Identification of novel toxin-producing cyanobacteria in Iowan lakes
Cyanobacteria is a diverse phylum of photosynthetic bacteria with many species harboring the ability to produce toxins. Toxin-producing cyanobacteria are a growing ecological concern in freshwaters that has garnered much attention from prominent health agencies due to their global distribution and potentially life-threatening health impacts. The cyanotoxin microcystin is routinely detected in Iowa lakes as part of the Iowa Department of Natural Resources (IDNR) Beach Monitoring program. This study focused analytical efforts on lesser studied toxins (anatoxin-a, cylindrospermopsin, and saxitoxin) and aimed to isolate and identify cyanobacteria that produce these cyanotoxins in Iowan lakes. While direct measurements of lake waters give insight into the presence and concentration of various toxins, determining which strains produce these toxins is complicated by several factors. Freshwater cyanobacteria grow in mixed communities, where many species are present. Different cyanobacterial strains are able to produce the same toxin types, and some strains produce multiple toxins. Many species have highly similar morphology, so microscopic identification cannot delineate toxic from non-toxic strains. Cyanobacteria were enriched through serial dilutions on a selective growth medium. Individual strains were further isolated using flow cytometry. Cyanobacterial cultures were screened for toxin functional genes (anaC, cyrJ, sxtI) via PCR, which revealed three cultures containing the sxtI gene involved in saxitoxin production. Cloning of the sxtI insert from one sample and a subsequent search of its amino acid residues using NCBI’s BLASTx showed the closest sequence match was a carbamoyltransferase enzyme belonging to an uncultured Nostoc species. Amplification, cloning, and Sanger sequencing of the 16S rRNA gene, followed by a nucleotide search in NCBI’s BLASTn indicated that the most abundant organisms in the culture were closely related to uncultured Nostoc strains. Microscopy revealed heterocysts and a complex life-cycle morphology, further corroborating the sequencing results that the culture contained organisms belonging to the genus Nostoc. The 16S rRNA and sxtI gene inserts showed the closest matches to uncultured Nostoc strains, indicating this organism is not yet taxonomically classified, and should be considered novel. Next steps include further purification and whole genome sequencing of the putatively toxic Nostoc enrichment culture to determine its species level classification. Verification of this strain’s ability to produce saxitoxin will be confirmed through searching its genome for the full sxt gene cluster, and through LC/MS/MS based analysis for detection of saxitoxin in the sample.