From the Cover: Are Pfiesteria species toxicogenic? Evidence against production of ichthyotoxins by Pfiesteriashumwayae -- Data Supplement - Supporting Information -- Proceedings of the National Academy of Sciences

Supporting information for Berry et al. (2002) Proc. Natl. Acad. Sci. USA, 10.1073/pnas.172221699

Supporting Text

In the interest of being completely thorough in our search for the genetic machinery for toxin production, we used primers for nonribosomal peptide synthase (NRPS) type I and II genes. The NRPS primers were designed from consensus sequences of adenylation domains of several known NRPSs including microcystin. For NRPSI, the anticipated product is 600 bp; for NRPSII, the anticipated product is 1,000 bp. As described in the published text, one of the bands found in these gels showed homology to a putative fatty acid synthase/polyketide synthase (PKS). The following text describes the experiments to characterize the PCR-amplification products.

Methods
Cloning and Sequencing of NRPSI and NRPSII Amplification Products.
Amplification products obtained using the NRPSII primers were cloned into the plasmid vector pCR2.1 using the TA cloning kit (Invitrogen). Plasmid DNA from overnight cultures of individual clones was obtained using the Qiagen (Valencia, CA) plasmid-prep protocol, and inserts were sequenced by simultaneous bidirectional cycle sequencing using the Thermo sequenase sequencing kit (Amersham Pharmacia) and infrared-labeled (IRD700 or IRD 800) M13 primers (LI-COR) following the manufacturer’s protocol.
PCR with Nested PKS Primers.
Based on the sequence of a 401-bp amplification product (GenBank accession no. AF525290) with homologies to PKSs, nested PCR primers were designed to amplify a 227-bp fragment. The primers RL-PshumPKSF and RL-PshumPKSR (5’-CAAGACCAGTGCGAAGTCCAAC-3’/5’-ACCAAAACCGAAGAGGAGACCC-3’) were used to attempt amplification from genomic DNAs and cDNAs isolated from the Center for Culture of Marine Phytoplankton (CCMP) 2089 culture, the Rhodomonas salina culture, and artificial seawater in which fish had been incubated to control for contaminating organisms potentially brought into the experiments with the fish. Amplification products obtained using the nested PKS primers were cloned and sequenced as described above. Results

Nested primers were designed based on the sequence of the PCR fragment with homology to PKSs to specifically target that DNA fragment. The primers were used to confirm the presence of this sequence in genomic DNA and cDNA isolations from Pfiesteria shumwayae and to test for the possibility that this amplification product was obtained from R. salina DNA or another contaminant DNA. Amplification products of the expected size (227 bp) were obtained from P. shumwayae genomic DNA, P. shumwayae cDNA, and cDNA isolated from the medium after exposure of fish to cultured P. shumwayae (Fig. 5). This product was not observed in genomic or cDNA isolated from the R. salina culture (data not shown) or in cDNA isolated from the artificial seawater after incubation with fish (Fig. 5). The sequence of the 227-bp amplification products obtained using the nested primers matched the sequence of the initial PCR product with homology to PKSs.

Discussion

We amplified a PKS-like gene fragment with the forward NRPSII primer and confirmed its presence (and expression) in the P. shumwayae genome using the nested primer pair. However, sequencing of the full-length gene and further characterization will be needed before the function of this enzyme can be deduced. For further discussion of the relevance of this finding, see the main article.