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Electronic Supplementary Material
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Maintenance of essential amino acid synthesis pathways in Blattabacterium
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symbionts of a wood-feeding cockroach
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Gaku Tokuda1*, Liam D. H. Elbourne2*, Yukihiro Kinjo1*, Seikoh Saitoh1, Zakee Sabree3,
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Masaru Hojo1, Akinori Yamada4, Yoshinobu Hayashi5, Shuji Shigenobu6, Claudio
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Bandi7, Ian T. Paulsen2, Hirofumi Watanabe8#, Nathan Lo5#
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TBRC, University of the Ryukyus, Okinawa, Japan1, Department of Chemistry and
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Biomolecular Sciences, Macquarie University, Sydney, Australia2, Department of
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Evolution, Ecology, and Organismal Biology, Ohio State University3, Department of
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Biological Sciences, Graduate School of Bioscience and Biotechnology, Tokyo Institute
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of Technology4, School of Biological Sciences, The University of Sydney, Sydney,
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Australia5, National Institute for Basic Biology, Aichi, Japan6, DIPAV, Universita’ di
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Milano, Milano, Italy7 National Institute of Agrobiological Sciences, Tsukuba, Japan8
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*equal first authors
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#Authors for correspondence:
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nathan.lo@sydney.edu.au
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hinabe@affrc.go.jp
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Supplementary Materials and Methods
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Genome sequencing and annotation. P. angustipennis individuals were collected on Mt.
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Tsukuba, Ibaraki, Japan, in January 2010. Four adult females and final-instar larvae
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were used for DNA preparations as previously described, omitting the pulse-field gel
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electrophoresis step (1). 128,725 reads from two 454 GS Junior runs were assembled
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into 29 contigs (N50, 89 Kb) with Newbler, ver. 2.6 (2) using default settings. Of these,
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19 were successfully mapped onto the genome of Blattabacterum sp. strain Bge using
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Projector 2 (3). For preparation of DNA library used for Illumina, a total DNA was
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extracted from the fat body dissected from a single female larva using Isoplant II
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(Nippon Gene, Tokyo) followed by further purification with columns in DNeasy Plant Mini
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Kit (Qiagen, Hilden, Germany). 61.3 M reads were obtained from a 100 bp paired-end
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library using an Illumina HiSeq2000. Illumina reads that could be matched with one of
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the 19 contigs from 454 (574,354 reads) were then successfully assembled into a linear
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632 kbp sequence using Newbler, ver. 2.7. A single gap consisting of sequence repeats
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was closed by PCR and Sanger sequencing. Annotation was performed using the
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Microbial Genome Annotation Pipeline in DDBJ and manually compared with other
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strains of Blattabacterium. 16S rRNA was also detected by manual comparison with
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genomes of other Blattabacterium strains, and some genes were detected using
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BLASTN nr, Rfam (4), and BRUCE (5). The genome has been deposited in
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DDBJ/EMBL/GenBank under accession number AP012548.
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Phylogenetic analysis. Protein sequences for EngA, RpsE, GidA, ValS, FusA, Tuf, GyrA,
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MutS, InfB, RpoC, RpoB, RplB, and RplK were obtained for 43
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Bacteroidales/Flavobacteriales species and individually aligned using MAFFT
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(v.6.624b), employing the 'linsi' algorithm (6). Columns containing gapped characters
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were removed with a custom Perl script and alignments for each species were
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concatenated, resulting in a total of 7,104 characters/species. RAxML (7) was used to
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perform a maximum-likelihood phylogenetic reconstruction (JTT+I+GAMMA) with 100
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bootstrapping replicates via the CIPRES Science Gateway (8). A phylogenetic analysis
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was also performed for host taxa, based on 18S rDNA sequences retrieved from
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GenBank (Periplaneta americana: AF220572; Cryptocercus punctulatus: AF220571;
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Mastotermes darwiniensis: AF220568; Blatella germanica: AF220573; Blaberus
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giganteus: EF363234; Panesthia angustipennis spadica: AB036190). Outgroups for this
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analysis were the phasmid Agathamera crassa (Z97561) and the orthopteran Tettigonia
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viridissima (Z97587). Alignment was based on secondary structure, as previously
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described (9). Modelgenerator was used to select an appropriate model of sequence
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evolution based on the Bayesian information criterion. Phylogenetic analysis was
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performed using MrBayes 3.2.1 (10). In the Bayesian phylogenetic analysis, posterior
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distributions of parameters, including the tree topology and branch lengths, were
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estimated using Markov chain Monte Carlo (MCMC) sampling. Samples from the
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posterior distribution were drawn every 1000 MCMC steps over a total of 10,000,000
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steps. The analysis was run using four chains, comprising one cold chain and three
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heated chains. The first 10% of samples were discarded as burn-in. Acceptable mixing
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and convergence to the stationary distribution were checked by inspection of samples
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from the posterior and by calculating the standard deviation of split frequencies among
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the tree topologies.
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Supplementary Results and Discussion
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The GC content BPAA is 26.41%, and coding sequence density is 94%, with 575 protein
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coding and 40 RNA-coding genes. The latter includes 34 tRNAs for transfer of all amino
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acids, tmRNA, a signal recognition particle RNA, RNase P, and an operon containing
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16S, 5S, and 23S. Figure S1 shows the inferred metabolic reconstruction of
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Blattabacterium strain BPAA based on genome data. Most genes essential for DNA
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replication as well as mRNA transcription and translation are present. Intracellular
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bacterial symbionts are dependent upon the major sigma factor RpoD for transcription,
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but they usually lack alternative sigma factors. BPAA, like other Blattabacterium strains,
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was found to encode the sigma factor RpoN, a transcriptional regulator of genes
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involved in nitrogen assimilation that is enhancer-activated. BPAA contains gene
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complements for the production of outer membrane and cell-wall components. It is
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inferred to generate carbon precursors and ATP respectively using gluconeogenesis
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and aerobic biosynthesis.
