Phylogenetic evidence for the early evolution of microcystin synthesis

Abstract

Cyanobacteria are a prolific source of secondary metabolites, including compounds with toxic and enzyme-inhibiting activities. Microcystins and nodularins are the end products of a secondary metabolic pathway comprised of mixed polyketide synthases and nonribosomal peptide synthetases. Both peptides are potent natural toxins produced by distantly related genera of cyanobacteria. Horizontal gene transfer is thought to play a role in the sporadic distribution of microcystin producers among cyanobacteria. Our phylogenetic analyses indicate a coevolution of housekeeping genes and microcystin synthetase genes for the entire evolutionary history of the toxin. Hence they do not corroborate horizontal transfer of genes for microcystin biosynthesis between the genera. The sporadic distribution of microcystin synthetase genes in modern cyanobacteria suggests that the ability to produce the toxin has been lost repeatedly in the more derived lineages of cyanobacteria. The data we present here strongly suggest that the genes encoding nodularin synthetase are recently derived from those encoding microcystin synthetase.

Fig. 1.

Comparison of the chemical structures of microcystins and nodularins and the hypothetical events leading to nodularin synthetase gene cluster. (A) The general structure of microcystins and nodularin. Microcystin is a cyclic peptide containing seven amino acids, d-Ala-X-d-MeAsp-Z-Adda-d-Glu-Mdha, where X and Z represent variable l-amino acids, d-Me-Asp is d-erythro-β-methylaspartic acid, Mdha is N-methyldehydroalanine, and Adda is β-amino acid 3-amino-9-methoxy-2,6,8-trimethyl-10-phenyldeca-4,6-dienoic acid. Nodularin differs from microcystins by lacking the amino acids d-Ala and X and having N-methyldehydrobutyrine (Mdhb) in place of Mdha. The dashed line indicates the two amino acids absent in nodularins. (B) A schematic diagram illustrating the peptide synthetase region (mcyA-mcyC)of Anabaena and the proposed organization of the homologous region of the gene cluster for nodularin biosynthesis in Nodularia if evolved by a mutation changing the substrate specificity of the first module of mcyA and the deletion of the last module of mcyA and the first module of mcyB (see text).

Fig. 2.

Congruence between the 16S rRNA and rpoC1 data set and the microcystin synthetase gene data set. (A) A maximum-likelihood tree based on the 16S rRNA and rpoC1 data set (–lnL = 8004.26493). (B) A maximum-likelihood tree based on the mcyA, mcyD, and mcyE data set (–lnL = 8781.50660). In both trees, branch lengths are proportional to sequence change. Minimum-evolution, maximum-parsimony, and maximum-likelihood bootstrap values from 2,000 bootstrap replicates are at the nodes. Outgroup taxa used in the construction of these trees are not shown.

Fig. 3.

A maximum-likelihood tree based on the 16S rRNA gene illustrating the sporadic distribution of cyanobacterial genera known to produce microcystins (–lnL = 17594.65327). Strains of the genera Planktothrix, Microcystis, Anabaena, and Nostoc produce microcystins (▪), whereas strains of the genus Nodularia produce nodularins (•). All strains in the lineage of cyanobacteria, which originally possessed microcystin synthetase genes, are contained within a light gray box. Support for this clade is given as follows: minimum evolution with LogDet distances, minimum evolution with maximum-likelihood distances, and maximum-parsimony bootstrap values. Outgroup taxa used in the construction of this tree are not shown.