AU2006254717B2 - Detection of hepatotoxic cyanobacteria - Google Patents

Abstract

The present invention relates to methods and kits for the detection of toxic cyanobacteria, in particular of hepatotoxin-producing cyanobacteria.

Description

WO 2006/128230 PCT/AU2006/000730 -1 Detection of Hepatotoxic Cyanobacteria Technical Field The present invention relates to methods and kits for the detection of toxic cyanobacteria, in 5 particular of hepatotoxin-producing cyanobacteria. Background of the Invention Cyanobacteria, also known as blue-green algae, are photosynthetic bacteria widespread in marine and freshwater environments worldwide. Of particular significance for water quality and 10 human and animal health are those cyanobacteria which produce toxic compounds. A diverse range of cyanobacterial genera are well known for the formation of toxic blue-green algal blooms on water surfaces (see, for example, Codd et al., 1999; Carmichael, 2001). Many of these blooms are harmful to humans and animals due to the production of hepatotoxins and neurotoxins by the bloom-forming organisms and the ability of blooms to flourish and expand in coastal waters, 15 streams, lakes, and in drinking water and recreational reservoirs. There is a need for rapid methods for the accurate detection of toxic cyanobacteria to enable an assessment of the potential health hazard of cyanobacterial blooms and to allow the implementation of effective water management strategies to minimize the effects of toxic bloom outbreaks. 20 Two hepatotoxins of particular concern are microcystin and nodularin. Both toxins are inhibitors of eukaryotic-type protein phosphatases 1 and 2A and in vertebrates toxicity is mediated via transport of the toxins into hepatocytes. Acute exposure to either toxin can lead to liver failure and death in animals, including humans. Further, subchronic exposure to microcystin and nodularin is associated with tumor promotion and, in the case of nodularin, tumor initiation (Hitzfeld 25 et al., 2000). Microcystin and nodularin are cyclic peptides synthesized non-ribosomally by large multi enzyme complexes consisting of different modules including non-ribosomal peptide synthetases (NRPS) and polyketide synthases (PKS) (Tillett et al., 2000; Moffitt and Neilan, 2001). These modules catalyze the activation, modification, and condensation of specific amino acids. 30 Microcystins form a large family of cyclic heptapeptides of the general formula cyclo-(D-alanine-X D-MeAsp-Z-Adda-D-glutamate-Mdha), where D-MeAsp is D-B-erythro-methyl-aspartatic acid, Mdha is N-methyldehydroalanine, and X and Z are variable L-amino acids. Nodularin is a cyclic pentapeptide of general formula cyclo-(D-MeAsp-L-arginine- Adda-D-glutamate-Mdhb), where Mdhb is 2-(methylamino)-2-dehydrobutyric-acid. The most unusual moiety of microcystin and 35 nodularin is Adda (3-amino-9-methoxy-2, 3, 8- trimethyl-10-phenyl-4, 6-decadienioc acid).

