Discovery of rare and highly toxic microcystins from lichen-associated cyanobacterium Nostoc sp. strain IO-102-I

Abstract

The production of hepatotoxic cyclic heptapeptides, microcystins, is almost exclusively reported from planktonic cyanobacteria. Here we show that a terrestrial cyanobacterium Nostoc sp. strain IO-102-I isolated from a lichen association produces six different microcystins. Microcystins were identified with liquid chromatography-UV mass spectrometry by their retention times, UV spectra, mass fragmentation, and comparison to microcystins from the aquatic Nostoc sp. strain 152. The dominant microcystin produced by Nostoc sp. strain IO-102-I was the highly toxic [ADMAdda(5)]microcystin-LR, which accounted for ca. 80% of the total microcystins. We assigned a structure of [DMAdda(5)]microcystin-LR and [d-Asp(3),ADMAdda(5)]microcystin-LR and a partial structure of three new [ADMAdda(5)]-XR type of microcystin variants. Interestingly, Nostoc spp. strains IO-102-I and 152 synthesized only the rare ADMAdda and DMAdda subfamilies of microcystin variants. Phylogenetic analyses demonstrated congruence between genes involved directly in microcystin biosynthesis and the 16S rRNA and rpoC1 genes of Nostoc sp. strain IO-102-I. Nostoc sp. strain 152 and the Nostoc sp. strain IO-102-I are distantly related, revealing a sporadic distribution of toxin production in the genus Nostoc. Nostoc sp. strain IO-102-I is closely related to Nostoc punctiforme PCC 73102 and other symbiotic Nostoc strains and most likely belongs to this species. Together, this suggests that other terrestrial and aquatic strains of the genus Nostoc may have retained the genes necessary for microcystin biosynthesis.

FIG. 1.

General structure of [(A)DMAdda]microcystin-X(Ha)R found in Nostoc sp. strain IO-102-I. In position 5, OX is −O(CO)CH₃ in ADMAdda and OH in DMAdda. In position 2, X is Leu, Leu with a modification in the side chain, an unidentified amino acid, H in Asp, and CH₃ in MeAsp. In position 4, the side chain with Arg has three CH₂ groups and with (H)Arg the side chain has four CH₂ groups.

FIG. 2.

Phylogenetic position of the Nostoc sp. strain IO-102-I. A Bayesian majority rule consensus tree inferred from 16S rRNA gene sequences shows the phylogenetic position of Nostoc sp. strain IO-102-I, which falls among symbiotic Nostoc strains. Nostoc sp. strain IO-102-I is distantly related to Nostoc sp. strain 152, the closest known microcystin producer. Bayesian posterior probabilities (≤1.0) are given at the nodes. The sequence accession number is in brackets after the strain code. ○, Aquatic; □, terrestrial; N/a, datum not available.

FIG. 3.

Congruence between the housekeeping genes 16S rRNA and rpoC1 and microcystin synthetase genes mcyA, mcyD, and mcyE. (A and B) Bayesian majority rule consensus tree inferred from mcyA, mcyD, and mcyE gene sequences (A) and from 16S rRNA and rpoC1 gene sequences (B). The outgroup (not shown) sequences are from Microcystis viridis strain NIES 102 and M. aeruginosa strain PCC 7806. Bayesian posterior probabilities (≤1.0) are given at the nodes.