Molecular characterization of the toxic cyanobacterium Cylindrospermopsis raciborskii and design of a species-specific PCR

Abstract

Cylindrospermopsis raciborskii is a toxic-bloom-forming cyanobacterium that is commonly found in tropical to subtropical climatic regions worldwide, but it is also recognized as a common component of cyanobacterial communities in temperate climates. Genetic profiles of C. raciborskii were examined in 19 cultured isolates originating from geographically diverse regions of Australia and represented by two distinct morphotypes. A 609-bp region of rpoC1, a DNA-dependent RNA polymerase gene, was amplified by PCR from these isolates with cyanobacterium-specific primers. Sequence analysis revealed that all isolates belonged to the same species, including morphotypes with straight or coiled trichomes. Additional rpoC1 gene sequences obtained for a range of cyanobacteria highlighted clustering of C. raciborskii with other heterocyst-producing cyanobacteria (orders Nostocales and Stigonematales). In contrast, randomly amplified polymorphic DNA and short tandemly repeated repetitive sequence profiles revealed a greater level of genetic heterogeneity among C. raciborskii isolates than did rpoC1 gene analysis, and unique band profiles were also found among each of the cyanobacterial genera examined. A PCR test targeting a region of the rpoC1 gene unique to C. raciborskii was developed for the specific identification of C. raciborskii from both purified genomic DNA and environmental samples. The PCR was evaluated with a number of cyanobacterial isolates, but a PCR-positive result was only achieved with C. raciborskii. This method provides an accurate alternative to traditional morphological identification of C. raciborskii.

FIG. 1

Morphotypes of C. raciborskii at ×268 magnification. (A) Straight; (B) coiled.

FIG. 2

Amino acid alignment of C. raciborskii, N. spumigena PCC73104, Anabaenopsis circularis, A. bergii ANA283A, and A. circinalis ANA118C rpoC1 sequences and additional rpoC1 sequences obtained from the GenBank database: Fischerella sp. strain PCC7414 (accession no. FSPRPOC1), Synechocystis (Syn.) sp. strain PCC6308 (accession no. U52344), Synechocystis sp. strain PCC7002 (accession no. U52345), Synechococcus sp. strain PCC7942 (accession no. SRPOC1), Dermocarpa sp. (accession no. U52341), E. coli (accession no. ECRPOBC), and P. putida (accession no. M38319). Amino acids identical to those of C. raciborskii are indicated by dots; the dashes represent gaps introduced into the alignment. The relative locations of primers cyl2 and cyl4 are also indicated (underlined).

FIG. 3

Phylogenetic position of C. raciborskii in relation to other cyanobacteria (sequences resulting from this work and obtained from the GenBank database) and to E. coli and P. putida based on analysis of aligned rpoC1 nucleotide sequences. The neighbor-joining tree was constructed by using corrected Jukes-Cantor distances. Boostrap percentages (calculated from 500 resamplings) are indicated for the nodes. I, cluster I.

FIG. 4

STRR sequence profile analysis of 13 isolates of C. raciborskii and other representative genera obtained by PCR amplification with primers STRR1F and STRR3F (A), STRR1F and STRR3R (B), and STRR1R and STRR3R (C). Lanes 1, CYP003A; lanes 2, CYP003K; lanes 3, CYP005E; lanes 4, CYP010A; lanes 5, CYP014A; lanes 6, CYP015A; lanes 7, CYP020A; lanes 8, CYP020B; lanes 9, CYP023A; lanes 10, CYP023E; lanes 11, CYP024C; lanes 12, CYP026J; lanes 13, AWT205; lanes 14, A. circinalis ANA173A; lanes 15, A. circinalis ANA118C; lanes 16, Microcystis aeruginosa PCC7806; lanes 17, N. spumigena PCC73104. Lane M, molecular size marker: 2,027, 1,904, 1,584, 1,375, 947, 831, and 564 bp.

FIG. 5

Graphical representation of the branching pattern of C. raciborskii STRR sequence profiles derived from total character differences by the neighbor-joining method. The isolates showing coiled morphology are underlined.

FIG. 6

C. raciborskii-specific PCR. Genomic DNA from laboratory cultures and environmental samples was amplified with primers cyl2 and cyl4 in a PCR spiked with ICF. Lane 1, AWT205; lane 2, CYP003A; lane 3, CYP003K; lane 4, CYP005E; lane 5, CYP005F; lane 6, CYP010A; lane 7, CYP010C; lane 8, CYP014A; lane 9, CYP015A; lane 10, CYP020A; lane 11, CYP020B; lane 12, CYP023A; lane 13, CYP023B; lane 14, CYP023D; lane 15, CYP023E; lane 16, CYP024C; lane 17, CYP025B; lane 18, CYP025E; lane 19, CYP026J; lane 20, brazil 1; lane 21, brazil 2; lane 22, A. circinalis ANA118C; lane 23, A. circinalis ANA173A; lane 24, Microcystis aeruginosa PCC7806; lane 25, M. aeruginosa; lane 26, N. spumigena PCC73104; lane 27, N. spumigena; lane 28, A. bergii ANA283A; lane 29, Anabaenopsis circularis; lane 30, environmental sample (Fred Haigh Dam, Queensland, Australia); lane 31, environmental sample (Currency Creek, South Australia, Australia); lane 32, ICF only; lane 33, CYP014A only. Lane M, molecular size marker: 587, 540, 504, 458, 434, 267, 234, 213, 192, and 184 bp.