WO 2006/128230 PCT/AU2006/000730 -2 Microcystin-producing species usually.produce a cocktail of different microcystin variants, however only one type will be predominately synthesized (Mikalsen et al., 2001). Only a few variants of nodularin have been identified to date. To date there have been reports of microcystin production by cyanobacterial species from s the four orders Chroococcales, Nostocales, Oscillatoriales, and Stignonematales, including Microcystis sp., Chroococcus dispersus (Chroococcales), Anabaena sp., Nostoc sp., Anabeanopsis sp. (Nostocales), Haphalosiphon, Phormidium sp., Planktothrix sp., and Oscillatoria sp. However, the genes for the production of nodularin have only been reported in N. spumigena and one N. harveyana strain (Moffitt and Neilan, 2001). 10 Due to this broad distribution of hepatotoxin production and sequence differences in cyanobacterial genera there is a lack of reliable molecular protocols that enable detection of all potentially hepatotoxin-producing cyanobacterial species. Most PCR-based detection methods are only based on the amplification of either microcystin or nodularin synthetase gene sequences from one genus or one species (for example Neilan, 1996; Moffitt et al., 2001; Tillet et al., 2000; is Christiansen et al., 2003; Vaitomaa et al., 2003; Kurmayer et al., 2003, 2004). Protocols based on more than one data set only target the most common microcystin producing bloom-forming species Microcystis, Planktothrix, and Anabaena (Hisbergues et al., 2003). The molecular detection of other microcystin producing species such as Anabaenopsis, Phormidium, and Nostoc has not previously been addressed. 20 Accordingly, there is a clear need for the development of a simple detection system for the identification of multiple hepatotoxin-producing cyanobacterial species and genera. The present inventors have now developed a molecular method that enables the detection of all known potentially microcystin and nodularin producing species with one single PCR reaction. 25 Summary of the Invention According to a first aspect of the present invention there is provided a method for the detection of toxic cyanobacteria, the method comprising the steps of: (a) obtaining a cyanobacterial sample; and (b) analyzing the sample for the presence of hepatotoxin-associated aminotransferase 30 domain sequences, wherein the presence of hepatotoxin-associated aminotransferase domain sequences are indicative of toxic cyanobacteria. The hepatotoxin-associated aminotransferase domain sequences may be derived from the mcyE open reading frame of the microcystin synthetase gene complex or an orthologue or WO 2006/128230 PCT/AU2006/000730 -3 homologue thereof and/or the ndaF open reading frame of the nodularin synthetase gene complex or an orthologue or homologue thereof. The analysis step (b) may comprise: (i) amplification of DNA from the sample using suitable primers; and 5 (ii) detection of amplified sequences. The primers may be oligonucleotide primers comprising the nucleotide sequences as set forth in any of SEQ ID Nos: 1 to 4. In one embodiment, the amplification may be performed using the primer pair comprising the nucleotide sequences as set forth in SEQ ID Nos: 1 and 2. In one embodiment, the amplification may be performed using the primer pair comprising the nucleotide to sequences as set forth in SEQ ID Nos: 1 and 4. In one embodiment, the amplification may be performed using the primer pair comprising the nucleotide sequences as set forth in SEQ ID Nos: 3 and 4. Detection of the amplified sequences may comprise gel electrophoresis and/or nucleic acid sequencing. is The cyanobacterial sample may comprise one or more isolated or cultured cyanobacterial organisms or may be an environmental sample containing one or more cyanobacterial organisms. The environmental sample may be a water sample or a sample from a blue-green algal bloom. According to a second aspect of the present invention there is provided a method for the detection of hepatotoxin-producing cyanobacteria, the method comprising the steps of: 20 (a) obtaining a cyanobacterial sample; and (b) analyzing the sample for the presence of hepatotoxin-associated aminotransferase domain sequences, wherein the presence of hepatotoxin-associated aminotransferase domain sequences are indicative of hepatoxin-producing cyanobacteria. 25 The hepatotoxin may be microcystin or nodularin. The hepatotoxin-associated aminotransferase domain sequences may be derived from the mcyE open reading frame of the microcystin synthetase gene complex or an orthologue or homologue thereof and/or the ndaF open reading frame of the nodularin synthetase gene complex or an orthologue or homologue thereof. 3D The analysis step (b) may comprise: (i) amplification of DNA from the sample using suitable primers; and (ii) detection of amplified sequences. The primers may be oligonucleotide primers comprising the nucleotide sequences as set forth in any of SEQ ID Nos: 1 to 4. In one embodiment, the amplification may be performed using 35 the primer pair comprising the nucleotide sequences as set forth in SEQ ID Nos: 1 and 2. In one WO 2006/128230 PCT/AU2006/000730 -4 embodiment, the amplification may be performed using the primer pair comprising the nucleotide sequences as set forth in SEQ ID Nos: 1 and 4. In one embodiment, the amplification may be performed using the primer pair comprising the nucleotide sequences as set forth in SEQ ID Nos: 3 and 4. 5 Detection of the amplified sequences may comprise gel electrophoresis and/or nucleic acid sequencing, The cyanobacterial sample may comprise one or more isolated or cultured cyanobacterial organisms or may be an environmental sample containing one or more cyanobacterial organisms. The environmental sample may be a water sample or a sample from a blue-green algal bloom. 10 According to a third aspect of the present invention there is provided a kit for the detection of toxic cyanobacteria, the kit comprising at least one primer designed to detect hepatotoxin associated aminotransferase domain sequences. The kit may comprise multiple primers designed to amplify the hepatotoxin-associated aminotransferase domain sequences. The primers may be oligonucleotide primers comprising the is nucleotide sequences as set forth in SEQ ID Nos: 1 to 4. The oligonucleotide primers may comprise the nucleotide sequences as set forth in SEQ ID Nos: 1 and 2. The oligonucleotide primers may comprise the nucleotide sequences as set forth in SEQ ID Nos: 1 and 4. The oligonucleotide primers may comprise the nucleotide sequences as set forth in SEQ ID Nos: 3 and 4. 20 According to a fourth aspect of the present invention there is provided a kit for the detection of hepatotoxin-producing cyanobacteria, the kit comprising at least one primer designed to detect hepatotoxin-associated aminotransferase domain sequences. The kit may comprise multiple primers designed to amplify the hepatotoxin-associated aminotransferase domain sequences. The primers may be oligonucleotide primers comprising the 25 nucleotide sequences as set forth in SEQ ID Nos: 1 to 4. The oligonucleotide primers may comprise the nucleotide sequences as set forth in SEQ ID Nos: 1 and 2. The oligonucleotide primers may comprise the nucleotide sequences as set forth in SEQ ID Nos: 1 and 4. The oligonucleotide primers may comprise the nucleotide sequences as set forth in SEQ ID Nos: 3 and 4. 30 Definitions The term "hepatotoxin-associated aminotransferase domain sequence" as used herein means a nucleotide sequence encoding an aminotransferase activity that functions in the addition of an amino group to a cyclic peptide hepatotoxin. Typically the aminotransferase domain 35 sequence is part of an open reading frame (ORF) encoding a multienzyme complex functioning in WO 2006/128230 PCT/AU2006/000730 -5 the production of the hepatotoxin, the ORF being physically located within the hepatotoxin biosynthetic gene cluster, as in the case of the mcyE ORF of the microcystin synthetase gene complex and the ndaF ORF of the nodularin synthetase gene complex. Alternatively the aminotransferase domain may be located within a separate gene encoding an aminotransferase s enzyme. In the context of this specification, the term "comprising" means "including principally, but not necessarily solely". Furthermore, variations of the word "comprising", such as "comprise" and "comprises", have correspondingly varied meanings. 10 Brief Description of the Drawings Embodiments of the present invention will now be described, by way of example only, with reference to the accompanying drawings. Figure 1. Alignment of the HEPF and HEPR primers with the aminotransferase target region from the characterized hepatotoxic species Nodularia NSOR10 (Moffitt and Neilan, 2004), 15 Anabaena strain 90 (Rouhiainen et a/,, 2004), M. aeruginosa PCC7806 (Tillett et al., 2000), M. aeruginosa K-139 (Nishizawa et al., 1999), and Planktothrix NIVA-CYA126/8 (Christiansen et al., 2003). Figure 2. Relative positions of primers HEPF, HEPR, HEPF2 and HEPR2 and approximate sizes (bp) of PCR products. 20 Figure 3. AMT domain-containing PCR products using HEPF/HEPR primers from blue green algal bloom samples separated by gel electrophoresis on a 2% agarose gel: M: 1kb+ DNA ladder. Lane 1: positive control (M. aeruginosa PCC7806), lane 2: Lake Spino (Italy) sample, lane 3: Lake Alexandria (Australia) sample, lane 4: Lake John Oldman Park (Australia) sample, lane 5: Swan River (Australia) sample. 25 Figure 4. Phylogenetic analysis of the 472 bp AMT domain-containing PCR fragment. The phylogenetic relationship was constructed using the Neighbor-Joining method, Bootstrap values greater than 500 (after 1000 data resampling events) are shown. The scale is 0.05 mutations per amino acid position. Bloom samples are indicated in bold. The nucleotide sequence of the oligonucleotide primer HEPF is set forth in SEQ ID No:1 30 and the nucleotide sequence of the oligonucleotide primer HEPR is set forth in SEQ ID No:2. The nucleotide sequence of the oligonucleotide primer HEPF2 is set forth in SEQ ID No:3. The nucleotide sequence of the oligonucleotide primer HEPR2 is set forth in SEQ ID No:4.

