Genetic characterization of Cylindrospermopsis raciborskii (cyanobacteria) isolates from diverse geographic origins based on nifH and cpcBA-IGS nucleotide sequence analysis

Abstract

Isolates of the toxic, N(2)-fixing species Cylindrospermopsis raciborskii from various geographic locations were analyzed with respect to their genetic diversity based on the nifH and cpcBA-IGS genes. Gene sequences clustered according to their geographic origin, with the nifH sequences separating into European, Australian, and American groups and the cpcBA-IGS sequences separating into American and European or Australian groups. PCR primers for both genes were designed to exclusively amplify DNA from Cylindrospermopsis species, and an additional primer set for cpcBA-IGS was designed to specifically amplify the American C. raciborskii strains.

FIG. 1.

Phylogenetic tree based upon nifH sequences of C. raciborskii isolates originating from different geographic locations. Bootstrap values (>50) are given by the corresponding nodes and were generated with distance methods. Anabaena oscillarioides (GenBank accession no. M63686) was used as the outgroup.

FIG. 2.

Phylogenetic tree based upon cpcBA-IGS sequences of C. raciborskii isolates originating from different geographic locations. Bootstrap values (>50) are given by the corresponding nodes and were generated with distance methods. Anabaena 7120 (GenBank accession no. X05239) was used as the outgroup.

FIG. 3.

PCR products amplified with the nifH and cpcBA-IGS primer sets. Each set of primers was used in a separate PCR, and then the two (for nifH) or three (for cpcBA-IGS) PCR products from each isolate were run together in the same lane of the gel. A φX174/HaeIII molecular size marker (M) was run on each gel. (A) For nifH, the 324-bp product was amplified by the general cyanobacterial primer set (cyano) which had been used in the original sequencing of the C. raciborskii isolates. Each of these samples was also amplified with the Cylindrospermopsis-specific primer set (cylindro), which resulted in a 225-bp product exclusively from C. raciborskii samples. The C. raciborskii isolates used were the following: Falls Lake NC, Florida G, Florida D, Brazil 1, A205, Germany 1, Bal 6, and Marau 1. (B) For cpcBA-IGS, the 685-bp product was amplified by the general cyanobacterial primer set (cyano) which had been used in the original sequencing of the C. raciborskii isolates. Each of these samples was also amplified with the Cylindrospermopsis-specific primer set (cylindro), which resulted in a 638-bp product exclusively from C. raciborskii samples, and an American strain-specific primer set (FL/Brazil) which amplified a 464-bp band exclusively from the Florida and Brazil isolates. The C. raciborskii isolates used were Florida F, Florida G, Florida D, Brazil 1, Brazil 2, A205, Germany 1, and Bal 6.