA. ci:rcinalis 7.1 BACKGROUND A. circinalis is a cyanobacterial species found world-wide. There is, however, a geographical segregation of toxin-producing strains, with only Australian isolates being able to produce PSP toxins (Section 1.2.2). The reason for this geographical segregation of neurotoxin production is not known. Adaptation to specific environmental pressures or genetic heterogeneity within the species A. circinalis are possible explanations. Recently, the phylogenetic structure of this species was determined by analysing the 16S rRNA and DNA-dependent RNA-polymerase gene sequences (Beltran and Neilan 2000, Fergusson and Saint 2000). A. circinalis was found to form a monophyletic group of worldwide distribution. Nevertheless, the PSP- and non-PSP toxin-producing isolates formed two clusters according to the 16S rRNA gene tree, with most of the toxic and non-toxic strains clustering separately, with few exceptions (Beltran and Neilan 2000). These data suggested that a certain degree of genomic divergence is present among toxic and non-toxic strains of this species. A phylogenetic analysis targeting STX encoding genes in A. circinalis would be more precise and ideal for both toxigenicity identification and population analysis. Unfortunately, DNA sequence information regarding these biosynthesis genes is currently unavailable. In this study, the cyanobacterial specific HIPl (highly iterated octameric palindrome 1) repeated sequence PCR was used to compare the genomes of phylogenetically closely related isolates of toxic and non-toxic A. circinalis. This technique is based on the genetic polymorphisms within defined cyanobacterial repetitive elements (Gupta et al. 1993, Robinson et al. 1995), and utilises HIPl directed PCR primers. HIPl is an octameric sequence (5'-GCGATCGC-3') abundant in the coding regions of cyanobacterial genomes (Robinson et al. 1995). HIP 1 PCR has been previously used to demonstrate genetic diversity among strains of the genera Anabaena and Nostoc (Smith et al. 1998, Zheng et al. 2002) and to distinguish between C. raciborskii isolates (Neilan et al. 2003, Saker and Neilan 2001). The aim here was to identify genomic differences that correlated with STX production in Australian strains of A. circinalis. Additionally, HIPl-generated libraries from STX-producing and non toxic cyanobacterial isolates were investigated by suppression subtractive hybridisation (SSH), to further recover possible toxic and non-toxic specific sequences. DNA 129 Chapter7 Tax ic-s-crain Specific Genes in A. ci:rrinalis fragments from both the HIPl and SSH analyses were screened by DNA microarray hybridisation with labelled toxic/non-toxic A. circinalis genomic DNA. A single toxic strain specific gene was identified by this multi-staged process. The application of this gene as a molecular probe for routine assessment of the potential risk associated with the presence of PSP toxin-producing A. circinalis in water reservoirs was also demonstrated in this study. The possible function of this gene is discussed with regards to the cellular homeostasis of these STX-producing planktonic bacteria. 7.2 EXPERIMENTAL PROCEDURES 7.2.1 Cyanobacteria PSP toxin-producing and non-toxic isolates of A. circinalis were maintained in JM medium, while Cylindrospermopsis raciborskii strains A WT205 and T3 were maintained in ASM-1 medium (Section 2.1.1). Cyanobacteria were grown without agitation or aeration in glass 250 mL flasks and cultures in mid-exponential growth phase were used for DNA extraction. A. circinalis strains used in this study were 131 C, 344B, 134C, 307C, 150A and 279B among the PSP-toxin producing together with the non-toxic 306A, 332H, 271C and 342D (Appendix A). Environmental samples of PSP toxin-producing and non-toxic cyanobacterial blooms in the Sydney catchment area were kindly provided by the NSW Department of Land and Water Conservation. 7.2.2 DNA extraction Cyanobacterial cultures were filtered through a 3.0 µm pore size filter (Millipore, Billerica, MA), and cells washed twice with sterile water. Genomic DNA was extracted from filtered and washed cyanobacterial cells, and environmental bloom samples, according to the procedure described in Section 2.6.1 and resuspended in TE. 130 Chapter7 Tax ic-strain Specific Gm:s in A. ci:rrinal,is 7.2.3 PCR amplifications and DNA sequencing HIPl PCR amplifications were performed using both primers Hip-CA and Hip TG (Smith et al. 1998, Saker and Neilan 2001) (Appendix C, Section C.2). Twenty microliter volume reactions contained 200 µM deoxynucleoside triphosphates (dNTP), 2.5 mM MgCh , Taq polymerase buffer, 5 pmol of each primer, 100 ng of DNA template and 1 U Taq polymerase. Reactions were cycled using a temperature profile of 95°C, 5 min; followed by 30 cycles of 95°C, 10 s; 40°C, 20 s; 72°C, 90 s; and concluded with one cycle of 72°C for 5 min. HIPl PCR products were separated by 4% polyacrylamide gel electrophoresis (PAGE) in TBE buffer according to standard protocols (Sambrook et al. 1989). Gels were photographed and the images were then used to construct a binary matrix based on the visual presence/absence of DNA bands on the electrophoresis gel. This binary matrix was subsequently used as the basis for the construction of a phylogenetic tree, achieved using the ClustalX program version 1.8 (Thompson et al. 1997), with C. raciborskii A WT205 and C. raciborskii T3 serving as outgroups. DNA was extracted from 4% PAGE bands following the standard "crush and soak" procedure (Sambrook et al. 1989) and re-amplified by HIP 1 PCR. PCR products were ethanol precipitated and cloned into pGEM-TE vector. Clones were amplified using the pGEM-TE vector-specific primers (mpf and mpR) and sequenced by PRISM Automated BigDye terminator sequencing and an ABI 373 sequencer (PE Applied Biosystems, Foster City, CA, USA), with reactions performed using 3 µl (-150 ng) of each PCR product and 10 pmol of each appropriate primer in a half-scale reaction as specified by the manufacturer. Amplification of the Na+ dependent transporter sequences was performed using 20 pmol each of the degenerate primers NaTF and NaTR (Appendix C, Section C.2) in a 20 µl reaction containing 200 µM dNTP, 2.5 mM MgCh, Taq polymerase Buffer, 100 ng of DNA template and 0.25 U Taq polymerase. PCRs were cycled using a temperature profile of 94°C, 3 min; followed by 30 cycles of 94°C, 10 s; 50 ± 5°C, 20 s; 72°C, 50 s; and concluded with one cycle of 72°C for 5 min. Specific primers for the Na+ dependent transporter were also designed, YZF and YZR (Appendix C, Section C.2), and 10 pmol of each utilised in 20 µ1 reactions as described above. The following 131 diapter7 T(IJC ic-strain Specific Gerl3 in A. ci:rcin:di.s protocol was used: 94°C, 3 min; 30 cycles of 94°C, 10 s; 55°C, 20 s; 72°C, 50 s; one cycle of 72°C, 5 min. To detect the presence of cyanobacteria and the gene encoding the Na+ dependent transporter simultaneously, a multiplex PCR was performed. This involved combining the primers YZF and YZR, with the cyanobacterial-specific 16S rRNA gene primers 27F and 809R (Appendix C, Section C.2) in the same PCR reaction. Primer concentrations and PCR cycling conditions were as above. All results were visualized by 1.5 or 2% agarose gel electrophoresis in T AE buffer according to standard protocols (Sambrook et al. 1989). PCR products were either ethanol precipitated or extracted from agarose using the QIAquick Gel Extraction Kit (QIAGEN, Germantown, Maryland, USA) and sequenced as mentioned above. Phylogenetic tree construction and sequence alignments were performed using the ClustalX program version 1.8 (Thompson et al. 1997). 7.2.4 SSH DNA libraries were generated from the HIPl PCR products as reported above and with the addition of fresh Pfu polymerase (Promega, Madison, WI, USA) at 0.5 U per 20 µL reaction volume at the end of the temperature cycles. Pfu polymerase reactions were carried out at 72°C for 20 min, the DNA precipitated with ethanol and resuspended in water following standard protocols (Sambrook et al. 1989). Subtraction of HIPl PCR libraries was achieved by means of a modified PCR-based subtractive hybridisation protocol descried in Section 2.7. Subtraction experiments were carried out using A. circinalis strains 332H (non-toxic) as the driver and 134C (toxic) as the tester, in one experiment, and A. circinalis strains 131 C (toxic) as the driver and 306A (non toxic) as the tester, in another experiment. Briefly, for each experiment, 1 µg of HIP 1- generated DNA library from each strain (driver and tester) were digested with Rsal and two different PCR adapters were ligated to two different aliquots of the tester DNA. Two hybridisations were then performed according to the standard procedure (Section 2.7). 132 Chapter7 Tax ic-strain Specific GerX:S in A. ci:rrina/,is 7.2.5 Microarray design and production Positive SSH clones (Section 7.2.4), together with the cloned Na+ dependent transporter fragment from A. circinalis l34C, were amplified with the vector specific primers mpf and mpR (Appendix C), purified using 96 well multi-screening membrane plates (Millipore) and resuspended in 70 µL water. Cloned and purified PCR products were spotted in duplicate with more than 300 other DNA fragments, including 16S rRNA of the investigated strains included as housekeeping genes (BGGM 1 microarray) as reported in Section 2.9. The complete BGGM1 array list of genes is reported in Appendix D. 7.2.6 Genomic DNA labelling and hybridisation One microgram of genomic A. circinalis DNA from strains 332H and 306A (non-toxic), 134C and 131 C (toxic) was digested with Rsal, extracted with phenol and precipitated with ethanol. Fluorescently labelled DNA was prepared indirectly by incorporating amino-allyl dUTP followed by coupling with the fluorescent dyes as described in Section 2.10. Labelled DNA samples were then combined according to the different experiments and evaporated to dryness. Each microarray hybridisation was performed in duplicate. Cy3-labeled genomic DNA (green) from the non-toxic strains 332H and 306A were hybridised with Cy5- labeled genomic DNA (red) from the toxic strains 134C and 131 C, respectively. For each single hybridisation, fluorescently labelled DNA was resuspended in 20 µL of hybridisation solution and the hybridisation performed overnight at 37°C as reported in Section 2.11. Array slides were washed, dried and kept in the dark prior to scanning (Section 2.11 ). 7 .2. 7 Microarray scanning, data acquisition and statistical analyses Clean slides were scanned and images quantified as described in Section 2.12. Erroneous spots were manually flagged and removed from the final data set, and the median Cy5/Cy3 ratio for each spot was used for subsequent analysis (Section 2.12). 133 Oiapter7 Tax ic-strain Specific Genes in A. cirrinal.is The ratio of medians were normalised to give a ratio measurement of 1 for the control sequences corresponding to the tested strains 16S rRNA gene (Appendix D). 7.2.8 Nucleotide sequence accession numbers The sequences determined in this study have been submitted to the GenBank nucleotide sequence database and assigned to accession numbers AY326655 to AY326688 (Appendix F). 7.3 RESULTS 7 .3.1 HIPl genomic polymorphism The HIP 1 PCR technique showed clear differences in banding patterns between toxic and non-toxic isolates of A. circinalis (Fig. 7.1). Using this approach, and resolving the banding patterns with 4% PAGE, 15 bands were identified that could be used to distinguish between strains of A. circinalis, from an overall of 22 genomic characters, including data from the two outgroup isolates of C. raciborskii (Fig. 7 .1 ). The phylogenetic tree constructed using the binary matrix based on the presence/absence of electrophoresis bands is shown in Fig. 7.2. PSP toxin-producing A. circinalis strains clustered into a monophyletic clade supported by a bootstrap resampling value of 79% (A). This cluster, characterized by toxic strains apart from the non-toxic 271C, branched from the deeper non-toxic lineage. Toxic strains also demonstrated more conserved HIPl genomic patterns compared to the non-toxic A. circinalis, as seen by the relative distances in the phylogenetic tree reconstruction (Fig. 7.2) and the actual DNA profiles. No toxic isolates were found to cluster within the group characterised as non-toxic A. circinalis. 134 Chapter7 Tax ic-strain Specific Genes in A. circinalis 10 16 Ml 342D 344B 131C 134C 307C A\VT205 Figure 7.1 Electrophoretic comparison of the PCR products formed in reactions primed with Hip-CA and Hip-TG primers for the nine isolates of A. circinalis and the two strains of C. raciborskii. M1 = qiX174 Haelll DNA marker; M2 = 1 Kb Plus DNA Ladder (lnvitrogen). Band 10, characteristic of the majority of non-toxic strains, and band 16, characteristic of the majority of STX-producing strains, are shown. 135 Chapter7 Taxic-strain Speafic Gerx:s in A. ci:ranaHs C. raciborkii A WT205 C. raciborkii T3 ~ A. circinalis 342D ~ A. circinalis 306A 965 - A. circinalis 332H 66( A. circinalis 344B 762 10011A, circinalis 307C 786 991 A. circinalis134C A -0.05 414 A. circinalis 279B 545 A. circinalis 271C ~ A. circinalis 131 C Figure 7.2 Phenogram constructed from analysis of electrophoresis gels resulting from the HIP-PCR in Fig. 7.1. A binary matrix was tabulated based on the presence or absence of bands and consisted of 22 characters across the 11 operational taxonomic units. Tree reconstruction procedures are described in the text. All bootstrap values (1,000 resampling events) are shown. A) STX-producing A. circinalis. By visual analysis of DNA bands, each of the two groups of A. circinalis strains, non-toxic and toxic, were delineated by the presence of a single unique DNA band. These bands were denoted 10 and 16, respectively (Fig. 7 .1 ). Acrylamide bands were excised and the DNA extracted, cloned, and sequenced. Band 10, characteristic of the majority of non-toxic strains, was extracted from the A. circinalis 306A electrophoresis profile. The DNA fragment (clone 4 7) encoded for an unknown conserved hypothetical protein, with the best BLASTX (Altschul et al. 1997) sequence analysis score of 77% identity and 89% similarity to locus ZP _00112177 from Nostoc punctiforme SAG69.79. Band 16, characteristic of the majority of STX-producing strains, was extracted from the A. circinalis 134C banding pattern. The recovered DNA fragment (clone 219) 136 Chapter7 Tax; ic-strain Specific Genes in A. ci:rrinal:is encoded for a putative Na+ dependent transporter protein, with the best BLASTX score of 71 % identity and 85% similarity to ORF alr5254 (accession number BAB76953) from Nostoc PCC7120 (Table 7.1). Clone 219 was spotted onto the DNA microarray for further investigation. 7.3.2 SSH of HIPl genomic libraries DNA sequences unique to the toxic strain 134C and the non-toxic strain 306A of A. circinalis were tentatively identified by the SSH of HIPl PCR generated libraries. Strains 332H and 131 C were used as the driver genomes. DNA containing putative tester-specific products was cloned and the insert size of 50 randomly selected clones per library estimated by PCR amplification. Insert size varied from 0.15 to 0.5 kb and, for each experiment, a total of 25 cloned DNA fragments with different sizes were purified and sequenced. Of the 50 clones analysed, 9 sequences were encountered more than once (18%) while 10 showed no significant similarity with any entry in the databases. General features of the putative tester-specific sequences detected in this study for A. circinalis 134C (toxic) and 306A (non-toxic) are reported in Tables 7.1 and 7.2, respectively. The library of possible toxic-strain sequences contained both hypothetical proteins of unknown function, membrane proteins, as well as enzymes that could be involved in both primary and secondary metabolic pathways, such as carbamoyl-phosphate synthase and methyltransferase (Table 7.1). The putative library of non toxic-strain specific fragments comprised mostly hypothetical proteins of unknown function and three defined enzymes, two of which were protein kinases (Table 7.2). 137 Chapter7 Taxic-strain Spttific GerK!S inA. arcinalis Table 7.1 A. circinalis 134C putative specific sequences with significant protein matches (Id: identity; Sim: similarity) in The National Center for Biotechnology Information (NCBI) protein database. a Values are the result of two independent hybridisations and are expressed as average ± SE of Cy5/Cy3 normalised ratios. Toxin-strain specificity levels are defined by Cy5/Cy3 ratios > 2, while non-toxic strain specificity is defined by Cy5/Cy3 ratios < 0.5. ND = not detected. Put. = putative. Clone Best BLASTX hit Organism % % Microarray Id Sim hybridisationa 219 putative Na+ dependent Nostoc sp. PCC7120 71 85 2.42 ± 0.12 transporter 194 hypothetical protein C. hutchinsonii 41 61 1.45 ± 1.03 147 hypothetical protein M magnetotacticum 47 50 1.17 ± 0.11 146 succinate dehydrogenase Nostoc sp. PCC7120 93 96 1.15 ± 0.01 flavoprotein 149 hypothetical protein B.fungorum 42 62 1.11 ± 0.03 153 carbamoyl-phosphate Synechocystis sp. 96 98 1.03 ± 0.02 synthase, pyrimidine- PCC6803 specific, large chain 154 hypothetical ORF S. cerevisiae 37 55 0.90 ± 0.15 129 mucin 1 precursor M. musculus 36 56 0.85 ± 0.12 130 dolichol-phosphate B. thetaiotaomicron VPI- 80 90 0.76 ± 0.10 mannosyltransferase 5482 155 put. phosphoglycerate B. thetaiotaomicron VPI- 50 72 0.61 ± 0.04 dehydro genase 5482 . . 145 morgan1c A. mediterranea 54 74 0.60 ± 0.02 pyrophosphatase 150 hypothetical protein V. parahaemolyticus 39 69 0.58 ± 0.03 RIMD 122 oligopeptide-binding Nostoc sp. PCC7120 31 63 0.50 ± 0.16 protein 138 Chapter7 Taxic-strain Specific Gerx:s in A. ci:rrina/.is Table 7.1-Continued Clone Best BLASTX hit Organism % % Microarray Id Sim hybridisationa 190 peptidoglycan anchored L. monocytogenes EGD-e 29 50 0.50 ± 0.14 protein 123 hypothetical protein T. erythraeum IMS 101 44 62 ND 192 S-adenosyl- B. thetaiotaomicron VPI- 45 59 ND methyltransferase mra W 5482 139 Chapter7 Tax ic-strain Specific Genes in A. ci:ranaHs Table 7.2 A. circinalis 306A putative specific sequences with significant protein matches (Id: identity; Sim: similarity) in the NCBI protein database. a Values are the result of two independent hybridisations and are expressed as in Table 7.1. Clone Best BLASTX hit Organism % % Microarray Id Sim hybridisationa 208 serine/threonine 0. javanicus 31 56 2.28 ± 0.22 kinase 158 hypothetical protein N punctiforme 59 76 1.86 ± 0.12 205 splicing factor Prp8 G. theta 31 53 1.59 ± 0.01 163 hypothetical protein R. sphaeroides 43 56 1.41 ± 0.26 210 hypothetical protein Nostoc PCC7120 37 68 1.11 ± 0.05 166 locus CG32796-PB D. me/anogaster 40 65 1.04 ± 0.06 162 hypothetical protein N punctiforme 47 54 1.00 ± 0.02 164 two-component sensor Nostoc PCC71120 75 91 0.92 ± 0.09 histidine kinase 143 DNA polymerase III B. thetaiotaomicron VPI- 80 85 0.66 ± 0.02 alpha subunit 5482 137 hypothetical protein C. hutchinsonii 33 61 0.65 ± 0.02 140 conserved hypothetical B. thetaiotaomicron VPI- 41 66 0.55 ± 0.01 protein 5482 207 conserved hypothetical Y. pestis KIM 47 65 0.51 ± 0.03 protein 202 hypothetical protein N punctiforme 55 77 0.48±0.18 206 hypothetical protein C. hutchinsonii 34 57 0.37 ± 0.05 204 hypothetical protein C. hutchinsonii 44 67 0.16 ± 0.03 213 cylM protein E. faecalis V583 42 57 ND 140 Chapter7 Taxic-strain Spedfic Genes inA. ci:rrina/,is 7.3.3 Microarray hybridisation The array was hybridised with labelled genomic DNA of the SSH testers and drivers. By microarray hybridisation, only one tester-specific sequence was identified for each strain (Tables 7 .1 and 7 .2). The only fragment with a Cy5/Cy3 ratio of means higher than 2 arising from the toxic strain 134C was the HIP 1 band corresponding to the Na+ dependent transporter. On the other hand, the non-toxic strain 306A revealed a candidate gene similar to a serine/threonine kinase (locus BABl 7220) from Oryzias javanicus (ratio of 2.28, Table 7.2). 