WO 2006/128230 PCT/AU2006/000730 -6 Best Mode of Performing the Invention The invention will now be described in more detail, including, by way of illustration only, with respect to the examples which follow. As described herein, the present inventors have developed a molecular tool enabling the s successful identification of potentially hepatotoxic cyanobacterial species and blue-green algal blooms. The tool resides in a single PCR reaction based on the presence/absence of an aminotransferase (AMT) domain within the hepatoxin synthesis gene cluster. The AMT domain is located between the polyketide synthase (PKS) and non-ribosomal peptide synthetase (NRPS) modules within the mcyE and ndaF open reading frames of the microcystin and nodularin 10 synthetase enzyme complexes, respectively. AMT plays a critical role in the biosynthesis of microcystins and nodularins for the transfer of an amino group to the Adda moiety (Tillett et al., 2000). Accordingly, one aspect of the present invention provides a method for the detection of toxic cyanobacteria, the method comprising the steps of: 15 (a) obtaining a cyanobacterial sample; and (b) analyzing the sample for the presence of hepatotoxin-associated aminotransferase domain sequences, wherein the presence of hepatotoxin-associated aminotransferase domain sequences are indicative of toxic cyanobacteria. 20 The present invention also provides kits for the detection of toxic cyanobacteria the kits comprising at least one primer designed to detect hepatotoxin-associated aminotransferase domain sequences. As exemplified herein, using the method according to an embodiment of the invention, hepatotoxic cyanobacteria cultures, as well as blue-green algal blooms, from the orders 25 Oscillatoriales, Chroococcales and Nostocales were successfully detected. Furthermore, the genera of the identified hepatotoxic cyanobacteria could be determined by sequence analysis due to the congruence of species and peptide hepatotoxin evolution in cyanobacteria. The methods and kits of the present invention provide a valuable tool for the detection of potentially toxic cyanobacterial isolates, for example, from water samples, thereby enabling the 30 assessment of the potential for a toxic bloom to form, the identification of toxic blooms, or an assessment of the toxic potential, and thus the health hazard posed by, a blue-green algal bloom. Accordingly, the methods and kits of the invention may form part of routine water quality monitoring procedures or be used as required in the event of a blue-green algal bloom outbreak or in conditions considered likely to support or be conducive to such an outbreak.

