Genetic identification of microcystin ecotypes in toxic cyanobacteria of the genus Planktothrix

Rainer Kurmayer1, Guntram Christiansen2, Marlies Gumpenberger1 and Jutta Fastner3

1 Austrian Academy of Sciences, Institute for Limnology, Mondseestraße 9, A-5310 Mondsee, Austria
2 University of Hawaii at Manoa, Department of Chemistry, 2545 McCarthy Hall, Honolulu, HI 96822, USA
3 Federal Environmental Agency, Corrensplatz 1, D-14195 Berlin, Germany

Correspondence
Rainer Kurmayer
rainer.kurmayer{at}oeaw.ac.at


   ABSTRACT
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Microcystins (MCs) are toxic heptapeptides which are synthesized by the filamentous cyanobacterium Planktothrix and other genera via non-ribosomal peptide synthesis. MCs share the common structure cyclo(-D-ala1-L-x2-D-erythro-{beta}-iso-aspartic acid3-L-z4-adda5-D-Glu6-N-methyl-dehydroalanine7) [Adda; (2S, 3S, 8S, 9S)-3-amino-9-methoxy-2,6,8-trimethyl-10-phenyldeca-4,6-dienoic acid], in which numerous MC variants have been reported. In general, the variation in structure is due to different amino acid residues in positions 7, 2 and 4 within the MC molecule, which are thought to be activated by the adenylation domains mcyAAd1, mcyBAd1 and mcyCAd, respectively. It was the aim of the study (i) to identify MC ecotypes that differed in the production of specific MC variants and (ii) to correlate the genetic variation within adenylation domains with the observed MC variants among 17 Planktothrix strains. Comparison of the sequences of mcyAAd1 revealed two distinctive Ad-genotypes differing in base pair composition and the insertion of an N-methyl transferase (NMT) domain. The mcyAAd1 genotype with NMT (2854 bp) correlated with N-methyl-dehydroalanine and the mcyAAd1 genotype without NMT (1692 bp) correlated with dehydrobutyrine in position 7. Within mcyBAd1, a lower genetic variation (0–4 %) and an exclusive correlation between one Ad-genotype and homotyrosine as well as another Ad-genotype and arginine in position 2 was found. The sequences of mcyCAd were found to be highly similar (0–1 % dissimilarity) and all strains contained arginine in position 4. The results on adenylation domain polymorphism do provide insights into the evolutionary origin of adenylation domains in Planktothrix and may be combined with ecological research in order to provide clues about the abundance of genetically defined MC ecotypes in nature.


Abbreviations: Adda, (2S, 3S, 8S, 9S)-3-amino-9-methoxy-2,6,8-trimethyl-10-phenyldeca-4,6-dienoic acid; Dhb, dehydrobutyrine (2-amino-2-butenoic acid); MC, microcystin; Mdha, N-methyl-dehydroalanine; NMT, N-methyl transferase

The GenBank/EMBL/DDBJ accession numbers for the sequences reported in this paper are AJ749248–AJ749302 and AJ863131–AJ863134, as indicated in Table 1.


   INTRODUCTION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Planktothrix is considered to be an important genus of harmful cyanobacteria as its members are regularly found to produce the hepatotoxin microcystin (MC). During a field survey, all samples dominated by either the red-pigmented Planktothrix rubescens or the green-pigmented Planktothrix agardhii were shown to contain MCs (Fastner et al., 1999). Both species are known to have a specific ecological niche in lakes of the temperate zone of the Northern hemisphere and are highly efficient in monopolizing resources and frequently dominate the phytoplankton community (Mur et al., 1999; Scheffer et al., 1997). Generally, red-pigmented, phycoerythrin-rich genotypes assigned to P. rubescens occur in deep, stratified and oligo- to mesotrophic waters, where they can build up metalimnetic layers. Green-pigmented, phycocyanin-rich genotypes frequently assigned to P. agardhii have a broader distribution and inhabit shallower, polymictic and mesotrophic to hypertrophic water bodies (Oliver & Ganf, 2000).