7.3.4 Amplification of genes encoding Na+ dependent transporters Degenerate primers (NaTF/R) were designed based on the conserved regions among the translated sequence of clone 219 from A. circinalis l34C and putative Na+ dependent transporter (NaDT) protein homologues from Nostoc sp. PCC7120 and Synechocystis sp. PCC6803. By NaTF/R PCR, amplicons of the expected size (750 bp) were produced from toxic A. circinalis strains. PCR products amplified from strains 134C and 279B were purified and sequenced. The data confirmed that the DNA fragment corresponded to the same putative NaDT. BLAST analysis also indicated the relationship of this sequence to members of the sodium bile acid symporter family (SBF) and arsenical resistance protein (ACR). Based on sequences from strains 134C and 279B, the specific primers YZF/R were designed to amplify a 602 bp DNA fragment of the A. circinalis putative Na+ dependent transporter. As seen in Fig. 7.3, the primer set amplified NaDT gene sequences only from toxic isolates of A. circinalis, with the exception of the non-toxic strain 271C. 141 Chapter 7 Tade-strain Specific Genes in A. ci,rci,nalis M 1 2 3 4 5 6 7 8 9 10 11 M Figure 7 .3 Detection of Na+ dependent transporter genes in toxic and non-toxic strains of A. circinalis by PCR amplification with YZF/R specific primers. Gel lanes 1-11 as follows: 344B (1 ), 131 C (2), 279B (3), 150A (4 ), 307C (5), 134C (6), 271 C (7), 306A (8), 342D (9), 332H (10), no DNA control (11 ). PCR products were loaded as a total of 4 µL per sample and run with 1 Kb Plus DNA Ladder (lanes M) as standard. Different stringencies were tested for the PCR reaction based on YZF/R, showing no product of the expected size being amplified from the other non-toxic DNA-templates even for the lowest stringency employed, corresponding to a primer annealing temperature of 45°C. 7.3.5 Phylogeny of Na+ dependent transporter proteins Phylogenetic tree reconstruction was performed using several NaDT proteins from the databases to define possible functional homologies of the A. circinalis gene (Fig 7.4). Three distinct phylogenetic groups were recognised. All the ACR-like proteins clustered (A), with no cyanobacterial counterparts. The second group (B) clustered with mrpF-like proteins including the B. subtilis and B. firmus mrpF genes, and the Na+ dependent transporters from A. circinalis 134C and 279B, Synechocystis PCC6803 (ORF slll428), and Nostoc PCC7120 (ORF arl5254). A third group (C) comprising various bacterial and cyanobacterial putative sequences together, clustered with a SBF protein from A. thaliana. Partial alignment comparison (Fig. 5) between the 142 Chapter7 Tax ic-strain Spedfic GerES in A. ci,rrina/,is Na+ dependent transporters of A. circinalis 279B, Synechocystis PCC6803, and Nostoc PCC7120, together with the mrpF genes from B. subtilis and B. firmus showed that loci of major similarity between the two Bacillus sequences were also conserved in the three cyanobacterial proteins. 7.3.6 Screening for STX-producing A. circinalis by multiplex PCR A multiplex PCR assay was developed with the aim of detecting the presence of potential PSP toxin-producing A. circinalis in the environment. Figure 7 .6 shows the validation of this method on toxic and non-toxic cyanobacterial isolates (Fig. 7.6A) and its application to natural samples of PSP-toxin producing and non-toxic blooms (Fig. 7.6B). The 16S rDNA band (782 bp) identified the occurrence of cyanobacteria in the sample, while the YZF/R band (602 bp) indicated the presence of the toxic-strain specific NaDT and the potentiality of STX-producing A. circinalis. 143 Chapter7 Taxic-strain Specific Genl5 inA. ci:rci:naHs ACR3 A. thaliana 1000 ACR3 B. anthracis Ames ACR3 B. cereus ATCC14579 A 573 ] ACRI S. cerevisiae Microbulbifer degradans2-40 544 .------mrpF B. subtilis ...... ,,.99""6,-----1 414 mrpF B.jirmus Magnetospirillum magnetotacticum 895 1000 .----Pseudomonas putida KT2440 ~--"-="'---I Pseudomonas aeruginosaPAO I B 879 .------Nostoc sp. PCC7120 1000 A. circinalis 279B 1000 A. circinalis 134C 925 820 Synechocystissp. PCC6803 875 Leptospira interrogansserovar Corynebacterium glutamicumATCC 13032 ,-8""7""2 ______Salmonella typhimurium L T2 4'-17------Methanosarcina mazeiGoe I 1000 .------Synechococcussp. WH8 l 02 .------Thermosynechococcus elongatusBP-I .______SBF A. thaliana 516 Streptomyces coelicolorA3(2) C ~51~5----- Brucella suis 1330 455 .______B. halodurans C-125 758 Bifidobacterium /ongum NCC2705 241 ~------Staphylococcus aureusMW2 455 .------Neisseria meningitidisZ249I Oceanobacillus iheyensisHTE83 I I 0.1 .______B. subtilis subtilis 168 Figure 7.4 Phylogenetic affiliations of prokaryotic homologues of A. circinalis 134C and 279B Na+ dependent transporters. A. tha/iana ACR3 and SBF proteins, together with S. cerevisiae ACR1 gene, are included as reference. The phenogram was reconstructed from a pairwise distance matrix (Jukes and Cantor, 1969) by the neighbor-joining method (Saitou and Nei 1987). The scale represents one substitution per 100 amino acid positions. Local bootstrap values (1,000 resampling cycles) are shown. A) ACR transporters; B) mrpF-like proteins; C) other Na+ dependent transporters. 144 Chapter 7 Twcic-s"train S-pecific Gerll5 inA. ci:rr:inalis A. circinalis 279B ------TAVILPLALAIIMLGMGLSLLPDDFLPVTKYPKAVAIGLIS Nostoc sp. PCC7120 ---MQANVFTNVILPLALAIIMLGMGLSLQIEDFKRITKYPKAVSIGSAT Synechocystis sp. PCC6803 ---MESNFLTTIFLPLALFLIMFGMGLGLTIKDFNRIWLEPKAVAIGLIA mrpF B. subtilis ------MFTL---ILQIALGI-MGVSTF----1------YVI mrpF B. firmus ------MFQS---ILMIVLVV-MSISLF---V------CFI ...... * A. circinalis 279B QLIFLPIIGFIIAKIIPMEPAIAMGLMIIALCPGGVSSNIITFLAKGDVA Nostoc sp. PCC7120 QLILLPIMGFLVAKVVPMQPEIAVGLVILSLCPGGPSSNMITYLAQGDVA Synechocystis sp. PCC6803 QLVMLPMVGFGLSSLFSLSPELAVGLMILAACPGGSTSNVITYLLKGNVA mrpF B. subtilis RVIKGPTVP---DRVVALD---AIGINLIAIT------ALVSILLKTSAF mrpF B. firmus RTLIGPTMS---DRIVALD---TFGINLIGFI------GVIMMLQETLAY * . . *. . . . . * . A. circinalis 279B LSVTLTAFSSLITVFTIPILGNLAYQXFIGKTETAAIGLPIGATILQIFL Nostoc sp. PCC7120 LSVTLTVVSSMVTIFTIPIFANLALQHFLG--QTAAIALPIGSTMLQIFL Synechocystis sp. PCC6803 LSITLTAISSLVTIITIPLVVNLAANYFMG--EQFALQLPFLKTVLQIAV mrpF B. subtilis LDIIL-----LLGILS--FIGTIAFSKFL---E------mrpF B. firmus SEVVL-----VISILA--FIGSIALSKFI---E------ * . . * *. A. circinalis 279B MTLLPISLGMIFRQILPDIALRLEKVTNRLAVAFLSTKLSFLVI-----I Nostoc sp. PCC7120 ITIVPIGLGMYIKRIFPATALRLEKATNRLAIAFLALIILILVIREWNRI Synechocystis sp. PCC6803 ITIIPVSLGMIFYHFLPKIAMVMEKNVKWLSLFFLGIIIVAILVKERENL mrpF B. subtilis -----K--GEIIEN------DRNR------mrpF B. firmus -----R--GVVFDR------G------ * Figure 7.5 Alignment of partial amino acid sequences corresponding to the cyanobacterial Na+ dependent transporters of A. circinalis 279B, Synechocystis PCC6803 and Nostoc PCC7120, together with mrpF gene products of B. subtilis and B. firmus. Regions of identity and similarity are shown: * =fully conserved positions; : = strongly conserved residue; . = weakly conserved residue. 145 Chapter 7 Tax u:-strain Specific Genes in A . circi:nalis A) M 1 2 3 4 5 6 7 8 M 16S rDNA~ NaDT~ B) M 1 2 3 4 5 6 7 M 16S rDNA~ NaDT~ Figure 7.6 Electrophoretic comparison of the PCR products formed in reactions primed with the 16S rDNA primers 27F/809R and the toxic-strain specific NaDT primers YZF/R. A) Multiplex PCR screening of PSP toxin-producing and non-toxic A. circinalis isolates. Gel lanes 1-8 as follows: 131C (1), 134C (2), 279B (3), 118C (4), 150A (5), 332H (6), 306A (7), 342D (8). B) Multiplex PCR screening of PSP toxin-producing and non-toxic cyanobacterial blooms. Gel lanes 1-7 as follows: strain 118C (positive control), lane 1; PSP toxin-producing cyanobacterial blooms, lanes 2-3-4; non-toxic cyanobacterial blooms, lanes 5-6-7. PCR products were loaded as a total of 6 µL per sample and run with 1 Kb Plus DNA Ladder (lanes M) as standard. 146 Chapter7 Tax ic-strain Specific Ger/13 in A. circi:nal.is 7.4 DISCUSSION In the present study the identification of genomic differences between toxic and non-toxic strains of A. circinalis is reported. This was achieved by the application of HIP! repeated sequence PCR. Since the HIP! element is present in more than 50% of cyanobacterial ORFs and is absent in rRNA and tRNA genes (Bhaya et al. 2000), it can be used to identify gene content and genomic structure in this phylum of bacteria. Electrophoresis of HIP! PCR products showed evident genetic diversity between closely related strains of A. circinalis (Fig. 7 .1 ). The number of bands detected in A. circinalis was comparable to previous reports using the HIP 1 molecular typing technique (Neilan et al. 2003, Saker and Neilan 2001). Cluster analysis of these data showed that the toxic phenotype in A. circinalis was somehow portrayed by a particular HIP! genomic DNA pattern (Fig. 7.2). The STX-producing strains formed a distinct clade, with the branch of non-toxic isolates appearing as ancestral to the toxic group of strains. In a previous study (Beltran and Neilan 2000) however, 16S rRNA phylogeny revealed that the majority of STX-producing and non-toxic isolates clustered in two distinct lineages. Strain 271C was one of the few non-toxic A. circinalis clustering with the toxic strains, as is reported in the present study using HIPI-typing. On the other hand, strain 134C, which was shown to be more closely related to non-toxic A. circinalis by 16S rRNA analysis (Beltran and Neilan 2000), clustered with the other STX-producing isolates by HIP! PCR-based phylogeny. These results suggested that whole genome typing of closely related A. circinalis strains provide more information on toxigenicity than the evolution of a single gene, such as 16S rDNA or DNA dependent RNA-polymerase (Beltran and Neilan 2000, Fergusson and Saint 2000). The differences in HIP! genomic profiles among isolates of A. circinalis were first indicated by the presence of a single unique DNA band in both toxic and non-toxic strains. This suggested consistent differences in composition and genomic organization between the two groups or evolutionary divergence of toxic strains over the non-toxic congeners. These bands were characterized as encoding a Na+ dependent transporter and a conserved hypothetical protein in the STX-producing and the non-toxic isolates 134C and 306A, respectively. 147 Oiapter7 Tax ic-strain Spaific Genes in A. arrinalis Genomic heterogeneity between toxic and non-toxic strains of A. circinalis was further explored by subtracting HIPl generated DNA pools using the SSH technique. The aim here was to highlight the diversity in HIP 1 libraries that could not be identified or resolved by gel electrophoresis. SSH has been used for the rapid identification of genetic differences between pathogenic bacteria (Akopyants et al. 1998, Harakava and Gabriel 2003), as well as to derive information on ecologically relevant genetic adaptations in closely related prokaryotes (Mavrodi et al. 2002, Nesb0 et al. 2002). The two groups of clones obtained here, though not exhaustive of the entire pool, were sufficiently representative of the whole SSH libraries compared to previous studies (Akopyants et al. 1998), since an average of 18% of the sequences were encountered more than once. However, SSH did not detect the two HIPl PCR products found to be characteristic of the toxic and non-toxic DNA banding patterns, possibly as a consequence of the number of clones analysed. Amongst the two categories of SSH putative tester-specific DNA fragments, of particular interest were the genes coding for carbamoyl-phosphate synthase and S adenosyl-methyltransferase. These two enzymes could be involved in regulation or production of STX in cyanobacteria (Shimizu 1993, Taroncher-Oldenburg and Anderson 2000). The specificity of tester recovered fragments was verified by m1croarray hybridisation, indicating that only one HIPl-SSH sequence was tester-specific: clone 208 of the non-toxic strain 306A which is a putative serine/threonine kinase (Table 7.2). One toxic-strain specific gene was, however, indicated by microarray hybridisation: the Na+ dependent transporter that was originally cloned from the HIPl PCR banding pattern of strain 134C. This gene was only found in STX-producing isolates with the exception of the non-toxic strain 271C, either using both degenerate and specific PCR (Fig. 7.3). Using YZF/R PCR amplification, as well as by HIPl typing, strain 271C was the only false positive observed in this study. Previously shown to cluster with toxic strains by 16S rRNA analysis (Beltran and Neilan 2000), it is possible that this non-toxic isolate could be a spontaneous natural mutant with regards to STX biosynthesis. The putative function of the recovered NaDT was investigated by comparing the translated amino acid sequences from A. circinalis 134C and 279B with several other 148 Chapter7 Tax ic-st:rain Specific Genes in A. ci:ranaHs similar proteins, showing A. circinalis genes clustering in a group characterised by other bacterial and cyanobacterial Na+ dependent transporters (Fig. 7.4). Together with conservation of particular peptide motifs (Fig. 7 .5), this data suggested functional homology between cyanobacterial Na+ dependent transporters and mrpF genes of Bacillus. The mrp (multiple resistance and pH adaptation) operon and its homologues are distributed among diverse prokaryotic genera and function in multiple processes involving ion-coupled transport reactions, including Na+-specific pH homeostasis. Thus far, only the mrp operon of B. subtilis has been studied in detail (Ito et al. 1999, 2000). The mrpF gene has been found to encode for a protein functioning in cholate and Na+ efflux, with MrpF activity independent of the expression of any other additional mrp gene product (Ito et al. 2000). The Na+ efflux catalysed by the independent transporter MrpF, coupled to solute efflux (e.g., endogenous cholate-like substrate and/or exogenous cholate-like compounds), has been suggested to be particularly important for coordinating a full Na+ cycle and achieving both substrate uptake and cytoplasmic pH regulation under alkaline pH conditions (Ito et al. 2000). Apart from Nostoc punctiforme (http://www.jgi.doe.gov/JGI_microbial/html/), homologues of mrpF are present as single copy genes in the known cyanobacterial genomes (Cyanobase: http://www.kazusa.or.jp/cyano/) and none are organized in a cluster similar to the Bacillus mrp operon. The putative transposases upstream and/or downstream of the coding region for the cyanobacterial Na+ dependent transporter homologues may indicate the possibility of a mobile genomic region comprising this transporter protein. Taken together, these data may indicate an essential metabolic difference between STX producing and non-toxic isolates of A. circinalis in terms of Na+ -dependent pH homeostasis and the Na+ cycle in these two groups of strains. In previous studies an intrinsic association between the variation of cellular Na+ levels and the regulation of STX metabolism has been demonstrated in the freshwater cyanobacterium C. raciborskii T3 (Chapters 3 and 4). STX production was strongly induced by alkaline pH and salt stress, while STX inhibited sodium uptake in C. raciborskii and A. circinalis (Chapter 5). These results suggested a potential advantage for PSP toxin-producing microorganisms over other non-toxic species under conditions of high pH or salt stress. Differences in this Na+-dependent pH homeostasis may also be 149 Chapter7 Tax ic-strain Spedfic GerK:S in A. ci:rri:nalis the reason for the geographic segregation of STX production in cyanobacteria, as an adaptation to specific environmental pressures such as natural cycles of flood and drought. Similar environmental conditions correlated with the dominance of STX producing A. circinalis blooms in Australian freshwaters (Bowling and Baker 1996). Therefore the toxic-strain specific gene NaDT was utilised to develop a PCR based screening assay to detect the presence of potential STX-producing A. circinalis in the environment. The multiplex PCR reaction was optimised to have an internal positive control (16S rDNA) to assess the occurrence of cyanobacterial DNA, as well as the YZF/R probe to indicate the presence of PSP toxin-producing A. circinalis (Fig. 7.6). Compared to previous approaches (Beltran and Neilan 2000), this multiplex technique based on NaDT has the advantage of detecting no false negatives, which represent a serious problem in water management strategies. Due to the sensitivity of the PCR (detection limit of-100 cells), this test may prove invaluable in the early detection of potentially toxic blooms. As bloom treatment can be environmentally and economically costly, the rapid and accurate nature of this test would allow for more logical assessment of bloom toxicity before treatment. In addition, by performing these predictive tests on sediment during the winter months areas of concern can be highlighted and thus treated prior to bloom proliferation in the warmer months. 7.5 CONCLUSIONS The present study reveals greater diversity among STX-producing and non-toxic A. circinalis strains than what was evident from studies on 16S rRNA or DNA dependent RNA-polymerase gene sequencing. HIPl PCR demonstrated to be a valid tool to investigate the toxigenicity of related A. circinalis strains, and allowed the identification of a toxic-strain specific gene corresponding to Na+ dependent transporter. This discovery further supports a link between the production of STX and the maintenance of sodium and pH homeostasis in cyanobacteria. Specific primers were designed and applied to laboratory and environmental screening of STX-producing A. circinalis. As these tests are based on PCR, the combination of HIPl typing and SSH requires only a small amount of original template DNA. Together with microarray 150 Chapter7 Toxic-strain Specific Genes in A. arri:nalis technology, this method may also represent an advantageous procedure for investigating heterogeneity in gene structure and metabolism between closely related slow-growing or environmental microorganisms. Nonetheless, the findings reported in this study could be applied to other PSP toxin-producing cyanobacteria and algae. 