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1 University of Oslo, Department of Molecular Biosciences, 0316 Oslo, Norway
2 University of Oslo, Department of Biology, Centre for Ecological and Evolutionary Synthesis (CEES), 0316 Oslo, Norway
3 University of Oslo, Microbial Evolution Research Group (MERG), 0316 Oslo, Norway
Correspondence
Kjetill S. Jakobsen
kjetill.jakobsen{at}bio.uio.no
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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Present address: Division of Pathology, Rikshospitalet University Hospital, 0027 Oslo, Norway.
The GenBank/EMBL/DDBJ accession numbers for the sequences determined in this work are shown in Table 1.
A supplementary table of primers and three supplementary figures are available with the online version of this paper.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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). Many strains produce several microcystin isoforms, but only one or two isoforms dominate in any single strain.
Microcystins are synthesized nonribosomally by a thiotemplate mechanism catalysed by microcystin synthetase (Marahiel et al., 1997). Microcystin synthetase gene clusters (mcy) include genes for peptide synthetases, polyketide synthases, mixed peptide/polyketide synthases and tailoring enzymes, and have been characterized in detail in Microcystis (Nishizawa et al., 1999, 2000; Tillett et al., 2000), Planktothrix (Christiansen et al., 2003) and Anabaena (Rouhiainen et al., 2004). Insertional gene knockout experiments have demonstrated that all microcystin variants produced by a strain are synthesized by a single enzyme complex encoded by a 55 kb gene cluster (Christiansen et al., 2003; Dittmann et al., 1997; Nishizawa et al., 1999).
The phylogenetic concurrence between a few housekeeping genes and microcystin synthetase genes from the same strains representing a wide selection of microcystin-producing genera suggests that mcy genes represent an ancestral state of many lineages (Rantala et al., 2004). Thus, within lineages producing microcystin, non-producing strains would be the result of inactivation either by point mutation, or by partial or complete deletion of mcy genes (Christiansen et al., 2006; Dittmann et al., 1997; Kurmayer et al., 2004; Mikalsen et al., 2003).
One of the most intriguing features of microcystin is the high number of isoforms, often associated with changes in the synthetases as a result of mutations in the gene cluster (Kurmayer et al., 2005; Mikalsen et al., 2003). Several independent investigations on Microcystis and Planktothrix strains have concluded that the natural variation in the gene cluster within each strain is often caused by recombination. The regions with highest site variation seem to be mcyA (Kurmayer et al., 2005; Tanabe et al., 2004) and mcyB (Kurmayer & Gumpenberger, 2006; Mikalsen et al., 2003). For example within Planktothrix spp., the typical N-methyltransferase (NMT)-containing adenylation (A) domain of the first module of McyA has been replaced by an A domain without NMT, leading to production of desmethyl7-microcystin isoforms (Kurmayer et al., 2005). For Microcystis Mikalsen et al. (2003) showed that a recombination event between segments encoding two different adenylation domains has led to the presence of a second type of A domain in McyB1. Strains containing the Arg-activating A domain in McyB1 (hereafter denoted as C-like due to properties similar to the A domain of McyC) produce mainly microcystin-RR (indicating a microcystin with Arg in both the X and Z position), while strains with the Leu-activating A domain in McyB1 (hereafter denoted as B-like) produce mainly microcystin-LR.
In the previous study of Mikalsen et al. (2003) only a part of mcyB (A1) was analysed in detail. We therefore expanded the investigation using the same 17 field-collected strains to include the NMT domain of McyA and the A domain of McyC, as well as the flanking regions on both sides of the gene cluster. We employed a combination of alignments, a phylogenetic approach using split decomposition analysis and various statistical software (GENECONV, RPD and MaxChi) to address the genomic processes leading to new microcystin gene cluster variants (and thus to new Microcystis chemotypes, or inactivation of the cluster), the genetic basis for desmethyl7-microcystin variants and the recombination events involving mcyB1 and mcyC.