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A key difference between BPAA and all other Blattabacterium genomes is the absence
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of any plasmid in the former. The three plasmid-based genes were present as a cluster
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in the BPAA chromosome. We confirmed this apparent integration by PCR and Sanger
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sequencing. BPAA has only two genes that are not found in any other Blattabacterium
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genome, both of which encode hypothetical proteins (BPAA_093 and BPAA_129). A
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total 9 genes in BPAA (nadD, mvaK, miaA, topA, murD, menC, rplR, rnpA, comEB)
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contain single nucleotide insertions that alter the downstream reading frame. It is
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possible that these genes no longer encode intact proteins; however, the mechanism of
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transcriptional slippage within homopolymeric regions may correct the reading frame in
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a subpopulation of transcripts that can be translated into functional enzymes (11).
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Phylogenetic analysis showed that BPAA is most closely related to strain BGIGA.
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The Blattabacterium clade was found to be a sister group to a clade containing strains of
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the endosymbiont Sulcia muelleri. Phylogenetic relationships of Blattabacterium host
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species of based on 18S rDNA were found to be equivalent to those of Blattabacterium
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strains (data not shown).
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Supplementary References
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1.
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genome characterization of the bacterial endosymbiont Blattabacterium cuenoti from the
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fat bodies of cockroaches. BMC Res Notes. 2008;1:118.
Tokuda G, Lo N, Takase A, Yamada Y, Watanabe H. Purification and partial
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et al. Genome sequencing in microfabricated high-density picolitre reactors. Nature.
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2005;437:376-380.
Margulies, M., Egholm, M., Altman, W. E., Attiya, S., Bader, J. S., Bemben, L. A.,
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3.
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mapping for efficient gap-closure of prokaryotic genome sequence assemblies. Nucleic
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Acids Res 2005;33:W560-W566.
van Hijum, S. A. F. T., Zomer, A. L., Kuipers, O. P. & Kok, J. Projector 2: contig
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4.
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Finn, R. D., Nawrocki, E. P., Kolbe, D. L., Eddy, S. R. & Bateman, A. Rfam: Wikipedia,
Gardner, P. P., Daub, J., Tate, J., Moore, B. L., Osuch, I. H., Griffiths-Jones, S.,
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clans and the “decimal” release. Nucleic Acids Res. 2011;39: D141-D145.
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transfer-messenger RNA genes in nucleotide sequences. Nucleic Acids Res.
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Laslett, D., Canback, B. & Andersson, S. BRUCE: a program for the detection of
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Analyses with Thousands of Taxa and Mixed Models”. Bioinformatics. 2006; 22:2688-
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Gateway for inference of large phylogenetic trees" in Proceedings of the Gateway
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Computing Environments Workshop (GCE), 14 Nov. 2010, New Orleans, LA pp 1 - 8.
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from multiple gene sequences indicates that termites evolved from wood-feeding
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cockroaches. Curr Biol 2000;10:801-4.
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under mixed models. Bioinformatics 2003; 19:1572-1574.
Katoh K, Asimenos G, Toh H. Multiple alignment of DNA sequences with MAFFT.
Stamatakis A. RAxML-VI-HPC: Maximum Likelihood-based Phylogenetic
Miller, M.A., Pfeiffer, W., and Schwartz, T. "Creating the CIPRES Science
Lo N, Tokuda G, Watanabe H, Rose H, Slaytor M, Maekawa K, et al. Evidence
Ronquist F & Huelsenbeck JP. MrBayes 3: Bayesian phylogenetic inference
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11.
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Gomez-Valero, L., Lundin, D., Poole, A. M. & Andersson, S. G. E. Endosymbiont gene
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functions impaired and rescued by polymerase infidelity at poly(A) tracts. PNAS.
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2008;105:14934-14939.
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Tamas, I., Wernegreen, J. J., Nystedt, B. r., Kauppinen, S. N., Darby, A. C.,
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Supplementary Figure Legends
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Figure S1. Inferred metabolic reconstruction of Blattabacterium strain BPAA based on
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genome data. Genes absent from predicted biosynthetic pathways are red. Orange,
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blue, green, and purple blocks indicate sufficient gene content to recycle nitrogen
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wastes and for production of indicated amino acids, metabolites, and vitamins,
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respectively. Asterisks indicate essential amino acid pathways missing in CPU and
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MADAR.
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Figure S2. Phylogenetic relationship of BPAA (in bold) to other Blattabacterium cuenoti
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strains as well as other members of the Flavobacteriales. Names of Blattabacterium
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strains are shown with host species. The tree was inferred from an alignment of 13
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protein coding genes from the strains shown. Bootstrap values for nodes <95% are
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indicated; all unlabeled nodes were supported with bootstrap values at or above 95%.
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Species for which only draft genomes are available are noted with asterisks. A
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phylogenetic analysis of host species for each Blattabacterium strain shown based on
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16S rRNA gene sequences provided an equivalent topology to that shown for
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Blattabacterium strains (data not shown).
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Figure S1
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Figure S2