A Comprehensive Description and Evolutionary Analysis of 22 Grouper (Perciformes, Epinephelidae) Mitochondrial Genomes with Emphasis on Two Novel Genome Organizations

<div><p>Groupers of the family Epinephelidae are a diverse and economically valuable group of reef fishes. To investigate the evolution of their mitochondrial genomes we characterized and compared these genomes among 22 species, 17 newly sequenced. Among these fishes we identified three distinct genome organizations, two of them never previously reported in vertebrates. In 19 of these species, mitochondrial genomes followed the typical vertebrate canonical organization with 13 protein-coding genes, 22 <i>tRNAs</i>, two <i>rRNAs</i>, and a non-coding control region. Differing from this, members of genus <i>Variola</i> have an extra <i>tRNA-Ile</i> between <i>tRNA-Val</i> and <i>16S rRNA</i>. Evidence suggests that this evolved from <i>tRNA-Val</i> via a duplication event due to slipped strand mispairing during replication. Additionally, <i>Cephalopholis</i><i>argus</i> has an extra <i>tRNA-Asp</i> in the midst of the control region, likely resulting from long-range duplication of the canonical <i>tRNA-Asp</i> through illicit priming of mitochondrial replication by tRNAs. Along with their gene contents, we characterized the regulatory elements of these mitochondrial genomes’ control regions, including putative termination-associated sequences and conserved sequence blocks. Looking at the mitochondrial genomic constituents, <i>rRNA</i> and <i>tRNA</i> are the most conserved, followed by protein-coding genes, and non-coding regions are the most divergent. Divergence rates vary among the protein-coding genes, and the three cytochrome oxidase subunits (<i>COI, II, III</i>) are the most conserved, while NADH dehydrogenase subunit 6 (<i>ND6</i>) and the ATP synthase subunit 8 (ATP8) are the most divergent. We then tested the phylogenetic utility of this new mt genome data using 12 protein-coding genes of 48 species from the suborder Percoidei. From this, we provide further support for the elevation of the subfamily Epinephelinae to family Epinephelidae, the resurrection of the genus <i>Hyporthodus</i>, and the combination of the monotypic genera <i>Anyperodon</i> and <i>Cromileptes</i> to genus <i>Epinephelus</i>, and <i>Aethaloperca</i> to genus <i>Cephalopholis</i><i>.</i></p> </div

Schematic structures of mitochondrial control regions.

<p>ETAS, extended termination associated sequences; CCD, central conserved domain; CBSs, conserved sequence blocks. This figure does not show the structure for the CR of <i>Cephalopholis argus</i>.</p

Sequence variations among mitochondrial genes.

<p>Genes were ranked by their sequence identity percentages from low to high (left to right). (A) Sequence identities of 22 tRNA genes. (B) NT% (dark grey) and deduced AA% (light grey) of 13 protein-coding genes. Genes were ranked by the AA%.</p

Gene maps for mitochondrial genomes.

<p>Genes encoded on the heavy and light strand are shown outside and inside the circle, respectively. The inner grey ring indicates the GC content. This genome map was constructed via OrganellarGenomeDRAW [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0073561#B81" target="_blank">81</a>] with manual modifications. (A) The mt gemone organization of <i>Aethaloperca rogaa</i>, <i>Anyperodon leucogrammicus</i>, <i>Cephalopholis sonnerati</i>, <i>Cromileptes altivelis</i>, <i>Epinephelus akaara</i>, <i>E</i>. <i>areolatus</i>, <i>E</i>. <i>awoara</i>, <i>E</i>. <i>bruneus</i>, <i>E</i>. <i>coioides</i>, <i>E</i>. <i>epistictus</i>, <i>E</i>. <i>fuscoguttatus</i>, <i>E</i>. <i>lanceolatus</i>, <i>E</i>. <i>moara</i>, <i>E</i>. <i>trimaculatus</i>, <i>Hyporthodus octofasciatus</i>, <i>H</i>. <i>septemfasciatus</i>, <i>Plectropomus areolatus</i>, <i>P</i>. <i>leopardus</i>, and <i>Triso dermopterus</i>. (B) The mt gemone organization of genera <i>Variola, V</i>. <i>albimarginata</i> and <i>V</i>. <i>louti</i>. (C) The mt gemone organization of <i>Cephalopholis argus</i>.</p

Phylogenetic tree of 22 groupers in family Epinephelidae and 29 representatives from other families in suborder Percoidei.