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METHODS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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) have previously been genetically typed using RAPD and REP fingerprinting (Mikalsen et al. 2003). Unialgal cultures were grown at the Norwegian Institute of Water Research (NIVA) as previously described (Skulberg & Skulberg, 1990) except for strain PCC 7806, which was kindly provided by H. Utkilen (National Institute of Public Health, Norway). Genomic DNA for Southern blotting and PCR was isolated as described earlier (Mikalsen et al., 2003).
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. Approximately 1 µg genomic DNA was digested overnight with 15 U HindIII (37 °C). Fragments were separated on a 1 % agarose gel and transferred to an Amersham Hybond-N membrane by Southern dry blotting overnight. Hybridization was performed at 68 °C by standard procedures (Galau et al., 1986). Radiolabelled probes were generated from NMT-F/NMT-R, dnaN-F/dnaN-R and uma1 F/uma1-R PCR products from strain PCC 7806 by standard MSPL (magnetic solid-phase labelling) random-primed synthesis (Espelund et al., 1990) using the Random Primed Labelling kit (Roche Diagnostics).
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. Primers were designed based on the publicly available mcy gene sequence of M. aeruginosa PCC 7806, accession no. AF183408 (Tillett et al., 2000). The primer hypX (Nishizawa et al., 2007) was used to investigate the genetic organization downstream of uma1 in two non-toxic strains. BD Advantage 2 polymerase (BD Biosciences) was used in PCRs. Amplicons were purified using the E.Z.N.A Gel Extraction kit (Omega Biotek) and sequenced directly. DNA sequencing was performed on both strands with the BigDye Terminator v3.1 Cycle Sequencing kit (Applied Biosystems) with a capillary electrophoresis sequencer (ABI 3730). Sequences have been submitted to GenBank; accession numbers are shown in Table 1.
Phylogenetic and recombination analyses.
Sequences were aligned with the publicly available mcy sequence of M. aeruginosa PCC 7806 (accession no. AF183408) using CLUSTAL W (Thompson et al., 1997) and manual editing. The sequences were used to infer the phylogeny in a Bayesian framework applying the program MrBayes v3.1 (Ronquist & Huelsenbeck, 2003). Analysis was performed with the following parameters: GTR model, gamma distribution, running 2 million generations and sampling trees every 100 generation, burn-in 3000 trees. The maximum-likelihood (ML) tree was estimated for mcyB1 and mcyC A domain sequences using PhyML (Guindon & Gascuel, 2003) under the GTR model, gamma distribution and with parameter values indicated by MrmodelTest (Nylander, 2004). The neighbour joining (NJ) tree was obtained under the default nucleotide substitution model using MEGA version 3.1 (Kumar et al., 2004). Mrmodelltest, PhyML and MrBayes analyses were performed on the Bioportal computer platform resources (http://www.bioportal.uio.no). Recombination was detected by split decomposition analysis using SplitsTree version 4.8 (Huson & Bryant, 2006) with default settings (uncorrectedP method) and 1000 bootstrap replicas, and Phi test for recombination (implemented to test split decomposition analysis reliability) (Bruen et al., 2006). In addition, statistical tests for detecting recombination were used: GENECONV (Padidam et al., 1999), RDP and MaxChi (Martin et al., 2005) analyses in the RDP version 2 b08 program package. For GENECONV, G-scale values 0 and 1 were used, for detecting recent and older recombination events, respectively. Recombination was also investigated by visual analysis of informative sites (variable sites where each variant occurs in at least two strains).
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RESULTS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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) was used to investigate the presence of this domain in 17 Microcystis strains (see Table 1 for description of the strains). Positive hybridization signals were obtained for all nine toxic strains (Fig. 2A), including the strains producing only desmethyl7-microcystin isoforms (N-C 57, N-C 228/1 and N-C 264) (Mikalsen et al., 2003). The same HindIII restriction pattern was obtained for all strains except N-C 118/2. No hybridization signal was observed for the non-toxic strain N-C 143, which possesses mcy genes, but is deficient in microcystin production (Mikalsen et al., 2003).