WO 2006/128230 PCT/AU2006/000730 -7 Those skilled in the art will appreciate that the methods and kits of the present invention may be used alone or in conjunction with other available testing in identifying toxic cyanobacterial species and blooms. It will be appreciated by those skilled in the art that the cyanobacteria to which the present 5 invention relates are any potentially hepatotoxic cyanobacteria, typically being those capable of production of microcystin or nodularin. For example, the cyanobacteria to which the present invention is applicable may be selected from the orders Oscillatoriales, Chroococcales, Nostocales and Stigonematales, For example, the cyanobacteria may be selected from the genera Anabaena, Nostoc, Microcystis, Planktothrix, Oscillatoria, Phormidium, and Nodularia. For example, the 10 cyanobacteria may be selected from the species Microcystis aeruginosa, Microcystis viridis, Microcystis wesenbergii, Nodularia harveyana and Nodularia spumigena. For example, the cyanobacteria may be selected from Microcystis aeruginosa PCC7806, Microcystis aeruginosa PCC7005, Microcystis viridis NIES 102, Microcystis wesenbergii NIES 107, Microcystis sp. strain UTEX 2667, Microcystis sp, strain UTEX 2664, Nodularia harveyana PCC7804, Nodularia is spumigena NROS 10, Nodularia spumigena BY1, Nodularia spumigena HEM, Anabaena sp. strain 202A2, Nostoc sp. strain 152, Oscillatoria sp. strain 18R, Oscillatoria sp. strain 195, Phormidium sp. isolate 2-26b, Phormidium sp. isolate 1-6c and Phormidium sp. isolate 4-19b. The methods and kits of the invention may also be employed for the discovery of novel hepatotoxic species or genera in culture collections or from environmental samples. 20 Methods and kits DNA to be analysed using methods and kits of the present invention may be extracted from cyanobacterial cells, either in mixed culture or as individual species or genus isolates. Accordingly, the cells may be cultured prior to DNA isolation or alternatively DNA may be extracted directly from 25 environmental samples, such as water samples or blue-green algal blooms. A number of suitable methods known to those skilled in the art may be used for the extraction and purification of DNA for the purposes of the present invention, for example as described in Neilan (1995) and Neilan et al. (2002) the disclosures of which are incorporated herein by reference. Those skilled in the art will readily appreciate that the present invention is not to be limited to the use of specific methods for 30 DNA isolation. Indeed the present invention may be performed without DNA isolation prior to DNA analysis. Typically, according to methods of the invention the analysis of DNA is carried out by PCR amplification. Amplified products may be further analysed by nucleic acid sequencing. PCR amplification may be conducted on DNA extracted from cyanobacterial isolates or environmental 35 samples as described above, or alternatively sequences may be amplified directly from organisms WO 2006/128230 PCT/AU2006/000730 -8 without the need for prior DNA extraction or purification steps. A variety of methods for direct PCR are known to those skilled in the art. The methods and reagents for use in PCR amplification reactions, subsequent fragment resolution, and nucleic acid sequencing are well known to those skilled in the art. In each case, 5 suitable protocols and reagents will largely depend on individual circumstances. Guidance may be obtained from a variety of sources, such as for example Sambrook et al., Molecular Cloning : A Laboratory Manual, Cold Spring Harbor, New York, 1989, and Ausubel et al., Current Protocols in Molecular Biology, Greene Publ. Assoc, and Wiley-Intersciences, 1992. A person skilled in the art would readily appreciate that various parameters of these procedures may be altered without 10 affecting the ability to achieve the desired product. For example, in the case of PCR amplification, the salt concentration may be varied or the time and/or temperature of one or more of the denaturation, annealing and extension steps may be varied. Similarly, the amount of DNA used a template may also be varied depending on the amount of DNA available or the optimal amount of template required for efficient amplification. 15 The primers for use in the methods and kits of the present invention are typically oligonucleotides of, generally, 15 to 30 bases in length. Such primers can be prepared by any suitable method, including, for example, direct chemical synthesis or cloning and restriction of appropriate sequences. Not all bases in the primer need reflect the sequence of the template molecule to which the primer will hybridize, the primer need only contain sufficient complementary 20 bases to enable the primer to hybridize to the template. A primer may also include mismatch bases at one or more positions, being bases that are not complementary to bases in the template, but rather are designed to incorporate changes into the DNA upon base extension or amplification. A primer may include additional bases, for example in the form of a restriction enzyme recognition sequence at the 5' end, to facilitate cloning of the amplified DNA. 25 As exemplified herein, suitable primers for the amplification of AMT domain-containing products for detection of potentially hepatotoxic cyanobacteria may comprise nucleotide sequences as set forth in SEQ ID Nos: 1 to 4. Suitable primer pairs for PCR amplification may comprise nucleotide sequences as set forth in SEQ ID No: 1 and SEQ ID No:2, SEQ ID No: 1 and SEQ ID No:4, or SEQ ID No: 3 and SEQ ID No:4. However those skilled in the art will appreciate that the 30 present invention is not limited to the use of the specific primers exemplified, but alternative primer sequences may also be used, provided the primers are designed appropriately so as to enable the amplification of AMT sequences from hepatoxic cyanobacteria. For example, in alternative embodiments, the nucleotide sequence of a primer may share at least 85%, at least 90%, or at least 95%, 96%, 97%, 98% or 99% identity with the sequence as set forth in SEQ ID Nos:1 to 4.. 3s Those skilled in the art will appreciate that one or base substitutions, additions or deletions of the WO 2006/128230 PCT/AU2006/000730 sequence(s) of these sequences may be made in generating a sequence of at least 85%, at least 90%, or at least 95%, 96%, 97%, 98% or 99% identity. Further, the location of suitable primers for the amplification of AMT sequences may be determined by such factors as G+C content and the ability for a sequence to form unwanted 5 secondary structures. Suitable primer sequences can be determined by those skilled in the art using routine procedures without undue experimentation. Suitable methods of analysis of the amplified DNA are well known to those skilled in the art, for example, gel electrophoresis which may or may not be preceded by restriction enzyme 10 digestion, and nucleic acid sequencing. Gel electrophoresis may comprise agarose gel electrophoresis or polyacrylamide gel electrophoresis, techniques commonly used by those skilled in the art for separation of DNA fragments on the basis of size, The concentration of agarose or polyacrylamide in the gel in large part determines the resolution ability of the gel and the appropriate concentration of agarose or polyacrylamide will therefore depend on the size of the 15 DNA fragments to be distinguished. Kits for use in detecting toxic cyanobacteria are also provided by the present invention. Typically, kits according to the present invention are designed specifically with components to enable the detection of AMT domain sequences and thereby facilitate the identification of potentially toxic cyanobacteria. 20 Accordingly, kits of the present invention may include one or more oligonucleotide primers that specifically hybridize to AMT domain sequences of cyanobacteria and enabling the detection of hepatotoxic cyanobacteria. In such kits, appropriate amounts of the primers may be provided in suitable containers. The oligonucleotide primers may be provided suspended in an aqueous solution or as a freeze-dried or lyophilized powder, for example. 25 The appropriate sequences of the primers may vary. In one embodiment a kit of the invention comprises a primer pair suitable for the PCR amplification of AMT sequences from cyanobacteria. The primer pair may comprise sequences as set forth in SEQ ID Nos: 1 and 2. The primer pair may comprise sequences as set forth in SEQ ID Nos: 1 and 4. The primer pair may comprise sequences as set forth in SEQ ID Nos: 3 and 4. The amount of each primer supplied in 30 the kit can be any appropriate amount, depending on the nature of the application, and typically may be an amount sufficient to prime at least several amplification reactions. A person skilled in the art would readily appreciate the appropriate amount of each primer to use in a single amplification reaction.

