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Complete genome sequence of Syntrophothermus lipocalidus type strain (TGB-C1T)
Standards in Genomic Sciences volume 3, pages267–275(2010)
Syntrophothermus lipocalidus Sekiguchi et al. 2000 is the type species of the genus Syntrophothermus. The species is of interest because of its strictly anaerobic lifestyle, its participation in the primary step of the degradation of organic maters, and for releasing products which serve as substrates for other microorganisms. It also contributes significantly to maintain a regular pH in its environment by removing the fatty acids through β-oxidation. The strain is able to metabolize isobutyrate and butyrate, which are the substrate and the product of degradation of the substrate, respectively. This is the first complete genome sequence of a member of the genus Syntrophothermus and the second in the family Syntrophomonadaceae. Here we describe the features of this organism, together with the complete genome sequence and annotation. The 2,405,559 bp long genome with its 2,385 protein-coding and 55 RNA genes is a part of the Genomic Encyclopedia of Bacteria and Archaea project.
Strain TGB-C1T (= DSM 12680) is the type strain of Syntrophothermus lipocalidus  which in turn is the type species of the genus Syntrophothermus . Currently, this is the only species placed in the genus Syntrophothermus. The genus name derives from the Greek words “syn”, together with, “trophos”, one who feeds, and “thermus”, hot, referring to a thermophilic bacterium growing in syntrophic association with hydrogenotrophic organisms at high temperature of around 55°C . The species epithet derives from the Greek word “lipos”, fat, and from the Latin adjective “calidus”, expert, referring to the organisms trait of specifically utilizing fatty acids . Strain TGB-C1T was isolated from granular sludge in a thermophilic upflow anaerobic sludge blanket (UASB) . No further cultivated strains belonging to the species S. lipocalidus have been described so far. Here we describe the features of this organism, together with the complete genome sequence and annotation.
Classification and features
The 16S rRNA gene sequence of strain TGB-C1T revealed an only distant relationship with the other representatives of the family Syntrophomonadaceae  (Figure1), with Thermosyntropha lipolytica  showing the highest degree of sequence similarity (88.1%). The sequence distances of strain TGB-C1T to other members of this family were 13.6% with Syntrophomonas wolfei subsp. wolfei, 14.0% with S. bryantii, and 14.8% with S. sapovorans, respectively . Further analysis showed 98% 16S rRNA gene sequence identity with an uncultured bacterium represented by clone AR80B63 (AB539943) from the high-temperature Yabase oil field in Japan. The sequence of the 16S rRNA gene of strain TGB-C1T is identical with two unclassified sequences from an hydrothermal vent metagenome LCHCB.C3615  and from human gut metagenome DNA (contig sequence: F2-Y_011332)  (status August 2010), indicating that members of the species, genus and even family are widely represented in the habitats screened so far.
A representative genomic 16S rRNA sequence of S. lipocalidus TGB-C1T was also compared using BLAST with the most resent release of the Greengenes database  and the relative frequencies of taxa and keywords, weighted by BLAST scores, were determined. The five most frequent genera were Moorella (44.1%), Syntrophomonas (33.8%), Clostridium (6.0%), Syntrophothermus (5.6%) and Carboxydocella (3.5%). The species yielding the highest score was Moorella thermoautotrophica. The five most frequent keywords within the labels of environmental samples which yielded hits were ‘microbial’ (5.5%), ‘anaerobic’ (4.2%), ‘rice’ (2.9%), ‘soil’ (2.8%) and ‘populations’ (2.8%). The three most frequent keywords within the labels of environmental samples which yielded hits of a higher score than the highest scoring species were ‘temperature’ (8.2%), ‘acetate, coupled, evidence, field, hydrogenotrophic, methanogenesis, oil, oxidation, petroleum, reservoir, syntrophic, yabase’ (5.0%) and ‘dependent, hot, muddy, reducing, sediment, southwestern, spring, succession, sulfate, taiwan’ (3.2%). These keywords largely fit to what is known about the ecology and physiology of strain TGB-C1T .
Figure 1 shows the phylogenetic neighborhood of S. lipocalidus TGB-C1T, in a 16S rRNA based tree. The sequences of the two 16S rRNA gene copies in the genome differ from each other by up to two nucleotides, and differ by up to two nucleotides from the previously published 16S rRNA sequence (AB021305).
