Open Access Research article

Loss of genes for DNA recombination and repair in the reductive genome evolution of thioautotrophic symbionts of Calyptogena clams

Hirokazu Kuwahara12, Yoshihiro Takaki1, Shigeru Shimamura1, Takao Yoshida1, Taro Maeda3, Takekazu Kunieda2 and Tadashi Maruyama1*

Author Affiliations

1 Marine Biodiversity Research Program, Japan Agency for Marine-Earth Science and Technology, Natsushima-cho, Yokosuka, Kanagawa 237-0061, Japan

2 Department of Biological Sciences, Graduate School of Science, The University of Tokyo, Hongo, Bunkyo-ku, Tokyo 113-0033, Japan

3 Graduate School of Marine Science and Technology, Tokyo University of Marine Science and Technology, Konan, Minato-ku, Tokyo 108-8477, Japan

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BMC Evolutionary Biology 2011, 11:285  doi:10.1186/1471-2148-11-285

Published: 3 October 2011

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Additional file 1:

Figure S1. Multiple sequence alignments of recA-amplicons from Calyptogena clam symbiont genomes. The recA-containing genome region (recA-amplicon) was amplified with a primer set [recA_F (5'-GATTGCATATCATTCATCTGATAACG-3'), recA_R (5'-AGTGGATTRGGATCAAGCATAGC-3')] from 9 Calyptogena clam symbionts using the PCR and from 2 symbiont genomes [Vesicomyosocius okutanii (Vok: accession # = AP009247) and Ruthia magnifica (Rma: accession # = CP000488)] in in silico PCR. Abbreviations of symbionts are shown in Table 3. Gray background-colored horizontal sequence in Cpha S, part of ribD; blue background-colored horizontal sequence in Cpha S, recA; light gray background horizontal sequence in Cpha S, recX; brown background horizontal sequence of Cpha S, part of ABC-t (ABC transporter ATP-binding protein gene). *Identical nucleotide in the aligned sequences. Gray background vertical column, gap in Cpha S sequence. Red letters, in-frame start codon of recA or mutated recA ORFs. Blue letters, in-frame stop codon.

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Additional file 2:

Figure S2. Multiple sequence alignment of mutY-amplicons from Calyptogena clam symbiont genomes. The mutY-containing genome region (mutY-amplicon) was amplified with a primer set [mutY_F and mutY_R (Table 2)] from 9 Calyptogena clam symbionts in PCR and from 2 symbiont genomes [Vesicomyosocius okutanii (Vok: accession # = AP009247) and Ruthia magnifica (Rma: accession # = CP000488)] in in silico PCR. Abbreviations of symbionts are shown in Table 3. Gray background horizontal sequence in Cpha S, part of metA (homoserine O-succinyltransferase); blue background sequence in Cpha S, mutY. *Identical nucleotide in the aligned sequences. Gray vertical column, gap in Cpha S sequence. Red letters, in-frame start codon of the original or mutated mutY ORFs. Blue letters, in-frame stop codon.

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Additional file 3:

Figure S3. Part A. 3D homology models reconstructed for RecA of the Calyptogena phaseoliformis symbiont. Homology modeling using the Swiss-Model Workspace (http://swissmodel.expasy.org/ webcite) was based on the 3D structure of Escherichia coli RecA (PDB accession number 1U94: [24]) as a template. A, Alignment of amino acid sequences of Calyptogena clam symbiont RecA and E. coli. RecA. Sequences were aligned with ClustalW. a, RecA sequence from Lys-6 to Pro-331 in E. coli; b, secondary structure of E. coli RecA (1U94). Red rectangles, α-helices; blue arrows, β-strands. c, RecA sequence from Lys-5 to Thr-329 in the symbiont of C. phaseoliformis. d, RecA sequence of N-terminal ORF in the symbiont of C. fausta. e, RecA sequence of C-terminal ORF in the symbiont of C. fausta.

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Additional file 4:

Figure S3. Parts B-E. 3D homology models reconstructed for RecA of the Calyptogena phaseoliformis symbiont. Homology modeling using the Swiss-Model Workspace (http://swissmodel.expasy.org/ webcite) was based on the 3D structure of Escherichia coli RecA (PDB accession number 1U94: [24]) as a template. B, Homology model reconstructed for C. phaeoliformis symbiont RecA. C, Homology model reconstructed for N-terminal and C-terminal amino acid peptides of RecA in C. fausta symbiont. N-terminal and C-terminal peptides are shown in violet and light blue, respectively. D, Crystal structure of E. coli RecA (accession # = 1U94); α-helices and β-sheets are indicated in red-green, and light blue, respectively. E, Merged 3D structures of RecAs of E. coli (D) and of C. phaseoliformis symbionts (B) showing that their 3D structures are nearly the same. This suggests that the C. phaseoliformis symbiont RecA is intact and functional.

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Additional file 5:

Figure S4. Part A. 3D homology models reconstructed for MutY of the C. phaseoliformis symbiont. The model was reconstructed using the Swiss-Model Workspace (http://swissmodel.expasy.org/ webcite) based on the crystal structure of Geobacillus stearothermophilus MutY [26] (PDB accession # = 3FSP) as a template. A, Alignment of MutY amino acid sequences of Calyptogena symbionts and G. stearothermophilus. Sequences were aligned using ClustalW. a, MutY sequence from Phe-8 to Ser-360 in G. stearothermophilus. b, Secondary structure of G. stearothermophilus MutY (3FSP). Red rectangles, α-helices; blue arrows, β-strands. c, MutY sequence from Val-1 to Asp-341 in the symbiont of C. phaseoliformis. d, MutY sequence of N-terminal ORF in Ruthia magnifica. e, MutY sequence of C-terminal ORF in R. magnifica.

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Additional file 6:

Figure S4. Parts B-E. 3D homology models reconstructed for MutY of the C. phaseoliformis symbiont. B, Homology model reconstructed for C. phaseoliformis symbiont MutY. C, Homology model reconstructed for N-terminal (violet) and C-terminal (light blue) amino acid peptides of MutY in R. magnifica. D, Crystal structure of G. stearothermophilus MutY (accession # = 3FSP). α-Helices and β-sheets are indicated as red-green and light blue, respectively. E, Merged 3D structures of MutY of G. stearothermophilus (D) and C. phaseoliformis symbionts (B).

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Additional file 7:

Figure S5. Phylogenetic tree of the Calyptogena clam symbionts including those of reported in Stewart et al. 2009 [28]. 16S and 23S rRNA gene sequences of the symbionts reported in the present study and of those reported in Stewart et al. (2009) [28] were concatenated and used for phylogenetic tree reconstruction. Topology of the tree constructed using the maximum likelihood method is shown with bootstrap values (> 50%) obtained from the neighbor joining and maximum likelihood methods at each node. Accession numbers of the sequences are shown in the tree. Names and abbreviations of the symbionts are the same as those in Table 3 of the present study or those in [28]. Abbreviations for generic names: C., Calyptogena; E., Ectenagena; V., Vesicomya. *Symbionts reported in Stewart et al. [28]; **symbionts reported in both Stewart et al. [28] and the present study.

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