Geographical segregation of the neurotoxin-producing cyanobacterium Anabaena circinalis

Abstract

Blooms of the cyanobacterium Anabaena circinalis are a major worldwide problem due to their production of a range of toxins, in particular the neurotoxins anatoxin-a and paralytic shellfish poisons (PSPs). Although there is a worldwide distribution of A. circinalis, there is a geographical segregation of neurotoxin production. American and European isolates of A. circinalis produce only anatoxin-a, while Australian isolates exclusively produce PSPs. The reason for this geographical segregation of neurotoxin production by A. circinalis is unknown. The phylogenetic structure of A. circinalis was determined by analyzing 16S rRNA gene sequences. A. circinalis was found to form a monophyletic group of international distribution. However, the PSP- and non-PSP-producing A. circinalis formed two distinct 16S rRNA gene clusters. A molecular probe was designed, allowing the identification of A. circinalis from cultured and uncultured environmental samples. In addition, probes targeting the predominantly PSP-producing or non-PSP-producing clusters were designed for the characterization of A. circinalis isolates as potential PSP producers.

FIG. 1

A. circinalis 16S rDNA distance tree. DNA sequences corresponding to the E. coli 16S rRNA gene positions 27 to 1494 were aligned using the programs PILEUP (10) and CLUSTAL W (39). A. circinalis strains in bold were found to produce PSPs as determined by HPLC and mouse bioassay (; P. Baker, personal communication). Also included were an additional five Anabaena 16S rDNA sequences including A. affinis NIES40, A. solitaria NIES80, and A. flos-aquae AWQC112D, NRC44-1, and NRC525-17. Genetic distances were calculated using the method of Jukes and Cantor, and the phylogenetic was tree reconstructed using the neighbor-joining algorithm of Saitou and Nei (34) as implemented within CLUSTAL W. The root of the tree was determined by using the 16S rRNA gene of A. cylindrica NIES19 as an outgroup. Local bootstrap support for branches present in more than 50% of 1,000 resampling events is indicated at the respective nodes (10).

FIG. 2

Comparison of the 16S rRNA-DNA sequences of the bp 576 to 594 region from representatives of different species of cyanobacteria and the consensus sequence of A. circinalis strains (a), the bp 247 to 268 region from the consensus sequence from branch I and branch II A. circinalis strains (b), and the bp 202 to 227 region from the consensus sequence from branch I and branch II A. circinalis strains (c). Oligonucleotides AC510R, ACB1F, and ACB2F were designed on the basis of these sequences. Nucleotide positions correspond to the numbering of the E. coli sequence.

FIG. 3

Analysis of the 16S rRNA gene region using A. circinalis branch I- and branch II-specific oligonucleotide primers. Lanes labeled Branch I and Branch II correspond to the PCR products from the A. circinalis strains; other lanes correspond to the PCR products obtained from the reference strains and to the negative control (-ve control). Amplification with the A. circinalis-specific primer AC510R plus the eubacterial universal primer 27F1 yields a 483-bp product. Amplification with the branch I or branch II primer pairs ACB1F plus AC510R and ACB2F plus AC510R yields 325- and 361-bp PCR products, respectively. The two PCR products from each sample were pooled, and a total of 6 μl was run on a 3% agarose gel in 1× TAE with 100 ng of φX174 HaeIII as DNA marker (lanes M).

FIG. 4

Analysis of 16S rRNA gene using the A. circinalis branch I- and branch II-specific oligonucleotides. Lanes 21/02 to 21/05 correspond to PCR fragments from the environmental bloom samples collected on the dates indicated (day/month). Lanes AWQC105A and AWQC306A correspond to positive controls belonging to branch I and branch II, respectively. Lane -ve control, the negative control. Amplification with the phycocyanin primer pair PCαF plus PCβF yields a 680-bp product. Amplification with the A. circinalis-specific primer AC510R plus the cyanobacterial universal primer 27F1 yields a 483-bp product. Amplification with the branch I or branch II primer pairs ACB1F plus AC510R and ACB2F plus AC510R yields a 325- or 361-bp PCR product, respectively. The three PCR products from each sample were pooled, and a total of 6 μl was run on a 3% agarose gel in 1× TAE with 100 ng of φX174 HaeIII as DNA marker (lanes M).