Cells of strain TGB-C1T are Gram-negative, slightly curved rods with round ends and weakly motile with flagella, 2.4–4.0 µm long and 0.4–0.5 µm wide (Figure 2 and Table 1) , occurring singly or in pairs. Roll-tube isolation revealed the presence of small white colonies, lens-shaped and 0.1–0.2 mm in diameter . The growth rate of the strain TGB-C1T on 10 mM crotonate was 0.93 ± 0.01 d−1. Strain TGB-C1T is strictly anaerobic . It grows on crotonate at temperatures between 45°C and 60°C, with the optimum at 55°C. The pH25°C range for growth is 5.8–7.5, with an optimum at 6.5–7.0 . Strain TGB-C1T metabolizes in two ways, in pure culture only in the presence of the unsaturated fatty acid crotonate and in co-culture with Methanobacterium thermoautotrophicum strain ΔH in the presence of saturated fatty acids . In pure culture, the fermentation products are acetate and butyrate in equimolar amounts. In co-culture with M. thermoautotrophicum, the substrates used are butyrate, straight-chain fatty acids from C4 to C10 and isobutyrate . By oxidizing fatty acids, S. lipocalidus produces acetate and hydrogen , the latter of which is then scavenged by the syntrophic methanogen M. thermoautotrophicum . Syntrophic hydrogenotrophic interactions with bacteria from the genus Methanobacterium have been also observed in the genome sequenced bacterium Aminobacterium colombiense strain ALA-1T from the phylum Synergistetes . S. lipocalidus is the only species in the family Syntrophomonadaceae that is able to metabolize isobutyrate . Neither yeast extract nor tryptone significantly stimulates growth . In the presence of butyrate as electron donor, the following compounds do not serve as electron acceptors: sulfate, nitrate, sulfite, thiosulfate, fumarate, Fe(III)-nitrilotriacetate . Cell growth is inhibited by ampicillin, chloramphenicol, kanamycin, neomycin, rifampin or vancomycin (each 50 µg ml−1) .
To date, no experimental reports have specified the lipid composition of the cell envelope of strain TGB-C1T. Nevertheless, the cell envelope of the strain TGB-C1T was Gram-negative stained, although electron micrographs and the 16S rRNA analysis showed that the strain was affiliated to the Gram-positive bacteria . This feature was also observed for another member of the family Syntrophomonadaceae, S. bryantii [22,27]. The cell envelope is composed of the cytoplasmic membrane, an electron-dense layer, which is most probably made of peptidoglycan, and an electron-dense outermost wall .
Genome sequencing and annotation
Genome project history
This organism was selected for sequencing on the basis of its phylogenetic position , and is part of the Genomic Encyclopedia of Bacteria and Archaea project . The genome project is deposited in the Genome OnLine Database  and the complete genome sequence is deposited in GenBank. Sequencing, finishing and annotation were performed by the DOE Joint Genome Institute (JGI). A summary of the project information is shown in Table 2.
Growth conditions and DNA isolation
S. lipocalidus TGB-C1T, DSM 12680, was grown anaerobically in DSMZ medium 870 (Syntrophothermus medium)  at 55°C. DNA was isolated from 0.5–1 g of cell paste using the Jetflex Genomic DNA Purification kit (GENOMED 600100) following the standard protocol as recommended by the manufacturer, with 30 min incubation at 58°C for cell lysis.