WO 2006/128230 PCT/AU2006/000730 -10 A kit according to the present invention may also include a suitable control template molecule and/or control primers for use in a control reaction. The design of suitable control templates and primers and of control reactions are well known to those skilled in the art. A kit according to the present invention may additionally include other components for 5 performing amplification reactions including, for example, DNA sample preparation reagents, appropriate buffers (e.g. polymerase buffer), salts (e.g. magnesium chloride), polymerase enzyme, and deoxyribonucleotides (dNTPs). The kit may further include the necessary reagents for carrying out analysis of the amplified DNA, such as an appropriate restriction enzyme(s), reaction buffer(s) for restriction enzyme digestion, and reagents for use in separating digested fragments (e.g. 10 agarose) and/or nucleic acid sequencing. Typically, a kit may also include containers for housing the various components and instructions for using the kit components to conduct amplification reactions according to the present invention. The present invention will now be further described in greater detail by reference to the is following specific examples, which should not be construed as in any way limiting the scope of the invention. Examples Example 1- PCR detection of cyclic peptide hepatotoxin genes 20 Oligonucleotide primers were designed to be suitable for the amplification of the aminotransferase (AMT) domain encoded within the mcyE microcystin synthetase and ndaF nodularin synthetase multienzyme complexes. The primer design was based on the four complete microcystin synthetase sequence of Microcystis aeruginosa PCC7806, Microcystis aeruginosa K 139, Anabaena strain 90, and Planktothrix sp. 128/6, as well as the nodularin synthetase gene of 25 Nodularia spumigena NSOR10 (see Figure 1). From these five sequences two conserved sites were identified that enabled the design of specific primers, HEPF and HEPR, as shown in Figure 1 and in SEQ ID Nos: 1 and 2 respectively. Altemate primers, designated HEPF2 and HEPR2 were also designed and synthesized, these primers being complementary to target sequences about 170 bp and 560 bp downstream of the target sequences for HEPR (see Figure 2). As illustrated in 30 Figure 2, PCR amplification was performed using the HEPF/HEPR primer set, the HEPF/HEPR2 primer set and the HEPF2/HEPR2 primer set to determine their ability to screen for cyanobacterial peptide hepatotoxin producers. The specificity of the primers was tested using hepatotoxic cyanobacterial species in culture as well as total bloom material.

WO 2006/128230 PCT/AU2006/000730 -11 17 hepatotoxic cultures were tested (Table 1), belonging to the genera Microcystis, Anabaena, Nostoc, Planktothrix, Oscillatoria, Phormidium, and Nodularia,. The presence of microcystin or nodularin in the investigated strains was known from previous studies. 12 non-toxic cultures were tested, from the genera Microcystis, Anabaena, Nostoc, Nodularia, Lyngbya, s Phormidium, Synechocystis, and Cylindrospermopsis. The dominant bloom-forming genera in these samples were Microcystis, Planktothrix, and Nodularia. Bloom samples screened (Table 2) were collected from Lake Alexandria, John Oldman Park and the Swan River, Australia. The bloom sample from Lake Spino (Italy) was supplied by Milena Bruno (Department of Environmental Hygiene, National Health Institute, Italy). Planktothrix 10 sp. bloom isolate (Lake Ammersee, Germany) and Phormidium sp. isolates were supplied by Daniel Dietrich (University of Konstanz, Germany), and George Izaguirre, (Water Quality Section, Metropolitan Water District of Southern California, USA), respectively. Other cyanobacterial strains were obtained from the School of Biotechnology and Biomolecular Culture collection, University of New South Wales, Australia (Table 1).