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), PCR products were obtained from all toxic Microcystis strains, but not from strain N-C 143 (Table 1), thus confirming the Southern hybridization results. Sequencing of the NMT region (see Fig. 1) showed that strain N-C 118/2 lacked the HindIII restriction site present in the other hybridizing strains, thus explaining the polymorphism seen for this strain.
The amino acid residues constituting the S-adenosylmethionine (SAM)-binding site of cyclosporin synthetase NMT domains (Velkov & Lawen, 2003) were identified in the deduced amino acid sequences of the NMT domains. The NMT domain sequence from Microcystis strain K-139 (AB019578), which exclusively produces [Dha7]-MC, was also included in the NMT dataset. The analysis revealed that invariant amino acid residues in the SAM-binding site (indicated in Fig. 3A) were intact in all strains producing only [Mdha7]-MC. In all methylation-defective strains one or more of these amino acid residues were altered (Fig. 3A). Interestingly, in strain N-C 324/1, which produces both [Mdha7]-MC and [Dha7]-MC (Mikalsen et al., 2003), the invariant Gln residue in the SAM-binding site was replaced by Arg (Fig. 3A).
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) – a term we will use in the following. The split decomposition analysis revealed a reticulate phylogeny (Fig. 3B) and the Phi test found statistically significant evidence for recombination (P<0.01). All three recombination detection programs used suggested recombination events in this dataset (Table 2).
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). An alignment of the sequences was used to identify the 3' breakpoint of the recombination event involving the A-domain-encoding region of mcyB reported by Mikalsen et al. (2003). The 5' breakpoint of this recombination event is located near the conserved motif A3 (Mikalsen et al., 2003). Recombination detection programs suggested a 3' breakpoint near the conserved motif A8 (at position 1209 in nucleotide alignment) (Table 2) in strain N-C 31. Visual inspection of amino acid alignment revealed a putative recombination breakpoint near the conserved motif A9 in strains N-C 118/2, N-C 161/1 and PCC 7806 (Fig. 4). This breakpoint could not be detected by Mikalsen et al. (2003) due to the shorter McyB1 sequences studied in that work.
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) and the Phi test found statistically significant evidence for recombination (P<0.01). However, no recombination events received statistical support by the recombination detection programs (Table 2). The C-like McyB1 A domain nucleotide sequences showed 2–5 % sequence variation. The split decomposition analysis revealed a reticulate phylogeny (Fig. 5) and statistically significant evidence for recombination was found by the Phi test (P<0.01). Recombination was also detected by the recombination detection programs (Table 2) and suggested by the mosaic structure of informative sites (Supplementary Fig. S1C).
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). The sequences showed 0–5 % sequence variation. Multiple recombination events were detected in this dataset by the mosaic structure of informative sites (Supplementary Fig. S1C); the reticulate phylogeny revealed by split decomposition (Fig. 5) was supported by the Phi test (P<0.01) and all recombination detection programs (Table 2).
A comparison of mcyB and mcyC A-domain-encoding regions from the same strain revealed relatively low sequence variation (9–13 %) in strains possessing the C-like A domain in McyB1. In strains with a B-like A domain in McyB1, these sequences were rather divergent, with 39–40 % sequence variation. Split decomposition analysis including all mcyB and mcyC sequences revealed a reticulate phylogeny within, but not between the clusters (P<0.01) (Fig. 5). The 161 informative sites found in the 1063 bp alignment of mcyB (C-like) and mcyC sequences showed a mosaic structure, indicating recombination also between these regions (Supplementary Fig. S1C). Putative recombination events involving the A domain regions of mcyB (C-like) and mcyC identified by the recombination detection programs are listed in Table 2. The phylogenetic analyses of all A domain amino acid sequences revealed three clades: McyB1 (B-like), McyB1 (C-like) and McyC (Supplementary Fig. S2).
