Open Access

Genome sequence of the phylogenetically isolated spirochete Leptonema illini type strain (3055T)

  • Marcel Huntemann
  • , Erko Stackebrandt
  • , Brittany Held,
  • , Matt Nolan
  • , Susan Lucas
  • , Nancy Hammon
  • , Shweta Deshpande
  • , Jan-Fang Cheng
  • , Roxanne Tapia,
  • , Lynne A. Goodwin,
  • , Sam Pitluck
  • , Konstantinos Liolios
  • , Ioanna Pagani
  • , Natalia Ivanova
  • , Konstantinos Mavromatis
  • , Natalia Mikhailova
  • , Amrita Pati
  • , Amy Chen
  • , Krishna Palaniappan
  • , Miriam Land,
  • , Manfred Rohde
  • , Sabine Gronow
  • , Markus Göker
  • , John C. Detter
  • , James Bristow
  • , Jonathan A. Eisen,
  • , Victor Markowitz
  • , Tanja Woyke
  • , Philip Hugenholtz,
  • , Nikos C. Kyrpides
  • , Hans-Peter Klenk
  • and Alla Lapidus
Corresponding author

DOI: 10.4056/sigs.3637201

Received: 25 May 2013

Accepted: 25 May 2013

Published: 15 June 2013

Abstract

Leptonema illini Hovind-Hougen 1979 is the type species of the genus Leptonema, family Leptospiraceae, phylum Spirochaetes. Organisms of this family have a Gram-negative-like cell envelope consisting of a cytoplasmic membrane and an outer membrane. The peptidoglycan layer is associated with the cytoplasmic rather than the outer membrane. The two flagella of members of Leptospiraceae extend from the cytoplasmic membrane at the ends of the bacteria into the periplasmic space and are necessary for their motility. Here we describe the features of the L. illini type strain, together with the complete genome sequence, and annotation. This is the first genome sequence (finished at the level of Improved High Quality Draft) to be reported from of a member of the genus Leptonema and a representative of the third genus of the family Leptospiraceae for which complete or draft genome sequences are now available. The three scaffolds of the 4,522,760 bp draft genome sequence reported here, and its 4,230 protein-coding and 47 RNA genes are part of the Genomic Encyclopedia of Bacteria and Archaea project.

Keywords:

Gram-negativeflexiblemotilecytoplasmatic tubulesnon-sporulatingaxial flagellaaerobicchemoorganotrophicLeptospiraceaeGEBA

Introduction

Strain 3055T was isolated from urine of a clinically healthy bull [1], and was first mentioned in the literature as a new Leptospira serotype, serovar illini [2,3], but as no name was proposed, it was not validly published. This occurred in the comparative study of Hovind-Hougen [4] who found morphological differences between ‘Leptospira illini’ strain 3005 and other members of Leptospira, i.e. the presence of cytoplasmatic tubules and the structure of the basal complex of the flagella. These differences, together with the finding of a higher DNA base composition and growth behavior [5] were used as criteria to taxonomically separate strain 3055 from Leptospira as Leptonema illini with strain 3055T (= DSM 21528 = NCTC 11301) as the type strain. This species is the only species of the genus. The family Leptospiraceae was created in the same publication [4], although the name was proposed before, though not effectively published [J Pilot, Ph D Thesis, University of Paris, Paris, France 1965]. Despite a description in the International Journal of Systematic Bacteriology the name Leptonema was not included in the Approved List of Bacterial Names [6]. The omission of this name was not in accordance with the Bacteriological Code (1990 Revision) Rule 24a, Note 1, but was corrected in Validation List No 10 [7].

