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 microcystinproducing 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.Microcystin-producing cyanobacteria are common nuisance organisms in harmful algal blooms in freshwaters around the world (4). This genetically diverse group (based on 16S rRNA, mcyA, mcyD, and mcyE gene sequences [6,10,15,16,22]) ranges in morphology from unicellular and colonial cocci to large filamentous strands. Many species can produce a variety of secondary metabolites that can act as hepatatoxins upon ingestion by animals (e.g., variants of microcystin) (4, 33). Microcystin production reduces the water quality in reservoirs used by human populations and fishery resources, and production of these toxins by this group of cyanobacteria makes them important organisms for continued observation and study (4,33,36). Much effort has been expended over the past 15 years to characterize the genomic and structural components of the microcystin (mcy) synthetase operon responsible for the production of microcystins. Several complete DNA sequences of the mcy synthetase operon are currently available in GenBank (3,11,29,31).Although the mechanisms of microcystin production are now better understood, recent analyses of mcyA gene fragments from Lakes Erie and Ontario indicated a microcystin toxin producer of unknown phylogeny (7,28). This discrepancy suggested a need for improved molecular characterization of naturally occurring microcystin producers, which spurred our research to identify the source of several unusual mcyA fragments from the cyanobacterial community (7, 28). It was apparent from initial sequence data that these mcyA gene fragments, termed Microcystis-like, were highly similar to those from Microcystis spp. (colonial or unicellular cocci). However, they contained a 6-nucleotide insert consistent with mcyA genes from filamentous cyanobacteria (e.g., Anabaena, Nostoc, and Planktothrix) (28). These preliminary findings suggested that these unusual mcyA fragments either came from (i) a novel species or strain, (ii) an ancestral Microcystis, (iii) the highly unlikely hybridization of colonial cocci and filamentous cyanobacteria, or (iv) a chimera of cocci and filamentous PCR products....

Population Turnover in a Microcystis Bloom Results in Predominantly Nontoxigenic Variants Late in the Season

“…This sequence (and four other cloned sequences) encodes an McyA protein with ThrPhe appearing between amino acids Lys-1610 and Ser-1611 of McyA encoded by M. aeruginosa PCC 7806 (GenBank accession number AM778952). mcyA genes with this Lys-Ser dipeptide were first reported from the Great Lakes (1,22) and remain uncommon in the database. Our study and a recent report (19) show that this mcyA variant is common in the Klamath watershed, suggesting a wider distribution in the United States.…”

“…Microcystis cells are able to produce microcystin, a nonribosomally synthesized cyclic heptapeptide hepatotoxin with potent inhibitory activity against mammalian protein phosphatases (27) whose synthesis is directed by the 55-kb mcy gene cluster (25). The Microcystis genus exhibits worldwide occurrence, although the extent of genetic differentiation between or within geographical regions is currently uncertain due to a relatively sparse database, in spite of a growing number of studies (1,2,9,11,26,28,29).…”

Molecular Enumeration of an Ecologically Important Cyanophage in a Laurentian Great Lake

“…The lake's large population base of over 30 million people, in addition to agricultural and industrial influences, has resulted in historically high biological productivity in this lake and subsequently ecosystem degradation (26). Excessive primary productivity has been the highlight of research for decades in this system; most recently, studies have focused on Lake Erie due to seasonal harmful algal blooms that form during the summer months (1,3,30). Factors promoting or constraining primary production in the lake are thus of great interest not only to the academic community but also to ecosystem managers.…”

Presence and genetic diversity of microcystin-producing cyanobacteria (Anabaena and Microcystis) in Lake Kotokel (Russia, Lake Baikal Region)

“…The prevalence of MC-RR in temperate Lake Kotokel conflicts with this conclusion. MC-YR is generally a minor component of the MC pool, for example, in the Great Lakes its contribution ranged between 9-23% of total MCs (Hotto et al, 2007;Dyble et al, 2008;Allender et al, 2009). The low content of the dominating, the least toxic MC-RR in Lake Kotokel was not likely to lead to animal or human disease, nevertheless deaths were reported in 2008-2009.…”