Open Access

Complete genome sequence of Syntrophothermus lipocalidus type strain (TGB-C1T)

  • Olivier Duplex Ngatchou Djao
  • , Xiaojing Zhang
  • , Susan Lucas
  • , Alla Lapidus
  • , Tijana Glavina Del Rio
  • , Matt Nolan
  • , Hope Tice
  • , Jan-Fang Cheng
  • , Cliff Han
  • , Roxanne Tapia
  • , Lynne Goodwin,
  • , Sam Pitluck
  • , Konstantinos Liolios
  • , Natalia Ivanova
  • , Konstantinos Mavromatis
  • , Natalia Mikhailova
  • , Galina Ovchinnikova
  • , Amrita Pati
  • , Evelyne Brambilla
  • , Amy Chen
  • , Krishna Palaniappan
  • , Miriam Land,
  • , Loren Hauser,
  • , Yun-Juan Chang,
  • , Cynthia D. Jeffries,
  • , Manfred Rohde
  • , Johannes Sikorski
  • , Stefan Spring
  • , Markus Göker
  • , John C. Detter
  • , Tanja Woyke
  • , James Bristow
  • , Jonathan A. Eisen,
  • , Victor Markowitz
  • , Philip Hugenholtz
  • , Nikos C. Kyrpides
  • and Hans-Peter Klenk
Corresponding author

DOI: 10.4056/sigs.1233249

Received: 15 December 2010

Published: 31 December 2010

Abstract

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.

Keywords:

anaerobicmotileGram-negativesyntrophism with methanogencrotonatebutyrateisobutyrateSyntrophomonadaceaeGEBA

Introduction

Strain TGB-C1T (= DSM 12680) is the type strain of Syntrophothermus lipocalidus [1] which in turn is the type species of the genus Syntrophothermus [2]. 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 [1]. 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 [1]. Strain TGB-C1T was isolated from granular sludge in a thermophilic upflow anaerobic sludge blanket (UASB) [1]. 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 [1] (Figure1), with Thermosyntropha lipolytica [10] 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 [1]. 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 [11] and from human gut metagenome DNA (contig sequence: F2-Y_011332) [12] (status August 2010), indicating that members of the species, genus and even family are widely represented in the habitats screened so far.

Figure 1

Phylogenetic tree highlighting the position of S. lipocalidus TGB-C1T relative to the type strains within the family Syntrophomonadaceae. The trees were inferred from 1,434 aligned characters [3,4] of the 16S rRNA gene sequence under the maximum likelihood criterion [5] and rooted in accordance with the current taxonomy [6]. The branches are scaled in terms of the expected number of substitutions per site. Numbers above branches are support values from 1,000 bootstrap replicates [7] if larger than 60%. Lineages with type strain genome sequencing projects registered in GOLD [8] are shown in blue, published genomes in bold [9].

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 [13] 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 [1].

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) [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 [1]. 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 [1]. 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 [1]. 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 [1]. 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 [1]. By oxidizing fatty acids, S. lipocalidus produces acetate and hydrogen [1], the latter of which is then scavenged by the syntrophic methanogen M. thermoautotrophicum [1]. 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 [26]. S. lipocalidus is the only species in the family Syntrophomonadaceae that is able to metabolize isobutyrate [2]. Neither yeast extract nor tryptone significantly stimulates growth [1]. 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 [1]. Cell growth is inhibited by ampicillin, chloramphenicol, kanamycin, neomycin, rifampin or vancomycin (each 50 µg ml-1) [1].

Figure 2

Scanning electron micrograph of S. lipocalidus TGB-C1T

Table 1

Classification and general features of S. lipocalidus TGB-C1T in according with the MIGS recommendations [14]

MIGS ID

   Property

    Term

     Evidence code

   Current classification

    Domain Bacteria

     TAS [15]

    Phylum Firmicutes

     TAS [16,17]

    Class Clostridia

     TAS [18,19]

    Order Clostridiales

     TAS [20,21]

    Family Syntrophomonadaceae

     TAS [22,23]

    Genus Syntrophothermus

     TAS [1]

    Species Syntrophothermus lipocalidus

     TAS [1]

    Type strain TGB-C1

     TAS [1]

   Gram stain

    negative

     TAS [1]

   Cell shape

    slightly curved rods with round ends

     TAS [1]