MCs are cyclic heptapeptides and share the common structure cyclo(-D-ala1-L-x2-D-measp3-L-z4-adda5-D-Glu6-Mdha7), where X and Z are variable L-amino acids (e.g. LR refers to leucine and arginine in the variable positions), D-measp is D-erythro-{beta}-iso-aspartic acid, Adda is (2S, 3S, 8S, 9S)-3-amino-9-methoxy-2,6,8-trimethyl-10-phenyldeca-4,6-dienoic acid and Mdha is N-methyl-dehydroalanine (Carmichael et al., 1988). MCs are synthesized, like other non-ribosomal peptides produced by bacteria and fungi, by the thiotemplate mechanism (Marahiel et al., 1997). The large enzyme complex encoded by the mcy gene cluster is composed of peptide synthetases, polyketide synthases and tailoring enzymes (Christiansen et al., 2003; Rouhiainen et al., 2004; Tillett et al., 2000). It has a modular structure, each module containing specific functional domains for activation, aminoacyl adenylation (Ad; adenylation domains) and thioesterification (thiolation domains) of the amino acid substrate and for the elongation (condensation domains) of the growing peptide (Tillett et al., 2000).

The structural organization of MC biosynthesis has been elucidated and it has been postulated that McyA, McyB and McyC are responsible for the collinear activation and incorporation of Mdha7, D-Ala1, L-X2, D-measp3 and L-Z4 during biosynthesis (Tillett et al., 2000). The first adenylation domain of McyA (mcyAAd1) is expected to activate amino acids occurring in the variable position 7, where three different residues [dehydroalanine, dehydrobutyrine (Dhb) and serine] have been reported from Planktothrix strains (Luukkainen et al., 1993; Sano & Kaya, 1995). mcyBAd1 is responsible for the activation of residues in position 2, where three different amino acids [leucine, arginine and homotyrosine (Hty); Sivonen & Jones, 1999] have been described in Planktothrix strains. The adenylation domain of McyC (mcyCAd) is correlated with the activation of amino acids in position 4, where only one amino acid (arginine) has been reported in Planktothrix strains (Sivonen & Jones, 1999). These reports are in agreement with field observations documenting that the most abundant variants are [Asp3]-variants of MC-LR, MC-RR and MC-HtyR (Henriksen & Moestrup, 1997; Fastner et al., 1999).

The extent to which the diversity of MC variants is genetically determined is not yet fully understood. Based on the gramicidin synthetase GrsA crystal structure from Brevibacillus brevis, the region forming the amino-acid-binding pocket of adenylation domains has been defined within the core motifs A3 to A6 and the role of critical side chains during substrate recognition in the adenylation domains has been demonstrated (Conti et al., 1997). Using in silico analyses, eight specific critical amino acids (signature sequences) have been correlated with amino acid substrates and the so-called specificity-conferring code of adenylation domains could be defined (Stachelhaus et al., 1999; Challis et al., 2000). Point mutational investigations of a few critical amino acids of the adenylation domain of the peptide synthetase (GrsA) demonstrated a change of substrate specificity accompanied by losses in activity (Stachelhaus et al., 1999). Another approach included the investigation of variations found in the mcy gene cluster and correlating this to the structural MC variants produced by natural strains (Kurmayer et al., 2002; Mikalsen et al., 2003). Those studies revealed considerable genetic variation within the mcyBAd1 gene of the cyanobacterium Microcystis sp. that have been linked to recombination events (Mikalsen et al., 2003), which have recently also been documented for mcyA (Tanabe et al., 2004). Some genetic variants were suggested to correlate with the production of MC variants, i.e. the mcyB (C) genetic variant correlated with the production of MC-RR and its derivatives (Mikalsen et al., 2003). However, analyses of more strains are needed in order to assess the contribution of genetic recombination events to the production of specific MC variants. It was the aim of this study to investigate whether specific MC variants are correlated with different adenylation domain (Ad) genotypes in cyanobacteria of the genus Planktothrix. This was done by aligning the translated amino acid sequences of mcyAAd1, mcyBAd1 and mcyCAd isolated from 17 Planktothrix sp. strains from different lakes and correlating these data with the MC variants synthesized by the strains. If a relationship between Ad-genotypes within the mcyABC cluster and the occurrence of MC variants can be found, this knowledge could be used to identify specific MC ecotypes in nature. According to the EMBL nucleotide sequence database, an ecotype is defined as a distinct population of organisms of a widespread species that has adapted genetically to its own local habitat (Stoesser et al., 2003). This knowledge is important to understand the wax and wane of specific mcy genotypes and the evolution of MC synthesis in our water bodies.


