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. 2007 Jan 16;104(3):696-701.
doi: 10.1073/pnas.0607816103. Epub 2006 Nov 27.

Two different domains of the luciferase gene in the heterotrophic dinoflagellate Noctiluca scintillans occur as two separate genes in photosynthetic species

Liyun Liu  1 , J Woodland Hastings
Affiliations

Two different domains of the luciferase gene in the heterotrophic dinoflagellate Noctiluca scintillans occur as two separate genes in photosynthetic species

Liyun Liu et al. Proc Natl Acad Sci U S A. .

Erratum in

  • Proc Natl Acad Sci U S A. 2011 Aug 23;108(34):14371

Abstract

Noctiluca scintillans, a heterotrophic unarmored unicellular bioluminescent dinoflagellate, occurs widely in the oceans, often as a bloom. Molecular phylogenetic analysis based on 18S ribosomal DNA sequences consistently has placed this species on the basal branch of dinoflagellates. Here, we report that the structural organization of its luciferase gene is strikingly different from that of the seven luminous species previously characterized, all of which are photosynthetic. The Noctiluca gene codes for a polypeptide that consists of two distinct but contiguous domains. One, which is located in the N-terminal portion, is shorter than but similar in sequence to the individual domains of the three-domain luciferases found in all other luminous dinoflagellates studied. The other, situated in the C-terminal part, has sequence similarity to the luciferin-binding protein of the luminous dinoflagellate Lingulodinium polyedrum, encoded there by a separate gene. Western analysis shows that the native protein has the same size (approximately 100 kDa) as the heterologously expressed polypeptide, indicating that it is not a polyprotein. Thus, sequences found in two proteins in the L. polyedrum bioluminescence system are present in a single polypeptide in Noctiluca.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.
Fig. 1.
A schematic representation showing the genomic structure of Ns lcf genes and the domain organization of their predicted proteins in comparison with the second domain of Lp LCF and Lp LBP. At least two copies of the Ns lcf genes are tandemly arranged in a head-to-tail fashion, each consisting of 4,360 bp. The precise boundary between the intergenic region and 5′ UTR was not precisely mapped, and together they account for 1,518 bp. The 3′ UTR of the Ns lcf is only 97 bp long, therefore, much shorter than the 3′ UTRs of lcf genes of seven photosynthetic dinoflagellates, which range from 194 to 432 bp. The largest ORF of Ns lcf predicts a protein of 914 aa with two major domains, an N-terminal luciferase-like (LCF-like) domain and a C-terminal luciferin-binding protein-like (LBP-like) domain. The LCF-like domain shares a 56% sequence identity with but is shorter at the N terminus by 60 aa residues than the individual domains of Lp LCF. The LBP-like domain of 591 aa residues is 41% identical with the comparable region of Lp LBP. No significant similarity was found between the most N-terminal sequence of 29 aa residues of the Ns LCF and any sequences in the database.
Fig. 2.
Fig. 2.
A distance tree based on the protein sequences and generated by using a MEGA program as described in Materials and Methods. (Scale bar: 0.05 substitutions per site.) The numbers at the nodes of the tree are bootstrap values, which measure the robustness of the support for each particular node.
Fig. 3.
Fig. 3.
An alignment of four internal repeats of Ns and Lp LBPs. The residues shaded in red are identical in a majority of the sequences, whereas those in green are conserved in a majority. Arrows mark the charged residues, which are present in all of the sequences compared.
Fig. 4.
Fig. 4.
A phylogenetic tree showing a relationship of duplicated repeats of Ns LBP-like domain and Lp LBP. On this tree, corresponding domains of the LBPs of different species are grouped together. All nodes are well supported except for the one that defines the clade of the second repeats, which has a bootstrap value of only 39%. (Scale bar: 0.1 substitutions per site.)
Fig. 5.
Fig. 5.
Western blotting analysis of the native and recombinant Ns LCFs. Native Ns LCF was isolated from frozen cells and, after SDS/PAGE separation and blotting onto the nitrocellulose membrane, was probed by either anti-Lp luciferase (lane 1) or anti-Lp LBP (lane 2). FLAG-tagged Ns full-length LCF (lane 3) and its LBP-like domain (lane 4) were expressed in SF21 cells by using a bacuolovirus expression system and extracted for Western blotting analysis by using anti-FLAG antibody. Each of the lanes received ≈10 μg of total protein. Relative intensities of the bands shown in lane 3 and 4 correspond to the colors indicated by the color scale lane.
Fig. 6.
Fig. 6.
pH dependence of native and recombinant Noctiluca luciferases. Luciferase activity of the cell extract is strongly pH-dependent (A), whereas activity of the expressed luciferase-like domain with Pl luciferin is less so (B).
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