The phylogenetic relatedness among spirochetes and the isolated position of L. illini was first elucidated by 16S rRNA cataloguing [8] and then by comparative sequence analysis of reverse-transcribed 16S rRNA sequences [9] and by rDNA analyses [10,11]. The moderate similarity values between L. illini and strains of Leptospira were later supported by the absence of significant DNA-DNA hybridization values between members of the two genera [12-14], 16S rRNA restriction fragment analysis [15] and PCR amplification of the 16S-23S ribosomal DNA spacer [16]. Application of a 16S rRNA gene real-time PCR assay to leptospiras [17] confirmed the presence of L. illini strains in kidneys of Indian rats and bandicoots. Here we present a summary classification and a set of features for L. illini strain 3055T together with the description of the complete genomic sequencing and annotation. The rationale for sequencing the genome of this non-pathogenic strain is based on its isolated position within the phylum Spirochaetes.

Classification and features

16S rRNA gene sequence analysis

The single genomic 16S rRNA gene sequence of L. illini 3055T was compared using NCBI BLAST [18,19] under default settings (e.g., considering only the high-scoring segment pairs (HSPs) from the best 250 hits) with the most recent release of the Greengenes database [20] and the relative frequencies of taxa and keywords (reduced to their stem [21]) were determined, weighted by BLAST scores. The most frequently occurring genera were Leptospira (53.4%), Anaeromyxobacter (31.6%), Leptonema (11.5%), Turneriella (1.3%) and Desulfomonile (0.8%) (96 hits in total). Regarding the three hits to sequences from members of the species, the average identity within HSPs was 99.7%, whereas the average coverage by HSPs was 97.4%. Among all other species, the one yielding the highest score was Leptospira wolbachii (AY631890), which corresponded to an identity of 86.4% and an HSP coverage of 76.8%. (Note that the Greengenes database uses the INSDC (= EMBL/NCBI/DDBJ) annotation, which is not an authoritative source for nomenclature or classification.) The highest-scoring environmental sequence was EF648066 (Greengenes short name 'dynamics during produced water treatment aerobic activated sludge clone HB63'), which showed an identity of 99.2% and an HSP coverage of 98.4%. The most frequently occurring keywords within the labels of all environmental samples which yielded hits were 'microbi' (5.2%), 'soil' (2.3%), 'anaerob' (2.3%), 'industri' (2.0%) and 'ecolog' (1.4%) (154 hits in total). The most frequently occurring keywords within the labels of those environmental samples which yielded hits of a higher score than the highest scoring species were 'microbi' (4.5%), 'cell' (3.1%), 'prmr' (3.0%), 'sediment' (3.0%) and 'coral' (3.0%) (12 hits in total). None of these keywords provides useful information about the close relatives of strain 3055T in the environment.

Figure 1 shows the phylogenetic neighborhood of L. illini in a 16S rRNA based tree. The sequence of the single 16S rRNA gene copy in the genome does not differ from the previously published 16S rRNA sequence (AY714984).

Figure 1

Phylogenetic tree highlighting the position of L. illini relative to the type strains of the other species within the phylum Spirochaetes. The tree was inferred from 1,325 aligned characters [22,23] of the 16S rRNA gene sequence under the maximum likelihood (ML) criterion [24]. Rooting was done initially using the midpoint method [25] and then checked for its agreement with the current classification (Table 1). The branches are scaled in terms of the expected number of substitutions per site. Numbers adjacent to the branches are support values from 550 ML bootstrap replicates [26] (left) and from 1,000 maximum-parsimony bootstrap replicates [27] (right) if larger than 60%. Lineages with type strain genome sequencing projects registered in GOLD [28] are labeled with one asterisk. Those also listed as 'Complete and Published' with two asterisks (see [29-35] and CP003155 for Sphaerochaeta pleomorpha, CP002903 for Spirochaeta thermophila, CP002696 for Treponema brennaborense, CP001841 for T. azotonutricium). The collapsed Treponema subtree contains three species formerly assigned to Spirochaeta that have recently been included in the genus Treponema, even though those names are not yet validly published [34].

Table 1

Classification and general features of L. illini 3055T according to the MIGS recommendations [36].