   METHODS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Source and cultivation of cyanobacteria.
Strains were isolated during a study by Kurmayer et al. (2004); in this study, 17 strains were selected to represent the two major groups of MC variants as described by Kurmayer et al. (2004): group 1, strains with demethylated variants of MC-RR; and group 2, strains with [Asp3]-MC-HtyR as the major variant. In addition, four strains (21/1, CCAP1459/14, CCAP1459/17, CCAP1459/31) assigned to group 2 by Kurmayer et al. (2004) were included for the sequencing of mcyBAd1 only (Table 1). Strains with novel MC variants (group 3) isolated selectively from Lake Schwarzensee (Upper Austria) will be described elsewhere (R. Kurmayer, K. Ishida, J. Fastner and T. Hemscheidt, unpublished results). According to PCR analysis and sequencing of the internal transcribed spacer region of the phycocanin operon (Kurmayer et al., 2004) and sequence information on the 16S rRNA gene provided by Suda et al. (2002), all of the strains of this study were assigned either to P. agardhii (green-pigmented) or P. rubescens (red-pigmented). All strains were cultivated in BG11 (Rippka, 1988) containing 2 mM NaNO3 plus 10 mM NaHCO3 at 15 °C and continuous light (5–10 µmol m–2 s–1; Osram type L30W/77 Fluora).


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Table 1. Planktothrix strains used in this study

Species are abbreviated as Rub (P. rubescens; red-pigmented) and Aga (P. agardhii; green-pigmented). Country codes are given in ISO format (AT, Austria; DE, Germany; DK, Denmark; FI, Finland; JP, Japan; NO, Norway; UK, United Kingdom). 16S rRNA gene sequence accession numbers were taken from Suda et al. (2002) and phycocyanin internal transcribed spacer (PC-ITS) sequence accession numbers from Kurmayer et al. (2004). Groups with different MC variants as identified by Kurmayer et al. (2004) are identified as MC group 1, strains with [Asp3]-MC-RR as the major variant, and group 2, strains with [Asp3]-MC-HtyR as the major variant. ND, No data available.

 
MC analysis.
Strains were grown simultaneously and analysed independently on three separate occasions (3 October 2002, 29 January 2004, 13 April 2004). All strains showed comparable growth and 3 weeks after inoculation cells were filtered on pre-weighed glass fibre filters (GF/C; Comesa), dried at 95 °C overnight and then reweighed to quantify the biovolume for extraction. On the day of harvest, the biovolume was between 0·1 and 0·3 mg dry weight l–1. MCs were extracted using 75 % (w/v) aqueous methanol and the extracts were analysed for MC by HPLC with diode array detection (HPLC-DAD) as described by Kurmayer et al. (2003). MC variants were quantified at 240 nm by their characteristic absorption spectra (original spectrum and first-order derivative) and retention times (Fastner et al., 1999). Fastner et al. (1999) reported that, using the gradient according to Lawton et al. (1994), [Asp3, Mdha7]-MC-RR eluted 0·8 min before [Asp3, Dhb7]-MC-RR (Fastner et al., 1999). In order to test for the sensitivity of detection of [Asp3, Mdha7]-MC-RR in the presence of [Asp3, Dhb7]-MC-RR and vice versa, the proportion of one variant was gradually increased relative to the other during pilot experiments. Results showed that both [Asp3]-MC-RR variants could be linearly detected down to a minimum proportion of <5 %. [MeAsp3, Mdha7]-MC-RR, MC-YR and MC-LR were used as external standards (Calbiochem). The concentrations of MC variants were determined as concentration equivalents of [MeAsp3, Mdha7]-MC-LR. In addition, dried HPLC fractions of putative MCs were dissolved in 20 µl 50 % aqueous methanol, sonicated for 10 min and left for 20 min. From 1 µl of this sample preparation, positive-ion mass spectra from 500 to 2000 Da were recorded using a MALDI-TOF mass spectrometer (Voyager DE-PRO; PerSeptive BioSystems) as described by Erhard et al. (1997). MC variants were identified by post-source decay (PSD) fragment structure analysis (Fastner et al., 1999).

