The hepatotoxin cylindrospermopsin is produced by several cyanobacteria species, which may flourish in tropical and sub-tropical lakes. Biosynthesis of cylindrospermopsin is poorly understood but its chemical nature, and feeding experiments with stable isotopes, suggested that guanidinoacetic acid is the starter unit and indicated involvement of a polyketide synthase. We have identified a gene encoding an amidinotransferase from the cylindrospermopsin producing cyanobacterium Aphanizomenon ovalisporum. This is the first report on an amidinotransferase gene in cyanobacteria. It is likely to be involved in the formation of guanidinoacetic acid. The aoaA is located in a genomic region bearing genes encoding a polyketide synthase and a peptide synthetase, further supporting its putative role in cylindrospermopsin biosynthesis.
Cylindrospermopsin is a water-soluble hepatotoxin. It is produced by several cyanobacteria species including Aphanizomenon ovalisporum, Cylindrospermopsis raciborskii, Anabaena bergii and Umesakia natans[1,2]. These cyanobacteria may flourish in tropical and sub-tropical regions around the world and therefore, their blooms constitute a health hazard. The role of specific residues essential for cylindrospermopsin toxicity is emerging . Apart from cylindrospermopsin toxicity primarily to the liver, kidney and other organs, carcinogenic activity due to the presence of the uracil and sulfinated guanidino moieties was recently suggested [4,5].
The mechanism of cylindrospermopsin biosynthesis was partly resolved by following cylindrospermopsin production in C. raciborskii cultures that were supplemented with stable isotopes of 13C, 18O, 15N and 3H . The feeding experiments suggested that five molecules of acetate are catalytically condensed by a polyketide synthase (PKS) to form the carbon skeleton of cylindrospermopsin. Two other carbons and one of the nitrogens of the guanidine moiety are donated by glycine. Guanidinoacetic acid was proposed as a source for two nitrogen atoms of the guanidino moiety and as a starter unit that initiates the biosynthetic process. The guanidinoacetic acid is probably formed by amidination of glycine, but the exact origin of the other N atoms in the guanidino moiety is not yet resolved . Formation of guanidinoacetic acid may be catalyzed by amidinotransferase. The latter transfers the amidino group from l-arginine to glycine during creatine biosynthesis in vertebrates; to inosamine phosphate for the formation of streptomycin by Streptomyces griseus; and to lysine during phaseolotoxin production in Pseudomonas syringae[7–9]. Another component of the cylindrospermopsin structure, the polyketide, is presumably catalyzed by a PKS. This is a family of multienzyme complexes which contain all the enzymatic activities required for the sequential decarboxylative condensation of short acyl units, analogous to the chain elongation step of fatty acid biosynthesis. The PKS contains all the following active sites: β-keto acyl synthase (KS), acyl transferase (AT), acyl carrier protein, dehydrogenases (DH) and the ‘swinging arms’ of the phosphopantetheine binding domain (PPB) . These enzymes, responsible for the formation of polyketides, are involved in the biosynthesis of a broad family of compounds including different antibiotics and cyanobacterial toxins [1,11].
In this study, we have identified a novel gene, designated aoaA, encoding amidinotransferase in the cylindrospermopsin producing cyanobacterium A. ovalisporum. The aoaA was found within a genomic region encoding PKS and peptide synthetase, indicating its potential role in cylindrospermopsin biosynthesis.
2Materials and methods
A toxic strain of A. ovalisporum was isolated from Lake Kinneret . In this study, we first obtained an axenic culture using the methods developed by Prof. P. Wolk . The axenic cultures were grown on a shaker (100 rpm) in 500-ml flasks containing medium BG11  supplemented with 20 mM TAPS–NaOH buffer, pH 9 and 10 mM NaHCO3. Light intensity was 25 μmol photons m−2 s−1, provided by cool white fluorescent lamps, temperature was 30°C.
The cells were collected by centrifugation, washed with TE and resuspended in 300 μl lysozyme buffer (20 mM EDTA, 150 mM NaCl, 15 mg lysozyme) at 37°C, for 3 h, followed by addition of lysis buffer (100 mM NaCl, 500 mM Tris–HCl, 5% SDS). After three freeze–thaw cycles, proteinase K was provided (final concentration was 100 μg ml−1) and incubation continued for 30 min at 37°C. The DNA was then extracted with phenol, phenol:chloroform and chloroform and subsequently precipitated by the addition of 2.5 M Na-acetate and an equal volume of cold isopropanol at −20°C for 30 min. After centrifugation the DNA was washed with cold 70% ethanol, dried and resuspended in sterile water. Further DNA techniques were carried out according to standard procedures .
2.3Preparation of a DNA library from A. ovalisporum
A genomic DNA library was raised by cloning size-fractionated (7–10 kb), partially Sau3A digested DNA, within a dephosphorylated (SAP, Boehringer-Mannheim) BamHI site of plasmid pUC118. Transformation to Epicurian Escherichia coli XL10-Gold ultracompetent cells (Stratagene), according to the manufacturer's instructions and selection on LB plates with 50 μg ml−1 carbamecylin. The plasmids were extracted by a Miniprep kit (Qiagen) according to the manufacturer's instructions.