7.6SUMMARY In this study HIP 1 octameric-palindrome repeated sequence PCR was used to compare the genomic structure of phylogenetically similar Australian isolates of A. circinalis. STX-producing and non-toxic cyanobacterial strains showed different HIPl DNA patterns and characteristic inter-repeat amplicons for each group were identified. Phylogenetic tree reconstruction based on the presence/absence of DNA bands indicated that toxic strains of A. circinalis formed a monophyletic clade. Suppression subtractive hybridisation (SSH) was performed using HIPl PCR generated libraries to further identify toxic-strain specific genes. A STX-producing and a non-toxic strain of A. circinalis were chosen as testers in two distinct experiments. The two categories of SSH putative tester specific sequences were characterized by different families of encoded proteins that may be representative of the differences in metabolism between STX producing and non-toxic A. circinalis. DNA-microarray hybridisation and genomic screening revealed a toxic strain specific HIPl fragment coding for a putative Na+ dependent transporter. Analysis of this gene demonstrated analogy to the mrpF gene of B. subtilis, whose encoded protein is involved in Na+ -specific pH homeostasis. The application of this gene as a molecular probe in laboratory and environmental screening for STX-producing A. circinalis was demonstrated. The possible role of this putative Na+ dependent transporter in the toxic cyanobacterial phenotype was also discussed, in light ofrecent physiological studies of STX-producing cyanobacteria. 151 Chapter 8 Searrhingfar STX-Genes in Cy:indw:teria CHAPTERS PCR-BASED POSITIVE HYBRIDISATION TO DETECT GENOMIC DIVERSITY ASSOCIATED WITH BACTERIAL SECONDARY METABOLISM Full many a gem of purest ray serene, The dark unfathom'd caves of ocean bear (Thomas Gray) 152 Chapter 8 Se.arrhingfor STX-Genes in Cyi~ 8.1 BACKGROUND One of the most striking aspects of bacterial secondary metabolites is the unforeseen ways in which some of these compounds are biosynthesised. These natural products, besides attracting attention because of their potential or actual toxicity, also represent a potentially rich source of new drug candidates. Many bacterial metabolites have chemical structures unprecedented in other aquatic or terrestrial organisms, and display new and interesting biological activity. In addition, the synthetic capability of bioactive metabolite production is not universal to a species, but is exceptional and often limited to certain strains. A PCR-based subtractive hybridisation approach was used here to explore the genetic diversity associated with the production of saxitoxin (STX) within phylogenetically closely related strains of toxic and non-toxic Anabaena circinalis. Although STX has been studied for more than 30 years, the molecular basis for the synthesis of this peculiar alkaloid is currently unknown. PCR-based genomic subtraction, also called suppress10n subtractive hybridisation (SSH), as been developed for the rapid identification of differences among pathogenic bacteria and first applied to strains of Helicobacter pylori (Akopyants et al. 1998, see also Section 2.12). SSH has also been employed to compare the genomes of virulent and avirulent strains of the aquatic pathogen Aeromonas hydrophila (Zhang et al. 2000), and to identify genomic differences between uropathogenic and nonpathogenic E. coli strains (Janke et al. 2001). The SSH technique has been successfully utilised to detect pathogenicity islands in infectious bacteria (Agron et al. 2002). It has also being used to derive information, via genomic diversity, regarding divergence in ecologically relevant genetic adaptations in closely related strains of Pseudomonas jluorescens and Thermotoga maritima (Mavrodi et al. 2002, Nesb0 et al. 2002). In this study SSH was used to identify potential differences between the genomes of two STX-producing and two non-toxic strains of A. circinalis. The suppression technique was modified to include a third hybridisation step, termed PCR based positive hybridisation (PPH), and designed to recover toxic strain-specific genome fragments that were in common between the two STX-producing strains (Fig. 8.1). The aim was to obtain genes associated with STX production in A. circinalis. In 153 Chapter8 Searching/or STX-Genes in Cy:tndw:teria concordance with previous work (Agron et al. 2002), the toxic-strain specificity of tester DNA fragments from the SSH and PPH libraries was analysed by quantitative DNA microarray hybridisation with labelled toxic/non-toxic A. circinalis genomic DNA and verified by PCR amplification. Tester 131C ·1 r Tester 3448 Driver ...... · : · : ...... Driver 306A 271C Un subtracted Unsubtracted control control SSH SSH Adaptor digestions' Adaptor' digestions I ,, . .' y Subtracted Unsub tracte PPH p PH DNA microarray spotting ' Tester Driver genomic DNA genomic DNA Microarray hybridisation Figure 8.1 Schematic diagram of the procedure applied in this study. Toxic A. circina/is strains were used as testers, non-toxic strains as drivers. Dashed lines indicate unsubtracted tester DNA. 154 Chapter 8 Seanhingfar STX-Genes in Cy:indw:teria 8.2 EXPERIMENTAL PROCEDURES 8.2.1 Cyanobacterial strains and growth conditions PSP-toxin producing and non-toxic isolates of A. circinalis were maintained in JM as described in Section 2.1.1. Cyanobacteria were grown without agitation or aeration in glass 250 mL flasks and cultures in mid-exponential growth phase were used for DNA extraction. The strains used in this study were A. circinalis 131 C, 344B, 134C, and 279B among the PSP-toxin producing together with the non-toxic 306A and 271C (Appendix A). 8.2.2 DNA extraction A. circinalis cultures were filtered through a 3.0 µm pore size filter (Millipore, Billerica, MA, USA), and cells washed twice with sterile water. Genomic DNA was extracted from filtered and washed cyanobacterial cells according to the procedure described in Section 2.6.1 and resuspended in TE. 8.2.3 SSH Subtraction of cyanobacterial genomes was achieved by means of the modified PCR-based subtractive hybridisation protocol described in Section 2.7. One microgram of genomic A. circinalis DNA from strains 306A and 271C (non-toxic), 131C and 344B (toxic) was digested with Rsal, extracted with phenol, and precipitated with ethanol. Subtraction experiments were carried out using A. circinalis strains 306A (non-toxic) as the driver and 131 C (toxic) as the tester, in one experiment, and A. circinalis strains 271C (non-toxic) as the driver and 344B (toxic) as the tester, in another experiment. Briefly, for each experiment, two different PCR adapters were ligated to two different aliquots of the restriction digested tester DNA. Two hybridisations were then performed (Fig. 8.2). 155 diapter8 Searching/or S TX-GerX!S in Cy:inmu:teria EXPERIMENTAL TESTER GENOMIC DNA ( Tester ) ( Tester ) • Mix with • Mix with Adaptor 1 Adaptor 2R Un subtracted tester control 'f Ligate to Ligate to Ligate to Adaptors Adaptor 1 Adaptor 2R 1 and 2R l l Add driver DNA Add driver DNA First hybridization First hybridization Add driver DNA Second hybridization PCR• PCR Figure 8.2 Standard SSH procedure, as reported by Akopyants et al. (1998) In the first hybridisation, an excess of driver DNA was added to each of the adapter-ligated tester DNAs. Each sample mixture was then denatured at 98°C for 2 min and allowed to reanneal at 63°C for 90 min. This hybridisation, will enable single stranded DNA to be enriched for tester-specific DNA, as DNA fragments that are not tester specific will form hybrid molecules with the driver DNA. In the second hybridisation, the two primary hybridisation reaction mixtures were combined without denaturing and allowed to anneal at 63°C overnight. Only the subtracted single-stranded tester-specific DNA should reassociate to make hybrids with the two different terminal adapters. Molecules with different adapters at each end were amplified exponentially 156 Chapter8 Seanhirgfar STX-Genes in C')U~ using PCR primers to the two adapter sequences (Appendix C, Section C. l ). PCR amplifications were performed as reported in Section 2.7.4. The putative tester-specific libraries were amplified using, as a positive control, aliquots of unsubtracted-tester DNA ligated with both terminal adapters (unsubtracted tester control, Fig. 8.2). PCR products obtained after SSH were cloned into the pGEM-TE vector (Promega) and clones amplified and sequenced as describe in Section 2.8. Sequences were analysed by the BLAST program (http://www.ncbi.nlm.nih.gov/BLAST/, Altschul et al. 1997). 8.2.4 PPH The PCR-based positive hybridisation step was performed on subtracted and unsubtracted libraries from A. circinalis 131C and 344B (Fig. 1). PPH was achieved using the same principles of the SSH protocol (Section 2. 7). With this novel technique however, no driver DNA was required and two tester DNAs were hybridised at high stringency, with each tester DNA ligated to a different terminal adapter (Fig. 8.3). After hybridisation, only common single-stranded sequences could reassociate to make hybrids having different adapters at each terminus. Only DNA fragments with both of the terminal adapters can be amplified exponentially using PCR primers to the two adapter sequences (Fig. 8.3). The procedure was carried out as follows. The second PCR amplification of the standard SSH protocol was performed in duplicate to obtain a total of 50 µL volume per reaction and DNA ethanol precipitated. These DNA libraries thus obtained (subtracted 131C, unsubtracted 131C; subtracted 344B, unsubtracted 344B) were resuspended in 40 µL of water with 10 µL aliquots (approximately 1.5 µg) utilised for restriction enzyme digestions (Fig. 8.3). In strain 131 C, the adapter 2R was excised from subtracted DNA and unsubtracted tester control by digestion with Eagl (New England Biolabs, Beverly, MA), while adapter 1 was removed from strain 344B subtracted and unsubtracted DNA using Natl (New England Biolabs). 157 Chapter8 Seardnr,g,far STX-Genes in Cy:indw:teria Tester DNA 1 Tester DNA2 Eag I - digestion Not I - digestion Tester DNA 1 with Adaptor 1 Tester DNA 2 with Adaptor 2R - ----···- hybridization a ••o---- =- b - C MM:::::J---~, M i Fill in the ends a e-:::::i---- b - - - C - I Add primers ..._ • Amplify by PCR a no amplification b linear amplification CMJ' 5·- and "" c exponential amplification* 3·.., CMli' Figure 8.3 The PPH method based on suppression PCR. Solid lines represent tester DNA fragments, boxes represent terminal adapters 1 and 2R. Adapter 2R was excised from tester DNA 1 (strain 131C) by digestion with Eagl, adapter 1 was removed from tester DNA 2 (strain 344B) with Notl. *Although there is an identical primer binding sequence on both ends of the type c molecules, the short overall homology at the two ends negates the suppression PCR effect (Akopyants et al. 1998). 158 Chapter8 Searrhing,far STX-Genes in Cyirdt.u:teria Together with the tester DNA, 50 µL reactions contained 15 U of restriction enzyme, 5 µL lOx NEB3 buffer (New England Biolabs) and 5 µg of BSA. Reactions were incubated at 37°C overnight, heat inactivated at 65°C for 10 min, the DNA extracted with phenol, precipitated with ethanol, and resuspended in 13 µL of water. The positive hybridisation was performed as shown in Fig. 8.3, combining the two subtracted libraries in one experiment, and the unsubtracted tester controls in another (Fig. 8.1 ). In a final 5 µL mixture, 1.5 µL aliquots of both 131 C DNA (tester DNA 1, adapter 1) and 344B DNA (tester DNA 2, adapter 2R) were combined with 1 µL of 5x hybridisation buffer (250 mM HEPES-Cl, pH 8.1; 2.5 M NaCl; 1 mM EDTA, pH 8.0) and 1 µL of water. The DNA was denatured at 98°C for 2 min and the hybridisation reactions incubated at 65°C for 1 h. Subsequently, 100 µL of dilution buffer (20 mM HEPES-Cl, pH 8.1; 50 mM NaCl; 0.2 mM EDTA, pH 8.0) was added to each mixture and the hybridisation completed by incubation at 65°C overnight. One microliter of each sample was diluted in 100 µL of water and 1 µL of this diluted hybridisation mixture used as template for the successive round of PCR amplification. PCR was performed essentially as the secondary amplification of the SSH protocol (Section 2.7), with the addition of an initial step at 72°C for 5 min to allow the Taq polymerase to fill any overhanging ends of hybrid DNA fragments. PCR products obtained after PPH for both subtracted and unsubtracted libraries were cloned and sequenced as described in Section 2.8. 8.2.5 PCR amplifications Amplification of sequences 179 and 109 was performed using 20 pmol each of the specific primers 179F and 179R, or 109F and 109R (Appendix C, Section C.2), in 20 µL reactions containing 200 µM dNTP, 2.5 mM MgC}z, Taq polymerase Buffer, 100 ng of genomic DNA template and 0.25 U Taq polymerase. PCRs were cycled using a temperature profile of 94°C, 3 min; followed by 30 cycles of 94°C, 10 s; 55°C, 20 s; 72°C, 30 s; and concluded with one cycle of 72°C for 5 min. All results were visualised by 2% agarose gel electrophoresis in TAE buffer according to standard protocols (Sambrook et al. 1989). 159 Cliapter 8 SeardJingfar S TX-Gerx:s in Cy:irdtuteria 8.2.6 Microarray design and production Candidate SSH and PPH clones amplified, purified and spotted onto a DNA microarray together with more than 200 other DNA fragments, including 16S rDNA of the investigated strains as housekeeping genes (BGGM 1 microarray), as described in Section 2.9. The complete BGGM1 array list of genes is reported in Appendix D. Each gene probe was spotted in duplicate and each glass slide contained two copies of the BGGM1 array. 8.2.7 Labelling of genomic DNA One microgram of genomic A. circinalis DNA from strains 271C and 306A (non-toxic), and 131 C and 344B (toxic) was prepared by digestion with Rsal, extracted with phenol and precipitated with ethanol. Fluorescently labelled DNA was prepared indirectly by incorporating amino-allyl dUTP followed by coupling with the fluorescent dyes according to the protocol described in Section 2.10. Labelled DNA samples were then combined according to the different experiments and evaporated to dryness. 8.2.8 Microarray hybridisation Each microarray hybridisation was performed in duplicate. Cy3-labeled genomic DNA (green) from the non-toxic strains 271C and 306A were hybridised with Cy5- labeled genomic DNA (red) from the toxic strains 344B and 131 C, respectively. For each single hybridisation, fluorescently labelled DNA was resuspended in 20 µL of hybridisation solution and the hybridisation performed overnight at 37°C as reported in Section 2.11. Array slides were washed, dried and kept in the dark prior to scanning (Section 2.11 ). 8.2.9 Microarray scanning, data acquisition and statistical analyses Clean slides were scanned and images quantified as described in Section 2.12. Erroneous spots were manually flagged and removed from the final data set, and the 160 Chapter 8 Searrhingfor STX-Genes in Cy:inth:uteria median Cy5/Cy3 ratio for each spot was used for subsequent analysis (Section 2.12). The ratio of medians were normalised to give a ratio measurement of 1 for the control sequences corresponding to the tested strains 16S rRNA gene (Appendix D). 8.2.10 Nucleotide sequence accession numbers The sequences determined in this study have been submitted to the GenBank nucleotide sequence database and assigned to accession numbers A Y 445143 to A Y 445188 (Appendix F). 8.3 RESULTS 8.3.1 Suppression subtractive hybridisation Putative DNA sequences specific to the toxic A. circinalis strains 131 C and 344B were cloned and the insert size of 50 randomly selected clones per library estimated by PCR amplification. Insert sizes varied from 0.15 to 0.5 kb and, for each experiment, a total of 20 cloned DNA fragments of different sizes were purified and sequenced. Of the 40 clones analysed comprising toxic-specific fragments from strains 13 lC and 344B, 7 sequences (17.5%) were encountered more than once (17.5%). General features of these SSH DNA fragments are reported in Tables 8.1 and 8.2 for A. circinalis 131 C and 344B, respectively. Each library was found to be composed of 11 putative toxic-specific sequences, with strain 131 C fragments mainly characterized by hypothetical proteins of unknown function (Table 8.1) and an average mol¾ GC of 58.4. Toxic sequences specific to strain 344B, however, comprised hypothetical proteins of unknown function together with defined enzymes and membrane proteins (Table 8.2), with an average mol¾ GC of 47.5. 161 Chapter8 Searrhingfar STX-Genes in Cy:tn,h:tcteria Table 8.1 A. circinalis 131C putative specific sequences with protein matches (Id: identity; Sim: similarity} in The National Center for Biotechnology Information (NCBI} protein database. Microarray experiments represent hybridisations between strains 131C (Cy5} and 306A (Cy3} using genomic DNA. Microarray values are expressed as average± SE of Cy5/Cy3 normalised ratios. Toxin-strain specificity levels are defined by Cy5/Cy3 ratios > 2, while non-toxic strain specificity is defined by Cy5/Cy3 ratios< 0.5. ND= not detected. Put.