DNA amplification and sequencing.
For DNA extraction, 2 ml culture was incubated for 1 h on ice and centrifuged at 13 000 r.p.m. for 10 min and the pellet was lyophilized in a vacuum centrifuge at 30 °C. DNA was extracted using a protocol described by Kurmayer et al. (2003). For PCR, DNA extracts were diluted 100-fold and 1·0 µl of the sample was pipetted into reaction tubes and incubated as described below. PCR amplifications were performed in a volume of 20 µl, containing 1x Qiagen PCR buffer, 3 mM MgCl2 (Qiagen), 300 µM each dNTP (MBI Fermentas), 0·5 µM each primer, 0·5 units Taq DNA polymerase (Qiagen), 13·1 µl sterile Millipore water and 1·0 µl DNA extract. Primers used for PCR and sequencing are listed in Table 2. For mcyAAd1 (product size 3022 bp), the PCR thermal cycling protocol included an initial denaturation step at 94 °C for 3 min, followed by 35 cycles at 94 °C for 30 s, annealing at 60 °C for 30 s and elongation at 72 °C for 3 min. For mcyBAd1 (product size 1692 bp) the cycling protocol was identical, but annealing was at 52 °C for 30 s and elongation at 72 °C for 2 min. For mcyCAd (product size 1416 bp) the cycling protocol was identical to that for mcyAAd1, but the elongation time was 2 min at 72 °C. PCR products (4 µl of the reaction mixture) were visualized by electrophoresis in 1·0 % agarose in 0·5x TBE with ethidium bromide staining. The amplification products of mcyABC were sequenced directly by standard automated fluorescence techniques (Applied Biosystems). These sequence data have been submitted to the DDBJ/EMBL/GenBank databases under the accession numbers AJ749248–AJ749302 and AJ863131–AJ863134 (see Table 1).


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Table 2. Oligonucleotide primers used for PCR (P) and sequencing (S)

All primers were designed during this study.

 
Sequence alignment and analysis.
Sequences were aligned using multiple sequence alignment (CLUSTAL W 1.8). Similarity values between amino acid sequences and the corresponding adenylation domain of strain CYA126/8 (GenBank accession no. AJ441056) were calculated using the program PROTDIST of the PHYLIP software package [version 3.6(alpha3); Felsenstein, 1993]. These values are the fractions of amino acid positions that are identical between the sequences (without weighting of protein positions).

Phylogenetic trees were constructed by (i) average linkage clustering (UPGMA) from the amino acid distance matrix using the approximation of Kimura (1983) to the Dayhoff PAM matrix using the programs PROTDIST and NEIGHBOR, (ii) the maximum-likelihood method (ML) using the program PROML using the Jones–Taylor–Thornton model of change between amino acids (Jones et al., 1992) and (iii) the maximum-parsimony (MP) method using PROTPARS from the PHYLIP software package. In general, sites were not weighted. The statistical significance of the branches was estimated by bootstrap analysis generating 1000 replicates of the original dataset. Finally, consensus trees following the 50 % majority rule were computed. For all of the genes, phylogenetic trees were congruent and the parsimonious trees and significant bootstrap values for all of the methods are presented.