Regions flanking the mcy gene cluster
Probes derived from the genes flanking the mcy gene cluster in strain PCC 7806, dnaN and uma1 (Fig. 1) (Tillett et al., 2000), were used for Southern hybridization analysis of flanking regions. All 17 strains gave positive hybridization signals with both probes (Fig. 2B, C). For the uma1 probe, all mcy-containing strains (Mikalsen et al., 2003) gave a 4.5 kb HindIII fragment similar to that from strain PCC 7806. The dnaN probe displayed a far more variable restriction pattern, with few shared bands between the strains.
The primer pairs dnaN-mcyJ and mcyC-uma1 (Fig. 1 and Supplementary Table S1) amplified flanking regions from all toxic strains. The mcy-containing non-toxic strain N-C 143 gave a PCR product with the mcyC-uma1 combination but not with the dnaN-mcyJ primer pair. The region between dnaN and mcyB could however be amplified using the dnaN-676R (mcyB-located) primer pair. Sequencing revealed a deletion of the whole mcyD–J gene cluster and a large part of mcyA (everything before position 43 852 in AF183408). Comparison of DNA sequences from the toxic strains showed a high degree of conservation of all coding regions. A 90 bp intergenic region between mcyC and uma1 was also conserved in all strains except strain N-C 118/2, which has a shorter (80 bp) region with no apparent similarity to the intergenic regions from the other strains (Fig. 6A). In contrast, the dnaN–mcyJ intergenic regions were highly different among the strains, with regard to both length (from 360 to 2192 bp) and sequence. Based on the alignments, the region was divided into segments A–D (Fig. 6A and Supplementary Fig. S3) that were assigned to subgroups containing similar sequences (colour coded in Fig. 6A). The dnaN–mcyJ sequence from strain N-C 118/2 was at the dnaN end highly similar to the corresponding sequences from strains N-C 161/1, N-C 169/7, N-C 264 and N-C 324/1, while it was most similar to the sequences from strains N-C 228/1, N-C 31, N-C 57 and PCC 7806 at the mcyJ end, indicating recombination.
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) and a single 1407 bp ORF transcribed in the same direction as mcyJ. A GATC methylation site known to play a modulating role in transposition activity (Mahillon & Chandler, 1998) was found in the left inverted repeat. The transposase encoded by ISMae7 contains a DDE motif known to be necessary for efficient DNA transposition (Mahillon & Chandler, 1998) and has probably retained transposition activity, since no stop codons were present within the ORF. Direct repeats associated with ISMae7 were also found in the dnaN–mcyJ intergenic spacer in strains N-C 118/2 and N-C 143. Interestingly, another 16 bp direct repeat not associated with ISMae7 was present in dnaN–mcyJ intergenic sequences from several strains (Fig. 6B).
Phylogenetic analysis of the mcy flanking regions gave fairly similar trees, except for strain N-C 324/1, which clustered with different clades at the two flanks, and the clade including strains N-C 31, N-C 57, N-C 228/1 and PCC 7806, which showed incongruent phylogenies (Fig. 7). Split decomposition analysis indicated recombination events for both flanks (Fig. 7), while the Phi test found statistically significant evidence for recombination only in the dnaN–mcyJ alignment (P<0.01).