= putative. % % Microarray ID Best BLASTX hit Organism Id Sim hybridisation SSH 65 Hypothetical protein Cytophaga hutchinsonii 57 81 5.3 ±0 70 TonB, ferric-siderophore B. thetaiotaomicron 42 66 4.9 ± 0.3 uptake VPI-5482 73 Alpha-amylase precursor C. acetobutylicum 47 58 4.6 ±2.6 71 NADH2 dehydrogenase C. elongatum 70 75 4.4 ± 0.7 175 GLP--- 291 11778 8566 G. lamblia ATCC 50803 29 43 4.5 ± 0.8 75 No similarity 3.9 ± 1.8 74 Hypothetical protein N. crassa 46 66 3.6 ± 1.9 69 Hypothetical protein B. Japonicum USDA 62 83 2.9±0 110 77 Sulfatase family protein N. aromaticivorans 42 57 2.8 ± 0.5 68 Hypothetical protein N. aromaticivorans 91 95 2.7 ± 0.2 72 Put. Integral membr. prot. S. coelicolor A3(2) 46 61 2.6 ± 0.5 Subtracted PPH 104 Hyp. protein, transcriptase C.elegans 32 48 6.8 ± 0.3 95 Putative hydrolase B. thetaiotaomicron 53 67 4.1 ± 0.3 VPI-5482 185 Novel antigenic To-ORF2 T. orientalis 94 94 1.8 ± 0 120 Hypothetical protein Nostoc sp. PCC7120 87 90 1.7 ± 0.3 119 50S ribosomal protein L33 P. lunula 80 96 1.6 ± 0.1 99 Hypothetical protein N. punctiforme 63 80 1.4 ± 0.5 162 Chapter 8 Searching/or STX-Gene; in Cy:mdmteria Table 8.1-Continued ID Best BLASTX hit Organism % % Microarray Id Sim hybridisation 186 Thiamin-phosphate Nostoc sp. PCC7120 70 81 1.2 ± 0.4 pyrophosphorylase 98 RNA polymerase ECF-type B. thetaiotaomicron VPI- 38 61 1.1 ± 0 sigma factor 5482 102 Unknown protein Nostoc sp. PCC7120 80 100 0.9 ± 0.2 97 Putative gluconate aldolase Microscilla sp. PREl 33 56 ND 106 Hypothetical protein C. hutchinsonii 67 72 ND 187 ABC transporter ATP- B. thetaiotaomicron VPI- 60 70 ND binding 5482 189 Phosphoglucomutase - C. hutchinsonii 58 69 ND Phosphomannomutase Unsubtracted PPH 108 Hypothetical protein C. hutchinsonii 41 61 6.6 ± 1.9 179 Put. Transposase Synechocystis sp. 35 60 5.6 ± 1.1 BO8402 109 Hypothetical protein B. thetaiotaomicron VPI- 23 43 4.9 ± 0.3 5482 112 Hypothetical protein T erythraeum IMS 101 44 65 3.5 ± 1.8 115 No similarity 3.4 ± 0.2 111 Hypothetical protein, FtsZ C. hutchinsonii 56 72 2.4 ± 0.8 107 No similarity 2.4 ± 0 183 Acetyltransferase B. thetaiotaomicron VPI- 60 82 2.4 ± 0.7 5482 178 Hypothetical protein Tfusca 35 57 2.1 ± 0.4 118 60 kDa chaperonin GroEL C. hutchinsonii 91 100 2 ± 0.4 110 Hypothetical protein, put. C. hutchinsonii 35 65 ND protease 163 Chapter 8 Searrmngfar STX-Gerl3 in Cytndxuteria Table 8.1-Continued % % Microarray ID Best BLASTX hit Organism Id Sim hybridisation 180 DNA polymerase III alpha B. thetaiotaomicron 78 84 ND subunit VPI-5482 182 Transport protein X axonopodis pv. citri 28 46 ND 306 164 Chapter 8 Sear Table 8.2 A. circinalis 344B putative specific sequences with protein matches {Id: identity; Sim: similarity) in the NCBI protein database. Genomic DNA experiments represent microarray hybridisations between strains 344B (Cy5) and 271 C (Cy3). Microarray values are expressed as in Table 8.1. ID Best BLASTX hit Organism % % Microarray Id Sim hybridisation SSH 91 Magnesium chelatase, subunit I B. thetaiotaomicron 55 84 8.7± 0.2 VPI-5482 89 Excinuclease ABC subunit A C. hutchinsonii 47 66 7.6 ± 1.3 88 50S ribosomal protein L33 P. lunula 78 93 6.8 ± 0.6 173 Hypothetical protein N. aromaticivorans 61 73 6.2 ± 0.2 84 Penicillin-binding protein B. subtilis 51 59 4.9 ± 0.1 90 Hypothetical protein B. thetaiotaomicron 36 59 4.1 ± 0 VPI-5482 85 Hypothetical protein A. thaliana 56 77 4± 0.1 86 Similar to chloride channel N. punctiforme 73 85 2.3 ± 0 87 Thiamin-phosphate Nostoc sp. PCC7120 77 89 0.8 ± 0.3 pyrophosphorylase 170 Unknown protein 0. sativa 28 41 ND 174 Putative hydrolase B. thetaiotaomicron 48 67 ND VPI-5482 Subtracted PPH 95 Putative hydrolase B. thetaiotaomicron 53 67 8.2 ± 0.7 VPI-5482 119 50S ribosomal protein L33 P. lunula 80 96 6.2 ± 0.6 98 RNA polymerase ECF-type B. thetaiotaomicron 38 61 5.9 ± 0.4 sigma factor VPI-5482 104 Hyp. protein, transcriptase C.elegans 32 48 4.1 ± 0.3 106 Hypothetical protein C. hutchinsonii 67 72 4.1 ± 0.1 185 Novel antigenic To-ORF2 T. orientalis 94 94 2.2 ± 0.2 165 Chapter 8 Searrhir,g,far STX-Genes in Cy:indxu:teria Table 8.2-Continued ID Best BLASTX hit Organism % % Microarray Id Sim hybridisation 99 Hypothetical protein N. punctiforme 63 80 1.3±0.1 186 Thiamin-phosphate Nostoc sp. PCC7120 70 81 0.8 ± 0.2 pyrophosphorylase 102 Unknown protein Nostoc sp. PCC7120 80 100 0.7± 0 97 Putative gluconate aldolase Microscilla sp. PREl 33 56 ND 120 Hypothetical protein Nostoc sp. PCC7120 87 90 ND 187 ABC transporter ATP-binding B. thetaiotaomicron 60 70 ND VPI-5482 189 Phosphoglucomutase - C. hutchinsonii 58 69 ND phosphomannomutase Unsubtracted PPH 179 Put. transposase Synechocystis sp. 35 60 22.6 ± 2 BO8402 108 Hypothetical protein C. hutchinsonii 41 61 18.7 ± 0.4 107 No similarity 13.8 ± 0.7 111 Hypothetical protein - FtsZ C. hutchinsonii 56 72 11.6± 1.7 178 Hypothetical protein T.fusca 35 57 11 ± 1.9 109 Hypothetical protein B. thetaiotaomicron 23 43 8.6 ± 0.1 VPI-5482 115 No similarity 6.6 ± 1.6 182 Transport protein X axonopodis pv. 28 46 6.5 ± 1.2 citri 306 183 Acetyltransferase B. thetaiotaomicron 60 82 1.6 ± 0.3 VPI-5482 110 Hypothetical protein C. hutchinsonii 35 65 ND 166 Chapter8 SeardJingfor STX-Genes in Cy:indw:teria Table 8.2-Continued ID Best BLASTX hit Organism % % Microarray Id Sim hybridisation 112 Hypothetical protein T erythraeum IMS 101 44 65 ND 118 60 kDa chaperonin GroEL C. hutchinsonii 91 100 ND 180 DNA polymerase III B. thetaiotaomicron VPI- 78 84 ND alpha subunit 5482 167 Chapter 8 Se.arrhingfar STX-Genes in Cy:;inducteria 8.3.2 PCR-based positive hybridisation Toxic-specific DNA sequences common to both A. circinalis strains 131 C and 344B were identified by the PPH of subtracted SSH libraries (subtracted-PPR) and unsubtracted tester controls (unsubtracted-PPH). The insert size of fifty randomly selected clones per library was estimated and, for each experiment, a total of 20 polymorphic DNA fragments were analysed. These sequences showed a mol¾ GC of 46.1 for the PPH of subtracted libraries, and 51.4 for the positive hybridisation of unsubtracted tester DNA. Among the 40 clones analysed, 13 distinct fragments were found to characterise each experiment. Two sequences were encountered more than once in the subtracted-PPR library as were three in the unsubtracted-PPH. No sequence was found to be in common between the subtracted and unsubtracted PPH libraries. Features of the putative toxic-specific sequences in common between A. circinalis 131 C and 344B are reported in Tables 8.1 and 8.2, with the two groups of clones comprising predicted enzymes, regulatory proteins, and hypothetical proteins. 8.3.3 DNA microarray analysis of SSH and PPH libraries All fragments recovered from A. circinalis 131 C by SSH were also shown to be toxic strain-specific by microarray hybridisation using genomic DNA from strains 131 C and 306A {Table 8.1 ). Among the library of putative toxic strain-specific sequences obtained from strain 344B, one ( clone 87) was found to be a false positive while another 2 fragments (clones 170 and 174) could not be detected by genomic DNA-microarray hybridisation of strains 344B and 271C (Table 8.2). Two SSH fragments from strain 344B (clones 88 and 89) demonstrated specificity also for strain 131 C. In the same microarray experiments, only 2 DNA fragments belonging to the subtracted-PPR library were shown to be toxic strain-specific and common to strains 131C and 344B (clones 95 and 104). Most of the other subtracted-PPR sequences were specific only for strain 344B, while clones 97, 187 and 189 could not be detected by microarray hybridisation of the cyanobacterial digested genomes. Two subtracted-PPR genes from strain 344B, a 50S ribosomal protein L33 (clone 119) and a thiamin- 168 Chapter 8 S eanhingfor STX-Genes in Cy:indt:uteria phosphate pyrophosphorylase ( clone 186) also recovered by SSH, was shown to be false positives, together with fragments 99 and 102 (Tables 8.1 and 8.2). Out of 10 distinct fragments, seven sequences obtained by unsubtracted-PPH were found to be toxic strain-specific and in common to both A. circinalis 131 C and 344B (clones 107,108,109,111,115,178 and 179). Two unsubtracted-PPH fragments ( clones 110-180) could not be detected (Tables 8.1 and 8.2). 8.3.4 PCR amplification of putative unsubtracted-PPH toxic-specific sequences The toxic-strain specificity of two unsubtracted-PPH fragments among the seven toxic-specific sequences in common between strains 131 C and 344B was verified by designing primers to clones 179 and 109. Amplicons of the expected size (164 bp for clone 179 and 298 bp for clone 109, respectively) were observed only for the two toxic strains 13 IC and 344B, and not for either of the other toxic (134C and 279B) or non toxic (306A and 271 C) A. circinalis isolates (Fig. 8.4 ). Similar results were obtained also for unsubtracted-PPH clones 111 (Cell division GTPase) and 107 (no similarity), found by microarray hybridisation to be toxic-strain specific. Clone 179 PCR Clone 109 PCR M 1 2 3 4 5 6 7 8 9 10 11 12 M Figure 8.4 Electrophoretic comparison of fragments 179 (lanes 1 to 6) and 109 (lanes 7 to 12) amplified by PCR from toxic and non-toxic strains of A. circinalis. Gel lanes 1-12 as follows: 131 C (1 and 7), 344B (2 and 8), 134C (3 and 9), 279B (4 and 10), 306A (5 and 11) and 271 C (6 and 12). PCR products were loaded as a total of 4 µL per sample and run with 1 Kb Plus DNA Ladder (lnvitrogen) as standard (lanes M). 169 Chapter8 Searr:hingfar STX-Gerx:s in Cy:lndxtcteria 8.4 DISCUSSION In this study a combined approach of subtractive and positive hybridisations was used to investigate the molecular basis for STX production in A. circinalis. The two groups of clones obtained by SSH, though not exhaustive of the entire pool, were sufficiently representative of the SSH library when compared to previous studies (Akopyants et al. 1998). The majority of DNA fragments recovered by SSH were toxic strain-specific but unique to each tester strain, either 131 C or 344B, and not common to both isolates (Tables 8.1 and 8.2) or to other STX-producing strains of A. circinalis ( data not shown). Therefore, a novel molecular tool was developed in this study, the PPH technique, with the aim of recovering toxin biosynthetic genes, hypothesised to be in common between the two tester A. circinalis strains. Subtracted-PPB libraries identified only 2 toxic-specific DNA fragments common to both 131 C and 344B, (clones 95 and 104). Most of the subtracted-PPB clones had a low toxic-strain specificity for A. circinalis 131 C (Table 2). Since SSH genes were found to be mainly characteristic of each distinct strain, it is possible that the percentage of common sequences in the final positive hybridisation reaction was relatively low. The subsequent single round of PCR may not have, therefore, sufficiently enriched sequences common to the two SSH libraries and thus resulting in the high percentage of false positives among the characterised libraries of subtracted-PPB clones. On the other hand, PPH of unsubtracted tester controls resulted in seven toxic strain-specific clones, with no fragment found to be in common to both subtracted and unsubtracted-PPH indicating no cross contamination between the two libraries (Tables 8.1 and 8.2). The subtracted-PPB library, compared to the unsubtracted-PPH, showed higher percentage of false positives and the presence of SSH sequences, suggesting that this technique was less effective than the PPH of unsubtracted tester DNA. Moreover, PCR amplification of unsubtracted-PPH fragments, including clones 109 and 179 (Fig. 8.4), indicated that similar genomic loci between two A. circinalis strains may not be in common with otherwise phylogenetically closely related (Beltran and Neilan 2000) toxic and non toxic strains of the same species. This consideration suggests that the unsubtracted-PPH method may have detected lateral gene transfer (LGT) events that have occurred between strains 131 C and 344B of A. circinalis. Alternatively, other phenomena may 170 Chapter8 Searchingfar STX-Genes in Cy:irdmteria explain these results rather than LGT, such as unusual rates of evolution or gene loss (Eisen and Fraser 2003) in A. circinalis isolates. During the course of the present investigation, an average mol% GC of 50 was found among all the fragments recovered by SSH and PPH. An average mol% GC of 43 has been estimated in the genome of A. circinalis (F. Pomati and R. Kellmann, unpublished results), which is consistent with what has been reported for other cyanobacteria of the order Nostocales, including Nostoc PCC7120 (41 %) and Nostoc punctiforme PCC73102 (42%) (Meeks et al. 2001). This evidence could suggest that the SSH-based techniques preferentially selected for genomic regions of A. circinalis with high mol% GC, although this phenomenon has not been observed in previous studies (Perrin et al. 1999, Nesb0 et al. 2002). Alternatively these methods, and in particular PPH, may have detected DNA fragments belonging to genomic regions of A. circinalis with an anomalous G+C content. This finding resembles the discrepancies in mol% GC found in bacterial pathogenicity islands (Pai) (Hacker et al. 1997). These are mobile genomic regions generally characterised by different mol% GC compared to the DNA of the host bacterium and by the presence of mobility genes, such as integrases and transposases (Hacker et al. 1997). Here we isolated, by unsubtracted-PPH, a toxic-strain specific putative transposase ( clone 179). This suggests the possibility of the presence of Pai in A. circinalis and supports the hypothesis that LGT may have occurred between toxic strains of this cyanobacterium. Recently, the presence of Pai-like DNA regions has been documented in the genome of the marine cyanobacterium Synechococcus sp. WH8102, and associated with the lateral acquisition of specific ecological and metabolic strategies (Palenik et al. 2003). Transposases have also been associated with toxin biosynthesis gene clusters in other cyanobacterial species, including the cyclic peptide toxin-producing Microcystis aeruginosa and Nodularia spumigena (Tillett et al. 2000, Moffitt 2003). It is possible that the molecular basis for STX production is encoded in a relatively large and mobile region of the A. circinalis genome. The SSH procedure is designed to select highly divergent genes, while the PPH method would instead select for highly conserved genomic sequences. These loci can be characterised by either a very low rate of mutation or, possibly, by their lateral acquisition in recent evolutionary times. STX, which is unique in its distribution as a bioactive secondary metabolite of 171 Chapter8 Searching/or S TX-GerK:S in C-yinclt:u:teria both prokaryotes and eukaryotes, may have conversely been acquired early in the evolution of these microorganisms. However, given the pathway proposed by Shimizu (1993), based on feeding experiments carried out with labelled precursors in dinoflagellates and cyanobacteria, one gene recovered by unsubtracted-PPH appears to be a possible candidate in STX biosynthesis. The toxic-strain specific clone 183 encodes a putative acetyltransferase, which could be implicated in the first and one of the more peculiar reactions proposed in STX synthesis: the Claisen-type condensation between the C2-carbon of arginine and Cl of acetate (Shimizu et al. 1984). This gene had, however, low specificity for the toxic A. circinalis strain 344B (Table 8.2). Since no biosynthetic pathway known to date is similar to the one proposed for STX, it could also be expected that some genes involved in STX biosynthesis would show very limited or no homology to other genes listed in the databases. Several hypothetical proteins and fragments with no similarity have been recovered by unsubtracted-PPH, which had high levels of toxic-strain specificity (Tables 8.1 and 8.2). The hypothetical proteins investigated in this study by PCR were mainly specific for strains 131 C ad 344B, rather then being in common with other STX-producing A. circinalis. Additionally, one other gene identified by the unsubtracted-PPH has the potential of being involved in STX regulation: a 60 kDa chaperonin GroEL ( clone 118). Recently, a 70 kDa chaperonin has been correlated with the regulation of STX biosynthesis in the heterotrophic bacterium Pseudomonas diminuta, a symbiont of the PSP-toxin producing dinoflagellate Alexandrium catenella (Cordova et al. 2002). Although toxic-strain specificity for strain 131 C was observed (Table 8.1), this gene could not be detected by microarray hybridisation using digested strain 344B genomic DNA (Table 8.2). 8.5 CONCLUSIONS The PCR-based positive hybridisation technique was employed here for the first time, with the aim of highlighting genomic DNA sequences associated with STX production in A. circinalis. The approach presented in this study allowed the identification of genes potentially involved in STX biosynthesis and regulation. This 172 Chapter8 Searching/or STX-Genes in Cy:irdmteria method could also be applied to phylogenetically distant taxa by ligating one terminal adapter directly to each digested genomic DNA, in order to recover genes with similar primary structure. PPH of two genomes could therefore be utilised as a new tool for the identification of genes that have been laterally transferred between bacterial strains. The application of this technique suggested the presence of Pai-like regions in the genome of A. circinalis. The combined SSH-PPH/DNA-microarray approach revealed a high degree of genomic diversity among phylogenetically closely related strains of A. circinalis and could be used for identifying molecular probes to detect STX-producing genotypes in the environment. PPH may also be useful for acquiring information on secondary metabolism and its regulation in other toxic microalgae. 8.6SUMMARY A PCR-based positive hybridisation (PPH) method was developed to explore toxic-specific genes in common between toxigenic strains of A. circinalis. The PPH technique is based on the same principles of suppression subtractive hybridisation (SSH), although with the former no driver DNA is required and two tester genomic DNAs are hybridised at high stringency. The aim was to obtain genes associated with cyanobacterial STX production. The genetic diversity within phylogenetically similar strains of A. circinalis was investigated by comparing the results of the standard SSH protocol to the PPH approach by DNA-microarray analysis. SSH allowed the recovery of DNA libraries that were mainly specific for each of the two STX-producing strains used. Several candidate sequences were found by PPH to be in common between both the STX-producing testers. The PPH technique performed using unsubtracted genomic libraries proved to be a powerful tool to identify DNA sequences possibly transferred laterally between two cyanobacterial strains that may be candidate(s) in STX biosynthesis. The approach presented in this study represents a novel and valid tool to study the genetic basis for secondary metabolite production in microorganisms. 173 Chapter9 Differential, Expression ifCandidate STX-Genes CHAPTER9 TRANSCRIPTIONAL ANALYSIS IN ANABAENA CIRCINALIS USING THE BGGM1 DNA-MICROARRAY: A COMPARATIVE STUDY OF TOXIC AND NON-TOXIC STRAINS AND THE EFFECTS OF LIDOCAINE HYDROCHLORIDE One of these nights, One of these crazy old nights. We're gonna find out, pretty mama, What turns on your lights. (D. Henley, G. Frey) 174 Ch.apter9 Differential, E xpressian ifGmdidate STX-Genes 9.1 BACKGROUND The BGGM1 DNA-microarray has been utilised in Chapters 7 and 8 to verify the toxic-strain specificity of subtracted and unsubtracted genomic-DNA fragments from A. circinalis. Also, the array has been designed with the aim of broadly summarising the known molecular biology involved in cyanobacterial toxigenicity and ecology. The array, which comprises more than 400 DNA fragments, includes 16S rDNA corresponding to the most common genera of potentially harmful cyanobacteria, in addition to the strains used in this study. Moreover, the BGGM1 DNA-microarray comprises all the known and candidate genes associated with the biosynthesis of cyanotoxins, as well as a range of genes involved in the metabolic pathways related to cyanobacterial bloom development in the environment. BGGM 1 was designed for laboratory screening of isolates and gene expression studies of toxin production in cultured cyanobacteria, and has been previously employed for transcriptional characterisation of Nodularia spumigena strains (Moffitt 2003). In this study, the BGGM1 DNA-microarray was used for a comprehensive characterisation of A. circinalis gene expression. Previously, the toxic-specificity of tester DNA fragments from the SSH and PPH libraries were analysed by DNA microarray hybridisation with labelled toxic/non-toxic A. circinalis genomic DNA (Chapter 8). Here, again with the intention of highlighting genes associated with STX production, the expression of spotted BGGM1 DNA fragments (Appendix D) was monitored under standard growth conditions and after exposure to 1 µM lidocaine hydrochloride. Lidocaine was previously shown to promote STX production strongly in the cyanobacterium Cylindrospermopsis raciborskii T3 (Chapter 3). 175 Chapter9 Differential, Expn:ssion ifCardidate STX-Gm:S 9.2 EXPERIMENTAL PROCEDURES 9.2.1 Cyanobacterial strains and growth conditions For this study, the toxic A. circinalis strains 131 C and 344B, together with the non-toxic 306A and 271 C, were utilised (Appendix A). PSP toxin-producing and non toxic isolates were maintained in JM medium as described in Section 2.1.1. Cyanobacterial cultures were grown in glass 250 mL flasks at a constant temperature of 26° C, under continuous irradiance of cool white light at an intensity of 15 µmol photon m-2 s- 1• Cultures in mid-exponential growth phase were used for total RNA extractions. For gene expression studies, 100 mL cultures of A. circinalis strains 131 C and 344B were grown to mid-exponential phase and adjusted to pH 8.1 by adding HEPES buffer to a final concentration of 10 mM. Lidocaine hydrochloride (Sigma-Aldrich Co., Dorset, UK) was supplemented to a final concentration of 1 µM, as previously reported (Chapter 3). Total RNA was extracted from cyanobacterial cultures immediately after the addition of lidocaine (time 0) and after 2 h of exposure under otherwise standard growth conditions. Experiments were performed in triplicate. 