   Motility

    weakly motile by flagella

     TAS [1]

   Sporulation

    None

     TAS [1]

   Temperature range

    45°C–60°C

     TAS [1]

   Optimum temperature

    55°C

     TAS [1]

   Salinity

    < 0.5% NaCl

     TAS [1]

MIGS-22

   Oxygen requirement

    obligately anaerobic

     TAS [1]

   Carbon source

    crotonate in pure culture; fatty acids with    4-10 carbon atoms including isobutyrate in syntrophy

     TAS [1]

   Energy source

    crotonate

     TAS [1]

MIGS-6

   Habitat

    not reported

     NAS

MIGS-15

   Biotic relationship

    syntrophic with methanogens

     NAS

MIGS-14

   Pathogenicity

    not reported

     NAS

   Biosafety level

    1

     TAS [24]

   Isolation

    granular sludge in a thermophilic    upflow anaerobic sludge blanket (UASB) reactor

     TAS [1]

MIGS-4

   Geographic location

    most probably Japan

     TAS [1]

MIGS-5

   Sample collection time

    2000 or before

     TAS [1]

MIGS-4.1MIGS-4.2

   Latitude   Longitude

    not reported

     NAS

MIGS-4.3

   Depth

    not reported

     NAS

MIGS-4.4

   Altitude

    not reported

     NAS

Evidence codes - IDA: Inferred from Direct Assay (first time in publication); TAS: Traceable Author Statement (i.e., a direct report exists in the literature); NAS: Non-traceable Author Statement (i.e., not directly observed for the living, isolated sample, but based on a generally accepted property for the species, or anecdotal evidence). These evidence codes are from of the Gene Ontology project [25]. If the evidence code is IDA, then the property was directly observed by one of the authors or an expert mentioned in the acknowledgements.

Chemotaxonomy

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 [1]. 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 [1].

Genome sequencing and annotation

Genome project history

This organism was selected for sequencing on the basis of its phylogenetic position [28], and is part of the Genomic Encyclopedia of Bacteria and Archaea project [29]. The genome project is deposited in the Genome OnLine Database [8] 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.

Table 2

Genome sequencing project information

MIGS ID

    Property

   Term

MIGS-31

    Finishing quality

   Finished

MIGS-28

    Libraries used

   Three genomic libraries:   454 pyrosequence standard library and;   paired end library (10.2 kb insert size);   Illumina standard library

MIGS-29

    Sequencing platforms

   454 GS FLX Titanium, Illumina GAii

MIGS-31.2

    Sequencing coverage

   103.3 × pyrosequence, 81.3 × Illumina

MIGS-30

    Assemblers

   Newbler version 2.1-PreRelease-4-28-2009,   Velvet, phrap

MIGS-32

    Gene calling method

   Prodigal 1.4, GenePRIMP

    INSDC ID

   CP002048

    Genbank Date of Release

   June 7, 2010

    GOLD ID

   Gc012392

    NCBI project ID

   37873

    Database: IMG-GEBA

   2502957035

MIGS-13

    Source material identifier

   DSM 12680

    Project relevance

   Tree of Life, GEBA

Growth conditions and DNA isolation

S. lipocalidus TGB-C1T, DSM 12680, was grown anaerobically in DSMZ medium 870 (Syntrophothermus medium) [30] 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 [31]. 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 [32] 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 [33] 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 [31], Dupfinisher, or sequencing cloned bridging PCR fragments with subcloning or transposon bombing (Epicentre Biotechnologies, Madison, WI) [34]. 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 [35]. 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.

Genome annotation

Genes were identified using Prodigal [36] as part of the Oak Ridge National Laboratory genome annotation pipeline, followed by a round of manual curation using the JGI [37]. 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 [38].

Genome properties

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.

Table 3

Genome Statistics

Attribute

   Value

    % of Total

Genome size (bp)

   2,405,559

    100.00%

DNA coding region (bp)

   2,078,709

    86.41%

DNA G+C content (bp)

   1,226,580

    50.99%

Number of replicons

   1

Extrachromosomal elements

   0

Total genes

   2,440

    100.00%

RNA genes

   55

    2.25%

rRNA operons

   2

Protein-coding genes

   2,385

    97.75%

Pseudo genes

   72

    2.95%

Genes with function prediction

   1,726

    70.74%

Genes in paralog clusters

   348

    14.26%

Genes assigned to COGs

   1,767

    72.42%

Genes assigned Pfam domains

   1,912

    78.26%

Genes with signal peptides

   603

    24.71%

Genes with transmembrane helices

   545

    22.34%

CRISPR repeats

   2

Figure 3

Graphical circular map of the genome. From outside to the center: Genes on forward strand (color by COG categories), Genes on reverse strand (color by COG categories), RNA genes (tRNAs green, rRNAs red, other RNAs black), GC content, GC skew.