<p>A species from the suborder Labrodei family Labridae, <i>Pseudolabrus sieboldi</i>, was selected as outgroup. Congruent tree topology was inferred from partitioned Maximum likelihood and Bayesian analyses using the concatenated nucleotide sequences of 12 mitochondrial protein-coding genes (excluding <i>ND6</i>). The Bayesian posterior probability values (top) and bootstrap values (bottom) are labeled at branch nodes. Branch length information from the Bayesian tree is shown. NCBI RefSeq or GenBank accession number of each species was listed on the right of the species name. Clade A indicates the derived epinephelid clade whose <i>ATP6</i> start codon is not ATG but CTG or TTG, different from most other teleosts and basal groupers.</p

The essential function of the Crb2 SQ/TQ cluster is to mediate a phosphorylation-dependent interaction with Chk1.

<p>(A and B) A 19-amino-acid peptide containing the two conserved SQ/TQ motifs, Crb2(67–85), can bind directly to Chk1 when either T73 or S80 is phosphorylated. Chk1 tagged at its C-terminus with YFP-Flag-His6 (YFH) tag was immunoprecipitated from fission yeast cell extract with anti-Flag beads, eluted with Flag peptide and incubated separately with four types of biotin-labeled Crb2(67–85) peptide, un-phosphorylated (un-P), phosphorylated on T73 alone, S80 alone or both. Peptides were pulled down by streptavidin Dynabeads and eluted by boiling in SDS loading buffer. (A) In one experiment, eluates and input of the peptide pull-down assay were analyzed by 4%–20% SDS-PAGE followed by Coomassie staining. (B) In another experiment, eluates and serial dilutions of Chk1-YFH input were examined by immunoblotting with an anti-GFP antibody. The strain used was DY485. (C) DNA damage sensitivity caused by <i>crb2-2AQ</i> mutation can be fully rescued by fusing Crb2 with Chk1 kinase. Spot assay was performed as in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002817#pgen-1002817-g002" target="_blank">Figure 2B</a>. Strains used were DY6508, DY6509, DY809, DY6507, DY6510 and DY6511.</p

Two conserved SQ/TQ motifs in the N-terminal region of Crb2 are essential for Chk1 recruitment and activation.

<p>(A) Sequence alignment of <i>S. pombe</i> Crb2 and its orthologs from three other fission yeast species revealed two conserved neighboring SQ/TQ motifs in the N-terminal region of Crb2. The positions of the two motifs in <i>S. pombe</i> Crb2 are labeled on top. (B) Mutations in Crb2 SQ/TQ cluster resulted in DNA damage hypersensitivity. Fivefold serial dilutions of cells were spotted on YES plates and incubated at 30°C. Photos were taken 2 d later for untreated, UV-treated, IR-treated and CPT-containing plates. The HU-containing plates were photographed 3 d later. Strains used were LD195, LD346, DY377, DY369, DY370 and DY371. (C) DNA damage-induced Chk1 phosphorylation is defective in Crb2 SQ/TQ cluster mutants. Cells were untreated or treated with 20 µM CPT for 2 h. Cell lysates were separated on SDS-PAGE and probed with an anti-Myc antibody by immunoblotting. Strains used were DY377, LD195, DY369, DY370 and DY371. (D) Mutations in Crb2 SQ/TQ cluster diminished Chk1 foci but not Crb2 foci. Cells expressing Chk1-GFP and CFP-Crb2 were challenged with S-phase IR treatment as in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002817#pgen-1002817-g001" target="_blank">Figure 1A</a> and examined by fluorescence microscopy. Arrows indicate dim Chk1 foci in <i>crb2-T73A</i> and <i>crb2-S80A</i> cells. Strains used were DY6503, DY6504, DY6505 and DY6506. Bar, 5 µm.</p

A model of how Crb2 mediates Chk1 activation.