   RESULTS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
MC variants
Analysis of the residue variation in position 7 revealed that either Mdha (nine strains) or Dhb (six strains) was part of the MCs (Table 3, Fig. 1). Residue variations in position 2 could be split into two groups: the first group exclusively produced one MC variant containing arginine (five strains). The second, larger group contained 10 strains, producing a mixture of MCs carrying arginine as the major variant (>=67 %) and leucine (<33 %) and homotyrosine (<2 %). Two strains (CCAP1460/5, CCAP1459/16) showed no arginine, but homotyrosine (>=69 %) and leucine (<30 %) were major variants. There were no residue variations seen in position 4 (arginine).


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Table 3. Similarity in the amino acid composition of adenylation domains of mcyAAd1, mcyBAd1 and mcyCAd and the production of MC variants measured for 17 strains of P. agardhii and P. rubescens

The presence (+) or absence (–) of the NMT within mcyAAd1 is indicated. Similarity is given in comparison with strain CYA126/8, sequenced for the mcy gene cluster by Christiansen et al. (2003). For a complete list of strains and strain origins, see Table 1 in Kurmayer et al. (2004). MC variants were identified from their retention times in HPLC: [Asp3, Mdha7]-MC-RR, 13·4–13·9 min; [Asp3, Dhb7]-MC-RR, 14·3–14·8 min; [Asp3]-MC-HtyR, 18·3–18·5 min; [Asp3]-MC-LR, 19·3–19·6 min. The MC content is given in equivalents of [MeAsp3, Mdha7]-MC-LR (mean±SEM).

 


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Fig. 1. Structure of [Asp3, Mdha7]-MC-RR and the multienzymes McyA, McyB and McyC believed to be responsible for the collinear activation and incorporation of Mdha7, D-Ala1, L-X2, D-measp3 and L-Z4 during biosynthesis (Tillett et al., 2000). Each rectangle represents a non-ribosomal peptide synthetase enzymic domain: A, aminoacyl adenylation; C, condensation; NMT, N-methyl transferase; Ep, epimerase; TE, thioesterase. Thiolation domains are shown in black. Braces indicate the adenylation domains sequenced from Planktothrix strains (see Table 1) and arrows denote the corresponding amino acids activated during biosynthesis.

 
Genetic variation within the mcyABC cluster
Using the same primers for mcyAAd1, a PCR product of either 3 kb (corresponding to the mcyAAd1 sequence of strain CYA126/8; GenBank accession no. AJ441056) or 1·5 kb was obtained. Most of the reduction in PCR product size was due to the lack of a DNA sequence encoding an N-methyl transferase (NMT, 416 aa). In all strains showing a PCR product of 3 kb (950 aa), the NMT was inserted between the core motifs A8 (KIRGXRIELGEIE) and A9 (LPXYM) defined by Marahiel et al. (1997). Within the remaining mcyAAd1 sequence (534 aa), the two distinctive Ad-genotypes of mcyA also differed significantly in amino acid sequence (44 % dissimilarity when compared to CYA126/8; Table 3). Phylogenetic analyses of mcyAAd1 sequences revealed a separation of Planktothrix strains that was found to be independent of the taxonomic distinction between P. agardhii and P. rubescens. Two main clades were observed: the mcyA (I) clade (lacking NMT) showed the lowest genetic dissimilarity (0–0·2 %), and the mcyA (II) clade (with NMT) showed higher genetic variation (0–3·2 %; Fig. 2). The mcyAAd1 genotype with NMT showed 63 % sequence identity with mcyAAd1 of Microcystis aeruginosa (BAA83992 and 65 % identity with mcyAAd1 of Anabaena sp. 90 (AAO62586. In contrast, the mcyAAd1 genotype without NMT (563 aa) revealed sequence identities (47–66 %) with putative non-ribosomal peptide synthetases from organisms of the cyanobacteria [Nostoc punctiforme PCC73102 Anabaena ATCC 2941, Anabaena sp. 90 (Rouhiainen et al., 2000) and Nodularia spumigena (Moffitt & Neilan, 2004)], the bacillales (Bacillus cereus), the proteobacteria (Pseudomonas spp., Chromobacterium violaceum and Ralstonia solanacearum) and the actinobacteria (Streptomyces avermitilis) (data not shown).