Genome sequencing and assembly
The genome was sequenced using a combination of Illumina and 454 sequencing platforms. All general aspects of library construction and sequencing can be found at the JGI website . Pyrosequencing reads were assembled using the Newbler assembler version 2.1-PreRelease-4-28-2009-gcc-3.4.6-threads (Roche). The initial Newbler assembly consisting of 16 contigs in one scaffold was converted into a phrap assembly by making fake reads from the consensus, collecting the read pairs in the 454 paired end library. Illumina GAii sequencing data (704 Mb) was assembled with Velvet  and the consensus sequences were shredded into 1.5 kb overlapped fake reads and assembled together with the 454 data. 454 draft assembly was based on 248.9 Mb 454 draft data and all of the 454 paired end data. Newbler parameters are -consed -a 50 -l 350 -g -m -ml 20. The Phred/Phrap/Consed software package  was used for sequence assembly and quality assessment in the following finishing process. After the shotgun stage, reads were assembled with parallel phrap (High Performance Software, LLC). Possible mis-assemblies were corrected with gapResolution , Dupfinisher, or sequencing cloned bridging PCR fragments with subcloning or transposon bombing (Epicentre Biotechnologies, Madison, WI) . Gaps between contigs were closed by editing in Consed, by PCR and by Bubble PCR primer walks (J.-F.Chang, unpublished). A total of 37 additional reactions were necessary to close gaps and to raise the quality of the finished sequence. Illumina reads were also used to correct potential base errors and increase consensus quality using a software Polisher developed at JGI . The error rate of the completed genome sequence is less than 1 in 100,000. Together, the combination of the Illumina and 454 sequencing platforms provided 184.6 × coverage of the genome. Final assembly contains 815,143 pyrosequence and 5,434,428 Illumina reads.
Genes were identified using Prodigal  as part of the Oak Ridge National Laboratory genome annotation pipeline, followed by a round of manual curation using the JGI . The predicted CDSs were translated and used to search the National Center for Biotechnology Information (NCBI) nonredundant database, UniProt, TIGRFam, Pfam, PRIAM, KEGG, COG, and InterPro databases. Additional gene prediction analysis and functional annotation was performed within the (IMG-ER) platform .
The genome consists of a 2,405,559 bp long chromosome with a 51.0% GC content (Table 3 and Figure 3). Of the 2,440 genes predicted, 2,385 were protein-coding genes, and 55 RNAs; 72 pseudogenes were also identified. The majority of the protein-coding genes (70.7%) were assigned with a putative function while the remaining ones were annotated as hypothetical proteins. The distribution of genes into COGs functional categories is presented in Table 4.
Sekiguchi Y, Kamagata Y, Nakamura K, Ohashi A, Harada H. Syntrophothermus lipocalidus gen. nov., sp. nov., a novel thermophilic, syntrophic, fatty-acid-oxidizing anaerobe which utilizes isobutyrate. Int J Syst Evol Microbiol 2000; 50:771–779. PubMed
Sobieraj M, Boone DR. Syntrophomonadaceae. The Prokaryotes 2006; 4:1041–1049. doi:10.1007/0-387-30744-3_37
Castresana J. Selection of conserved blocks from multiple alignments for their use in phylogenetic analysis. Mol Biol Evol 2000; 17:540–552. PubMed
Lee C, Grasso C, Sharlow MF. Multiple sequence alignment using partial order graphs. Bioinformatics 2002; 18:452–464. PubMed doi:10.1093/bioinformatics/18.3.452
Stamatakis A, Hoover P, Rougemont J. A rapid bootstrap algorithm for the RAxML web servers. Syst Biol 2008; 57:758–771. PubMed doi:10.1080/10635150802429642
Yarza P, Richter M, Peplies J, Euzeby J, Amann R, Schleifer KH, Ludwig W, Glöckner FO, Rosselló-Móra R. The All-Species Living Tree project: A 16S rRNA-based phylogenetic tree of all sequenced type strains. Syst Appl Microbiol 2008; 31:241–250. PubMed doi:10.1016/j.syapm.2008.07.001
Pattengale ND, Alipour M, Bininda-Emonds ORP, Moret BME, Stamatakis A. How many bootstrap replicates are necessary? Lect Notes Comput Sci 2009; 5541:184–200. doi:10.1007/978-3-642-02008-7_13
Liolios K, Mavromatis K, Tavernarakis N, Kyrpides NC. The Genomes On Line Database (GOLD) in 2007: status of genomic and metagenomic projects and their associated metadata. Nucleic Acids Res 2008; 36:D475–D479. PubMed doi:10.1093/nar/gkm884
Sieber JR, Sims DR, Han C, Kim E, Lykidis A, Lapidus AL, McDonnald E, Rohlin L, Culley DE, Gunsalus R, et al. The genome of Syntrophomonas wolfei: new insights into syntrophic metabolism and biohydrogen production. Environ Microbiol 2010; 12:2289–2301.
