A cultivation-independent approach for the genetic and cyanotoxin characterization of colonial cyanobacteria

To bypass the constraint of cyanobacterial strain isolation and cultivation, a combination of whole genome amplification (WGA) and enzyme-linked immunoassay (ELISA) for microcystin toxins (MCs) was tested on individual colonies of Microcystis and Woronichinia, taken directly from aquatic environments. Genomic DNA of boiled cells was amplified by multiple strand displacement amplification (MDA), followed by several specific PCR reactions to characterize the genotype of each colony. Sequences of 3 different housekeeping genes (ftsZ, gltX, and recA), of 3 MC biosynthesis genes (mcyA, mcyB, and mcyE), and the Internal Transcribed Spacer (ITS) were analyzed for 11 colonies of Microcystis. MCs were detected and quantified by ELISA in 7 of the 11 Microcystis colonies tested, in agreement with the detection of mcy genes. Sequence types (ST) based on the concatenated sequences of housekeeping genes from cyanobacterial colonies from Belgian water bodies appeared to be endemic when compared to those of strains described in the literature. One colony appeared to belong to a yet undiscovered lineage. A similar protocol was used for 6 colonies of the genus Woronichinia, a taxon that is very difficult to cultivate in the laboratory. The 16S rRNA sequences of 2 colonies were obtained and were quasi identical to that of W. naegeliana 0LE35S01. For one Woronichinia colony, the mcyE PCR gave a non-specific PCR product. The corresponding amino acid sequence was 50% identical to a Microcystis ketoacyl carrier protein transferase. This approach for the simultaneous detection and quantification of MCs with mcy genotyping, at single colony level, offers potential for the ecotoxicological characterization of environmental populations of cyanobacteria without the need for strain isolation and culture.