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Fig. 2. Phylogenetic tree based on MP analysis calculated from mcyAAd1 sequences (950 aa) from 17 Planktothrix strains. Significant bootstrap percentages obtained from 1000 replicates are indicated for mcyA clades I, II, III and IV at the top left for MP, UPGMA clustering (UPG) and ML. Bootstrap values for clades with lower support are given for MP only. mcyAAd1 from Microcystis PCC7806 (AAF00960 was used as an outgroup. The suffixes r and a after the strain name indicate the species P. rubescens (r) and P. agardhii (a). The corresponding amino acids in position 7 determined in the MC molecule are given (ND, not determined). Bar, 4 substitutions per 10 amino acids. [Asp3, Dhb7]-MC-HtyR has been isolated from strain CCAP1459/16 (indicated by an asterisk) by Sano & Kaya (1998).

 
Within mcyBAd1, the genetic variation was lower and no polymorphism in sequence length was observed (0–4 %; Table 3, Fig. 3). Phylogenetic analysis of mcyBAd1 sequences revealed the occurrence of three clades (I, II, III), with the mcyB (I) clade consisting of both P. rubescens and P. agardhii, the mcyB (II) clade consisting of P. agardhii and the mcyB (III) clade consisting of P. rubescens only. The mcyB (II) clade (strains CCAP1459/11A and CCAP1459/21) showed the most significant deviation in amino acid sequence (4 %). Genetic variation was lowest within mcyCAd (0–1 %; Table 3), and no consistent branching using any of the three phylogenetic methods was detected (data not shown).



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Fig. 3. Phylogenetic tree based on MP analysis calculated from mcyBAd1 sequences (513 aa) from 21 Planktothrix strains. Bootstrap percentages obtained from 1000 replicates are indicated for mcyB clades I, II, III at the bottom left for MP, UPGMA clustering (UPG) and ML. Bootstrap values for clades with lower support are given for MP only. mcyBAd1 from Microcystis PCC7806 (AAF00961 was used as an outgroup. The suffixes r and a after the strain name indicate the species P. rubescens (r) and P. agardhii (a). The corresponding amino acids in position 2 determined in the MC molecule are given. Bar, 0·3 substitutions per 10 amino acids.

 
In summary, no correlation was found between mcy genotype and taxonomic distinction between P. agardhii and P. rubescens. In addition, no correlation between the mcy genotype distribution and the origin of isolation was found. For example, identical mcyBAd1 genotypes were isolated from habitats with the largest geographical distance observed in this study, i.e. Blelham Tarn, UK (strain CCAP1459/16) and Lake Kasumigaura, Japan (strain CCAP1460/5).

Genetic variation, signature sequences and MC variants
The mcyAAd1 genotype with NMT had a signature sequence DVWHISLI that matched exactly (identity 8/8) with the reference sequence of nostopeptolide synthetase (gb|AAF15891.2|) and the prediction of serine as the amino acid substrate. This prediction correlated perfectly with the presence of Mdha in [D-Asp3, Mdha7]-MC-RR in nine of the nine strains (Table 3, Fig. 2). The mcyAAd1 genotype without NMT had a signature sequence DFWNIGMV that matched exactly (identity 8/8) with the reference sequence of exochelin synthetase (gb|AAC82550.1|), pyoverdine synthetase D (gb|AAB60198.1|), fengycin synthetase (emb|CAA09819.1|, emb|CAA84361.1|) and coelichelin synthetase (gi|5763943), all predicting threonine as the amino acid substrate. This prediction correlated with Dhb occurring in [D-Asp3, Dhb7]-MC-RR (six strains).