Svetlitshnyi V, Rainey F, Wiegel J. Thermosyntropha lipolytica gen. nov., sp. nov., a lipolytic, anaerobic, alkalitolerant, thermophilic bacterium utilizing short- and long-chain fatty acids in syntrophic coculture with a methanogenic archaeum. Int J Syst Bacteriol 1996; 46:1131–1137. PubMed doi:10.1099/00207713-46-4-1131
Brazelton WJ, Baross JA. Abundant transposases encoded by the metagenome of a hydrothermal chimney biofilm. ISME J 2009; 3:1420–1424. PubMed doi:10.1038/ismej.2009.79
Kurokawa K, Itoh T, Kuwahara T, Oshima K, Toh H, Toyoda A, Takami H, Morita H, Sharma VK, Srivastava TP, et al. Comparative metagenomics revealed commonly enriched gene sets in human gut microbiomes. DNA Res 2007; 14:169–181. PubMed doi:10.1093/dnares/dsm018
DeSantis TZ, Hugenholtz P, Larsen N, Rojas M, Brodie EL, Keller K, Huber T, Dalevi D, Hu P, Andersen GL. Greengenes, a chimera-checked 16S rRNA gene database and workbench compatible with ARB. Appl Environ Microbiol 2006; 72:5069–5072. PubMed doi:10.1128/AEM.03006-05
Field D, Garrity G, Gray T, Morrison N, Selengut J, Sterk P, Tatusova T, Thomson N, Allen MJ, Angiuoli SV, et al. The minimum information about a genome sequence (MIGS) specification. Nat Biotechnol 2008; 26:541–547. PubMed doi:10.1038/nbt1360
Woese CR, Kandler O, Wheelis ML. Towards a natural system of organisms: proposal for the domains Archaea, Bacteria, and Eucarya. Proc Natl Acad Sci USA 1990; 87:4576–4579. PubMed doi:10.1073/pnas.87.12.4576
Garrity GM, Holt JG. The Road Map to the Manual. In: Garrity GM, Boone DR, Castenholz RW (eds), Bergey’s Manual of Systematic Bacteriology, Second Edition, Volume 1. Springer, New York 2001:119–169.
Gibbons NE, Murray RGE. Proposals concerning the higher taxa of Bacteria. Int J Syst Bacteriol 1978; 28:1–6. doi:10.1099/00207713-28-1-1
Validation list 132. List of new names and new combinations previously effectively, but not validly, published. Int J Syst Evol Microbiol 2010; 60:469–472. doi:10.1099/ijs.0.022855-0
Rainey FA. 2009. Class II. Clostridia class nov., p. 736. In P. De Vos, G. Garrity, D. Jones, N.R. Krieg, W. Ludwig, F.A. Rainey, K.H. Schleifer, and W.B. Whitman (ed.), Bergey’s Manual of Systematic Bacteriology, Second Edition ed, vol. 3. Springer, New York.
Prévot AR. in: P. Hauduroy, G. Ehringer, G. Guillot, J. Magrou, A. R. Prévot, Rosset and A. Urbain: Dictionnaire des Bactéries Pathogènes, 2nd ed., Masson, Paris, 1953, pp. 1–692.
Skerman VBD, McGowan V, Sneath PHA. Approved Lists of Bacterial Names. Int J Syst Bacteriol 1980; 30:225–420. doi:10.1099/00207713-30-1-225
Zhao H, Yang D, Woese CR, Bryant MP. Assignment of fatty acid-β-oxidizing syntrophic bacteria to Syntrophomonadaceae fam. nov. on the basis of 16S rRNA sequence analyses. Int J Syst Bacteriol 1993; 43:278–286. PubMed doi:10.1099/00207713-43-2-278
Jumas-Bilak E, Roudiere L, Marchandin H. Description of ‘Synergistetes’ phyl. nov. and emended description of the phylum ‘Deferribacteres’ and of the family Syntrophomonadaceae, phylum ‘Firmicutes’. Int J Syst Evol Microbiol 2009; 59:1028–1035. PubMed doi:10.1099/ijs.0.006718-0
Classification of bacteria and archaea in risk groups. http://www.baua.de TRBA 466.
