Open Access

Complete genome sequence of Sebaldella termitidis type strain (NCTC 11300T)

  • Miranda Harmon-Smith1,
  • Laura Celia2,
  • Olga Chertkov3,
  • Alla Lapidus1,
  • Alex Copeland1,
  • Tijana Glavina Del Rio1,
  • Matt Nolan1,
  • Susan Lucas1,
  • Hope Tice1,
  • Jan-Fang Cheng1,
  • Cliff Han1, 3,
  • John C. Detter1, 3,
  • David Bruce1, 3,
  • Lynne Goodwin1, 3,
  • Sam Pitluck1,
  • Amrita Pati1,
  • Konstantinos Liolios1,
  • Natalia Ivanova1,
  • Konstantinos Mavromatis1,
  • Natalia Mikhailova1,
  • Amy Chen4,
  • Krishna Palaniappan4,
  • Miriam Land1, 5,
  • Loren Hauser1, 5,
  • Yun-Juan Chang1, 5,
  • Cynthia D. Jeffries1, 5,
  • Thomas Brettin1, 3,
  • Markus Göker6,
  • Brian Beck2,
  • James Bristow1,
  • Jonathan A. Eisen1, 7,
  • Victor Markowitz4,
  • Philip Hugenholtz1,
  • Nikos C. Kyrpides1,
  • Hans-Peter Klenk6 and
  • Feng Chen1
Standards in Genomic Sciences20102:2020220

https://doi.org/10.4056/sigs.811799

Published: 30 April 2010

Abstract

Sebaldella termitidis (Sebald 1962) Collins and Shah 1986, is the only species in the genus Sebaldella within the fusobacterial family ‘Leptotrichiaceae’. The sole and type strain of the species was first isolated about 50 years ago from intestinal content of Mediterranean termites. The species is of interest for its very isolated phylogenetic position within the phylum Fusobacteria in the tree of life, with no other species sharing more than 90% 16S rRNA sequence similarity. The 4,486,650 bp long genome with its 4,210 protein-coding and 54 RNA genes is part of the Genomic Encyclopedia of Bacteria and Archaea project.

Keywords

anaerobic mesophile nonmotile non-sporeforming Gram-negative termite intestine Fusobacteria Leptotrichiaceae GEBA

Introduction

Strain NCTC 11300T (= ATCC 33386TM = NCTC 11300) is the type strain of the species Sebaldella termitidis [1]. The strain was first isolated from posterior intestinal content of Reticulitermes lucifugus (Mediterranean termites) by the French microbiologist Madeleine Sebald [1,2], and was initially classified as Bacteroides termitidis [3]. The unusually low G+C content, as well as biochemical features which did not correspond to those known for the other members of the genus Bacteroides [4], and the subsequently described novel 16S rRNA sequences [5] made the position of B. termitidis within the genus Bacteroides appear controversial, and guided Collins and Shah in 1986 to reclassify B. termitidis as the type strain of the novel genus Sebaldella [1]. Here we present a summary classification and a set of features for S. termitidis NCTC 11300T, together with the description of the complete genomic sequencing and annotation.

Classification and features

NCTC 11300T represents an isolated species, with no other cultivated strain known in the literature belonging to the species. An uncultured clone with identical 16S rRNA sequence was identified in a mesophilic anaerobic digester that treats municipal wastewater sludge in Clos de Hilde, France [6], and another uncultured clone, PCD-1 (96.1% 16S rRNA sequence identity), was reported from the digestive tract of the ground beetle Poecilus chalcites [7]. The closest related type strains are those of the genus Leptotrichia, which share 85.9 to 89.96% 16S rRNA sequence similarity [8]. Neither environmental screenings nor metagenomic surveys provided any 16S rRNA sequence with significant sequence similarity to NCTC 11300T, indicating that members of the species S. termitidis and the genus Sebaldella are not very frequent in the environment (status February 2010).