MIGS ID

      Property

      Term

    Evidence code

      Domain Bacteria

    TAS [37]

      Phylum Spirochaetes

    TAS [38]

      Class Spirochaetes

    TAS [39,40]

      Current classification

      Order Spirochaetales

    TAS [41,42]

      Family Leptospiraceae

    TAS [4,14,42]

      Genus Leptonema

    TAS [4,7]

      Species Leptonema illini

    TAS [4,7]

MIGS-7

      Subspecific genetic lineage (strain)

      3055T

    TAS [4]

MIGS-12

      Reference for biomaterial

      Hovind-Hougen, 1979

    TAS [4]

      Gram stain

      negative

    TAS [4]

      Cell shape

      helical rods

    TAS [4]

      Motility

      motile

    TAS [4]

      Sporulation

      non-sporulating

    TAS [4

      Temperature range

      mesophile

    TAS [4]

      Optimum temperature

      29° C

    TAS [4]

      Salinity

      not reported

MIGS-22

      Relationship to oxygen

      aerobe

    TAS [4]

      Carbon source

      long-chain fatty acids

    TAS [4]

      Energy metabolism

      chemoorganotroph

    TAS [4]

MIGS-6

      Habitat

      not specified

MIGS-6.2

      pH

      not reported

MIGS-15

      Biotic relationship

      free living

    TAS [4]

MIGS-14

      Known pathogenicity

      opportunistic infections

    TAS [43]

MIGS-16

      Specific host

      Bos taurus (cow)

    TAS [4]

MIGS-18

      Health status of host

      healthy

    TAS [4]

      Biosafety level

      1

    TAS [44]

MIGS-19

      Trophic level

      not reported

MIGS-23.1

      Isolation

      urine of a bull

    TAS [4]

MIGS-4

      Geographic location

      Iowa

    TAS [5]

MIGS-5

      Time of sample collection

      1965

    TAS [1]

MIGS-4.1

      Latitude

      not reported

MIGS-4.2

      Longitude

      not reported

MIGS-4.3

      Depth

      not reported

MIGS-4.4

      Altitude

      not reported

Evidence codes - 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). Evidence codes are from the Gene Ontology project [45].

Morphology and physiology

The unicellular cells of strain 3055T stain Gram negatively and are of helical shape (13-21 µm long and 0.1 µm wide) [4] [Figure 2]. Most cells have hook-shaped ends and display a typical leptospiral morphology [46]. The wavelength of the coils within the helix is about 0.6 µm with an amplitude of about 0.1 µm. A single flagellum is inserted at each pole and in well-preserved cells the flagellum is entwined with the helical body within the periplasmatic cell for about four to six turns of the helix (not visible in Figure 2). Rotation of the flagella by a flagellar motor induces changes in the cell morphology that drives motility [47]. In cells treated with Myxobacter Al-1 protease [48] bundles of three to four cytoplasmic tubules are observed which originate close to the insertion point of each of the two flagella. The bundles are located close to the inner site of the cytoplasmic membrane just underneath the flagellum. As bundles and flagella are shorter than the total length of the cell, the middle part is devoid of both. Flagella, released by the AL-1 protease, are often found as spirals. Each flagellum consists of a core (diameter 10 nm), covered by a sheath (diameter 16 nm). One of the arguments to classify strain 3055 as the type of a new genus was the structure of the insertion part of the flagellum, similar to those of Gram-positive bacteria in L. illini while other leptospiras possess the Gram-negative type insertion [4].

Figure 2

Scanning electron micrograph of L. illini 3055T

Serum and long-chain fatty acids are required for growth, no serum is required in trypticase soy broth. The organism is chemoorganotrophic and aerobic. Long-chain fatty acids (>14 carbons) are used as source of carbon and energy. Ammonia, in the form of inorganic salts rather than amino acids is used as a nitrogen source. Purines, but not pyrimidines, are utilized. Strain 3005T is non-pathogenic for hamsters, mice, gerbils, guinea pigs and cattle [15], although it may cause opportunistic infections, as it has been isolated from the blood of a HIV-infected patient [43].