9.2.2 Total RNA extraction Cyanobacterial cells were collected from 50 mL culture samples by filtration through 3.0 µm pore size filters (Millipore, two filters for each 50 mL aliquot), washed twice with sterile water and immediately frozen in liquid nitrogen. Total RNA was then extracted as described in Section 2.6.2. 9.2.3 Labelling of total RNA The extracted and purified total RNA was retro-transcribed and labelled according to the protocols reported in Sections 2.10.2 and 2.10.3. Labelled cDNA samples were then dried to -20 µL, combined according to the different experiments (Section 9.2.4), evaporated to dryness and hybridised on the DNA microarray. 176 Chapter9 Differential, Exprr5sion ifGirdidate STX-Genes 9.2.4 Microarray hybridisation Each microarray hybridisation was performed in duplicate. Cy3-labeled cDNA (green) from the non-toxic strains 271C and 306A were hybridised with Cy5-labeled cDNA (red) from the toxic strains 344B and 131 C, respectively. Investigations of the lidocaine hydrochloride effect on toxic strains 131 C and 344B were performed by comparing gene expression profiles between time O (Cy3-labeled cDNA) and after 2 h exposure (Cy5-labeled cDNA) to the agent. Microarray hybridisations were carried out as described in Section 2.11, array slides washed, spun dry, and kept in the dark prior to scannmg. 9.2.5 Microarray scanning, data acquisition and statistical analyses Microarray slides were scanned and spots analysed as illustrated in Section 2.12. Data were filtered to remove erroneous spots and the median Cy5/Cy3 values obtained normalised to give an overall ratio measurement of 1 across each microarray slide. Normalised data sets were assembled and analysed by hierarchical clustering analysis as described in Section 2.12. 9.3 RESULTS 9.3.1 Comparative analysis of gene expression in toxic and non-toxic strains Among all the DNA fragments obtained by SSH and PPH, only one hypothetical protein (clone 120) was preferentially expressed by the toxic strain 131 C in reference to strain 306A, although this result was not statistically significant (Table 9 .1 ). On the other hand, while most of the SSH genes showed no change, the majority of subtracted and unsubtracted PPH fragments were apparently expressed in a preferential manner by the non-toxic strain 306A. Similarly, a number of putative toxic-strain specific 177 Chapter9 Differential, Expressim ifGirdidate STX-Genes sequences were preferentially expressed in the non-toxic strain 271C when compared to the toxic 344B, with a few genes showing no significant difference in their transcription patterns between the two strains (Table 9.2). Clones 119 and 98, corresponding to a 50S ribosomal protein L33 and a RNA polymerase extracytoplasmic function (ECF)-type sigma factor, respectively, were apparently expressed at higher levels in both non-toxic strains compared to toxic A. circinalis, together with a number of hypothetical proteins obtained by unsubtracted PPH (Tables 9.1 and 9.2). Among the other genes spotted on the BGGM1 DNA-microarray, the gas vesicle protein GVP-A was shown to be highly expressed by the two STX-producing A. circinalis strains 131C and 344B in comparison to the non-toxic strains 306A and 271C (Tables 9.1 and 9.2). The heat-shock protein HSP-70, nitrogen fixation genes, phosphopantetheinyl transferases (PPTs), an amidinotransferase, and the iron response gene isiA also displayed higher levels of transcription in the non-toxic A. circinalis strains. 9.3.2 Effect of lidocaine on gene expression using BGGM1 DNA microarray Exposure to lidocaine at 1 µM seemed to have no statistically significant effect on the regulation of SSH and PPH genes in strain 131 C, although a hypothetical protein (clone 112) was shown to be slightly down-regulated (Table 9.1). The same gene in strain 344B was also shown to be the only SSH or PPH microarray fragment significantly affected by lidocaine in strain 344B, being repressed equally in both the toxic A. circinalis isolates 131 C and 334B (Table 9.2). Lidocaine dosing, however, promoted the expression of phycocyanin and GVP A genes in both toxic strains 131C and 344B (Tables 9.1 and 9.2). Several other genes including amidinotransferases, phosphopantetheiyl transferases, a heat-shock protein and an iron responsive chlorophyll-a binding protein isiA, were down-regulated in response to lidocaine in these two cultures. Many subtraction clones were not detected during transcript analysis of the lidocaine effect (Tables 9.1 and 9.2). 178 Chapter9 Differential, Expression ifG:trdidate STX-Gm:s Table 9.1 A. circinalis 131 C putative specific sequences with protein matches in The National Center for Biotechnology Information (NCBI) protein database. Microarray experiments represent hybridisations between strains 131C (Cy5) and 306A (Cy3) using total RNA (RNA). The effect of lidocaine hydrochloride at 1 µM on gene expression {LIDOCAINE) was investigated hybridising total RNA extracted from strain 131C at O h (Cy3) and 2 h (Cy5) exposure. Microarray values are expressed as average ± SE of Cy5/Cy3 normalised ratios. Increased gene-expression levels are defined by Cy5/Cy3 ratios > 2, while decreased gene expression are defined by Cy5/Cy3 ratios< 0.5. ND= not detected. SSH and PPH refer to DNA fragments discussed in Chapter 8. Put.= putative. ID Gene Organism Microarray Microarray RNA LIDOCAINE SSH 77 Sulfatase family protein A. circinalis 131 C 0.6±0 2.2 ± 1.8 175 GLP 291 - 11778 8566 A. circinalis 131 C 0.7 ±0 2.2 ± 1.4 69 Hypothetical protein A. circinalis 131 C 0.9±0 1.9 ± 0.8 65 Hypothetical protein A. circinalis 131 C 1 ± 0.1 1.9 ± 0.7 74 Hypothetical protein A. circinalis 131 C 0.7 ±0 1.3 ± 0.9 72 Put. Integral membr. A. circinalis 131 C 0.9 ± 0.1 1.2 ± 0.1 prot. 73 Alpha-amylase precursor A. circinalis 131 C 0.7 ± 0.5 1.1 ± 0.1 71 NADH2 dehydrogenase A. circinalis 131 C 0.5 ± 0.3 1 ± 0.5 75 No similarity A. circinalis 131 C 0.9± 0.6 0.7 ± 0.3 68 Hypothetical protein A. circinalis 131 C 0.5 ±0 0.5 ± 0.1 70 TonB, ferric-siderophore A. circinalis 131 C 0.4± 0 ND uptake Subtracted PPH 185 Novel antigenic ToORF2 A. circinalis 131C-344B 0.6 ± 0.1 1.2 ± 0.3 187 ABC transporter ATP- A. circinalis 131 C-344B 0.2 ± 0.1 0.9 ± 1.2 binding 186 Thiamin-phosphate A. circinalis 131 C-344B 0.8 ± 0.1 0.9 ± 0.2 pyrophosphorylase 179 Chapter9 Differential, ExpY£5sion ifGirdidate STX-Genes Table 9.1-Continued ID Gene Organism Microarray Microarray RNA LIDOCAINE 99 Hypothetical protein A. circinalis 131 C-344B 0.7 ± 0.3 0.7 ±0.2 95 Putative hydrolase A. circinalis 131 C-344B 0.3 ± 0.1 0.6 ± 0.3 119 SOS ribosomal protein A. circinalis 131 C-344B 0.3 ± 0.1 0.6 ± 0.3 L33 98 RNA polymerase ECF- A. circinalis 131 C-344B 0.3 ± 0 0.5 ± 0.2 type sigma factor 120 Hypothetical protein A. circinalis 131 C-344B 2.1 ± 1.3 ND 102 Unknown protein A. circinalis 131 C-344B 0.9 ± 0.1 ND Unsubtracted PPH 118 60 kDa chaperonin A. circinalis 131 C-344B 0.5 ± 0.4 1.2 ± 0.8 GroEL 178 Hypothetical protein A. circinalis 131 C-344B 0.3 ± 0.1 1 ± 0.7 111 Hypothetical protein, A. circinalis 131 C-344B 0.2 ± 0 0.7 ± 0.2 FtsZ 179 Put. transposase A. circinalis 131 C-344B 0.2±0 0.6 ± 0.1 112 Hypothetical protein A. circinalis 131 C-344B 0.6 ± 0.1 0.4± 0 108 Hypothetical protein A. circinalis 131 C-344B 0.5 ±0 ND 109 Hypothetical protein A. circinalis 131 C-344B 0.3 ± 0 ND 110 Hypothetical protein, A. circinalis 131 C-344B 0.4± 0 ND put. protease Other genes BAN7 Gas vesicle protein A. circinalis 344B 2.6 ± 0.1 4.1 ± 2.5 GYP-A BAN15 Gas vesicle protein A. circinalis 332H 2.5 ± 0.4 3.6 ± 1.3 GYP-A TS35 Phycocyanin a/~ A. circinalis 279B 1.7 ± 0 2.5 ± 1.6 TS16 16S rRNA (27 /809) A. circinalis 306A 1.1 ± 0.2 1.4 ± 0.3 180 Chapter9 Differential, E xpressim ifCardidate STX-Genes Table 9.1-Continued ID Gene Organism Microarray Microarray RNA LIDOCAINE TS12 16S rRNA (27/809) A. circinalis 131 C 1.2 ± 0.3 1.4 ± 0.3 FP194 Putative viral coat prot. A. circinalis 134C 0.4± 0 1.1 ± 0.2 FP88 50S ribosomal protein L33 A. circinalis 344B 0.4± 0 1 ±0.7 BANl Nitrogen fixation NifA C. raciborskii T3 0.2 ± 0 0.6 ± 0.1 BANS Nitrogen fixation Nif J A. circinalis 344B 0.3 ± 0 0.5 ± 0 FP89 Excinuclease ABC - A A. circinalis 344B 0.4± 0 0.4 ± 0.1 RM12 Amidinotransferase A. circinalis 150A 0.3 ±0 0.4 ± 0.1 JC39 Put. Phosphopantetheinyl- A. circinalis 279B 0.4± 0 0.3 ± 0.1 transferase BAN12 Heat shock protein HSP-70 N spumigena 0.4 ± 0 0.3 ± 0.1 NSORl0 JC44 Put. Phosphopantetheinyl- Cylindrospermum 0.7 ± 0.1 0.2 ± 0.1 transferase CENA33 TS66 Chl-a binding protein isiA N spumigena 0.1 ± 0 0.2 ± 0.1 NSORl0 RM15 Amidinotransferase C. raciborskii T3 0.1 ±0 0.2±0 FP130 Dolichol-phosphate A. circina/is l 34C 0.2 ± 0 ND mannosyltransferase 181 Chapter9 Differential, Exprr5sion ifCarxlidate STX-Genes Table 9.2 A. circinalis 344B putative specific sequences with protein matches in NCBI protein database. Total RNA (RNA) experiments represent microarray hybridisations between strains 344B (Cy5) and 271 C (Cy3). The effect of lidocaine hydrochloride at 1 µM on gene expression (LIDOCAINE) was investigated hybridising total RNA extracted from strain 344B at O h (Cy3) and 2 h (Cy5) exposure. Microarray values are expressed as in Table 9.1. SSH and PPH refer to DNA fragments discussed in Chapter 8. ID Gene Organism Microarray Microarray RNA LIDOCAINE SSH 86 Similar to chloride channel A. circinalis 344B 0.4 ± 0.2 0.9 ± 0.4 174 Putative hydrolase A. circinalis 344B 0.9 ± 0.1 0.9 ± 0.1 90 Hypothetical protein A. circinalis 344B 0.3 ± 0 0.9 ± 0.1 84 Penicillin-binding protein A. circinalis 344B 0.3 ± 0.1 0.8 ± 0.3 85 Hypothetical protein A. circinalis 344B 0.7 ± 0.1 0.7 ± 0.5 87 Thiamin-phosphate A. circinalis 344B 0.7±0 0.7 ± 0.3 pyrophosphorylase 89 Excinuclease ABC sub. A A. circinalis 344B 0.6 ± 0.3 0.6 ± 0.1 173 Hypothetical protein A. circinalis 344B 0.8 ±0 0.5 ± 0 88 50S ribosomal protein L33 A. circinalis 344B 0.3 ± 0.1 0.4 ± 0.3 170 Unknown protein A. circinalis 344B 0.8 ± 0.1 ND 91 Magnesium chelatase, A. circinalis 344B 0.2 ± 0 ND subunit I Subtracted PPH 186 Thiamin-phosphate A. circinalis 131 C-344B 0.6 ± 0.1 1.1 ± 0.1 pyrophosphorylase 99 Hypothetical protein A. circinalis 131 C-344B 0.5 ±0 1 ± 0.1 189 Phosphoglucomutase - A. circinalis 131C-344B 0.6±0 0.9±0 Phosphomannomutase 182 Chapter9 Differential, Exjm5sinn ifGurlidate STX-Genes Table 9.2-Continued ID Gene Organism Microarray Microarray RNA LIDOCAINE 102 Unknown protein A. circinalis 131 C-344B 1.5 ± 0.1 0.7 ± 0.3 187 ABC transporter A. circinalis 131 C-344B 1.2 ± 0.1 0.6 ± 0.3 ATP-binding 119 50S ribosomal prot. A. circinalis 131 C-344B 0.2 ± 0.1 0.5 ± 0.1 L33 185 Novel antigenic To- A. circinalis 131 C-344B 0.6 ± 0.1 ND ORF2 120 Hypothetical protein A. circinalis 131 C-344B 0.5 ± 0.1 ND 95 Putative hydrolase A. circinalis 131 C-344B 0.3 ±0 ND 98 RNA polymerase A. circinalis 131 C-344B 0.1 ±0 ND ECF-type sigma factor Unsubtracted PPH 111 Hypothetical protein A. circinalis 131C-344B 0.4 ± 0 0.8 ± 0.1 FtsZ 109 Hypothetical protein A. circinalis 131 C-344B 0.3 ± 0 0.7 ± 0.3 182 Transport protein A. circinalis 131 C-344B ND 0.4 ± 0.2 118 60 kDa chaperonin A. circinalis 131 C-344B 0.5 ± 0.4 0.4 ± 0.1 GroEL 112 Hypothetical protein A. circinalis 131 C-344B 0.5 ±0.2 0.4± 0 108 Hypothetical protein A. circinalis 131 C-344B 0.7 ± 0.1 ND 179 Put. transposase A. circinalis 131 C-344B 0.4 ± 0 ND 178 Hypothetical protein A. circinalis 131 C-344B 0.3 ± 0.1 ND 110 Hypothetical protein A. circinalis 131 C-344B 0.3 ± 0 ND 183 Chapter9 Differential, Expnssim ifGindidate STX-Genes Table 9.2-Continued ID Gene Organism Microarray Microarray RNA LIDOCAINE Other genes BAN15 Gas vesicle protein A. circina/is 332H 6 ± 1.3 8.2 ± 1.9 GYP-A BAN7 Gas vesicle protein A. circinalis 344B 6.6 ± 2.7 7.4 ± 1 GYP-A TS24 Phycocyanin a/~ A. circina/is 332H 2.1 ± 1.3 2.4 ± 0.4 BAN12 Heat shock protein N. spumigena 0.4 ± 0.1 1.2 ± 0.1 HSP-70 NSORl0 TS15 16S rRNA (27/809) A. circina/is 271C 0.9 ± 0.4 0.9± 0.2 TS18 16S rRNA (27/809) A. circinalis 344B 1.2 ± 0.2 0.9 ± 0.1 BANll Heat shock protein A. circinalis 344B 0.2±0 0.8 ±0 HSP-70 BANS Nitrogen fixation nifJ A. circinalis 344B 2.2 ± 1.7 0.7 ± 0.2 JC39 Put. Phosphopantetheinyl- A. circinalis 279B 0.4 ±0 0.4 ± 0.2 transferase RM15 Amidinotransferase C. raciborskii T3 0.4± 0 0.4 ± 0.2 TS66 Chl-a binding protein isiA N. spumigena 0.4 ± 0.1 0.4± 0 NSORl0 FP122 Oligopeptide-binding A. circina/is 134C 0.5 ±0 0.3 ± 0.2 protein JC44 Put. Cylindrospermum 0.3 ± 0.1 0.3 ± 0.1 Phosphopantetheinyl- CENA33 transferase FP194 Putative viral coat prot. A. circinalis l 34C 0.8 ± 0.1 ND FP130 Dolichol-phosphate A. circinalis l 34C 0.4 ± 0.2 ND mannosyltransferase 184 Chapter 9 Differential Expression ef Candidate STX-Genes 9.4 DISCUSSION Differential gene expression of candidate genes associated with STX production in A. circinalis was investigated by means of DNA-microarray hybridisation (Fig 9 .1). The BGGM1 DNA-microarray, employed here for transcriptional studies, has been utilised for genomic screening in Chapters 7 and 8 and the reliability of this gene array has been previously validated (Chapter 7 and 8, Moffitt 2003). Hierarchical cluster analysis also showed that the range of microarray results was due to either the toxic strain specificity of DNA fragments or to their expression profiles (Appendix E, Fig. E), which further validates the experimental data sets obtained by microarray hybridisation. Figure 9.1 Laser scan corresponding to one of the four blocks of the BGGM1 DNA-microarray. The hybridisation displayed corresponds to the effect of 1 µM lidocaine on A. circinalis 3448 gene expression. The DNA fragments recovered by SSH (Chapter 8) were not found to be preferentially expressed in toxic versus non-toxic strains, or to significantly alter expression after exposure to lidocaine at 1 µM. These data suggest that such genes may not be involved in the production of STX by A. circinalis. According to the model of STX biosynthesis highlighted in Section 1.3 .5.5 and based on previous studies (Shimizu 1993, 1996), no enzyme potentially associated with this function was identified among SSH genes. Alternatively, it is possible that exposure to 1 µM lidocaine affects STX Chapter9 Differential, Expression ifG:lrdidate STX-GerK!S metabolism in A. circinalis to a different extent compared to C. raciborskii T3 (Chapter 3). The majority of DNA fragments recovered by PPH showed poor toxic-strain specific expression and no statistically significant change in transcription levels after dosing with 1 µM lidocaine. A number of the fragments recovered either by SSH or PPH displayed, after cDNA hybridisation, low signal values for both lasers (595 and 685 nm) when compared to other BGGM1 microarray genes. The transcript analysis of lidocaine's effect revealed that many unsubtracted-PPH fragments could not be detected. This may indicate that SSH and PPH genes were not expressed or that they are transcribed at very low levels. Clone 112, obtained by unsubtracted-PPH (Chapter 8) and encoding a hypothetical protein, however, was observed to be repressed 2 h after the initial exposure to lidocaine. Given the effect of lidocaine on STX accumulation in C. raciborskii T3 (Chapter 3), an increased expression would be expected for this gene if it was implicated in the production of PSP toxins, or a decrease in transcripts if it was involved in toxin degradation. The biosynthetic pathway proposed for STX is to date unique and the proteins responsible for its regulation unknown (Shimizu et al. 1984, Shimizu 1996). It cannot be excluded, therefore, that a hypothetical protein could be implicated in the regulation of STX metabolism. Lidocaine supplementation significantly affected the expression of several other BGGM1 microarray genes other than SSH and PPH DNA fragments. A number of genes discussed below have been amplified and spotted on the array using template DNA belonging to other cyanobacteria rather than A. circinalis. Such genes were, however, obtained from cyanobacterial species belonging to the same order of Nostocales, that also comprises A. circinalis. These sequences also represent ORFs that are likely to be evolutionarily conserved among different taxonomic groups within the same order (nitrogen fixation genes, heat shock proteins, etc., Tables 9.1 and 9.2). It was assumed, therefore, that the DNA probes spotted on the microarray hybridised with the corresponding homologous genes of A. circinalis. ORFs either involved in cyanobacterial stress responses or m secondary metabolic pathways showed lidocaine-induced changes at the level of transcription. For both strains tested, the expression of a heat shock protein HSP-70 and the iron 186 Chapter9 Differential, Expmsion ifGtrdidate STX-Gem; responsive isiA gene were found to be repressed (Tables 9.1 and 9.2). Increasing temperature can result in a decrease in alkaloid production by cyanobacteria, including the synthesis of STX (see Section 1.3.5.4). This may suggest that heat shock proteins have a role in the regulation of STX production. Repression of HSP-70 could therefore be a critical component of enhanced STX biosynthesis as induced by lidocaine. The chlorophyll-a binding protein isiA functions in cyanobacteria as a non radiative dissipator of energy under conditions of iron deficiency or oxidative stress (Jeanjean et al. 2003, Sandstorm et al. 2003). Although there is no evidence in the literature linking iron limitation or oxidative stress to the production of STX in cyanobacteria, iron depletion is known to affect the biosynthesis of cyanobacterial hepatotoxins (see Section 1.3.3). In this study, lidocaine addition was also shown to repress the transcription of amidinotrasferases (AT) and PPTs, enzymes that are potential candidates in the production or regulation of PSP-toxins. An AT can be putatively implicated in the second hypothesised reaction of STX biosynthesis, characterised by the transfer of an amidino group from a molecule of arginine to the incomplete skeleton of STX (see Section 1.3.5.5). PPTs are enzymes that post-translationally modify and activate PKS and NRPS modules responsible for the production of polyketide natural products, both in primary and secondary metabolic pathways (Watanabe et al. 2003). The participation of a mixed NRPS/PKS enzyme with an adenylation and a condensation domain has also been hypothesised in the first reaction of STX biosynthesis (Section 1.3.5.5). The evidence presented here, however, may suggest that PPT-mediated activation of PKSINRPS modules, as well as AT activity, are not involved in the biosynthesis of STX. Alternatively, it is possible that the genes encoding STX biosynthetic enzymes are very different in nucleotide sequence from their homologues involved in other pathways, including the probes corresponding to AT and PPTs present on the DNA m1croarray. The transcription of two genes linked to cyanobacterial ecophysiology was observed to increase in consequence to lidocaine treatment (Tables 9.1 and 9.2). Phycocyanin and GYP-A, involved in the regulation of photosynthetic efficiency and buoyancy, play a central role in the formation of blooms in water bodies. The gas vesicle structural protein GYP-A, in particular, is also known to be overexpressed in 187 Chapter9 Differential, Exprrssinn response to increasing salinity in the moderately halophilic archaeon Haloferax mediterranei (Jager et al. 2002). Considering that the effect of lidocaine on cyanobacterial cell physiology mimics the consequence of salt stress (Chapters 4 and 5), these results suggest that salt stress may also contribute to the formation of bloom and surface scums of A. circinalis in the environment, in addition to a possible stimulation of STX production (Chapters 3 and 4). This hypothesis is also consistent with the previously published data regarding the dominance of STX-producing A. circinalis in surface blooms that are exposed to conditions of high salinity (Bowling and Baker 1996). 