Figure 1 shows the phylogenetic neighborhood of S. termitidis NCTC 11300T in a 16S rRNA based tree. The sequences of the four identical copies of the 16S rRNA gene in the genome do not differ from the previously published 16S rRNA sequence generated from ATCC 3386 (M58678), which is missing two nucleotides and contains 30 ambiguous base calls.
Figure 1.

Phylogenetic tree highlighting the position of S. termitidis NCTC 11300T relative to the other type strains within the family ‘Leptotrichiaceae’. The tree was inferred from 1,422 aligned characters [9,10] of the 16S rRNA gene sequence under the maximum likelihood criterion [11] and rooted in accordance with the current taxonomy. 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 if larger than 60%. Lineages with type strain genome sequencing projects registered in GOLD [12] are shown in blue, published genomes in bold, e.g. the recently published GEBA genomes from Leptotrichia buccalis [13], and Streptobacillus moniliformis [14].

Cells of strain NCTC 11300T are Gram-negative, obligately anaerobic, nonmotile, nonspore-forming rods of 0.3 to 0.5 x 2 to 12 µm with central swellings (Figure 2 and Table 1) [1]. Cells occur singly, in pairs, as well as in filaments [1]. Colonies on surface are transparent to opaque, circular measuring 1–2 mm in diameter, whereas colonies in deep agar are non pigmented and lenticular [1].
Figure 2.

Scanning electron micrograph of S. termitidis NCTC 11300T. (J. Carr, CDC, Atlanta, Georgia). More EM photos of the organism can be found at http://phil.cdc.gov/phil.

Table 1.

Classification and general features of S. termitidis NCTC 11300T according to the MIGS recommendations [15]

MIGS ID

Property

Term

Evidence code

 

Current classification

Domain Bacteria

TAS [16]

 

Phylum Fusobacteria

TAS [17]

 

Class Fusobacteria

TAS [17]

 

Order Fusobacteriales

TAS [17]

 

Family Leptotrichiaceae

TAS [18]

 

Genus Sebaldella

TAS [1,19]

 

Species Sebaldella termitidis

TAS [1,19]

 

Type strain NCTC 11300

TAS [1]

 

Gram stain

Gram negative

TAS [1]

 

Cell shape

rod-shaped, with central swellings; occur singly, in pairs and in filaments

TAS [1]

 

Motility

nonmotile

TAS [1]

 

Sporulation

nonsporulating

TAS [2]

 

Temperature range

mesophile

NAS

 

Optimum temperature

not determined

 
 

Salinity

not reported

 

MIGS-22

Oxygen requirement

obligate anaerobic

TAS [1]

 

Carbon source

glucose and other sugars

TAS [1]

 

Energy source

fermentation of glucose and other sugars

TAS [1]

MIGS-6

Habitat

bacterial flora of termite gastrointestinal tract

TAS [1]

MIGS-15

Biotic relationship

unknown

 

MIGS-14

Pathogenicity

none reported

NAS

 

Biosafety level

2

TAS [20]

 

Isolation

posterior intestinal content of termites

TAS [2]

MIGS-4

Geographic location

unknown

 

MIGS-5

Sample collection time

1962 or before

TAS [1,2]

MIGS-4.1

Latitude

not reported

 

MIGS-4.2

Longitude

 

MIGS-4.3

Depth

not reported

 

MIGS-4.4

Altitude

not reported

 

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 [21]. If the evidence code is IDA, then the property was directly observed for a live isolate by one of the authors or an expert mentioned in the acknowledgements.

The major end products of the glucose metabolism by strain NCTC 11300T are acetic and lactic acids (with some formic acid) as opposed to succinic and acetic acids dominating in members of the genus Bacteroides [1]. Enzymes of the hexose-monophosphate-shunt are missing, while present in members of the genus Bacteroides [1,4]. A list of additional sugars and alcohols used or not-used for fermentation is provided by Collins and Shah [1].