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). PCR with the hypX (Nishizawa et al., 2007) and uma1 primer pair gave a PCR product for one of the two remaining non-toxic strains, N-C 43 (Table 1). In three strains (N-C 123/1, N-C 144 and N-C 279), dnaN was found to be situated close to uma1. The intergenic region between these genes showed no similarity to the intergenic regions in the toxic strains, except for a 80 bp segment close to uma1 that was similar to the region between mcyC and uma1 in strain N-C 118/2 (Fig. 7A). In three other non-toxic Microcystis strains, the genetic organization downstream of uma1 was different: in strains N-C 166 and N-C 172/5, an ORF encoding a protein homologous to FtsH (Kaneko et al., 1995) and in strain N-C 43, a hypothetical protein (HypX) (Nishizawa et al., 2007) was found (Fig. 6A). The flanking regions of strain N-C 122/2 could not be amplified using these primer pairs, indicating differences in the flanking regions of its mcy gene cluster, and therefore this strain is not included in Fig. 6(A). |
DISCUSSION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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) and Anabaena (Fewer et al., 2008). Our results indicate that the production of desmethyl7-microcystin by five of our Microcystis strains is not due to deletion of the NMT domain, but rather to specific point mutations altering amino acid residues in the cofactor SAM-binding site. Not all mutations were associated with total inactivation of the NMT domain, as shown for strain N-C 324/1, where replacement of invariant Gln with Arg (Fig. 3A) was associated with co-production of methylated and non-methylated microcystin isoforms (Mikalsen et al., 2003). The replacement of the second conserved Gly residue (as in strain N-C 57, Fig. 3A) has previously been shown to inactivate the NMT domain of pyochelin synthetase in Pseudomonas aeruginosa (Patel & Walsh, 2001). The effect of the remaining amino acid residues in the SAM-binding site has not been verified by mutations. However, residues which form the SAM-binding site (Velkov & Lawen, 2003) were found to be replaced only in Microcystis strains producing desmethyl7-microcystin, and always with residues with different physicochemical properties (Fig. 3A). Although the effects of these replacements on the methyltransferase activity have not yet been demonstrated, the results suggest that the NMT domain may be inactivated by point mutations, in addition to the complete deletion of this domain described in other nonmethylating strains (Fewer et al., 2008; Kurmayer et al., 2005).
Inter- and intragenomic recombinations within and between domains
Previously, we have shown recombination involving the A domain of McyB1 resulting in altered substrate specificity (Mikalsen et al., 2003). Here we show that this recombination included the A domain segment between the conserved motifs A3 and A8/A9 (Table 2, Fig. 4). Notably, Fewer et al. (2007) have described a similar recombination in Anabaena spp. and Hapalosiphon hibernicus, involving only the A domain regions of mcyB1 and mcyC and not affecting the condensation (C) domains in these modules. In both the Planktothrix and the Nostocales (Anabaena and Hapalosiphon) case the recombinations involve larger, non-identical functional units (different A domains) and affect essentially an entire domain. For Microcystis the 5' recombination breakpoint (near the conserved motif A3) is the same in all strains, but the downstream breakpoint has two different locations (near conserved motifs A8 in strain N-C 31 and A9 in strains N-C 118/2, N-C 161/1 and PCC 7806), separated by about 120 bp (Fig. 4). This might indicate two independent recombination events. However, as seen from the high similarity between the conserved A8 and A9 regions of McyB1 and McyC in strains PCC 7806, K-139 and UV027 (AP183408, AB019578 and AP458094, respectively), the variable location of the 3' breakpoint may alternatively be the result of a recombination event between two different A domain sequences followed by intragenomic or intergenomic recombinations spanning the regions encoding the conserved motifs A8 and A9.
Our data also indicate several recombination events between the A domain regions of mcyB (C-like) and mcyC, covering almost the whole segment between the conserved motifs A3 and A8 (as in strain N-C 264) or shorter segments of the A domain (Supplementary Fig. S1C and Table 2) and mainly resulting in the replacement of McyB1 A domain segments with corresponding McyC segments. Recombination between these two A-domain-encoding gene regions has been shown also for cyanobacteria from other genera (Fewer et al., 2007; A. Tooming-Klunderud, D. P. Fewer, T. Rohrlack, J. Jokela, L. Rouhiainen, K. Sivonen, T. Kristensen & K. S. Jakobsen, unpublished).