<p>In step 1, DSB formation induces the phosphorylation of H2A (<b>γ</b>-H2A) on surrounding chromatin. Upon DSB resection, Rad3 and 9-1-1 are recruited to single-stranded DNA and single-strand/double-strand junction, respectively. Rad4/Cut5 is also recruited via binding to Rad9. In step 2, through its interactions with modified histones and Rad4/Cut5, Crb2 relocalizes to the DSB and becomes phosphorylated at the SQ/TQ cluster by Rad3. In step 3, phosphorylated SQ/TQ cluster interacts with Chk1 and promotes its phosphorylation by Rad3. In step 4, the activated Chk1 molecule leaves the DSB to fulfill its effector function and allows further rounds of Chk1 activation to occur.</p

Fusing the Crb2(67–85) peptide with Rad4/Cut5 is sufficient for Chk1 recruitment and activation in the absence of endogenous Crb2.

<p>(A) Fusing Crb2(67–85) with Rad4/Cut5 bypasses Crb2 for Chk1 focus formation. Cells were challenged with S-phase IR treatment as in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002817#pgen-1002817-g001" target="_blank">Figure 1A</a> and then examined by fluorescence microscopy. Strains used were DY6512, DY6513 and DY6514. Bar, 5 µm. (B) Fusing Crb2(67–85) with Rad4/Cut5 bypasses Crb2 for checkpoint activation. The <i>cdc25-22</i> block-and-release assay was performed as in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002817#pgen-1002817-g005" target="_blank">Figure 5F</a>. Strains used were DY6546, DY6547, DY6548 and DY6549. (C) DNA damage sensitivity of <i>crb2Δ</i> is fully rescued by Cut5-Crb2(67–85) fusion. Spot assay was performed as in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002817#pgen-1002817-g002" target="_blank">Figure 2B</a>. Strains used were DY6517, DY6518, DY6519, DY6520, DY6512, DY6513 and DY6514. (D) Chk1 phosphorylation defect of <i>crb2Δ</i> is rescued by Cut5-Crb2(67–85) fusion. Cells were treated with 20 µM CPT for 2 h. Cell lysates were separated on SDS-PAGE and probed with anti-Myc antibody. Strains used were DY6521, DY6522, DY6523 and DY6524.</p

DNA damage-induced Chk1 focus formation requires the N-terminal 275 amino acids of Crb2.

<p>(A) Chk1-GFP forms nuclear foci at IR- and HO-induced DSBs. For IR treatment, cells expressing Chk1-GFP and CFP-Crb2 were either treated with 80 Gy IR and incubated for 3 h, or first arrested in early-S phase by a 4-h treatment of 20 mM hydroxyurea (HU), and then treated with 80 Gy IR before releasing into HU-free medium and incubated for 3 h (S-phase IR treatment). For HO endonuclease induction, cells expressing Chk1-GFP and Rad22-CFP were shifted to thiamine-free medium for 16 h to induce the expression of HO, which is under the control of the thiamine-repressible <i>nmt1</i> promoter. Strains used were DY6498 and DY6502. Bar, 5 µm. Inset, higher magnification of cells containing Chk1 foci. (B) Quantitation of Chk1 foci in (A). About 200 nuclei were scored for each condition. (C) The N-terminal region of Crb2 is required for Chk1-GFP foci. Cells expressing Chk1-GFP and Rad22-mCherry in wild type (WT), <i>crb2Δ</i>, <i>crb2(1–358)-LZ</i> or <i>crb2(276–778)</i> background were challenged with S-phase IR treatment as in (A). Strains used were DY6498, DY6497, DY6499 and DY6500. Bar, 5 µm.</p