Chemotaxonomy

The cell wall structure of strain NCTC 11300T has not yet been reported. Nonhydroxylated and 3-hydroxyated fatty acids were present [1]. The major long chain fatty acids are saturated and monounsaturated straight chain acids: C16:0 (37%) and C18:1 (41%), with methyl branched acids being absent [1], as opposed to straight-chain saturated, anteiso- and iso-methyl branched-chain acids in members of the genus Bacteroides, which are missing the monounsaturated acids [1]. Menaquinones were not detected, as opposed to members of the genus Bacteroides [1].

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 [22]. The genome project is deposited in the Genome OnLine Database [12] 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

One genomic 8kb pMCL200 library, one 454 pyrosequence library and one Illumina library

MIGS-29

Sequencing platforms

Sanger, 454 Titanium, Illumina

MIGS-31.2

Sequencing coverage

9.2× Sanger; 30.3× 454 Titanium

MIGS-30

Assemblers

Newbler, phrap

MIGS-32

Gene calling method

Prodigal, GenePRIMP

 

INSDC ID

CP001739 (chromosome), CP001740, CP001741 (plasmids)

 

Genbank Date of Release

November 19, 2009

 

GOLD ID

Gc01144

 

NCBI project ID

29539

 

Database: IMG-GEBA

2501846314

MIGS-13

Source material identifier

ATCC 33386

 

Project relevance

Tree of Life, GEBA

Growth conditions and DNA isolation

S. termitidis NCTC 11300T, ATCC 33386TM, was grown anaerobically in ATCC medium 1490 (Modified chopped meat medium) [23] at 37°C. DNA was isolated from cell paste using a basic CTAB extraction and then quality controlled according to JGI guidelines.

Genome sequencing and assembly

The genome was sequenced using a combination of Sanger and 454 sequencing platforms. All general aspects of library construction and sequencing can be found at http://www.jgi.doe.gov/. 454 Pyrosequencing reads were assembled using the Newbler assembler version 1.1.02.15 (Roche). Large Newbler contigs were broken into 4,966 overlapping fragments of 1,000 bp and entered into assembly as pseudo-reads. The sequences were assigned quality scores based on Newbler consensus q-scores with modifications to account for overlap redundancy and to adjust inflated q-scores. A hybrid 454/Sanger assembly was made using the parallel phrap assembler (High Performance Software, LLC). Possible mis-assemblies were corrected with Dupfinisher [24] or transposon bombing of bridging clones (Epicentre Biotechnologies, Madison, WI). Gaps between contigs were closed by editing in Consed, custom primer walk or PCR amplification. A total of 796 Sanger finishing reads were produced to close gaps, to resolve repetitive regions, and to raise the quality of the finished sequence. Illumina reads were used to improve the final consensus quality using an in-house developed tool (the Polisher, unpublished). The error rate of the completed genome sequence is less than 1 in 100,000. Together all sequence types provided 39.5× coverage of the genome. The final assembly contains 45,934 Sanger and 760,187 pyrosequence reads.

Genome annotation

Genes were identified using Prodigal [25] as part of the Oak Ridge National Laboratory genome annotation pipeline, followed by a round of manual curation using the JGI GenePRIMP pipeline [26]. 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 manual functional annotation was performed within the Integrated Microbial Genomes Expert Review (IMG-ER) platform [27].

Genome properties

The genome consists of a 4,418,842 bp long chromosome, and two plasmids with 54,160 bp and 13,648 bp length, respectively, with a 33.4% GC content (Table 3 and Figure 3). Of the 4,264 genes predicted, 4,210 were protein-coding genes, and 54 RNAs; 59 pseudogenes were identified. The majority of the protein-coding genes (60.4%) were assigned with a putative function while those remaining were annotated as hypothetical proteins. The distribution of genes into COGs functional categories is presented in Table 4.
Figure 3.

Graphical circular maps of the chromosome and the two plasmids. 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 3.

