Comparative gene expression in toxic versus non-toxic strains of the marine dinoflagellate Alexandrium minutum

Abstract

Background: The dinoflagellate Alexandrium minutum typically produces paralytic shellfish poisoning (PSP) toxins, which are known only from cyanobacteria and dinoflagellates. While a PSP toxin gene cluster has recently been characterized in cyanobacteria, the genetic background of PSP toxin production in dinoflagellates remains elusive.

Results: We constructed and analysed an expressed sequence tag (EST) library of A. minutum, which contained 15,703 read sequences yielding a total of 4,320 unique expressed clusters. Of these clusters, 72% combined the forward-and reverse reads of at least one bacterial clone. This sequence resource was then used to construct an oligonucleotide microarray. We analysed the expression of all clusters in three different strains. While the cyanobacterial PSP toxin genes were not found among the A. minutum sequences, 192 genes were differentially expressed between toxic and non-toxic strains.

Conclusions: Based on this study and on the lack of identified PSP synthesis genes in the two existent Alexandrium tamarense EST libraries, we propose that the PSP toxin genes in dinoflagellates might be more different from their cyanobacterial counterparts than would be expected in the case of a recent gene transfer. As a starting point to identify possible PSP toxin-associated genes in dinoflagellates without relying on a priori sequence information, the sequences only present in mRNA pools of the toxic strain can be seen as putative candidates involved in toxin synthesis and regulation, or acclimation to intracellular PSP toxins.

Figure 1

KOG-based functional classification of 1005 contigs as obtained by BLAST-identified similarities.

Figure 2

Increase of cell number and culture toxin content in strain AL3T over 24 hours. Grey boxes = dark phase, circles = cell numbers, diamonds = total PSP toxins mL^-1.

Figure 3

Extracted ion traces of LC-MS/MS chromatograms of the A. minutum strains AL1T, AL3T and AL9T. Top: transition 396 > 288 for GTX2/3 (retention time GTX3: 14.1 min); bottom: transition 412 > 314 for GTX1/4 (retention time GTX4 13.3 min)

Figure 4

Comparison of expression ratios based on microarray- and qPCR- data. Array data: more than 15× higher expressed in toxic strains, ++; more than 5×, +; more than 5× higher expressed in non-toxic strain, -; 2-5× higher expressed in non-toxic strain, (-); raw data see supplementary table 1. qPCR: Ct < 25, ++; Ct<27, +; Ct>27, -; N.D., not detected at Ct < 35.