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
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ABSTRACT |
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The GenBank/EMBL/DDBJ accession numbers for the sequences reported in this paper are AJ749248AJ749302 and AJ863131AJ863134, as indicated in Table 1.
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INTRODUCTION |
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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--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.
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METHODS |
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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 AJ749248AJ749302 and AJ863131AJ863134 (see Table 1
).
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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 JonesTaylorThornton 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.
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RESULTS |
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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.
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DISCUSSION |
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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 m2 s1) as opposed to the culture conditions used in this study (15 °C, 510 µmol m2 s1), 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 m2 s1; R. Kurmayer, unpublished).
Genetic recombination of the mcyABC cluster
In this study, the mcyAAd1 genotype without NMT (563 aa) revealed extensive sequence identity (4766 %) with non-ribosomal peptide synthetase genes from other cyanobacterial and bacterial genera. The genetic variation was lowest among strains of this genotype (00·2 %), 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.
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ACKNOWLEDGEMENTS |
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Received 23 November 2004;
revised 14 February 2005;
accepted 14 February 2005.
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