Evidence for positive selection acting on microcystin synthetase adenylation domains in three cyanobacterial genera

Abstract

Background: Cyanobacteria produce a wealth of secondary metabolites, including the group of small cyclic heptapeptide hepatotoxins that constitutes the microcystin family. The enzyme complex that directs the biosynthesis of microcystin is encoded in a single large gene cluster (mcy). mcy genes have a widespread distribution among cyanobacteria and are likely to have an ancient origin. The notable diversity within some of the Mcy modules is generated through various recombination events including horizontal gene transfer.

Results: A comparative analysis of the adenylation domains from the first module of McyB (McyB1) and McyC in the microcystin synthetase complex was performed on a large number of microcystin-producing strains from the Anabaena, Microcystis and Planktothrix genera. We found no decisive evidence for recombination between strains from different genera. However, we detected frequent recombination events in the mcyB and mcyC genes between strains within the same genus. Frequent interdomain recombination events were also observed between mcyB and mcyC sequences in Anabaena and Microcystis. Recombination and mutation rate ratios suggest that the diversification of mcyB and mcyC genes is driven by recombination events as well as point mutations in all three genera. Sequence analysis suggests that generally the adenylation domains of the first domain of McyB and McyC are under purifying selection. However, we found clear evidence for positive selection acting on a number of amino acid residues within these adenylation domains. These include residues important for active site selectivity of the adenylation domain, strongly suggesting selection for novel microcystin variants.

Conclusion: We provide the first clear evidence for positive selection acting on amino acid residues involved directly in the recognition and activation of amino acids incorporated into microcystin, indicating that the microcystin complement of a given strain may influence the ability of a particular strain to interact with its environment.

Figure 1

Organization of the mcyABC gene cluster (A). Adenylation domains investigated in the present study are indicated in red and green. The relative positions of primers (arrows) are shown. Genus-specific mcyB and mcyC primers are listed in Table 8. (B) The structure of microcystin-LR. Amino acid residues activated by the adenylation domains of McyB1 and McyC are indicated by red and green, respectively. Mdha is N-methyl-dehydroalanine, D-MeAsp is 3-methyl-aspartic acid and Adda is 3-amino-9-methoxy-2,6,8,-trimethyl-10-phenyl-4,6-decandienoic acid.

Figure 2

(A) Phylogenetic analysis of adenylation domains of McyB1 and McyC. The Bayesian tree is shown with support from maximum likelihood tree (1000 replicates and neighbor-joining tree (1000 replicates). Bayesian posterior probability/ML bootstrap/NJ bootstrap values are shown. Only bootstrap values above 50% are shown. Adenylation domains of McyB1 and McyC from all genera are indicated by red and green, respectively.

Figure 3

Informative sites in Anabaena, Microcystis and Planktothrix mcyB1C data sets. Informative sites are defined as positions with at least two different nucleotides in which each of the variants occurs at least twice. Identical nucleotides have the same colour and the colours thus display phylogenetic affinity.

Figure 4

Splits decomposition analysis of adenylation domain encoding sequences of mcyB1. Shown are Anabaena (A), Microcystis (B) and Planktothrix (C). Bootstrap values over 50% are shown.

Figure 5

Splits decomposition analysis of adenylation domain encoding sequences of mcyC. Shown are Anabaena (A), Microcystis (B) and Planktothrix (C). Bootstrap values over 50% are shown.

Figure 6

Splits decomposition analysis of adenylation domain encoding sequences of mcyB1 and mcyC. Shown are Anabaena (A), Microcystis (B) and Planktothrix (C). mcyB1 and mcyC sequences are indicated by red and green, respectively. Bootstrap values above 50% are shown. Within mcyC sequences of Microcystis, all branches have bootstrap values ranging from 88–100%.

Figure 7

Alignment of adenylation domain sequences of McyB1 and McyC in Anabaena, Microcystis and Planktothrix strains. Identical amino acid residues within genus sequences are indicated by •. Positions of the conserved motifs [2] are shown and binding pocket residues [32] are indicated by red diamonds. Amino acid residues undergoing positive selection are shown in dark blue boxes. Numbering of amino acid residues according to GrsA (swissprot: P0C061).