WO 2006/128230 PCT/AU2006/000730 -12' - ,-'00 9\ C> C> 0 ~C NN cl 0 CD CO ', 0 / 0 0 c CL (C) o C ) I'D C=> CCdC o 00 C) C) C4 C) C)0I C- t- Ln V) -0 C-4 C 0 CL U o )t CO U CZ ) z caN 't- ~ ~ WO 2006/128230 PCT/AU2006/000730 -13 ca C:, C. Or.N -~~C C d-~C C C4' m U ) -~Cd '0 CIS- Cd Cd w 0 0 I' CC 00 Cao Cao Ca- 4 ' (D U) U ) U C) co C) 2 Z 0~ Lla I~ 6, u IZ + I+ + I i 3 co cnC ;*0 WO 2006/128230 PCT/AU2006/000730 -14 Table 2. Origins of bloom samples screened Blue-Green Algal Location Hepatotoxic Hepatotoxin Reference Bloom (dominant genus) Microcystis sp.* John Oldman + microcystin Neilan (unpublished) Park Lake, Australia Microcystis sp. Swan River, + microcystin Neilan (unpublished) Australia Planktothrix sp. Ammersee, + microcystin Ernst et al 2001 Germany Planktothrix sp.* Lake Spino, + microcystin Viaggiu B. (personal Italy communication) Nodularia sp.* Lake + nodularin Moffitt & Neilan 2001 Alexandria, Australia All bloom samples tested using primer pair HEPF/HEPR. Only those indicated (*) were tested using primer pair HEPFIHEPR2 or HEPF2/HEPR2, 5 Total genomic DNA of strains and bloom material was extracted using XS buffer (Neilan et al, 2002). Approximately 200 mg of cyanobacterial cells was used for each extraction. The cells were combined with 600 pl XS extraction buffer (1% potassium-methylxanthogenate; 800 mM ammonium acetate; 20 mM EDTA; 1% SDS; 100 mM Tris-HCI, pH7.4). The mixture was vortex 10 mixed and incubated at 65 0 C for 2 h and the extracts were cooled for 10 min on ice. Cell debris was removed by centrifugation at 12000 x g for 10 min. DNA was precipitated by the addition of 1 volume of isopropanol and 1/10 volume of 4 M ammonium acetate for 15 min at 4*C. The precipitated DNA was pelleted by centrifugation at 12000 x g for 10 min and washed with 70% ethanol. The extracted DNA was resuspended in 100 pl of sterile water, 1s PCR amplification was performed using various combinations of the primers HEPF (5' TTTGGGGTTAACTTTTTTGGGCATAGTC-3') (SEQ ID No:1), HEPR (5' AATTCTTGAGGCTGTAAATCGGGTT-3') (SEQ ID No:2), HEPF2 (5' AGTATGATCTGCGGTAAAGCAGATTTCT-3') (SEQ ID No:3) and HEPR2 (5' AAACAAACTCGTTTTTCCCATGT-3') (SEQ ID No:4), as outlined above (see Figure 2). 20 All PCR reactions were performed using 0.2 unit Taq polymerase (Fischer Biotech, Perth, Australia) in a 20 pl reaction mix containing 2.5 mM MgCl2, 1x Taq-polymerase buffer (Fischer Biotech), 0.2 mM dNTPs (Fischer Biotech), 0.5 pmol of forward and reverse primer. PCR was WO 2006/128230 PCT/AU2006/000730 -15 performed using 1 pl of template DNA at a concentration of approximately 100 ng plH. Thermal cycling was in a GeneAmp PCR System 2400 thermocycler (Perkin Elmer, Norwalk, CT). The annealing temperature used was 52"C for the HEPF/HEPR primers, 570C for HEPF/HEPR2 and 530C for HEPF2/HEPR2. An initial denaturation step at 920C for 2 min was followed by 35 cycles 5 of 920C for 20 sec, 52*C/57"Ci53"C (as appropriate) for 30 sec, and 720C for 1 min, with a final extension step at 72*C for 5 min. PCR products were analyzed on 1% or 2% agarose gels with 1x TAE-buffer. The PCR products were stained with ethidium bromide (1 pg/ml) for 10 min. For photo documentation a Gel-DOC Bio-RAD System with Quantity One 4.1 R software (BIO-RAD, USA) was used. 10 Sequencing was performed using HEPF, HEPR and HEPF2 primers for each amplification product. Automated sequencing was performed using the PRISM Big Dye cycle sequencing system and ABI 3730 Capillary Applied Biosystem (Foster City, California). The identities of the sequenced amplicons were determined using a BLAST search on GenBank. A fragment of approximately 472 bp was amplified by PCR using the HEPF/HEPR primer 15 set from DNA of all hepatotoxic strains belonging to Microcystis, Anabaena, Nostoc, Planktothrix, Phormidium, and Nodularia, whereas no PCR product was obtained from any of the non-toxic strains belonging to Microcystis, Anabaena, Nostoc, Phormidium, Nodularia, Lyngbya, Synechocystis, and Cylindrospermopsis (data not shown). Thus the PCR results are completely consistent with the designation, as toxic or non-toxic, of all of the species listed in Table 1. 20 All PCR products were sequenced to confirm the identity of the amplified fragments. Sequence analysis of the amplicons revealed that they represented the expected AMT gene fragment from orthologs of either mcyE or ndaF. The HEPFIHEPR PCR also successfully amplified the 472 bp fragment from all hepatotoxic cyanobacterial bloom samples (see Figure 3). These fragments were also sequenced and compared with the sequence data from the cultured 25 cyanobacteria. Further, a fragment of approximately 1000 bp was amplified by PCR using the primer-set HEPF/HEPR2 and about 400 bp using the primer-set HEPF2/HEPR2 from DNA of hepatotoxic strains belonging to Microcystis, Anabaena, Nostoc, Planktothrix, Phormidium, and Nodularia, whereas no PCR product was obtained from of the non-toxic strain Microcystis sp. HUB 53 (see 30 Table 3). These two primer pairs also successfully amplified the fragments from the hepatotoxic cyanobacterial bloom samples originating from John Oldman Park Lake, Lake Spino and Lake Alexandria (see Table 2), consistent with the results obtained using the HEPF/HEPR primer set (data not shown). The PCR products obtained from the strain M. aeruginosa PCC7806 using the HEPF/HEPR2 and HEPF2/HEPR2 primer sets were sequenced to confirm the identity of the 35 amplified fragments. Sequence analysis of the amplicons revealed that they represented the WO 2006/128230 PCT/AU2006/000730 -16 expected AMT gene fragment from mcyE (M. aeruginosa PCC7806, accession number: AAF00958). Table 3. Cyanobacterial isolates screened using HEPFIHEPR2 and HEPF2/HEPR2 Species Strain Hepatotoxic HEPFI HEPF2/ Hepatoxin HEPR2 HEPR2 Chroococcales Microcystis aeruginosa PCC7806 + + + microcystin Microcystis aeruginosa HUB53 Nostocales Anabaena sp. strain 202A2 + + + microcystin Anabaena sp. strain 90 + + + microcystin Nodularia spumigena NROS1O + + + nodularin Nostoc sp. strain 152 + + + microcystin Oscillatoriales Oscillatoria sp. strain 18R + + + microcystin Oscillatoria sp. strain 195 + + + microcystin Phormidium sp. isolate 2-26b + + + microcystin Phormidium sp. isolate 1-6c + + + microcystin Phormidium sp. isolate 4-19b + + + microcystin 5 Example 2- Evolution of cyclic peptide hepatotoxin genes Sequences from the cultures were aligned and phylogenetically analyzed according to their translated amino acid sequences. The glutamate-1-semialdehyde aminotransferase from Aquifex sp. (GenBank Accession No. AE000709) was chosen as the outgroup. Sequences obtained from 10 total bloom DNA was also included in the analysis when the sequences were considered high quality such as the samples Swan River (Australia), Lake Alexandria (Australia), Lake John Oldman Park (Australia), and Lake Spino (Italy). Sequence data was analyzed using the Applied Biosystem Auto-Assembler computer program. The sequences were compared in a multiple alignment using Clustal x (1.8) and the 15 PAM-Dayhoff matrix. Phylogenetic trees were obtained using the Neighbor-Joining methods and confidence levels were calculated via bootstrapping using a resampling number of 1000. Reference sequences were obtained from GenBank (NCBI).