9.5 CONCLUSIONS In this study, low levels of expression were observed for the DNA fragments recovered by either SSH or PPH, and also in response to lidocaine stimulation. These results suggested that none of these genes can be directly correlated to the production of STX in A. circinalis. The general transcript analysis performed using the BGGM1 DNA microarray under lidocaine supplementation suggested the exclusion of PPTs and AT in the production of STX. The DNA microarray analysis allowed the identification of genes potentially implicated in the development of STX-producing A. circinalis blooms. Since lidocaine exposure was shown to imitate salt stress in its effect on cyanobacterial physiology, the data presented also indicated that this condition may induce the formation of toxic A. circinalis surface scums. BGGM1 DNA microarray was confirmed to be a useful tool for gene expression studies of toxin production in cultured cyanobacteria. The BGGM1 DNA-microarray could also be applied for the validation of designed molecular probes for the environmental screening and analysis of toxic cyanobacterial blooms. 188 C1l.apter9 Differential, Expression ifG:trrlidate STX-Gm:s 9.6SUMMARY In this study the transcriptional profiles of PSP toxin-producing and non-toxic strains of A. circinalis were investigated by means of a DNA microarray approach. Additionally, gene expression was studied after exposure of A. circinalis cultures to lidocaine hydrochloride at 1 µM for 2 h. Under standard growth conditions, a limited number of DNA fragments recovered by either SSH or PPH were preferentially expressed in toxic versus non-toxic strains. The same genes did not significantly change their expression after exposure to 1 µM lidocaine, conditions previously shown to up regulate STX production in Cylindrospermopsis raciborskii T3. Other genes, however, showed significant variation in their expression profiles due to lidocaine supplementation. Open reading frames (ORFs) potentially involved in physiological adaptive responses and bloom formation in cyanobacteria were enhanced in their transcription, such as the gas vesicle structural protein A and phycocyanin. The heat shock protein HSP-70 and the chlorophyll-a binding protein isiA were significantly repressed by lidocaine exposure. Stress response proteins and genes implicated in secondary metabolism were repressed, including amidinotrasferases and phosphopantetheinyl transferases. The BGGM1 DNA microarray, used in this study, was shown to be suitable for gene expression studies in cultured toxic cyanobacteria and allowed the detection of gene transcripts associated with surface scum formation by toxic A. circinalis. 189 Chapter 10 D-iscussinn CHAPTER10 DISCUSSION The mystery of life isn't a problem to solve, but a reality to experience. (Frank Herbert) 190 Chapter 10 DisatSsinn 10.1 GENERAL DISCUSSION PSP toxin-producing algal blooms represent a worldwide concern due to their negative effects on the management of water sources, and on human health through contaminated seafood. Efficient prevention and control of harmful blooms requires a fundamental understanding of the factors associated with bloom formation and of the molecular basis of algal toxicity. In this study the production of the alkaloid neurotoxin STX was investigated with the ultimate aim of identifying the ecological and molecular basis of STX biosynthesis in cyanobacteria. To achieve this challenging task an approach of combined physiological and molecular biology studies was used. A schematic diagram summarising the entire logic structure underlying this research project is shown in Fig. 10.1. Physiology Molecular Biology study study New method: ! }- PPH What regulates Candidate STX production genes Possible+ role of +Molecular STX in probes cyanobacteria LExpression Study J Application: I Application: .L PCR-assay for STX-genes new bioassay STX-producing A. for STX circinalis Figure 10.1 Flow-chart diagram of the studies undertaken in this project. 191 Cbapter 10 DisatSsion The eco-physiological investigations, presented in Chapters 3, 4, 5 and 6, were carried out in C. raciborskii T3. The high levels of Cl +2 and STX synthesised by this cyanobacterium compared to other species, including A. circinalis, allowed the more precise detection of changes in toxin production under varying growth conditions. The specific aim of these studies was to understand whether the production of STX can be regulated by particular environmental factors. The information acquired led to the hypothesis of a possible interaction of STX with cyanobacterial ion fluxes, and in particular with Na+ uptake (Chapter 4). This hypothesis was verified by developing a bacterial bioassay for STX that was also applied to a standardised bioluminescent bacteria toxicity test for the detection of PSP-toxins in the environment (Chapter 5). On the other hand, molecular biology techniques were employed to identify genomic differences across several STX-producing and non-toxic A. circinalis strains (Chapters 7 and 8). Hypothesising the lateral acquisition of STX biosynthetic capability by toxic strains of this species (Section 1.3.5.6), a novel method was developed to recover genes with a common primary structure in toxic A. circinalis isolates (Chapter 8). The aim was to obtain candidate genes in STX production. These genes were utilised to prepare a DNA microarray and verified for their toxic-strain specificity by microarray hybridisation. One of the toxic-strain specific genes, encoding a Na+ dependent transporter involved in the maintenance of pH and Na+ homeostasis, was successfully applied as a probe for laboratory and environmental screening of STX producing A. circinalis (Chapter 7). Candidate genes involved in STX biosynthesis and other genes implicated in cyanobacterial ecophysiology were assayed for their expression profiles by inducing the physiological conditions previously found to modulate STX production (Chapter 9). The aim was to highlight candidate toxic-strain specific genes that correlated with the expected increase in STX biosynthesis. The following is a summary of the major findings reported in this study. The conclusions for each area of the work can be found in each section, and this discussion links together key issues that have been addressed during the course of this investigation. Some of the possible future directions of this research are also proposed. 192 Chapter 10 Dismssion 10.2 THE PHYSIOLOGY OF STX PRODUCTION IN CYANOBACTERIA PSP toxins have been long considered among the most mysterious microbial natural products, for several reasons. These neurotoxins represent one of the most potent classes of venoms, with a highly specific effect targeting voltage-gated sodium channels in excitable cells. Previous to this study, the factors influencing the production of these toxins were poorly understood, especially in cyanobacteria (Section 1.3.5.4). No specific stimuli inducing or repressing STX production were known in these microorganisms, and the metabolic role of PSP toxins was thought to be related to a possible defence strategy against unknown predators. In this study, evidence was found for a linkage between STX production and the maintenance of cyanobacterial homeostasis under alkaline pH or Na+ stressed conditions. Intracellular STX levels were responsive to changes in total cellular Na+ content and by influencing Na+ fluxes it was possible to induce a corresponding modulation of STX production (Chapter 4). The blockage of Na+ uptake by STX was demonstrated in bacterial and cyanobacterial strains (Chapter 5), suggesting that this inhibition could represent a possible mechanism that PSP toxin-producing cyanobacteria employ to cope with conditions of elevated pH and salinity. As mentioned in Section 1.3.5.4, these environmental features are not uncommon in rivers and water-bodies characterised by the seasonal occurrence of STX-producing cyanobacterial blooms. Taken together, these considerations suggest that the production of PSP-toxins could represent a potential evolutionary advantage to survive in certain drastic environmental conditions. It is a general and reasonable principle that toxins of all kinds should play a beneficial role in the producing microorganism. The biosynthesis of toxins requires precious cellular energy, and it would seem unlikely that evolution would be forgiving enough to tolerate wasted metabolism. For a number of microbial neurotoxins, the identity of this biological profit still remains a mystery. The microorganisms producing these toxins, and often those living in the environment around them, do not possess nerves nor many of the molecular systems that characterise neural transmission, 193 Chapter 10 Dis(J{Ssi.an however, ion channels are a common feature distinguishing all biological membranes. The function of ion channels range from motility or nutrient uptake in bacteria to the highest levels of complex neural transmission in the animal central nervous system. While commonly having different structures across phylogenetically unrelated organisms, ion channels often share similar specificity and cell function. Here support was found for the interaction of STX with bacterial ion channels. The identification and characteristics of the prokaryotic channel(s) inhibited by STX are still to be described, however, this study legitimises the hypothesis of an interaction of this natural neurotoxin with the ion channels of the producing microorganisms and/or their environmentally contiguous bacterial competitors. 10.3 THE MOLECULAR BIOLOGY OF STX PRODUCTION IN CYANOBACTERIA Many speculations have been made, through the years, as to how PSP toxins are synthesised by the producing microorganisms (Section 1.3.5.5). The molecular basis of STX production, however, is a captivating closed book. The origin and the evolution of PSP toxins production remains an enigma. To date, very few studies have been undertaken on the genetic bases of saxitoxin synthesis in dinoflagellates, and more scarcely in bacteria and cyanobacteria. The scattered toxigenicity of STX producing microorganisms across two of the three kingdoms of life ( or maybe even all, who knows?) suggests the hypothesis of a lateral exchange of STX biosynthetic genes by dinoflagellates, cyanobacteria and bacteria from, probably, an original prokaryotic source. This hypothesis is validated by the fact that the synthetic capability of PSP toxin production is limited to certain strains and not universal to a species. In this study evidence was found for possible LGT events to have occurred between toxic strains of A. circinalis. LGT is considered a relatively rare event in the evolution of microorganisms, particularly if it involves exchange of genes between phylogenetically distant species (Eisen and Fraser 2003). This may happen under particularly critical growth conditions, as the laterally transferred genes can be 194 Chapter 10 DisCJISSUJn maintained by the receiving microorganism since they code for a function essential for cell survival. Genetic heterogeneity was observed in this study among toxic and non-toxic isolates of A. circinalis. The results presented strongly suggest that the genetic differences between STX-producing and non-toxic A. circinalis strains indicated distinctive adaptation to specific environmental characteristics. Similarly, as mentioned in the previous Section (10.2), the genetic heterogeneity of A. circinalis seemed to be explained, to some extent, by genes associated with the maintenance of Na+ homeostasis. If STX biosynthetic capability represents a crucial adaptation to drastic environmental conditions, this could be the reason for the unusual acquisition of STX genes by microorganisms across phylogenetically distant taxa. This further suggests that STX, unique as a bioactive secondary metabolite of both prokaryotes and eukaryotes, may have been attained early in the evolution of these microorganisms. There are indications in the literature of high levels of salinity in the early oceans, from 1.5 to 2 times the modem value (Knauth 1998). These conditions persisted for a prolonged evolutionary period in which LGT genes may have acquired and maintained, and elevated salinity did not decline significantly until surprisingly late in Earth's geologic history (Knauth 1998). By fossil records, Precambrian aquatic environments were shared by ancestors of dinoflagellates, cyanobacteria and bacteria. If correct, this theory could explain the evolution and distribution of PSP toxin biosynthetic genes. 10.4 STX "GENES" AND STX "ENZYMES" Shimizu and coworkers established a milestone in the field of manne neurotoxins research. More than 20 years ago, they investigated the production of STX in dinoflagellate and cyanobacteria by feeding these microorganisms with labelled hypothetical precursors of PSP toxins (Section 1.3.5.5). The sequence of biochemical reactions found to correlate with STX biosynthesis was unprecedented in the literature and it remains a unique study. No biosynthetic enzymes have yet been identified and shown to be involved in STX production. Several years of speculation, however, has led to a list of potential candidate enzymes in the synthesis of STX (Section 1.3.5.5). 195 Chapter 10 Dismssian In this study, genes putatively encoding some of these previously hypothesised proteins have been isolated using molecular biology techniques. Exploring the differences between genomes of STX-producing and non-toxic A. circinalis strains, a carbamoyl-phosphate synthase, a S-adenosyl-methyltransferase, a transposase, an acetyltransferase and several toxic-strain specific hypothetical and regulatory proteins (such as a 60 kDa chaperonin GroEL) were recovered (Chapters 7 and 8). It was not possible during the course of the present investigation, however, to demonstrate the involvement of any of those genes in the production of STX by cyanobacteria. On the other hand, some of the present studies afforded the putative exclusion of some candidate genes and/or enzymes from the proposed STX-biosynthetic pathway. By microarray hybridisation, the transcription of ATs and PPTs was found to be repressed in toxic A. circinalis strains after lidocaine exposure (Chapter 9). Since the cell function of PPTs is to activate PKS and NRPS modules, these data suggest that hybrid PKS/NRPS enzymes may not be involved in the biosynthesis of STX. This hypothesis was confirmed here by the indication that STX biosynthesis is catalysed by enzymes different in function and half-life from NRPS and PKS (Chapter 6). Therefore, a hypothetical model for the STX biosynthetic pathway can be extrapolated based on present and previous studies. The Claisen-type condensation between the arginine and acetate could be catalysed by either an aminolevulinate synthase, a biosynthetic arginine decarboxylase or an acetyltransferase. A novel undescribed enzyme may transfer an amidino group from a molecule of arginine to the backbone of STX, followed by the cyclisation of the molecule activated by a thioesterase. A S-adenosylmethionine-dependent methyltransferase could actuate the methylation of the STX skeleton while a cytochrome P450 is likely to oxidise the methyl group. A STX specific carbamoyl-phosphate synthase may provide the substrate for the transferase implicated, together with a dioxygenase, in the last steps of STX biosynthesis. Sulfotransferases could be involved in the modification of the STX basic structure to produce the sulphated derivatives. Whether this putative biosynthetic pathway is common to the production of different PSP toxin analogues, and whether this list of hypothesised enzymes is conserved among the different PSP toxin-producing species, are still open questions. In this study a preliminary indication was obtained, however, that the production of Cl +2 196 Chapter 10 Dismssinn toxins and STX were operated by C. raciborskii T3 via distinct pathways (Chapter 6). Alternatively, the sequence of reactions leading to the synthesis of different STX derivatives in cyanobacteria may contrast with the one described in dinoflagellates (Sako et al. 2001). The former hypothesis could explain the observable natural differences in toxin profiles between various PSP toxin-producing isolates. The existence of distinct gene clusters, each encoding biosynthetic enzymes that are specific for each PSP-toxin category (carbamate, decarbamoyl, singly sulphated, doubly sulphated, etc.) and using different primary substrates, is highly unlikely. Considering that STX analogues are mainly characterised by different substitutions applied to the same chemical structure (Section 1.3.5.1), it is more plausible that STX biosynthetic enzymes have adapted to utilise the most appropriate arginine derivative in each producing microorganism. Other modifying enzymes belonging to primary metabolic pathways may then mediate the appropriate substitutions to the STX basic structure in order to produce each specific PSP toxin. 