Within mcyBAd1, the signature sequences had no clear precedent in the database. The mcyB (I) clade (six strains) was derived from one genotype only and showed the signature sequence DALLFGFV. An exclusive correlation with homotyrosine and leucine, but with no arginine, as major residues in position 2 was recognized (Fig. 3). Notably, all other strains reported to contain homotyrosine and leucine in position 2 as major residues by Kurmayer et al. (2004) (21/1, CCAP1459/14, CCAP1459/17, CCAP1459/31) were found exclusively to have one mcyBAd1 genotype. The mcyB (II) clade (two strains) showed the signature sequence DAWAFGLV and the mcyB (III) clade (three strains) showed the signature sequence DALFFGVV, and both clades correlated exclusively with arginine. The remaining 10 strains were found to be without clear genetic differentiation, showed the signature sequence DALFFGLV and produced a mixture of MCs carrying arginine and leucine as major variants.

Genetic differentiation was lowest within mcyCAd; one signature sequence, DPWGFGLV, without a precedent in the database was found and no variation in position 4 was found (Table 3). In summary, both Ad genotypes of mcyAAd1 were found to be specific for the amino acid composition in position 7. The Ad genotypes of mcyBAd1 were found to be both specific and unspecific, the latter correlating with the activation of two (or three) amino acids during MC biosynthesis.


   DISCUSSION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Genetic variation within adenylation domains and correlation with the synthesis of MC variants
In this study, a significant correlation between mcyAAd1 genotypes with NMT and mcyAAd1 genotypes without NMT and the occurrence of either Mdha or Dhb in position 7 of the MC molecule was found. According to Tillett et al. (2000), mcyAAd1 is responsible for the incorporation of L-serine into the growing molecule. L-Serine is N-methylated by the corresponding NMT domain and transformed by dehydration into Mdha prior to or following the condensation reaction. Analogous to L-serine, L-threonine is transformed by dehydration into Dhb (Rinehart et al., 1994). Whether strains CCAP1460/5 and CCAP1459/16, containing mcyAAd1 without NMT, indeed produced [Asp3, Dhb7]-MC-HtyR was not tested explicitly in this study, but [Asp3, Dhb7]-MC-HtyR has been isolated from strain CCAP1459/16 by Sano & Kaya (1998).

Within mcyBAd1, a significant correlation between mcyB (I) clade and homotyrosine in position 2 was found. Even the strains from Lake Schwarzensee (Kurmayer et al., 2004) were of the same mcyBAd1 genotype and contained homotyrosine as the major residue in position 2 (R. Kurmayer, K. Ishida, J. Fastner and T. Hemscheidt, unpublished results). In addition, two mcyB clades (II, III) correlated with arginine exclusively in position 2. mcyB (III) clade was found in 12 strains (<1·5 % dissimilarity) and all strains contained arginine in position 2 (Gumpenberger, 2004). In summary, the genetic differences in the mcyBAd1 genotypes correlated only partly with the observed differences in the structure of the MC variants. Corresponding to this study, the majority of mcyBAd1 sequences from a number of single colonies from Microcystis sp. did not correlate with differences in MC amino acid composition (Kurmayer et al., 2002). This result corresponds to the observation that Microcystis strain HUB524, with the same mcyBAd1 sequence, produces three different MCs simultaneously containing either leucine, arginine or tyrosine in position 2 (Fastner et al., 1999). Those results indicate the potential of mcyBAd1 to activate a variety of amino acids during MC biosynthesis. The results correspond to the general view that adenylation domains activating hydrophobic amino acids (e.g. mcyBAd1) possess a lower selectivity when compared to adenylation domains activating polar amino acids (e.g. mcyAAd1; Challis et al., 2000).

It has been suggested that the synthesis of specific MC variants in a particular strain depends on the physiological conditions; for instance, Rapala et al. (1997) found an increasing proportion of MC-RR at the expense of MC-LR with increasing temperature. According to the present authors' unpublished measurements, at a higher temperature along with high light conditions (20 °C, 40 µmol m–2 s–1) as opposed to the culture conditions used in this study (15 °C, 5–10 µmol m–2 s–1), unaltered synthesis of either [D-Asp3, Mdha7] or [D-Asp3, Dhb7] variants of MC-RR was observed. In addition, the occurrence of MC-RR only in some of the strains (CCAP1459/11A, CCAP1459/21, 3, 64, 111) was found to be unaltered at higher temperature and high light conditions (20 °C, 40 µmol m–2 s–1; R. Kurmayer, unpublished).