Chemotaxonomy

No data are available for fatty acids, quinones or polar lipids. The G+C content of the DNA was previously reported with 51-53 mol% [49], which is below the value inferred from the genome sequence (see genome statistics table).

Genome sequencing and annotation

Genome project history

This organism was selected for sequencing on the basis of its phylogenetic position [50], and is part of the Genomic Encyclopedia of Bacteria and Archaea project [51]. The genome project is deposited in the Genomes OnLine Database [28] and the complete genome sequence is deposited in GenBank. Sequencing, finishing and annotation were performed by the DOE Joint Genome Institute (JGI) using state of the art sequencing technology [52]. 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

      Improved high quality draft

MIGS-28

      Libraries used

      Three genomic libraries: one 454 pyrosequence standard library,       two 454 PE library (13 kb insert size), one Illumina library

MIGS-29

      Sequencing platforms

      Illumina GAii, 454 GS FLX Titanium

MIGS-31.2

      Sequencing coverage

      1,276.9 × Illumina; 35.5 × pyrosequence

MIGS-30

      Assemblers

      Newbler version 2.3, Velvet 1.0.13, phrap version SPS - 4.24

MIGS-32

      Gene calling method

      Prodigal 1.4, GenePRIMP

      INSDC ID

      AHKT00000000

      GenBank Date of Release

      January 24, 2012

      GOLD ID

      Gi04604

      NCBI project ID

      60435

      Database: IMG

      2506783010

MIGS-13

      Source material identifier

      DSM 21528

      Project relevance

      Tree of Life, GEBA

Growth conditions and DNA isolation

L. illini strain 3055T, DSM 21528, was grown in DSMZ medium 1113 (Leptospira Medium) at 30°C. DNA was isolated from 1-1.5 g of cell paste using MasterPure Gram-positive DNA purification kit (Epicentre MGP04100) following the standard protocol as recommended by the manufacturer with modification st/DL for cell lysis as described in Wu et al. 2009 [51]. DNA is available through the DNA Bank Network [53].

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 [54]. Pyrosequencing reads were assembled using the Newbler assembler (Roche). The initial Newbler assembly consisting of 140 contigs in tree scaffolds was converted into a phrap [55] assembly by making fake reads from the consensus, to collect the read pairs in the 454 paired end library. Illumina GAii sequencing data (5,940 Mb) was assembled with Velvet [56] and the consensus sequences were shredded into 1.5 kb overlapped fake reads and assembled together with the 454 data. The 454 draft assembly was based on 179 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 [55] was used for sequence assembly and quality assessment in the subsequent finishing process. After the shotgun stage, reads were assembled with parallel phrap (High Performance Software, LLC). Possible mis-assemblies were corrected with gapResolution [54], Dupfinisher [57], or sequencing cloned bridging PCR fragments with subcloning. Gaps between contigs were closed by editing in Consed, by PCR and by Bubble PCR primer walks (J.-F. Chang, unpublished). A total of 103 additional reactions and one shatter library 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 [58]. 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 1,312.4 × coverage of the genome. The final assembly contained 488,975 pyrosequence and 75,603,747 Illumina reads.

Genome annotation

Genes were identified using Prodigal [59] as part of the DOE-JGI genome annotation pipeline [60], followed by a round of manual curation using the JGI GenePRIMP pipeline [61]. The predicted CDSs were translated and used to search the National Center for Biotechnology Information (NCBI) non-redundant database, UniProt, TIGR-Fam, Pfam, PRIAM, KEGG, COG, and InterPro databases. Additional gene prediction analysis and functional annotation was performed within the Integrated Microbial Genomes - Expert Review (IMG-ER) platform [62].

