| Heterosigma akashiwo is a toxic golden brown raphidophytic stramenopile that undergoes rapid proliferation in response to favorable environmental cues. Blooms of Heterosigma visibly alter the color of marine or estuarine areas to a muddy brown. Heterosigma chloroplasts modulate transcript levels for many chloroplast-encoded genes in accordance with a diurnal (12 hour light:12 hour dark) light cycle. One possible mechanism for achieving this is through regulation of transcript initiation by “two component” or “His-Asp” transcriptional regulators, which recently have been found to be encoded in plastid genomes of non-chlorophyll-b-containing algae. A putative His-Asp response regulator gene (trg1) that is 693 nucleotides in length and encodes a putative 28 kDa protein of 231 amino acids was discovered in the Heterosigma chloroplast genome. TRG1 is homologous to bacterial response regulators within the “ompR” subfamily of His-Asp transducers, and is organized identically to the prokaryotic homologues, including all of the completely conserved residues shown to be functionally constrained.; Quantitative competitive reverse transcriptase polymerase chain reaction (RT-PCR) showed that Heterosigma trg1 is transcribed at a low abundance (5 g/g total RNA), and is equally abundant at all points in the Heterosigma cell cycle. In contrast, psbA transcript is highly transcribed (60 mg/g total RNA), and most abundant in the middle of the light portion of the diel cycle.; If TRG1 protein function is similar to the well-studied ompR protein from E. coli, then DNA binding activity likely is central to its putative function as a transcriptional regulator. To test this hypothesis, we investigated whether (A) TRG1 protein is present in Heterosigma, and (B) TRG1 protein interacts with DNA. Antibodies to TRG1 detected a 28 kDa protein in protein preparations from Heterosigma . When purified overexpressed TRG1 protein was tested for DNA binding ability using random-sequence oligomer fragments, TRG1 caused the DNA fragments to become immobile. The resulting set of sequences revealed a six to seven base putative DNA binding consensus motif. This sequence appears in the Heterosigma chloroplast genome, grouped within the photosystem II genes, suggesting that TRG1 may interact with this region of the Heterosigma genome in-vivo. |