Genetic recombination of the mcyABC cluster
In this study, the mcyAAd1 genotype without NMT (563 aa) revealed extensive sequence identity (47–66 %) with non-ribosomal peptide synthetase genes from other cyanobacterial and bacterial genera. The genetic variation was lowest among strains of this genotype (0–0·2 %), suggesting a relatively recent recombination event. Recombinations involving adenylation domains of the mcy gene cluster in Microcystis have been suggested for mcyB (Mikalsen et al., 2003) and the NMT region of mcyA (Tanabe et al., 2004). Recombinations and deletions involving the condensation domain in ndaA have been reported by Moffitt & Neilan (2004). Recombination therefore seems to be a general feature in mcy genes, and these findings may be important in understanding how new structural variants of MCs are created.

The same mcyAAd1 primers were used to amplify mcyAAd1 genotypes both with and without NMT in Planktothrix spp. Notably, ndaA (AAO64403, which shows 61 % identity to the mcyAAd1 genotype without NMT, consists of not only a threonine adenylation domain but also an NMT domain (Moffitt & Neilan, 2004). So far, an mcyAAd1 threonine adenylation domain with NMT has not been found in Planktothrix strains. It is possible that the NMT has been lost after the transfer of the mcyAAd1 threonine adenylation domain into mcyA of Planktothrix.

Ecological implications
In this study, it could be shown that DNA polymorphisms within specific regions of adenylation domains are associated with the synthesis of specific MC variants. The process by which secondary metabolic pathways evolve is probably a result of modifications and combinations of reactions from existing pathways. Through the process of natural selection, the producer of new structures will only increase in number relative to the producers of the older structures if the production of the new structure is advantageous. Assuming that the observed recombination within mcyAAd1 and the resulting [D-Asp3, Dhb7]-MC-RR ecotype originated from a single DNA recombination event, it must be assumed that natural selection favoured the increase of [D-Asp3, Dhb7]-MC producers relative to [D-Asp3, Mdha7]-MC producers. Notably, the first quantitative results showed that (i) in a few lakes, the mcyAAd1 genotype without NMT dominated, while, in other populations, mcyAAd1 genotypes both with and without NMT were found to co-occur over several years and (ii) the mcyBAd1 genotype producing [Asp3]-MC-HtyR occurred much less frequently in Lake Irrsee (Upper Austria) when compared with Lake Mondsee (Upper Austria; R. Kurmayer, unpublished). In the field, [D-Asp3, Dhb7]-MC-RR has been reported as the dominant variant in P. rubescens (Blom et al., 2001; Fastner et al., 1999), while [D-Asp3, Mdha7]-MC-RR was found to be most abundant in phytoplankton samples dominated by P. agardhii (Fastner et al., 1999). It is speculated that specific environmental conditions may not only influence the absolute abundance of MC-producing genotypes via the dominance of P. rubescens over P. agardhii (e.g. Kurmayer et al., 2004) but may also influence the proportion of specific MC ecotypes. The quantification of MC ecotypes as well as transplantation in their natural context will deliver important clues on the function of MCs in ecosystems.


   ACKNOWLEDGEMENTS
 
Johanna Schmidt provided valuable assistance in the culturing of strains and microcystin analysis in the laboratory. We would also like to thank Martin Meixner for sequencing. We appreciate the comments of two anonymous reviewers. This study was supported by the Austrian Science Fund (P15709) CYTOGENE (Linking CYanTOxin production to GENEtic diversity) and by the EU project PEPCY (QLK4-CT-2002-02634). The European Community is not responsible for any use that might be made of data appearing herein.


   REFERENCES
TOP
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METHODS
RESULTS
DISCUSSION
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Received 23 November 2004; revised 14 February 2005; accepted 14 February 2005.



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