Genome properties

The genome statistics are provided in Table 3 and Figure 3. The assembly of the draft genome sequence consists of three scaffolds with 4,325,094 bp, 184,087 bp and 13,579 bp length, respectively, and a G+C content of 54.3%. Of the 4,277 genes predicted, 4,230 were protein-coding genes, and 47 RNAs; 69 pseudogenes were also identified. The majority of the protein-coding genes (60.3%) were assigned 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)

      4,522,760

     100.00

DNA coding region (bp)

      4,079,818

     90.21

DNA G+C content (bp)

      2,453,341

     54.26

Number of scaffolds

      3

Extrachromosomal elements

      unknown

Total genes

      4,277

     100.00

RNA genes

      47

     1.10

rRNA operons

      1

tRNA genes

      41

     0.96

Protein-coding genes

      4,230

     98.90

Pseudo genes

      69

     1.61

Genes with function prediction

      2,579

     60.30

Genes in paralog clusters

      1,764

     41.24

Genes assigned to COGs

      2,805

     65.58

Genes assigned Pfam domains

      2,865

     66.99

Genes with signal peptides

      1,481

     34.63

Genes with transmembrane helices

      1,089

     25.46

CRISPR repeats

      0

Figure 3

Graphical map of the largest scaffold. From bottom to the top: 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 (purple/olive).

Table 4

Number of genes associated with the general COG functional categories

Code

     Value

    %age

       Description

J

     156

    5.0

       Translation, ribosomal structure and biogenesis

A

     0

    0.0

       RNA processing and modification

K

     201

    6.5

       Transcription

L

     194

    6.3

       Replication, recombination and repair

B

     4

    0.1

       Chromatin structure and dynamics

D

     34

    1.1

       Cell cycle control, cell division, chromosome partitioning

Y

     0

    0.0

       Nuclear structure

V

     61

    2.0

       Defense mechanisms

T

     303

    9.8

       Signal transduction mechanisms

M

     226

    7.3

       Cell wall/membrane/envelope biogenesis

N

     108

    3.5

       Cell motility

Z

     0

    0.0

       Cytoskeleton

W

     0

    0.0

       Extracellular structures

U

     74

    2.4

       Intracellular trafficking, secretion, and vesicular transport

O

     119

    3.8

       Posttranslational modification, protein turnover, chaperones

C

     160

    5.2

       Energy production and conversion

G

     111

    3.6

       Carbohydrate transport and metabolism

E

     189

    6.1

       Amino acid transport and metabolism

F

     60

    1.9

       Nucleotide transport and metabolism

H

     139

    4.5

       Coenzyme transport and metabolism

I

     131

    4.2

       Lipid transport and metabolism

P

     128

    4.1

       Inorganic ion transport and metabolism

Q

     43

    1.4

       Secondary metabolites biosynthesis, transport and catabolism

R

     401

    12.9

       General function prediction only

S

     260

    8.4

       Function unknown

-

     1,472

    34.4

       Not in COGs

Declarations

Acknowledgements

We would like to gratefully acknowledge the help of Sabine Welnitz for growing L. illini cultures, and Evelyne-Marie Brambilla for DNA extraction and quality control (both at DSMZ). 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.