Ashburner M, Ball CA, Blake JA, Botstein D, Butler H, Cherry JM, Davis AP, Dolinski K, Dwight SS, Eppig JT, et al. Gene Ontology: tool for the unification of biology. Nat Genet 2000; 25:25–29. PubMed doi:10.1038/75556
Chertkov O, Sikorski J, Brambilla E, Lapidus A, Copeland A, Rio TGD, Nolan M, Lucas S, Tice H, Cheng JF, et al. Complete genome sequence of Aminobacterium colombiense type strain (ALA-1T). Stand Genomic Sci 2010; 2:280–289. doi:10.4056/sigs.902116
McInerney MJ, Bryant MP, Hespell RB, Costerton JW. Syntrophomonas wolfei gen. nov. sp. nov., an anaerobic, syntrophic, fatty acid-oxidizing bacterium. Appl Environ Microbiol 1981; 41:1029–1039. PubMed
Klenk HP, Göker M. En route to a genome-based classification of Archaea and Bacteria? Syst Appl Microbiol 2010; 33:175–182. PubMed doi:10.1016/j.syapm.2010.03.003
Wu D, Hugenholtz P, Mavromatis K, Pukall R, Dalin E, Ivanova NN, Kunin V, Goodwin L, Wu M, Tindall BJ, et al. A phylogeny-driven genomic encyclopaedia of Bacteria and Archaea. Nature 2009; 462:1056–1060. PubMed doi:10.1038/nature08656
List of growth media used at DSMZ: http://www.dsmz.de/microorganisms/media_list.php.
DOE Joint Genome Institute http://www.jgi.doe.gov
Zerbino DR, Birney E. Velvet: algorithms for de novo short read assembly using de Bruijn graphs. Genome Res 2008; 18:821–829. PubMed doi:10.1101/gr.074492.107
Phrap and Phred for Windows, MacOS, Linux, and Unix. http://www.phrap.com
Sims D, Brettin T, Detter JC, Han C, Lapidus A, Copeland A, Glavina Del Rio T, Nolan M, Chen F, Lucas S, et al. Complete genome sequence of Kytococcus sedentarius type strain (541T). Stand Genomic Sci 2009; 1:12–20. doi:10.4056/sigs.761
Lapidus A, LaButti K, Foster B, Lowry S, Trong S, Goltsman E. POLISHER: An effective tool for using ultra short reads in microbial genome assembly and finishing. AGBT, Marco Island, FL, 2008.
Hyatt D, Chen GL, Locascio PF, Land ML, Larimer FW, Hauser LJ. Podigal: prokaryotic gene recognition and translation initiation site identification. BMC Bioinformatics 2010; 11:119. PubMed doi:10.1186/1471-2105-11-119
Pati A, Ivanova N, Mikhailova N, Ovchinikova G, Hooper SD, Lykidis A, Kyrpides NC. GenePRIMP: A gene prediction improvement pipeline for microbial genomes. Nat Methods 2010; 7:455–457. PubMed doi:10.1038/nmeth.1457
Markowitz VM, Ivanova NN, Chen IMA, Chu K, Kyrpides NC. IMG ER: a system for microbial genome annotation expert review and curation. Bioinformatics 2009; 25:2271–2278. PubMed doi:10.1093/bioinformatics/btp393
We would like to gratefully acknowledge the help of Maren Schröder (DSMZ) in cultivation of the strain. This work was performed under the auspices of the US Department of Energy Office of Science, Biological and Environmental Research Program, and by the University of California, Lawrence Berkeley National Laboratory under contract No. DE-AC02-05CH11231, Lawrence Livermore National Laboratory under Contract No. DE-AC52-07NA27344, and Los Alamos National Laboratory under contract No. DE-AC02-06NA25396, UT-Battelle, and Oak Ridge National Laboratory under contract DE-AC05-00OR22725, as well as German Research Foundation (DFG) INST 599/1-2.
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Ngatchou Djao, O.D., Zhang, X., Lucas, S. et al. Complete genome sequence of Syntrophothermus lipocalidus type strain (TGB-C1T). Stand in Genomic Sci 3, 267–275 (2010). https://doi.org/10.4056/sigs.1233249
- syntrophism with methanogen