Our data suggest multiple recombination events involving smaller segments within all examined domains of mcyABC operon. These recombinations lead to a mosaic pattern of phylogenetic affinities in the alignments covering nearly the entire analysed sequence of the NMT domain and the McyB1A domain. As in other studies of the NMT domain of Microcystis (Tanabe et al., 2004) and the McyB1A domain of Planktothrix (Kurmayer et al., 2005; Kurmayer & Gumpenberger, 2006) many of these recombinations do not change the amino acid sequence. Within the A domain of McyC, however, such a pattern was mainly found upstream of the conserved motif A3 (Table 2). The apparent lack of recombination in the region coding for the substrate-binding part of this domain may be explained by a more restricted function of the Arg-activating McyC A domain compared to the A domain of McyB1, which will activate several amino acids (Mikalsen et al., 2003).
Evidence indicates that all microcystin variants are synthesized by a single enzyme complex. Given a single mcy operon in each strain, the observed recombinations (Kurmayer & Gumpenberger, 2006; Tanabe et al., 2004; present study) most likely represent intergenomic processes and are thus manifestations of horizontal gene transfer (HGT) between strains. The frequent HGT between closely related cyanobacterial strains was originally suggested by Rudi et al. (1998) and recently reinforced by whole-genome analysis of cyanobacteria (Zhaxybayeva et al., 2006) and in a general bacterial context (Papke et al., 2007). HGT may be the result of uptake of free DNA through natural transformation, since Microcystis is naturally competent (Dittmann et al., 1997), or DNA shuttling catalysed by cyanophages through transduction, or possibly due to other transposable elements.
The mcy flanking sequences do not indicate frequent transfer of complete mcy gene clusters between Microcystis strains
In all microcystin-producing strains investigated here the same two genes (dnaN and uma1) flank the mcy operon, in agreement with Tillett et al. (2001), who previously have shown that uma1 is located upstream of mcyC in toxic Microcystis strains. Thus, it seems likely that the genomic location of the mcy gene cluster in Microcystis spp. is the same in all strains examined, in agreement with another recent study (Nishizawa et al., 2007). These findings do not support the idea of frequent transfer of complete mcy gene clusters between Microcystis strains. The same two genes (dnaN and uma1) were also found to be neighbours in three of the non-toxic Microcystis strains, as also reported by Nishizawa et al. (2007), indicating that loss of the mcy gene cluster was not accompanied by further rearrangements in this genomic region. Partial loss of the mcy gene cluster in strain N-C 143 clearly illustrates that loss of microcystin production can be caused by deletion (Fig. 6A).
A gene encoding a putative transposase (uma4) has previously been identified in the mcy region of Microcystis strain PCC 7806 (Tillett et al., 2000). Here we report the presence of an IS element (ISMae7) in the intergenic region between mcyJ and dnaN in strain N-C 228/1 (Fig. 6). Recently, two other genes coding for different types of transposases have been identified between dnaN and mcyJ in several Microcystis strains (Nishizawa et al., 2007), but none of these reported transposases are similar to the one encoded by uma4. The additional direct repeats detected by us in this intergenic region in several strains may also indicate an additional, now lost IS element. According to these results, the intergenic region between dnaN and mcyJ seems to be a recombinatorial ‘hot spot’ while the mcyC–uma1 region is more conserved.
The frequent recombinations – representing both intragenomic and intergenomic rearrangements – give rise to novel mcy gene cluster variants, most of which encode peptide synthetases with the same biosynthetic properties, due to a constant selective pressure. Changes in selection will, however, favour some variants within a particular ecosystem. The results presented here along with some previous studies (Kurmayer et al., 2005; Kurmayer & Gumpenberger, 2006; Mikalsen et al., 2003; Tanabe et al., 2004) suggest that the function of microcystins needs to be understood within the frame of the natural ecosystems of the bacterial strains. It seems likely that the processes promoting variation within the microcystin synthetase genes are crucial for adaptivity of a given population within a particular habitat.
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ACKNOWLEDGEMENTS |
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Edited by: K. Forchhammer
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Received 14 December 2007;
revised 16 April 2008;
accepted 17 April 2008.
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