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References

  1. Tripathy DN and Hanson LE. Colonial and morphologic variations of Leptospira illini strain 3055. Am J Vet Res. 1972; 33:1723-1727PubMed
  2. Tripathy DN and Hanson LE. Studies of Leptospira illini, strain 3055: pathogenicity for different animals. Am J Vet Res. 1973; 34:557-562PubMed
  3. Tripathy DN and Hanson LE. Studies of Leptospira illini, strain 3055: immunological and serological determinations. Am J Vet Res. 1973; 34:563-565PubMed
  4. Hovind-Hougen K. Leptospiraceae, a new family to include Leptospira Noguchi 1917 and Leptonema gen. nov. Int J Syst Bacteriol. 1979; 29:245-251 View Article
  5. Hanson LE, Tripathy DN, Evans LB and Alexander AD. An unusual Leptospira, serotype illini (a new serotype). Int J Syst Bacteriol. 1974; 24:355-357 View Article
  6. Skerman VBD, McGowan V and Sneath PHA. Approved Lists of Bacterial Names. Int J Syst Bacteriol. 1980; 30:225-420 View Article
  7. . 10. Int J Syst Bacteriol. 1983; 33:438-440 View Article
  8. Paster BJ, Stackebrandt E, Hespell RB, Hahn CM and Woese CR. The phylogeny of the spirochetes. Syst Appl Microbiol. 1984; 5:337-351 View Article
  9. Paster BJ, Dewhirst FE, Weisburg WG, Tordoff LA, Fraser GJ, Hespell RB, Stanton TB, Zablen L, Mandelco L, Woese CR. Phylogenetic analysis of the spirochetes. J Bacteriol. 1991; 173:6101-6109PubMed
  10. Paster BJ and Dewhirst FE. Phylogenetic foundation of spirochetes. J Mol Microbiol Biotechnol. 2000; 2:341-244PubMed
  11. Ramadass P, Jarvis BDW, Corner RJ, Cinco M and Marshall RB. DNA relatedness among strains of Leptospira biflexa. Int J Syst Bacteriol. 1990; 40:231-235 View ArticlePubMed
  12. Morey RE, Galloway RL, Bragg SL, Steigerwalt AG, Mayer LW and Levett PN. Species-specific identification of Leptospiraceae by 16S rRNA gene sequencing. J Clin Microbiol. 2006; 44:3510-3516 View ArticlePubMed
  13. Ramadass P, Jarvis BDW, Corner RJ, Penny D and Marshall RB. Genetic characterization of pathogenic Leptospira species by DNA hybridization. Int J Syst Bacteriol. 1992; 42:215-219 View ArticlePubMed
  14. Levett PN, Morey RE, Galloway R, Steigerwalt AG and Ellis WA. Reclassification of Leptospira parva Hovind-Hougen et al 1982 as Turneriella parva gen. nov., comb. nov. Int J Syst Evol Microbiol. 2005; 55:1497-1499 View ArticlePubMed
  15. Hookey JV. Characterization of Leptospiraceae by 16S DNA restriction fragment length polymorphisms. J Gen Microbiol. 1993; 139:1681-1689 View ArticlePubMed
  16. Woo TH, Smythe LD, Symonds ML, Norris MA, Dohnt MF and Patel BK. Rapid distinction between Leptonema and Leptospira by PCR amplification of 16S-23S ribosomal DNA spacer. FEMS Microbiol Lett. 1996; 142:85-90 View ArticlePubMed
  17. Woo TH, Patel BKC, Cinco M, Smythe LD, Symonds ML, Norris MA and Dohnt MF. Real-time homogeneous assay of rapid cycle polymerase chain reaction product for identification of Leptonema illini. Anal Biochem. 1998; 259:112-117 View ArticlePubMed
  18. Altschul SF, Gish W, Miller W, Myers EW and Lipman DJ. Basic local alignment search tool. J Mol Biol. 1990; 215:403-410PubMed
  19. Korf I, Yandell M, Bedell J. BLAST, O'Reilly, Sebastopol, 2003.
  20. 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
  21. Porter MF. An algorithm for suffix stripping. Program: electronic library and information systems 1980; 14:130-137.
  22. Lee C, Grasso C and Sharlow MF. Multiple sequence alignment using partial order graphs. Bioinformatics. 2002; 18:452-464 View ArticlePubMed