Table 4

Number of genes associated with the general COG functional categories

Code

   value

   %age

   Description

J

   144

   7.4

   Translation, ribosomal structure and biogenesis

A

   0

   0.0

   RNA processing and modification

K

   113

   5.8

   Transcription

L

   123

   6.3

   Replication, recombination and repair

B

   4

   0.2

   Chromatin structure and dynamics

D

   32

   1.6

   Cell cycle control, cell division, chromosome partitioning

Y

   0

   0.0

   Nuclear structure

V

   33

   1.7

   Defense mechanisms

T

   107

   5.5

   Signal transduction mechanisms

M

   96

   4.9

   Cell wall/membrane/envelope biogenesis

N

   81

   4.1

   Cell motility

Z

   0

   0.0

   Cytoskeleton

W

   0

   0.0

   Extracellular structures

U

   66

   3.4

   Intracellular trafficking and secretion, and vesicular transport

O

   74

   3.8

   Posttranslational modification, protein turnover, chaperones

C

   144

   7.4

   Energy production and conversion

G

   67

   3.4

   Carbohydrate transport and metabolism

E

   144

   7.4

   Amino acid transport and metabolism

F

   58

   3.0

   Nucleotide transport and metabolism

H

   112

   5.7

   Coenzyme transport and metabolism

I

   98

   5.0

   Lipid transport and metabolism

P

   70

   3.6

   Inorganic ion transport and metabolism

Q

   26

   1.3

   Secondary metabolites biosynthesis, transport and catabolism

R

   205

   10.5

   General function prediction only

S

   158

   8.1

   Function unknown

-

   673

   27.6

   Not in COGs

Declarations

Acknowledgements

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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References

