Identifying the source of unknown microcystin genes and predicting microcystin variants by comparing genes within uncultured cyanobacterial cells

Abstract

While multiple phylogenetic markers have been used in the culture-independent study of microcystin-producing cyanobacteria, in only a few instances have multiple markers been studied within individual cells, and in all cases these studies have been conducted with cultured isolates. Here, we isolate and evaluate large DNA fragments (>6 kb) encompassing two genes involved in microcystin biosynthesis (mcyA2 and mcyB1) and use them to identify the source of gene fragments found in water samples. Further investigation of these gene loci from individual cyanobacterial cells allowed for improved analysis of the genetic diversity within microcystin producers as well as a method to predict microcystin variants for individuals. These efforts have also identified the source of the novel mcyA genotype previously termed Microcystis-like that is pervasive in the Laurentian Great Lakes and they predict the microcystin variant(s) that it produces.

FIG. 1.

Maps indicating sample collection sites in Lake Erie (A), Lake Ontario (B) and North Carolina (C). Geographical coordinates (in decimal degrees): Hamilton Harbor (43.295, −79.813); station 589 (42.134, −80.101); station 971 (41.948, −83.054); station 974 (41.730, −83.157); station 1163 (41.469, −82.715); Waterville reservoir (35.695, −83.049). Maps generated using the Planiglobe web mapping service.

FIG. 2.

Phylogenetic trees for the condensation domain in mcyA and the adenylation domain in mcyB1. (A) Bayesian analysis for mcyA, indicating placement of 6-kb fragments among cultures and clones. Posterior probabilities between 0.5 and 1 are listed. (Panels 1a to 1c) Ancestral Microcystis genotypes, formerly known as the Microcystis-like sequences (6, 26) containing 6 nucleotides that were not present among Microcystis cultures, except for Microcystis sp. strain N-C 118-2. (Panel 2) Microcystis spp. clade, including most existing cultures, which all exhibit a common 6-nucleotide deletion. (Panel 3) Anabaena and Nostoc spp. (Panel 4) The filamentous cyanobacteria including 6-kb fragments LF5 to LF7, which cluster with Planktothrix cultures. (B) Bayesian analysis for mcyB1, indicating placement of 6-kb fragments among cultures with listing of posterior probabilities between 0.5 and 1. Bacillus brevis was used as an outgroup, as described in reference . (Panel 5) Corresponding to Microcystis genotype mcyB1(B) known to produce microcystin-LR, -LA, and -YR (15). Leucine was predicted to be incorporated at site 2 of the resulting microcystin molecule for each member based on an adenylation domain prediction tool (see text). (Panel 6) Corresponding to Microcystis genotype mcyB1(C), known to produce microcystin-RR and many others (16). All members, including 6-kb fragments LF8 to LF10, were predicted to incorporate arginine into site 2 of the microcystin molecule. (Panel 7) Planktothrix spp. including 6-kb fragments LF5 to LF7. Each member was predicted to incorporate a leucine into site 2 of the microcystin molecule.