  23. Castresana J. Selection of conserved blocks from multiple alignments for their use in phylogenetic analysis. Mol Biol Evol. 2000; 17:540-552 View ArticlePubMed
  24. Stamatakis A, Hoover P and Rougemont J. A rapid bootstrap algorithm for the RAxML web-servers. Syst Biol. 2008; 57:758-771 View ArticlePubMed
  25. Hess PN and De Moraes Russo CA. An empirical test of the midpoint rooting method. Biol J Linn Soc Lond. 2007; 92:669-674 View Article
  26. 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
  27. Swofford DL. PAUP*: Phylogenetic Analysis Using Parsimony (*and Other Methods), Version 4.0 b10. Sinauer Associates, Sunderland, 2002.
  28. Pagani I, Liolios K, Jansson J, Chen IM, Smirnova T, Nosrat B, Markowitz VM and Kyrpides NC. The Genomes OnLine Database (GOLD) v.4: status of genomic and metagenomic projects and their associated metadata. Nucleic Acids Res. 2012; 40:D571-D579 View ArticlePubMed
  29. Abt B, Han C, Scheuner C, Lu M, Lapidus A, Nolan M, Lucas S, Hammon N, Deshpande S and Cheng JF. Complete genome sequence of the termite hindgut bacterium Spirochaeta coccoides type strain (SPN1T), reclassification in the genus Sphaerochaeta as Sphaerochaeta coccoides comb. nov. and emendations of the family Spirochaetaceae and the genus Sphaerochaeta. Stand Genomic Sci. 2012; 6:194-209 View ArticlePubMed
  30. Han C, Gronow S, Teshima H, Lapidus A, Nolan M, Lucas S, Hammon N, Deshpande S, Cheng JF and Zeytun A. Complete genome sequence of Treponema succinifaciens type strain (6091T). Stand Genomic Sci. 2011; 4:361-370 View ArticlePubMed
  31. Mavromatis K, Yasawong M, Chertkov O, Lapidus A, Lucas S, Nolan M, Glavina del Rio T, Tice H, Cheng JF and Pitluck S. Complete genome sequence of Spirochaeta smaragdinae type strain (SEBR 4228T). Stand Genomic Sci. 2010; 3:136-144PubMed
  32. Pati A, Sikorski J, Gronow S, Lapidus A, Copeland A, Glavina del Rio T, Nolan M, Lucas S, Chen F and Tice H. Complete genome sequence of Brachyspira murdochii type strain (56-150T). Stand Genomic Sci. 2010; 2:260-269 View ArticlePubMed
  33. Fraser CM, Casjens S, Huang WM, Sutton GG, Clayton RA, Lathigra R, White O, Ketchum KA, Dodson R and Hickey EK. Genomic sequence of a Lyme disease spirochaete, Borrelia burgdorferi. Nature. 1997; 390:580-586 View ArticlePubMed
  34. Abt B, Göker MG, Scheuner C, Han C, Lu M, Misra M, Lapidus A, Nolan M, Lucas S and Hammon N. Genome sequence of the thermophilic fresh-water bacterium Spirochaeta caldaria type strain (H1T), reclassification of Spirochaeta caldaria and Spirochaeta stenostrepta in the genus Treponema as Treponema caldaria comb. nov. and Treponema stenostrepta comb. nov., revival of the name Treponema zuelzerae comb. nov., and emendation of the genus Treponema. Stand Genomic Sci. 2013; 8:88-105 View ArticlePubMed
  35. Stackebrandt E, Chertkov O, Lapidus A, Nolan M, Lucas S, Hammon N, Deshpande S, Cheng JF, Tapia R and Goodwin LA. Genome sequence of the free-living aerobe spirochaete Turneriella parva type strain (HT), end emendation of Turneriella parva. Stand Genomic Sci. 2013; (this issue).
  36. 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
  37. 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
  38. 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, p. 119-169.
  39. Ludwig W, Euzeby J, Whitman WG. Draft taxonomic outline of the Bacteroidetes, Planctomycetes, Chlamydiae, Spirochaetes, Fibrobacteres, Fusobacteria, Acidobacteria, Verrucomicrobia, Dictyoglomi, and Gemmatimonadetes Taxonomic Outline 2008.Web Site