  1. Sekiguchi Y, Kamagata Y, Nakamura K, Ohashi A and 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; 50:771-779PubMed
  2. Sobieraj M and Boone DR. Syntrophomonadaceae. The Prokaryotes. 2006; 4:1041-1049 View Article
  3. Castresana J. Selection of conserved blocks from multiple alignments for their use in phylogenetic analysis. Mol Biol Evol. 2000; 17:540-552PubMed
  4. Lee C, Grasso C and Sharlow MF. Multiple sequence alignment using partial order graphs. Bioinformatics. 2002; 18:452-464 View ArticlePubMed
  5. Stamatakis A, Hoover P and Rougemont J. A rapid bootstrap algorithm for the RAxML web servers. Syst Biol. 2008; 57:758-771 View ArticlePubMed
  6. Yarza P, Richter M, Peplies J, Euzeby J, Amann R, Schleifer KH, Ludwig W, Glöckner FO and 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 View ArticlePubMed
  7. Pattengale ND, Alipour M, Bininda-Emonds ORP, Moret BME and Stamatakis A. How many bootstrap replicates are necessary? Lect Notes Comput Sci. 2009; 5541:184-200 View Article
  8. Liolios K, Mavromatis K, Tavernarakis N and 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 View ArticlePubMed
  9. Sieber JR, Sims DR, Han C, Kim E, Lykidis A, Lapidus AL, McDonnald E, Rohlin L, Culley DE and Gunsalus R. The genome of Syntrophomonas wolfei: new insights into syntrophic metabolism and biohydrogen production. Environ Microbiol. 2010; 12:2289-2301
  10. Svetlitshnyi V, Rainey F and 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 View ArticlePubMed
  11. Brazelton WJ and Baross JA. Abundant transposases encoded by the metagenome of a hydrothermal chimney biofilm. ISME J. 2009; 3:1420-1424 View ArticlePubMed
  12. Kurokawa K, Itoh T, Kuwahara T, Oshima K, Toh H, Toyoda A, Takami H, Morita H, Sharma VK and Srivastava TP. Comparative metagenomics revealed commonly enriched gene sets in human gut microbiomes. DNA Res. 2007; 14:169-181 View ArticlePubMed
  13. DeSantis TZ, Hugenholtz P, Larsen N, Rojas M, Brodie EL, Keller K, Huber T, Dalevi D, Hu P and Andersen GL. Greengenes, a chimera-checked 16S rRNA gene database and workbench compatible with ARB. Appl Environ Microbiol. 2006; 72:5069-5072 View ArticlePubMed
  14. Field D, Garrity G, Gray T, Morrison N, Selengut J, Sterk P, Tatusova T, Thomson N, Allen MJ and Angiuoli SV. The minimum information about a genome sequence (MIGS) specification. Nat Biotechnol. 2008; 26:541-547 View ArticlePubMed
  15. Woese CR, Kandler O and 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 View ArticlePubMed
  16. 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.
  17. Gibbons NE and Murray RGE. Proposals concerning the higher taxa of Bacteria. Int J Syst Bacteriol. 1978; 28:1-6 View Article
  18. 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 View Article
  19. 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.
  20. 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.
  21. Skerman VBD, McGowan V and Sneath PHA. Approved Lists of Bacterial Names. Int J Syst Bacteriol. 1980; 30:225-420 View Article
  22. Zhao H, Yang D, Woese CR and 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 View ArticlePubMed
  23. Jumas-Bilak E, Roudiere L and 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 View ArticlePubMed
  24. Classification of bacteria and archaea in risk groups. TRBA 466.Web Site
  25. Ashburner M, Ball CA, Blake JA, Botstein D, Butler H, Cherry JM, Davis AP, Dolinski K, Dwight SS and Eppig JT. Gene Ontology: tool for the unification of biology. Nat Genet. 2000; 25:25-29 View ArticlePubMed
  26. Chertkov O, Sikorski J, Brambilla E, Lapidus A, Copeland A, Rio TGD, Nolan M, Lucas S, Tice H and Cheng JF. Complete genome sequence of Aminobacterium colombiense type strain (ALA-1T). Stand Genomic Sci. 2010; 2:280-289 View Article
  27. McInerney MJ, Bryant MP, Hespell RB and Costerton JW. Syntrophomonas wolfei gen. nov. sp. nov., an anaerobic, syntrophic, fatty acid-oxidizing bacterium. Appl Environ Microbiol. 1981; 41:1029-1039PubMed
  28. Klenk HP and Göker M. En route to a genome-based classification of Archaea and Bacteria? Syst Appl Microbiol. 2010; 33:175-182 View ArticlePubMed
  29. Wu D, Hugenholtz P, Mavromatis K, Pukall R, Dalin E, Ivanova NN, Kunin V, Goodwin L, Wu M and Tindall BJ. A phylogeny-driven genomic encyclopaedia of Bacteria and Archaea. Nature. 2009; 462:1056-1060 View ArticlePubMed
  30. List of growth media used at DSMZ: Web Site
  31. DOE Joint Genome Institute. Web Site
  32. Zerbino DR and Birney E. Velvet: algorithms for de novo short read assembly using de Bruijn graphs. Genome Res. 2008; 18:821-829 View ArticlePubMed
  33. Phrap and Phred for Windows, MacOS, Linux, and Unix. Web Site
  34. Sims D, Brettin T, Detter JC, Han C, Lapidus A, Copeland A, Glavina Del Rio T, Nolan M, Chen F and Lucas S. Complete genome sequence of Kytococcus sedentarius type strain (541T). Stand Genomic Sci. 2009; 1:12-20 View Article
  35. 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.
  36. Hyatt D, Chen GL, Locascio PF, Land ML, Larimer FW and Hauser LJ. Podigal: prokaryotic gene recognition and translation initiation site identification. BMC Bioinformatics. 2010; 11:119 View ArticlePubMed
  37. Pati A, Ivanova N, Mikhailova N, Ovchinikova G, Hooper SD, Lykidis A and Kyrpides NC. GenePRIMP: A gene prediction improvement pipeline for microbial genomes. Nat Methods. 2010; 7:455-457 View ArticlePubMed
  38. Markowitz VM, Ivanova NN, Chen IMA, Chu K and Kyrpides NC. IMG ER: a system for microbial genome annotation expert review and curation. Bioinformatics. 2009; 25:2271-2278 View ArticlePubMed