197 Chapter 10 Disa1Ssian 10.5 CONCLUSIONS AND SIGNIFICANCE In conclusion, this study has increased the general understanding of PSP toxin production in cyanobacteria. In addition, the present investigation has provided evidence for substantial genomic differences between STX-producing and non-toxic cyanobacterial strains. The major findings were: a. STX production is enhanced by Na+ stress and alkaline pH b. STX production can be modulated by influencing Na+ fluxes c. STX can block bacterial Na+-K+ fluxes d. A bacterial bioassay can be used to detect PSP-toxins e. There is a significant genomic diversity between STX-producing and non-toxic cyanobacteria f. A toxic-strain specific gene, coding for a Na+ dependent transporter protein involved in the maintenance of pH and Na+ homeostasis, was identified g. Adapter mediated PCR can be used for the identification of genes that have been laterally transferred between bacterial strains h. Several candidate genes in STX biosynthesis were identified The scope of this work required the implementation of methods from the fields of chemistry, biochemistry, physiology, microbiology, and molecular biology, and demonstrated the importance of a multi-disciplinary approach to the investigation of scientific problems, an approach which is essential in modem science. By adopting such a combined approach, it ensured achieving the objectives of these studies, which, had the focus been on only one type of discipline, would not have been otherwise possible. The potential impact of this research in the present and related fields of microbial toxins ensures that the findings of this study will continue to contribute to our understanding of these environmentally and pharmacologically important compounds. The increasing concern of public opinion on the hazard associated with toxic cyanobacterial bloom and red tides makes the present research very significant for this relevant environmental issue. The results of this project led to a deeper knowledge of the conditions that trigger neurotoxin production in cyanobacteria and will be crucial for 198 Chapter 10 Discusswn the management and control of toxic bloom events. A clearer understanding of the physiological and ecological role of PSP toxins, and of the influence of human and natural factors on their production, are the only tools for the possible prediction of toxic blooms both in freshwater and in marine environments. Moreover, this type of knowledge may suggest possible treatments for areas affected by toxic blooms. Molecular biology studies provided the necessary information for designing probes to identify STX-producing strains in the environment, and to further explore the biosynthesis of PSP toxins in cyanobacteria. Beside the environmental significance, this research can also have a future pharmaceutical application. The eventual genetic knowledge of saxitoxin biosynthesis could be the background for the development of either STX-based drugs, targeting excitable cells and different functional properties of the nervous system, or possibly the production of novel antibiotics. 10.6 FUTURE DIRECTIONS There are particular areas of research that could be followed up from the present study to obtain more information about the ecology of STX-producing cyanobacteria, the effect of STX on prokaryotic organisms and the molecular basis of STX biosynthesis. While the results of the present study have identified an association between STX production and the maintenance of Na+ homeostasis, further studies are required to completely characterise this relationship. A possible approach could be the assembly of a database of ecological parameters and toxicity data associated with cyanobacterial blooms, to additionally investigate the conditions that influence STX production in the environment. The same data could be coupled by a molecular biology analysis of bloom samples using the BGGM1 DNA microarray that was developed in this study, in order to integrate the ecological information with the presence/absence or expression of toxic genes. Focusing on the biochemistry of STX synthesis, a future investigation could target the enzymes associated with the production of osmolites through the urea cycle. Arginine is a key substrate in these pathways, which are activated in response to salt 199 Chapter 10 Disrussi.an stress. Alternatively, the production of PSP toxins by crude cyanobacterial extracts represents a promising basis for the development of an in vitro STX biosynthesis assay. Further research in this field may allow the identification, in a controlled in vitro system, of the substrates, cofactors and biochemical parameters required for STX production. In addition, such an assay may permit the selection of a cyanobacterial protein fraction able to operate STX biosynthesis. This procedure could facilitate the discovery, by chromatography or 2D-PAGE analysis, of the enzymes responsible for STX production. In this study, the blockage of Na+-K+ fluxes by STX was demonstrated. Additional studies are necessary, however, to elucidate the nature of this interaction and the membrane proteins involved. During the course of this research project, the identification of a STX-binding protein associated with cyanobacterial membranes has been attempted (data not shown). Using radioactively labelled STX, emission was measured by scintillation counting in fractions of cyanobacterial extracts. This endeavour was unsuccessful due to a number of reasons, including the quenching of photon-emission by cyanobacteria pigments and the presence of competing endogenous STX in the extracts. In light of the findings reported, it is strongly suggested to redo this experiment using non-toxic/non-pigmented bacteria. Toxic-strain specific and candidate genes recovered from A. circinalis during the course of this investigation have the potential of being used as probes for future additional genomic screening. A cosmid library of a STX-producing strain of this species could be prepared and clones analysed, by Southern hybridisation or PCR amplification, for the presence of multiple probes on the same cosmid. The occurrence of more toxic-strain specific genes in the same clone could indicate a toxic-strain specific genomic region being inserted, and possibly characterised by coding regions associated with STX production. PPH, a novel technique able to identify genes that have been laterally transferred between bacterial strains was also developed in this study. This method has the potential of being applied to both phylogenetically closely related and distant taxa, in order to recover genes with similar primary structure. With the aim of recovering STX biosynthetic genes, and based on the hypothesis that the corresponding nucleotide sequences would be the most similar among unrelated microorganisms, the genomes of 200 Chapter 10 Dismssi.on a toxic cyanobacterium and a toxic dinoflagellate could be, in a future experiment, hybridised at high stringency by ligating one terminal adapter directly to each digested genomic DNA. This approach may allow the recovery of STX-genes, if they have a conserved primary structure. Additionally, the same technique could be useful in the investigation of LGT events that have occurred in complex and outermost microbial communities, such as those characterising stromatolites or early stages of life on other planets. 201 Appendix A C')tl,rv:huterial strains used in dis study APPENDIX A CYANOBACTERIAL STRAINS USED IN THIS STUDY Species Strain Toxin Reference A. circinalis AWQC-118C PSPs Llewellyn et al. 2001 A. circinalis AWQC-131C PSPs Llewellyn et al. 2001 A. circinalis AWQC-134C PSPs Llewellyn et al. 2001 A. circinalis AWQC-150A PSPs Llewellyn et al. 2001 A. circinalis AWQC-279B PSPs Llewellyn et al. 2001 A. circinalis AWQC-307C PSPs Llewellyn et al. 2001 A. circinalis AWQC-344B PSPs P. Baker pers. comm. A. circinalis AWQC-271C Non-toxic P. Baker pers. comm. A. circinalis AWQC-306A Non-toxic Llewellyn et al. 2001 A. circinalis AWQC-332H Non-toxic Llewellyn et al. 2001 A. circinalis AWQC-342D Non-toxic P. Baker pers. comm. C. raciborskii AWT205 CYLN Hawkins et al. 1997 C. raciborskii T3 PSPs Lagos et al. 1999 M aeruginosa PCC7806 MCYST Moffitt 2003 N. spumigena NSORl0 NODLN Moffitt 2003 PSPs = PSP toxins; CYLN = cylindrospermopsin; MCYST = microcystin; NODLN = nodularin 202 AppendixB APPENDIXB GROWTH MEDIA PREPARATION MLA Stock solution Stock concentration mL perL MgS04 49.4 g/L 1 NaN03 85 g/L 2 K2HP04 6.96 g/L 5 H3B03 2.47 g/L 1 Vitamin Stock * 1 Micronutrients * 1 NaHC03 16.9 g/L 1«I> CaCb 29.4 g/L 1 * = see specific recipe «I>= add 10 mL per L for modified MLA-E VITAMIN STOCK Vitamin solution Concentration mg/L B12 0.5 Thiamin 100 Biotin 0.5 Add 10 mg of biotin to 50 mL of Milli-Q water, make up to a final volume of 100 mL con sterile Milli-Q, filter sterilise (solution A) - Repeat same procedure with B12 (solution B) - Add to 50 mL ofMilli-Q 10 mg ofthiamin, 50 µL of solution A and 50 µL of solution B, make up to a final volume of 100 mL with sterile Milli-Q, filter sterilise 203 AppendixB MICRO NUTRIENTS Micron u trien t Concentration g/L Primary addition Concentration g/L mL/L primary addition Na2EDTA 4.36 FeCb-6H20 1.58 NaHC03 0.6 MnCh-4H20 0.36 CuS04-SH20 10 1 ZnS04-7H20 10 2.2 C0Ch-6H20 10 1 NaMo04-2H20 10 0.6 ASM-1 Micronutrient Concentration Addition g/ 250 mL mL/L NaN03 21.25 2.0 KH2P04 4.35 1.0 Na2HP04 3.55 1.0 MnCh·6H20 10.15 1.0 MgS04·7H20 12.33 1.0 CaCh·2H20 7.35 1.0 FeCb·6H20 0.27 1.0 H3B03 0.618 1.0 MnCh·4H20 0.342 1.0 ZnCh 0.11 1.0 Na2EDTA 1.66 1.0 204 AppendixB 0.054 0.1 CuClz·2H2O 0.003 0.1 The pH should be 7.6 after sterilisation. JM Micronutrient Concentration Addition g/ 200 mL mL/L Ca(NO3)2·4H2O 4.0 1.0 KH2PO4 2.48 1.0 MgSO4·7H2O 10.0 1.0 NaHCO3 3.18 1.0 EDTAFeNa 0.45 1.0 EDTANa2 0.45 1.0 H3BO3 0.496 1.0 MnClz·4H2O 0.278 1.0 (NH4)6Mo1O24·4H2O 0.20 1.0 Cyanocobalamin 0.008 1.0 Thiamine HCl 0.008 1.0 Biotin 0.008 1.0 NaN03 16.0 1.0 Na2HPO4· 12H2O 7.2 1.0 Deionised water to l.0L The pH should be 7.8 after sterilisation. 205 AppendixB BG-11 Micronutrient Addition NaNO3 1.5 g K2HPO4 0.04g MgSO4·7H2O 0.075 g CaCli·2H2O 0.036 g Citric acid 0.006 g Ferric ammonium citrate 0.006 g EDTA (disodium salt) 0.001 g NaCO3 0.02 g Trace metal mix l.0mL Agar (if needed) 10.0 g Distilled water l.0L The pH should be 7 .1 after sterilisation. TRACE METAL MIX: Micronutrient Addition H3BO3 2.86 g MnC!i·4H2O 1.81 g ZnSO4·7H2O 0.222 g NaMoO4·2H2O 0.39 g CuSO4·5H2O 0.079 g Co(NO3)2·6H2O 49.4 mg Distilled water l.0L 206 AppendixC Teminal Adaptors ard PCR Prim:rs APPENDIXC Section C.1 PCR-BASED GENOME SUBTRACTION: ADAPTER AND PRIMER SEQUENCES Srfl/Sma I • T7 Promoter Not I ~~~ Rsa 11/2-site Adapter 1 [ 5'-CTAATACGACTCACTATAGGGCTCGAGCGGCCGCCCGGGCAGGT-3' L A 3 '-GGCCCGTCCA~ PCR Primer1 5 '-CTAATACGACTCACTATAGGGC-3' 5'-TCGAGCGGCCGCCCGGGCAGGT-3' ~ Nested Primer 1 l fag 1/Eae I Rsa 11/2-site Adapter2R [ 5'-CTAATACGACTCACTATAGGGCAGCGTGGTCGCGGCCGAGGT-3' T7 Promoter l 3'-GCCGGCTCCA-5' \~_~\ 5'-AGCGTGGTCGCGGCCGAGGT-3' Nested Primer 2R * When the adapters are ligated to Rsa I-digested DNA, the Rsal site is restored. 207 AppendixC Terminal A daptars and PCR Pnm;tS Section C.2 PCR PRIMERS USED IN THIS STUDY Designation Sequence a Reference mpF CCCAGTCACGACGTTGTAAAACG Section 2.8 mpR AGCGGATAACAATTTCACACAGG Section 2.8 Hip-CA GCGATCGCCA Section 7.2.3 Hip-TG GCGATCGCTG Section 7 .2.3 NaTF AT(ATC)AT(ATC)ATG(TC)TNGGNATGGG Section 7.2.3 NaTR ATNGCNGCAGGAATNGCCAT Section 7.2.3 vzFb AGCTGTGGCCATTGGCTTAA Section 7 .2.3 YZRb GCAATACAGATTTGCTGACG Section 7 .2.3 27F AGAGTTTGATTTACGCGACA Section 7.2.3 809R GCTTCGGCACGGCTCGGGTC Section 7.2.3 179F AATACCAATGCCTCCACTCC Section 8.2.5 179R AAAGACGGTGAAACACCTGC Section 8.2.5 109F ACAGGTGCGATGCGACCATT Section 8.2.5 109R TTGGTTGAGTGCGCTCCAAC Section 8.2.5 a Oligonucleotide sequences are given in 5' to 3' orientation b These primers have been proudly named after the author's motorcycle, a Yamaha YZF-Rl called "The General" 208 AppendixD BGGM1 Gene List APPENDIXD BGGM1 DNA-MICROARRAY GENE LIST And what have you got at the end of the day? what have you got to take away? a bottle of whisky and a new set of lies blinds on the windows and a pain behind the eyes (M Knopfler) LEGEND: NS = Not sequenced Cl= Clone PK = Polyketide Synthase UP = Unknown protein PS = Peptide Synthethase HP= Hypothetical protein AT= Amidino transferase HS = Heat shock protein AMT = Amino transferase PPT = Phosphopantetheinyl transferase PEOPLE THAT CONTRIBUTED TO BGGM1: Tim Salmon, Toby Mills, Ralf Kellmann, Michelle Moffitt, Michelle Allen, Leanne Pearson, Janine Copp, Francesco Pomati, Brendan Burns, Brett Neilan. 209 AppendixD BGGM1 Gene List SIZE Spot ID GENE ORGANISM number (bp) 1 TS 9a 1467 16S rDNA (27/1494) Synechocystis PCC6803 2 TS9 782 16S rDNA (27/809) Lyngbya CCAP 1446/4 3 TS Ba 1467 16S rDNA (27/1494) Streptomyces DSMZ 40785 4 TS8 782 16S rDNA (27/809) A. circinalis AWQC344 5 TS 7a 1467 16S rDNA (27/1494) Planctomyces DSM3776 6 TS 7 782 16S rDNA (27/809) A. circinalis AWQC332 7 TS69 Chi-a binding protein {lsiA) Lyngbya BAN TS01 8 TS67 Chi-a binding protein {lsiA) Nodularia 19661 93-1 9 TS66 Chi-a binding protein {lsiA) Nodularia NSOR10 10 TS65 Chi-a binding protein {lsiA) Synechocystis PCC6803 11 TS64 Chi-a binding protein {lsiA) Lyngbya BAN TS01 12 TS 63 Chi-a binding protein (lsiA) Lyngbya CCAP 1446/4 13 TS 62 782 16S rDNA (27/809) C. raciborskii T3 14 TS 61 782 16S rDNA (27/809) M. aeruginosa PCC7806 15 TS60 Phycocyanin intergenic spacer Lyngbya BAN TS01 16 TS6a 1467 16S rDNA (27/1494) Lactococcus /actis 17 TS6 782 16S rDNA (27/809) A. circinalis AWQC306 18 TS 5a 1467 16S rDNA (27/1494) C. g/utamicum DSM20300 19 TS5 782 16S rDNA (27/809) A. circinalis AWQC279 20 TS46 1467 16S rDNA (27/1494) C. glutamicum DSM20300 21 TS45 1467 16S rDNA (27/1494) C. g/utamicum DSM20300 22 TS44 1467 16S rDNA (27/1494) Lactococcus /actis 23 TS42 1467 16S rDNA (27/1494) Thermus thermophilus HB27 24 TS 41 1467 16S rDNA (27/1494) Thermus thermophilus Th104 25 TS40 1467 16S rDNA (27/1494) Thermus thermophilus DSM579 26 TS4a 1467 16S rDNA (27/1494) Thermus thermophilus HB27 27 TS4 782 16S rDNA (27/809) A. circinalis AWQC271 28 TS38a Phycocyanin intergenic spacer Nodularia NSOR10 29 TS38 1467 16S rDNA (27/1494) E.coli K12 30 TS 37a Phycocyanin intergenic spacer M. aeruginosa PCC7806 31 TS 37 1467 16S rDNA (27/1494) Zymomonas mobilis ZM4 32 TS 36a Phycocyanin intergenic spacer C. raciborskii AWT205 33 TS 36 1467 16S rDNA (27/1494) Synechocystis PCC6803 34 TS 35 Phycocyanin intergenic spacer A. circinalis AWQC279 35 TS34 Phycocyanin intergenic spacer A. circinalis AWQC332 36 TS 33 Phycocyanin intergenic spacer A. circinalis AWQC306 37 TS 32 Phycocyanin intergenic spacer A. circinalis AWQC271 38 TS 31 Phycocyanin intergenic spacer A. circinalis AWQC131c 39 TS30a 782 16S rDNA (27/809) Synechocystis PCC6803 40 TS 30 Phycocyanin intergenic spacer C. raciborskii AWT205 41 TS3a 1467 16S rDNA (27/1494) E. co/i K12 42 TS3 782 16S rDNA (27/809) A. circinalis AWQC148c 43 TS29a 782 16S rDNA (27/809) Nodularia NSOR10 44 TS29 Phycocyanin intergenic spacer Nodularia 19661 93-1 45 TS28a 782 16S rDNA (27/809) Nodularia 1983 46 TS28 Phycocyanin intergenic spacer Nodularia NSOR10 47 TS27a 782 16S rDNA (27/809) Nodularia 19661 93 -1 48 TS27 Phycocyanin intergenic spacer Synechocystis PCC6803 49 TS26a 782 16S rDNA (27/809) M. aeruginosa PCC7806 50 TS26 Phycocyanin intergenic spacer M. aeruginosa PCC7806 51 TS25 782 16S rDNA (27/809) Lyngbya BAN TS21 52 TS24 Phycocyanin intergenic spacer A. circinalis AWQC332 53 TS23 Phycocyanin intergenic spacer A. circinalis AWQC306 54 TS22 Phycocyanin intergenic spacer A. circinalis AWQC279 55 TS 21 Phlcoclanin inter9enic seacer A. circinalis AWQC271 210 AppendixD BGGM1 Gene List SIZE Spot ID GENE ORGANISM number (be) 56 TS20a 782 16S rDNA (27/809) C. raciborskii AWT205 57 TS20 Phycocyanin intergenic spacer A. circinalis AWQC131c 58 TS2a 1467 16S rDNA (27/1494) Thermus thermophilus DSMZ579 59 TS2 782 16S rDNA (27/809) A. circinalis AWQC134 60 TS 19 782 16S rDNA (27/809) A. circinalis AWQC279 61 TS 18a 782 16S rDNA (27/809) A. circinalis AWQC344 62 TS 18 782 16S rDNA (27/809) C. raciborskii AWT205 63 TS 17a 782 16S rDNA (27/809) A. circinalis AWQC332 64 TS 17 782 16S rDNA (27/809) Nodularia 1983 65 TS 16a 782 16S rDNA (27/809) A. circinalis AWQC306 66 TS16 782 16S rDNA (27/809) Nodularia 19661 93-1 67 TS 15a 782 16S rDNA (27/809) A. circinalis AWQC271 68 TS 15 782 16S rDNA (27/809) Nodularia NSOR10 69 TS 14a 782 16S rDNA (27/809) A. circinalis AWQC148c 70 TS 14 782 16S rDNA (27/809) Synechocystis PCC6803 71 TS 13 782 16S rDNA (27/809) A. circinalis AWQC134 72 TS 12a 782 16S rDNA (27/809) A. circinalis AWQC131c 73 TS 12 782 16S rDNA (27/809) Lyngbya BAN TS21 74 TS 11a 1467 16S rDNA (27/1494) Zymomonas mobilis ZM4 75 TS 11 782 16S rDNA (27/809) Lyngbya BAN TS02 76 TS 10a 1467 16S rDNA (27/1494) ThermusthermophHus104 77 TS 10 782 16S rDNA (27/809) Lyngbya BAN TS01 78 TS 1a 1467 16S rDNA (27/1494) C. g/utamicum DSMZ20300 79 TS 1 782 16S rDNA (27/809) A. circinalis AWQC131 80 TM.2 NS PK? C. raciborskii AWT205 81 TM.1 NS PK C. raciborskii AWT205 82 RM9 800 AT 84 Cl 1 A. circinalis AWQC150 83 RM? 1000 AT 83 Cl 1 A. circinalis AWQC150 84 RM6 1000 AT 81 Cl 4 A. circinalis AWQC134 85 RM55 1200 PSCl3 C. raciborskii T3 86 RM54 1200 PSCl2 C. raciborskii T3 87 RM53 1200 PS Cl 1° C. raciborskii T3 88 RM52 1200 PSCl3 A. circinalis AWQC150 89 RM51 1200 PS Cl 1 A. circinalis AWQC150 90 RM50 1200 PS Cl 5° A. circinalis AWQC134 91 RM5 1000 AT 81 Cl 3 A. circinalis AWQC134 92 RM49 1200 PS Cl 4° A. circinalis AWQC134 93 RM48 1200 PSCl3 A. circinalis AWQC134 94 RM46 900 PKCl2 A. circinalis AWQC306 95 RM45 900 PKCl6 A. circinalis AWQC150 96 RM44 900 PKCI 3 A. circinalis AWQC150 97 RM43 900 PK Cl 30 C. raciborskii AWT205 98 RM42 900 PK Cl 18 C. raciborskii AWT205 99 RM41 900 PK Cl 17 C. raciborskii AWT205 100 RM40 900 PK Cl 16 C. raciborskii AWT205 101 RM39 900 PK Cl 15 C. raciborskii AWT205 102 RM38 900 PK Cl 14 C. raciborskii AWT205 103 RM37 900 PK Cl 13 C. raciborskii AWT205 104 RM36 900 PK Cl 12 C. raciborskii AWT205 105 RM35 900 PK Cl 11 C. raciborskii AWT205 106 RM34 900 PK Cl 10 C. raciborskii AWT205 107 RM33 900 PKCl8 C. raciborskii AWT205 108 RM32 900 PKCl6 C. raciborskii AWT205 109 RM31 900 PKCl5 C. raciborskii AWT205 110 RM30 900 PKCl4 C. raciborskii AWT205 211 AppendixD BGGM1 Gene List Spot ID SIZE GENE ORGANISM number (bp) 111 RM3 1000 AT B1 Cl 4 A. circinalis AWQC131c 112 RM29 900 PKCl3 C. raciborskii AWT205 113 RM28 900 PKCl2 C. raciborskii AWT205 114 RM27 900 PKCI 1 C. raciborskii AWT205 115 RM26 900 PK Cl 26 C. raciborskii T3 116 RM25 900 PK Cl 15 C. raciborskii T3 117 RM24 900 PK Cl 11 C. raciborskii T3 118 RM23 900 PK Cl 10 C. raciborskii T3 119 RM22 900 PKCl7 C. raciborskii T3 120 RM21 900 PKCl6 C. raciborskii T3 121 RM20 900 PKCl4 C. raciborskii T3 122 RM2 1000 AT B1 Cl 3 A. circinalis AWQC131c 123 RM19 900 PKCl3 C. raciborskii T3 124 RM18 900 PKCl2 C. raciborskii T3 125 RM17 900 PKCI 1 C. raciborskii T3 126 RM16 650 ATCl8 C. raciborskii T3 127 RM15 650 ATCI 1 C. raciborskii T3 128 RM12 800 AT B4 Cl 4 A. circinalis AWQC150 129 RM11 800 AT B4 Cl 3 A. circinalis AWQC150 130 RM10 800 AT B4 Cl 2 A. circinalis AWQC150 131 RM1 1200 AT B1 Cl 2 A. circinalis AWQC131c 132 MM.9 1kb O-methyl transferase (NdaE) Nodularia NSOR1 O 133 MM.8 1kb A4 PS (NdaF) Nodularia NSOR10 134 MM.7 700bp AMT (NdaF) Nodularia NSOR10 135 MM.6 1kb pk3 PK (NdaF) Nodularia NSOR10 136 MM.55 1.5kb NADH ubiquinone Brazil Fischerella 19 oxidoreductase 137 MM.54 NS PSCl5 Brazil Fischerella 19 138 MM.53 NS PSCl2 Brazil Fischerella 19 139 MM.52 NS PS Cl 1 Brazil Fischerella 19 140 MM.51 NS PS Cl 1 D2 Pseudoalteramonas 141 MM.5 1kb PK2 (NdaD) Nodularia NSOR10 142 MM.49 PK upstream pyrroline Nodularia NSOR10 carboxylase 143 MM.48 800bp heterocyst glycolipid PK Nostoc AWT203 144 MM.45 UP downstream Mey-cluster M. aeruginosa PCC7806 145 MM.44 794 UP downstream Mey-cluster M. aeruginosa PCC7806 146 MM.43 833 UP downstream Mey-cluster M. aeruginosa PCC7806 147 MM.41 198 McyC PSA5 M. aeruginosa PCC7806 148 MM.40 790 McyB PSA4 M. aeruginosa PCC7806 149 MM.4 1.7kb PK1 (NdaC) Nodularia NSOR10 150 MM.39 707 McyB PSA3 M. aeruginosa PCC7806 151 MM.38 1128 McyAPSA2 M. aeruginosa PCC7806 152 MM.37 800bp McyA N-methyl transferase M. aeruginosa PCC7806 153 MM.36 694 McyD PK M. aeruginosa PCC7806 154 MM.35 1073 McyEAMT M. aeruginosa PCC7806 155 MM.34 1825 McyE PS M. aeruginosa PCC7806 156 MM.33 700bp McyF M. aeruginosa PCC7806 157 MM.3 1kb Nods A3 (NdaB) Nodularia NSOR10 158 MM.28 400bp dnaN M. aeruginosa PCC7806 159 MM.27 800bp PK Cl 2/9 Nodularia NSOR10 160 MM.26 700bp pyrroline carboxylase Nodularia NSOR10 161 MM.25 1.1kb ABC transporter Cl 1 Nodularia NSOR10 162 MM.23 1kb PSCl6 Nodularia NSOR10 163 MM.22 1kb PSCl4 Nodularia NSOR10 212 AppendixD BGGM1 Gene List Spot SIZE ID GENE ORGANISM number (b~) 164 MM.21 1kb PSCl2 Nodularia NSOR10 165 MM.20 1kb upstream#4 Nda-cluster (ORF4) Nodularia NSOR10 166 MM.2 800bp Nods A2 (NdaA) Nodularia NSOR10 167 MM.19 1431 upstream Nda-cluster (ORF6-8) Nodularia NSOR10 168 MM.18 1197 3-oxoacyl ketolase (ORF6) Nodu/aria NSOR10 169 MM.17 Heat regulator (ORF5) Nodularia NSOR10 170 MM.16 HP downstream Nda-cluster Nodularia NSOR10 (ORF4) 171 MM.15 500bp b-carotene ketolase (ORF3) Nodularia NSOR10 172 MM.14 200bp HLIP (ORF2) Nodularia NSOR10 173 MM.13 1kb? Transposon (ORF1) Nodularia NSOR10 174 MM.12 600bp NodS Racemase (NdaG) Nodularia NSOR10 175 MM.11 1.1kb Nods ABC-transporter (Ndal} Nodularia NSOR10 176 MM.10 600bp Nods D-3 (NdaH) Nodularia NSOR10 177 MM.1 1kb Nods A1 Nodularia NSOR10 178 MA"9" 754 16S rDNA Hapalosiphon welwitschii Stromatolite sample 179 MA"8" 754 16S rDNA Hapa/osiphon welwitschii Stromatolite sample 180 MA"5" 754 16S rDNA Cyanospira rippkae Stromatolite sample 181 MA"10" 754 16S rDNA Hapalosiphon welwitschii Stromatolite sample 182 MA"?" 