  40. . The nomenclatural types of the orders Acholeplasmatales, Halanaerobiales, Halobacteriales, Methanobacteriales, Methanococcales, Methanomicrobiales, Planctomycetales, Prochlorales, Sulfolobales, Thermococcales, Thermoproteales and Verrucomicrobiales are the genera Acholeplasma, Halanaerobium, Halobacterium, Methanobacterium, Methanococcus, Methanomicrobium, Planctomyces, Prochloron, Sulfolobus, Thermococcus, Thermoproteus and Verrucomicrobium, respectively. Opinion 79. Int J Syst Evol Microbiol. 2005; 55:517-518 View ArticlePubMed
  41. Buchanan RE. Studies in the nomenclature and classification of bacteria. II. The primary subdivisions of the Schizomycetes. J Bacteriol. 1917; 2:155-164PubMed
  42. Skerman VBD, McGowan V and Sneath PHA. Approved Lists of Bacterial Names. Int J Syst Bacteriol. 1980; 30:225-420 View Article
  43. Rocha T, Cardoso EA, Terrinha AM, Nunes JFM, Hovind-Hougen K and Cinco M. Isolation of a new serovar of the genus Leptonema in the family Leptospiraceae. Zbl. Bakt.. 1993; 279:167-172
  44. BAuA 2010 – 2012 update, Classification of bacteria and archaea in risk groups. TRBA 466, p. 19.Web Site
  45. 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
  46. Hovind-Hougen K. Determination by means of electron microscopy of morphological criteria of value for classification of some spirochetes, in particular treponemes. Acta Pathol Microbiol Scand. Sect. B. Suppl.. 1976; 255:28-30PubMed
  47. Wolgemuth CW, Charon NW, Goldstein SF and Goldstein RE. The flagellar cytoskeleton of the spirochetes. J Mol Microbiol Biotechnol. 2006; 11:221-227 View ArticlePubMed
  48. Hovind-Hougen K and Birch-Andersen A. Electron microscopy of endoflagella and microtubules in Treponema Reiter. Acta Pathol Microbiol Scand Sect B. 1971; 79:37-50PubMed
  49. Brendle JJ, Rogul M and Alexander AD. Deoxyribonucleic acid hybridization among selected leptospiral serotypes. Int J Syst Bacteriol. 1974; 24:205-214 View Article
  50. 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
  51. 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
  52. Mavromatis K, Land ML, Brettin TS, Quest DJ, Copeland A, Clum A, Goodwin L, Woyke T, Lapidus A and Klenk HP. The fast changing landscape of sequencing technologies and their impact on microbial genome assemblies and annotation. PLoS ONE. 2012; 7:e48837 View ArticlePubMed
  53. Gemeinholzer B, Dröge G, Zetzsche H, Haszprunar G, Klenk HP, Güntsch A, Berendsohn WG and Wägele JW. The DNA Bank Network: the start from a German initiative. Biopreserv Biobank. 2011; 9:51-55 View Article
  54. JGI website. Web Site
  55. The Phred/Phrap/Consed software package. Web Site
  56. 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
  57. Han C, Chain P. Finishing repeat regions automatically with Dupfinisher. In: Proceeding of the 2006 international conference on bioinformatics & computational biology. Arabnia HR, Valafar H (eds), CSREA Press. June 26-29, 2006: 141-146.
  58. 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.
  59. Hyatt D, Chen GL, LoCascio PF, Land ML, Larimer FW and Hauser LJ. Prodigal: prokaryotic gene recognition and translation initiation site identification. BMC Bioinformatics. 2010; 11:119 View ArticlePubMed
  60. Mavromatis K, Ivanova NN, Chen IM, Szeto E, Markowitz VM and Kyrpides NC. The DOE-JGI Standard operating procedure for the annotations of microbial genomes. Stand Genomic Sci. 2009; 1:63-67 View ArticlePubMed
  61. Pati A, Ivanova NN, Mikhailova N, Ovchinnikova G, Hooper SD, Lykidis A and Kyrpides NC. GenePRIMP: a gene prediction improvement pipeline for prokaryotic genomes. Nat Methods. 2010; 7:455-457 View ArticlePubMed
  62. 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