2.4Identification and cloning of a keto acyl synthase (KS)
A genomic DNA fragment bearing conserved region of KS was amplified by polymerase chain reaction (PCR) using the following degenerate primers:
5′-GGRTCNCCIARYTGIGTICCIGTICCRTGIGC-3′ (forward) and
5′-MGIGARGCIYTIGCIATGGAYCCICARCARMG-3′ (reverse) . The PCR protocol was: 94°C 5 min, 40 cycles of (94°C 1 min, 57°C 1.5 min, 72°C 1 min), 72°C 5 min, 10°C (hold). A fragment of 650 bp was synthesized, cloned and sequenced and showed high similarity to KS in a BLAST search (see Fig. 1). The KS fragment was labeled with [32P]dCTP by random priming and used as probe to screen the genomic library of A. ovalisporum by colony hybridization. Three colonies were selected for further analysis including subcloning, purification of DNA and plasmids  and sequencing and mapping. Additionally, the KS fragment and the amidinotransferase identified here were used as probes for Southern hybridization, using a non-radioactive probe (NEN) according to the manufacturer's instructions, at 65°C.
Sequence analysis was performed using the ABI 377 Prism DNA sequencer. Putative amino acid sequences encoded by the open reading frames found here were compared with those in the databases through the NCBI website (http://www.ncbi.nlm.nih.gov/blast/Blast). Multiple alignments were performed with the aid of the program CLUSTALW.
3Results and discussion
The polyketide nature of cylindrospermopsin and the feeding experiments  suggested that a PKS of either type I or II is involved in its assembly. A 650-bp fragment was amplified from the genomic DNA of A. ovalisporum using degenerate primers designed according to a conserved region of KS. The sequence of this fragment showed high homology to the KS regions within PKSs in various bacteria and fungi (Fig. 1). Screening of the genomic library of A. ovalisporum by colony hybridization, using the 650-bp fragment as a probe, revealed several different positive colonies. Three of them, designated j2b, j1a and j1c, were selected for further analysis.
Sequence analysis of clone j2b (included in accession number AF395828) revealed a 1177-bp fragment highly homologous to genes encoding amidinotransferases in various organisms (Fig. 2). This gene was designated aoaA for Aphanizomenon ovalisporumamidinotransferase A. This is the first report on the presence of an amidinotransferase encoding gene in cyanobacteria. We predict that it is also present in other cylindrospermopsin producing species.
There are two structurally well characterized amidinotransferases: the prokaryotic one from S. griseus, involved in the formation of streptomycin, and the eukaryotic type involved in the creatine biosynthetic pathway [7,8]. In these enzymes, structural differences were correlated with variations in the amidino acceptor, inosamine phosphate or glycine in the prokaryotic and eukaryotic forms, respectively. The structure of amidinotransferase and the nature of the amidino acceptor in A. ovalisporum are not yet resolved, but sequence comparisons showed higher similarity of this prokaryotic-originated gene to the eukaryotic type.
Genomic DNA was hybridized sequentially to two probes, the 650-bp fragment bearing part of the KS and a 492-bp fragment containing part of aoaA (from position −70 to 422 in the coding region), using the same membrane (Fig. 3). The Southern analysis showed that the genomic region bearing conserved KS motifs extends to about 35 kb, as revealed from the sum of the fragments in the HindIII digest (Fig. 3A). Both probes hybridized to a 12-kb fragment (Fig. 3B) suggesting that the aoaA might be located within the PKS region. This was confirmed by sequence analysis. The Southern analyses also showed a 8-kb HindIII fragment which hybridized with both aoaA and the conserved KS region for a reason not yet known.
In prokaryotes, genes involved in the same physiological process or biochemical pathway are often clustered. Figure 4 shows that genes encoding amidinotransferase, PKS and peptide synthetase are clustered in the same genomic region in A. ovalisporum, as revealed by sequence analysis (accession number AF395828). The conserved regions, indicated in Fig. 4, were identified on the basis of very high homology to identical motifs in other genes. Conserved domains found here include: KS, AT and DH. The aoaA, encoding the amidinotransferase, is located upstream of the PKS, on the reverse strand. The PKS found here bear several active sites within one reading frame, a characteristic of type I PKS. These are large multidomain proteins that carry all the active sites required for polyketide synthesis and are encoded by a single open reading frame. In type II, the active sites are distributed among several smaller polypeptides that are transcribed by different sequential genes . Recently, partial PKS sequences from C. raciborskii and A. bergii were reported . Downstream of aoaA, we found a peptide synthetase fused to a PKS bearing motif, encoding an AMP-binding domain (AMPB), the PPB, two KS domains and an AT domain. Some of these domains were also detected in genes encoding other cyanobacterial toxins such as the mcy genomic region encoding various types of microcystins in Microcystis[2,17–19].
This study identified genes encoding amidinotransferase, PKS and peptide synthetase in A. ovalisporum, all essential for cylindrospermopsin biosynthesis. Based on sequence homology and clustering of these genes at the same genomic region, it is likely that they participate in cylindrospermopsin formation. However, unequivocal evidence that this is indeed the case would have to rest on further analysis including gene inactivation (in progress).
We thank Prof. P. Wolk for the isolation of axenic cultures of A. ovalisporum. This study was supported by Infrastructure Grant 1319-1-98 from the Israel Ministry of Science and Arts and by the Israel Science Foundation (ISF).