WO 2006/128230 PCT/AU2006/000730 -17 For phylogenetic comparison, the amplified AMT regions from cyanobacterial cultures as well as total bloom material clustered into distinctively different clusters, A and B (Figure 3). Cluster A comprised strains of the genera Anabaena, Nostoc, and Nodularia belonging to the order Nostocales, while cluster B contained the genera Oscillatoria/Planktothrix and Phormidium, as well 5 as the hepatotoxic Microcystis strains. The sequence data obtained from the Microcystis bloom samples grouped with the Microcystis strains and the Nodularia bloom material aligned with N. spumigena NSOR10. Sequence data for the McyE AMT from the different Oscillatoria/Planktothrix strains was identical, The same was observed for the three Phormidium isolates, however the McyE data for the Microcystis, Anabaena, and the NdaF sequence data of Nodularia strains were 10 not 100% similar. Phylogenetic analysis indicated that NdaF evolved from McyE. Further, the AMT of the nodularin synthetase showed the closest relationship to the AMT from heterocyst-forming microcystin producers and was situated as a sub-branch of AMT of the Nostoc strain 152 (Figure 4). 15 The phylogeny of AMT revealed clustering according to the genera of the investigated species and therefore these results support the hypothesis that microcystin and nodularin are ancestral relics that have been lost in non-toxic strains. This would suggest the co-evolution of microcystin synthetase, nodularin synthetase, and 16S rDNA. 20 References Bolch, C. J. S. et al. 1999. Genetic, morphological, and toxicological variation among globally distributed strains of Nodularia (cyanobacteria). J. Phycol. 35:339-355. Carmichael, W. W. 2001. Health effects of toxin-producing cyanobacteria: The CyanoHABs. Human and ecological risk assessment 7:1393-1407. 25 Christiansen, G., J. et al. 2003. Microcystin biosynthesis in Planktothrix: genes, evolution, and manipulation. J. Bacteriol. 185:564-572. Codd, G, A. et al, 1999. Cyanobacterial toxins, exposure routes and human health. Europ. J. Phycol. 34:405-415. Dempsey, L, C. 1977. The isolation and characterization of Microcystis aeruginosa Kotzing 30 emend. Elenkin 1924 from the Lake Winnebago Pool. M.S. thesis. University of Wisconsin, Oshkosh. Doers, E. P., and D. L. Parker. 1988. Properties of Microcystis aeruginosa and M. flos aquae (Cyanophyta) in culture: taxonomic implications. J. Phycol. 24:502-508.

WO 2006/128230 PCT/AU2006/000730 - 18 Eloff, J. N., and A. J. Van der Westhuizen. 1981. Toxicological studies on Microcystis, p. 343-364. In W. W. Carmichael (ed.), The Water Environment - Algal Toxins and Health. Plenum, Press, New York. Ernst et al. 2001. Presence of Planktothrix sp. and cyanobacterial toxins in Lake s Ammersee, Germany and their impact on whitefish (Corregonus lavaretus L.). Environ. Tox. 16:483-488. Hawkins, P. R. et a/. 1997. Isolation and toxicity of Cylindrospermopsis raciborskil from an ornamental lake, Toxicon 35:341-346. Hisbergues, M. et al. 2003. PCR-based identification of microcystin-producing genotypes of 10 different cyanobacterial genera. Arch. Microbiol, 180:402-410. Hitzfeld, B. et al. 2000. Cyanobacterial toxins: Removal during drinking water treatment, and risk assessment. Environ. Health Perspect. 108:113-122. Izaguirre, G. et al. 2004. A benthic Phormidlum species that produces microcystin-LR, isolated from three reservoirs in Southern California. 6th International Conference on Toxic is Cyanobacteria. Kurmayer, R. et al. 2004. Abundance of active and inactive microcystin genotypes in populations of the toxic cyanobacterium Planktothrix spp. Environ. Microbiol. 6:831-841. Kurmayer, R., and T. Krutzenberger. 2003. Application of real-time PCR for quantification of microcystin genotypes in a population of the toxic cyanobacterium Microcystis sp. App. Environ. 20 Microbiol. 69:6723-6730, Lehtimski, J. et al. 1994. The effects of incubation time, temperature, light, salinity, and phosphorus on growth and hepatotoxin production by Nodularia strains. Arch, Hydrobiol. 130:269 282. Lyra, C. et a!. 2001. Molecular characterization of planktonic cyanobacteria of Anabaena, 25 Aphanizomenon, Microcystis and Planktothrix genera. Int. J. Syst. Evol. Microbiol. 51:513-526. Mikalsen, B. et al. 2003. Natural variation in the microcystin synthetase operon mcyABC and impact on microcystin production in Microcystis strains. J. Bacteriol. 185:2774-2785. Moffitt, C. M., and B. A. Neilan 2004. Characterization of the nodularin synthetase gene cluster and proposed evolution of cyanobacterial hepatotoxins. Appi. Environ. Microbiol. 70:6353 30 6362. Moffitt, M. C., and B. A. Neilan 2001. On the presence of peptide synthetase and polyketide synthetase genes in the cyanobacterial genus Nodularia. FEMS Microbiol. Lett. 196:207-214. Neilan, B. A. 1995. Identification and phylogenetic analysis of toxigenic cyanobacteria by multiplex randomly amplified polymorphic DNA PCR. App. Environ. Microbiol. 61:2286-2291.