754 16S rDNA Hapa/osiphon welwitschii Stromatolite sample 183 MA"3" 754 16S rDNA Mastidocladus laminosus Stromatolite sample 184 MA"2" 754 16S rDNA Fischerella sp. CENA 19 Stromatolite sample 185 LP.47 UP downstream Mey-cluster M. aeruginosa PCC7806 186 LP.46 UP downstream Mey-cluster M. aeruginosa PCC7806 187 LP.328 2058 McyG PKS M. aeruginosa PCC7806 188 LP.32A 2058 McyG PKS M. aeruginosa PCC7806 189 LP.31C 1.5kb McyH M. aeruginosa PCC7806 190 LP.318 1.5kb McyH M. aeruginosa PCC7806 191 LP.31A 1.5kb McyH M. aeruginosa PCC7806 192 LP.30 1kb Mcyl M. aeruginosa PCC7806 193 LP.29 800bp McyJ M. aeruginosa PCC7806 194 LP.1 F2f/R A. circinalis AWQC150 195 JC.50 550 Putative PPT M. aeruginosa PCC7806 196 JC.49 200 PPT Nostoc NIES19 197 JC.48 1500 Putative PPT C. raciborskii T3 198 JC.47 200 Putative PPT Nostoc piscinale CENA 21 199 JC.46 700 Putative PPT A. circinalis AWQC131c 200 JC.45 1500 Putative PPT A. circinalis AWQC131c 201 JC.44 200 PPT Cylindrospermum CENA 33 202 JC.43 200 PPT Nodu/aria NSOR10 203 JC.42 400 Putative Het I Synechocystis PCC6803 204 JC.41 700 Putative PPT Synechocystis PCC6803 205 JC.40 400 Putative Aldehyde reductase M. aeruginosa PCC7806 206 JC.39 200 Putative PPT A. circinalis AWQC279 207 JC.38 700 ferredoxin reductase M. aeruginosa PCC7806 208 JC.37 200 Putative PPT A. circinalis AWQC307 209 JC.36 800 Putative McyE Yellowstone Park env. sample 210 JC.35 800 HP Yellowstone Park env. samele 213 AppendixD BGGM1 Gene List SIZE Spot ID GENE ORGANISM number (bp) 211 JC.34 800 Putative PK Yellowstone Park env. sample 212 JC.33 800 Putative lsoleucyl tRNA Yellowstone Park env. sample synthase 213 JC.32 800 Putative McyE Yellowstone Park env. sample 214 JC.31 800 HP Yellowstone Park env. sample 215 JC.30 800 Putative McyD Yellowstone Park env. sample 216 JC.29 800 Putative PK Yellowstone Park env. sample 217 JC.28 800 Putative 6-phosphofructokinase Antarctic env. sample 218 JC.27 800 HP Antarctic env. sample 219 JC.26 800 HP Antarctic env. sample 220 JC.25 800 Putative Pytoene synthase Antarctic env. sample 221 JC.24 800 Putative Transposase Antarctic env. sample 222 JC.23 800 Putative NosB Antarctic env. sample 223 JC.22 800 Putative yersiniabactin synthase Antarctic env. sample 224 JC.21 800 UDP-Galactopyranose mutase Antarctic env. sample 225 JC.20 800 Putative 6-phosphofructokinase Antarctic env. sample 226 JC.19 800 UP Antarctic env. sample 227 JC.18 800 UP Antarctic env. sample 228 JC.17 800 Putative H+/Ca+ Exchanger Antarctic env. sample 229 JC.16 800 Putative PK Antarctic env. sample 230 JC.15 800 Putative PS Antarctic env. sample 231 JC.14 800 HP Antarctic env. sample 232 JC.13 800 Putative lturin A synthase Antarctic env. sample 233 JC.12 800 Putative PK Antarctic env. sample 234 JC.11 800 HP Lyngbya BAN TS01 235 JC.10 800 Putative PK Lyngbya BAN TS01 236 JC.09 800 Putative amidase Lyngbya BAN TS01 237 JC.08 800 Acetyl Co-A actetyltransferase Lyngbya BAN TS01 238 JC.07 800 Mb associated lipoprotein Lyngbya BAN TS01 precusor 239 JC.06 800 FKBP-type isomerase Lyngbya BAN TS01 240 JC.OS 800 2-component sensor Lyngbya BAN TS01 241 JC.04 800 Zeta-carotine desaturase Lyngbya BAN TS01 242 JC.03 800 HP Lyngbya BAN TS01 243 JC.02 800 HP Lyngbya BAN TS01 244 JC.01 800 Dihydroxypyrimidine Lyngbya BAN TS01 dehydrogenase 245 FP.99 509 HP A. circinalis AWQC131c/344 246 FP.98 277 RNA pol. ECF-type sigma factor A. circinalis AWQC131c/344 247 FP.97 520 Putative gluconate aldolase A. circinalis AWQC131c/344 248 FP.96 271 SOS Rib prot L33, UP A. circinalis AWQC131c/344 249 FP.95 443 Putative hydrolase A. circinalis AWQC131c/344 250 FP.94 469 Putative hydrolase A. circinalis AWQC344 251 FP.93 469 Putative hydrolase A. circinalis AWQC344 252 FP.92 276 SOS Rib prot L33, UP A. circinalis AWQC344 253 FP.91 543 Magnesium chelatase, subunit I A. circinalis AWQC344 254 FP.90 386 HP, PPE protein A. circinalis AWQC344 255 FP.89 469 Excinuclease ABC subunit A A. circinalis AWQC344 256 FP.88 276 SOS Rib prot L33, UP A. circinalis AWQC344 257 FP.87 273 Thiamin-phospahte A. circinalis AWQC344 pyrophosphorilase 258 FP.86 399 Similar to chloride channel A. circinalis AWQC344 259 FP.85 411 Conserved HP A. circinalis AWQC344 260 FP.84 389 Penicillin-binding protein A. circinalis AWQC344 261 FP.83 276 SOS Rib prot L33, UP A. circinalis AWQC344 214 AppendixD BGGM1 Gene List SIZE Spot ID GENE ORGANISM number (bp) 262 FP.82 162 NADH dehydrogenase A. circinalis AWQC131c 263 FP.81 232 Alpha-amylase A. circinalis AWQC131c 264 FP.80 232 Alpha-amylase A. circinalis AWQC131c 265 FP.79 269 UP conserved A. circinalis AWQC131c 266 FP.78 274 No similarita A. circina/is AWQC131c 267 FP.77 418 Sulfatase family protein A. circinalis AWQC131c 268 FP.76 162 NADH dehydrogenase A. circinalis AWQC131c 269 FP.75 274 No similarita A. circinalis AWQC131c 270 FP.74 221 Hypothetical protein A. circinalis AWQC131c 271 FP.73 232 Alpha-amylase precursor A. circinalis AWQC131c 272 FP.72 269 Put. Integral membr. prot. A. circinalis AWQC131c 273 FP.71 162 NADH2 dehydrogenase A. circinalis AWQC131c 274 FP.70 402 TonB, ferric-siderophore uptake A. circinalis AWQC131c 275 FP.69 596 HP transmembrane A. circinalis AWQC131c 276 FP.68 291 HP A. circinalis AWQC131c 277 FP.67 269 UP A. circinalis AWQC131c 278 FP.65 321 HP A. circinalis AWQC131c 279 FP.240 350 Putative helicase A. circinalis AWQC131c 280 FP.239 400 Putative helicase A. circinalis AWQC131c 281 FP.238 1000 Cation efflux protein A. circinalis AWQC131c 282 FP.237 900 Cation efflux protein A. circinalis AWQC134 283 FP.236 900 Cation efflux protein A. circinalis AWQC306 284 FP.235 800 Sodium ATPase sub. J A. circinalis AWQC306 285 FP.234 800 Sodium ATPase sub. J A. circinalis AWQC271 286 FP.233 500 HP A. circinalis AWQC271 287 FP.220 513 Carbamoyl phosphate synthase A. circinalis AWQC131c 288 FP.219 424 Put. Na+ dependent transporter A. circinalis AWQC134 289 FP.218 328 HP, AcylOrnitine A. circinalis AWQC134/306 Am inotransferase 290 FP.217 352 UP A. circinalis AWQC134/306 291 FP.216 357 UP, putative cytochrome A. circinalis AWQC134/306 292 FP.215 405 Putative oxidoreductase A. circinalis AWQC134/306 293 FP.214 378 UP A. circinalis AWQC134/306 294 FP.213 324 CylM protein A. circinalis AWQC306 295 FP.212 420 HS protein GroEL A. circinalis AWQC306 296 FP.211 417 Thiamin-phosphate A. circinalis AWQC306 pyrophosphorilase 297 FP.210 532 HP A. circinalis AWQC306 298 FP.209 262 UP, transporter A. circinalis AWQC306 299 FP.208 351 Serine/threonine kinase A. circinalis AWQC306 300 FP.207 264 HP A. circinalis AWQC306 301 FP.206 347 HP A. circinalis AWQC306 302 FP.205 414 Splicing factor Prp8 A. circinalis AWQC306 303 FP.204 541 HP A. circinalis AWQC306 304 FP.202 330 HP A. circinalis AWQC306 305 FP.199 461 Oligopeptide ABC-trans, UP A. circinalis AWQC134 306 FP.197 234 PS A. circinalis AWQC134 307 FP.196 249 Transporter, UP A. circinalis AWQC134 308 FP.194 457 HP A. circinalis AWQC134 309 FP.193 370 RNA polymerase beta subunit A. circinalis AWQC134 310 FP.192 510 S-adenosyl-methyltransferase A. circinalis AWQC134 mraW 311 FP.190 439 Peptidoglicane anchored protein A. circinalis AWQC134 312 FP.189 476 Phosphoglucomutase A. circinalis AWQC131c/344 313 FP.188 374 Thiamin-phosphate A. circinalis AWQC131c/344 215 AppendixD BGGM1 Gene List Spot SIZE ID GENE ORGANISM number (bp) pyrophosphorilase 314 FP.187 372 ABC-trans ATP binding A. circinalis AWQC131c/344 315 FP.186 381 Thiamin-phosphate A. circinalis AWQC131c/344 pyrophosphorilase 316 FP.185 299 Novel antigenic ToORF2 A. circinalis AWQC131c/344 317 FP.184 200 HS groEL A. circinalis AWQC131c/344 318 FP.183 526 Acetyltransferase A. circinalis AWQC131c/344 319 FP.182 664 Transporter, HP A. circinalis AWQC131c/344 320 FP.181 366 Peptidase-ribonucl reductase, A. circinalis AWQC131c/344 UP 321 FP.180 309 DNA pol 111 alpha sub. A. circinalis AWQC131c/344 322 FP.179 433 Put. transposase A. circinalis AWQC131c/344 323 FP.178 372 UP, Acyl-CoA dehydrogenase A. circinalis AWQC131c/344 324 FP.177 353 Alpha amylase, HP A. circinalis AWQC131c 325 FP.175 286 GLP_291_11778_8566 A. circinalis AWQC131c 326 FP.174 302 Putative hydrolase A. circinalis AWQC344 327 FP.173 271 Hypothetical protein A. circinalis AWQC344 328 FP.172 288 Thiamin-phosphate A. circinalis AWQC344 pyrophosphorilase 329 FP.171 418 50S Rib prot L33, UP A. circinalis AWQC344 330 FP.170 540 UP A. circinalis AWQC344 331 FP.169 274 UP A. circinalis AWQC344 332 FP.168 157 HP A. circinalis AWQC306 333 FP.167 348 HP A. circinalis AWQC306 334 FP.166 120 locus CG32796-PB A. circinalis AWQC306 335 FP.165 258 Thiamin-phosphate A. circinalis AWQC306 pyrophosphorilase 336 FP.164 243 Sensory Histidine Kinase A. circinalis AWQC306 337 FP.163 232 HP A. circinalis AWQC306 338 FP.162 210 HP A. circinalis AWQC306 339 FP.161 276 50S Rib prot L33, UP A. circinalis AWQC306 340 FP.160 230 HP A. circinalis AWQC306 341 FP.159 222 Ribonucleotide reductase A. circinalis AWQC306 related prot 342 FP.158 331 HP A. circinalis AWQC306 343 FP.157 260 Cons HP A. circinalis AWQC134 344 FP.156 193 No similarity A. circinalis AWQC134 345 FP.155 176 Phosphoglycerate A. circinalis AWQC134 dehydrogenase 346 FP.154 263 HP A. circinalis AWQC134 347 FP.153 399 Carbamoyl-phosph synthase A. circinalis AWQC134 (pyrim) 348 FP.152 152 Similar to shikimate kinase A. circinalis AWQC134 349 FP.151 133 HP A. circinalis AWQC134 350 FP.150 260 HP A. circinalis AWQC134 351 FP.149 280 HP A. circinalis AWQC134 352 FP.148 190 H+ transloc pyrophosph A. circinalis AWQC134 synthase, HP 353 FP.147 176 HP A. circinalis AWQC134 354 FP.146 377 Succinate dehydrogenase A. circinalis AWQC134 355 FP.145 180 inorganic pyrophosphatase A. circinalis AWQC134 356 FP.144 332 HS groEL A. circinalis AWQC306 357 FP.143 187 DNApol 111 alpha subunit A. circinalis AWQC306 358 FP.142 240 Am inotransferase A. circinalis AWQC306 359 FP.141 272 Thiamin-phosphate A. circinalis AWQC306 216 AppendixD BGGM1 Gene List Spot SIZE ID GENE ORGANISM number (bp) pyrophosphorilase 360 FP.140 376 HP A. circinalis AWQC306 361 FP.139 267 UP A. circinalis AWQC306 362 FP.138 240 hypothetical protein A. circinalis AWQC306 363 FP.137 240 hypothetical protein A. circinalis AWQC306 364 FP.135 186 HP A. circinalis AWQC306 365 FP.134 332 HS groEL A. circinalis AWQC306 366 FP.133 267 UP A. circinalis AWQC306 367 FP.132 267 UP A. circinalis AWQC134 368 FP.131 332 HS groEL A. circinalis AWQC134 369 FP.130 177 Mannosyltransferase A. circinalis AWQC134 370 FP.129 202 Mucin 1 precursor A. circinalis AWQC134 371 FP.128 273 50S Rib prot L33, UP A. circinalis AWQC134 372 FP.126 324 carbon-nitrogen hydrolase A. circinalis AWQC134 373 FP.125 274 Thiamin-phosphate A. circinalis AWQC134 pyrophosphorilase 374 FP.124 273 50S Rib prot L33, UP A. circinalis AWQC134 375 FP.123 203 HP A. circinalis AWQC134 376 FP.122 298 Oligopeptide binding protein A. circinalis AWQC134 377 FP.120 217 HP A. circinalis AWQC131c/344 378 FP.119 271 50S Rib prot L33, UP A. circinalis AWQC131c/344 379 FP.118 327 HS groEL A. circinalis AWQC131c/344 380 FP.116 256 UP A. circinalis AWQC131c/344 381 FP.115 89 No similarity A. circinalis AWQC131c/344 382 FP.114 327 HS groEL A. circinalis AWQC131c/344 383 FP.113 256 UP A. circinalis AWQC131c/344 384 FP.112 205 HP A. circinalis AWQC131c/344 385 FP.111 191 Cell division protein FtsZ A. circinalis AWQC131c/344 386 FP.110 269 Hypothetical protein, put. A. circinalis AWQC131c/344 Protease 387 FP.109 371 HP A. circinalis AWQC131c/344 388 FP.108 319 UP A. circinalis AWQC131c/344 389 FP.107 121 No similarity A. circinalis AWQC131c/344 390 FP.106 142 HP A. circinalis AWQC131c/344 391 FP.104 288 HP, transcriptase A. circinalis AWQC131c/344 392 FP.103 271 50S Rib prot L33, UP A. circinalis AWQC131c/344 393 FP.102 236 UP A. circinalis AWQC131c/344 394 FP.101 271 50S Rib prot L33, UP A. circinalis AWQC131c/344 395 FP.100 205 HP membrane A. circinalis AWQC131c/344 396 CTP.9 Putative protease A. circinalis AWQC118c 397 CTP.7 Sensory transduction system A. circinalis AWQC118c 398 CTP.6 Probable aldehyde A. circinalis AWQC118c dehydrogenase 399 CTP.4 HP A. circinalis AWQC118c 400 CTP.2 HP A. circinalis AWQC118c 401 CTP.16 UP A. circinalis AWQC118c 402 CTP.14 UP A. circinalis AWQC118c 403 CTP.13 Ammonium transporter A. circinalis AWQC118c 404 CTP.12 Tn5706 transposase A. circinalis AWQC 118c 405 CTP.11 Putative protease A. circinalis AWQC118c 406 CTP.10 Putrescine oxidase A. circinalis AWQC118c 407 CTP.1 HP A. circinalis AWQC118c 408 CT9 Arginine decarboxylase C. raciborskii 409 CT8 Arginine decarboxylase A. circinalis AWQC148c 410 CT7 Arginine decarboxylase A. circinalis AWQC148c 217 AppendixD BGGM1 Gene List Spot SIZE ID GENE ORGANISM number (bp) 411 CT6 Sulfo transferase A. cylindrica 412 CTS Sulfo transferase Anabaena NIES525 413 CT4 Sulfo transferase A. circinalis AWQC148c 414 CT3 Sulfo transferase A. affinis 415 CT21 Sulfo transferase Anabaena NIES525 416 CT20 Sulfo transferase Anabaena NIES525 417 CT2 Sulfo transferase Anabaena PCC7108 418 CT19 Sulfo transferase Anabaena NIES44-1 419 CT18 Ornitine decarboxylase Anabaena L-7 420 CT17 Ornitine decarboxylase Anabaena PCC7108 421 CT16 Ornitine decarboxylase A. circinalis AWQC306 422 CT15 Ornitine decarboxylase A. circinalis AWQC131c 423 CT14 Ornitine decarboxylase A. circinalis AWQC118c 424 CT13 Ornitine decarboxylase A. spiroides 425 CT12 Ornitine decarboxylase A. cylindrica 426 CT11 Ornitine decarboxylase A. circinalis AWQC148c 427 CT10 Ornitine decarboxylase A. affinis 428 CT1 Sulfo transferase A. spiroides 429 BB.5 NS 16S rDNA Archea isolate 430 BB.4 NS Poliphosphate kinase Nodu/aria NSORIO 431 BB.3 NS Poliphosphate kinase Cylindrospermopsis AWT205 432 BB.2 NS Poliphosphate kinase M. aeruginosa PCC7806 433 BB.1 NS 16S rDNA Archea isolate 434 BAN9 NS RecA recombination protein C. raciborskii T3 435 BANS NS RecA recombination protein Nodularia NSORIO 436 BAN? Gvp structural gas vesicle prot A. circinalis AWQC344 437 BAN6 270 Gvp structural gas vesicle prot Nodularia NSORIO 438 BANS NifJ nitrogen fixation A. circinalis AWQC344 439 BAN4 NifJ nitrogen fixation Nodularia NSORIO 440 BAN3 270 NifH nitrogenase reductase Nodularia NSORIO 441 BAN2 NS NifH nitrogen fixation C. raciborskii T3 442 BAN15 265 Gvp structural gas vesicle prot A. circinalis AWQC332 443 BAN14 265 Gvp structural gas vesicle prot C. raciborskii T3 444 BAN13 484 DNAk type chaperone, HSP?0-2 C. raciborskii T3 445 BAN12 266 DNAk type chaperone, HSP?0-1 Nodularia NSORIO 446 BAN11 NS HS A. circinalis AWQC344 447 BAN10 NS RecA recombination protein A. circinalis AWQC 344 448 BAN1 NS NifA nitrogen fixation C. raciborskii T3 218 Appendix£ Bioi,rfarm:itic anal')5is APPENDIXE EXAMINATION OF COMBINED MICROARRAY DATASETS After all the jacks are in their boxes, and the clowns have all gone to bed (J. Hendrix) Hierarchical analysis results, displaying all m1croarray data presented in Chapters 8 and 9, were performed as illustrated in Section 2.12 and are reported in Figure-E. For bi-dimensional clustering, individual genes were normalised as reported in Sections 8.2.9 and 9.2.5 prior to running the clustering algorithm. Red indicated toxic specific DNA fragments or up-regulated genes, blue designated repressed genes or DNA fragments with no toxic specificity, while grey boxes signify non-detected spots. Experiment data sets are also represented: strains 131 C and 306A hybridisation of genomic DNA (A), total RNA (B) and effect of lidocaine at 1 µM on 131 C gene expression (C); strains 344B and 271C hybridisation of genomic DNA (D), total RNA (E) and effect of lidocaine at 1 µM on 344B gene expression (F). Microarray hybridisation data sets grouped depending on the different typology of the performed experiments (genomic DNA -Chapter 8- and gene expression studies - Chapter 9), and not due to strain specificity. Genes clustered according to their toxic specificity or their expression profiles across all experiments in three major clades: the majority of strain 344B toxic-specific sequences (Fig. E-1 ), the majority of strain 131 C toxic-specific sequences (Fig. E-2), toxic-specific sequences with no expression profile or false positives (Fig. E-3). 219 Appendix E Bioinform atic ana/ysis Figure E 1 FP.75 FP.185 BAN6 FP.109 BAN1 FP.70 FP.65 FP.74 2 TS1 6/15 3 FP.1 02 T S1 2/18 J C .44 FP.1 20 FP.192 0 0.5 1 FP.106 FP.170 FP.174 Expression ADBCFE ' "' I"\ AppendixF Nudeatide Sequerm A aBSion Nurrhets APPENDIXF NUCLEOTIDE SEQUENCES GENBANK ACCESSION NUMBERS GenBank: http://www.ncbi.nlm.nih.gov/Genbank/ FP219 AY326655 FP210 AY326681 FP130 AY326656 FP213 AY326682 FP122 AY326657 FP158 AY326683 FP123 AY326658 FP162 AY326684 FP129 AY326659 FP163 AY326685 FP190 AY326660 FP164 AY326686 FP192 AY326661 FP166 AY326687 FP194 AY326662 NaDT Anabaena 2798 AY326688 FP145 AY326663 FP65 AY445143 FP146 AY326664 FP68 AY445144 FP147 AY326665 FP69 AY445145 FP149 AY326666 FP70 AY445146 FP150 AY326667 FP71 AY445147 FP153 AY326668 FP72 AY445148 FP154 AY326669 FP73 AY445149 FP155 AY326670 FP74 AY445150 FP47 AY326671 FP77 AY445151 FP140 AY326672 FP175 AY445152 FP143 AY326673 FP84 AY445153 FP137 AY326674 FP85 AY445154 FP202 AY326675 FP86 AY445155 FP204 AY326676 FP87 AY445156 FP205 AY326677 FP88 AY445157 FP206 AY326678 FP89 AY445158 FP207 AY326679 FP90 AY445159 FP208 AY326680 FP91 AY445160 221 AppendixF N udeatide S«jU£YK13 A a:ession Nwrhets FP170 AY445161 FP173 AY445162 FP174 AY445163 FP95 AY445164 FP97 AY445165 FP98 AY445166 FP99 AY445167 FP102 AY445168 FP104 AY445169 FP106 AY445170 FP119 AY445171 FP120 AY445172 FP185 AY445173 FP186 AY445174 FP187 AY445175 FP189 AY445176 FP108 AY445177 FP109 AY445178 FP110 AY445179 FP111 AY445180 FP112 AY445181 FP118 AY445182 FP178 AY445183 FP179 AY445184 FP180 AY445185 FP181 AY445186 FP182 AY445187 FP183 AY445188 222 Referma:s LITERATURE CITED o Agron P. 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