WO 2006/128230 PCT/AU2006/000730 -19 Neilan, B. A. et al. 1999. Nonribosomal peptide synthesis and toxigenicity of cyanobacteria. J. Bacteriol 181:4089-97. Neilan, B. A.. et al. 2002. Molecular identification of cyanobacteria associated with stromatolites from distinct geographical locations. Astrobiol. 2:271-280. 5 Nishizawa, T. et al. 1999. Genetic analysis of the peptide synthetase genes for a cyclic heptapeptide microcystin in Microcystis spp. J. Biochem. 126:520-529. Rippka, R., and M. Herdman, 1992. Pasteur Culture Collection (PCC) of cyanobacterial strains in axenic culture, vol.1, catalogue of strains, Paris, France; Institut Pasteur. Rouhiainen, L. et al. 2004. Genes coding for hepatotoxic heptapeptides (microcystins) in 1o the cyanobacterium Anabaena strain 90. Apple. Environ. Microbiol. 70:686-692. Starr, R. C., and J. A. Zeikus. 1993. UTEX-the Culture Collection of Algae at The University of Texas at Austin. J. Phycol. 29 (Suppl.):1-106. Tillet, D. et al. 2000. Structural organization of microcystin biosynthesis in Microcystis aeruginosa PCC7806: an integrated peptide-polyketide synthetase system. Chem. Biol. 7:753-764. 15 Vaitomaa, J. et al. 2003. Quantitative real-time PCR for determination of microcystin synthetase E copy numbers for Microcystis and Anabaena in lakes, Appl. Environ. Microbiol. 69:7289-7297.

Claims (20)

1. A method for the detection of toxic cyanobacteria, the method comprising the steps of: (a) obtaining a cyanobacterial sample; and 5 (b) analyzing the sample for the presence of hepatotoxin-associated aminotransferase domain sequences, wherein the presence of hepatotoxin-associated aminotransferase domain sequences are indicative of toxic cyanobacteria,

2. The method of claim 1 wherein the hepatotoxin-associated aminotransferase to domain sequences are derived from the mcyE open reading frame of the microcystin synthetase gene complex or an orthologue or homologue thereof and/or the ndaF open reading frame of the nodularin synthetase gene complex or an orthologue or homologue thereof.

3. A method for the detection of hepatotoxin-producing cyanobacteria, the is method comprising the steps of: (a) obtaining a cyanobacterial sample; and (b) analyzing the sample for the presence of hepatotoxin-associated aminotransferase domain sequences, wherein the presence of hepatotoxin-associated aminotransferase domain sequences 20 are indicative of hepatotoxin-producing cyanobacteria.

4. The method of claim 3 wherein the hepatotoxin is microcystin or nodularin.

5. The method of claim 3 or 4 wherein the hepatotoxin-associated aminotransferase domain sequences are derived from the mcyE open reading frame of the microcystin synthetase gene complex or an orthologue or homologue thereof and/or the 25 ndaF open reading frame of the nodularin synthetase gene complex or an orthologue or homologue thereof,

6. The method of any one of claims I to 5 wherein the analysis step (b) comprises: (i) amplification of DNA from the sample using suitable primers; and 30 (ii) detection of amplified sequences.

7. The method of claim 6 wherein the primers are oligonucleotide primers comprising the nucleotide sequences as set forth in SEQ ID Nos: I and 2.

8. The method of claim 6 wherein the primers are oligonucleotide primers comprising the nucleotide sequences as set forth in SEQ ID Nos: I and 4. 21

9. The method of claim 6 wherein the primers are oligonucleotide primers comprising the nucleotide sequences as set forth in SEQ ID Nos: 3 and 4.

10. The method of any one of claims 6 to 9 wherein the detection of the amplified sequences comprises gel electrophoresis and/or nucleic acid sequencing. 5

11. The method of any one of claims I to 10 wherein the cyanobacterial sample comprises one or more isolated or cultured cyanobacterial organisms.

12. The method of any one of claims I to 10 wherein the cyanobacterial sample comprises an environmental sample containing one or more cyanobacterial organisms.

13. The method of claim 12 wherein the environmental sample is a sample of salt 10 water or freshwater.

14. The method of claim 12 wherein the environmental sample is a sample from a blue-green algal bloom.

15. A kit for the detection of toxic cyanobacteria, the kit comprising at least one primer designed to detect hepatotoxin-associated aminotransferase domain sequences. is

16. The kit of claim 15 comprising multiple primers designed to amplify the hepatotoxin-associated aminotransferase domain sequences.

17. The kit of claim 16 wherein the primers are oligonucleotide primers comprising the nucleotide sequences as set forth in SEQ ID Nos: I and 2, SEQ ID Nos: 1 and 4, or SEQ ID Nos: 3 and 4. 20

18. A kit for the detection of hepatotoxin-producing cyanobacteria, the kit comprising at least one primer designed to detect hepatotoxin-associated aminotransferase domain sequences.

19. The kit of claim 18 comprising multiple primers designed to amplify the hepatotoxin-associated aminotransferase domain sequences. 25

20. The kit of claim 19 wherein the primers are oligonucleotide primers comprising the nucleotide sequences as set forth in SEQ ID Nos: I and 2, SEQ ID Nos: I and 4, or SEQ ID Nos: 3 and 4. Dated 14 November, 2011 NewSouth Innovations Pty Limited Patent Attorneys for the Applicant/Nominated Person SPRUSON & FERGUSON