Genome Statistics

Attribute

Value

% of Total

Genome size (bp)

4,486,650

100.00%

DNA coding region (bp)

3,918,335

87.33%

DNA G+C content (bp)

1,497,450

33.38%

Number of replicons

3

 

Extrachromosomal elements

2

 

Total genes

4,264

100.00%

RNA genes

54

1.27%

rRNA operons

4

 

Protein-coding genes

4,210

98.73%

Pseudogenes

59

1.38%

Genes with function prediction

2,576

60.41%

Genes in paralog clusters

1,253

29.39%

Genes assigned to COGs

2,299

60.95%

Genes assigned Pfam domains

2,787

65.36%

Genes with signal peptides

801

18.79%

Genes with transmembrane helices

901

21.13%

CRISPR repeats

1

 
Table 4.

Number of genes associated with the general COG functional categories

Code

value

%age

Description

J

152

3.6

Translation, ribosomal structure and biogenesis

A

0

0.0

RNA processing and modification

K

265

6.3

Transcription

L

130

3.1

Replication, recombination and repair

B

0

0.0

Chromatin structure and dynamics

D

22

0.5

Cell cycle control, cell division, chromosome partitioning

Y

0

0.0

Nuclear structure

V

47

1.1

Defense mechanisms

T

96

2.3

Signal transduction mechanisms

M

155

3.7

Cell wall/membrane biogenesis

N

17

0.4

Cell motility

Z

0

0.0

Cytoskeleton

W

0

0.0

Extracellular structures

U

41

1.0

Intracellular trafficking, secretion and vesicular transport

O

71

1.7

Posttranslational modification, protein turnover, chaperones

C

128

3.0

Energy production and conversion

G

468

11.1

Carbohydrate transport and metabolism

E

219

5.2

Amino acid transport and metabolism

F

93

2.2

Nucleotide transport and metabolism

H

106

2.5

Coenzyme transport and metabolism

I

59

1.4

Lipid transport and metabolism

P

105

2.5

Inorganic ion transport and metabolism

Q

32

0.8

Secondary metabolites biosynthesis, transport and catabolism

R

403

9.6

General function prediction only

S

241

5.7

Function unknown

-

1,665

39.5

Not in COGs

Declarations

Acknowledgements

We would like to gratefully acknowledge the help of Janice Carr (Centers of Disease Control, Atlanta, Georgia) for providing the EM photo of S. thermitidis NCTC 11300T. This work was performed under the auspices of the US Department of Energy’s 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, Los Alamos National Laboratory under contract No. DE-AC02-06NA25396, and Oak Ridge National Laboratory under contract DE-AC05-00OR22725

Authors’ Affiliations

(1)
DOE Joint Genome Institute
(2)
ATCC - American Type Culture Collection
(3)
Bioscience Division, Los Alamos National Laboratory
(4)
Biological Data Management and Technology Center, Lawrence Berkeley National Laboratory
(5)
Oak Ridge National Laboratory
(6)
DSMZ - German Collection of Microorganisms and Cell Cultures GmbH
(7)
University of California Davis Genome Center

References

  1. Collins MD, Shah HN. Reclassification of Bacteroides termitidis Sebald (Holdeman and Moore) in a new genus Sebaldella termitidis comb. nov. Int J Syst Bacteriol 1986; 36:349–350. doi:10.1099/00207713-36-2-349View ArticleGoogle Scholar
  2. Sebald M. Etude sur les bacteries anaérobes gramnégatives asporulées. Thèse de l’Université Paris. Imprimerie Barnéoud S.A. Laval, France, 1962.Google Scholar
  3. Holdeman LV, Kelly RW, Moore WEC. Genus Bacteroides, p. 604–631. In: Krieg NR, Holt JG (eds) Bergey’s manual of systematic bacteriology, Vol. 1. The Williams & Wilkins Co., Baltimore. 1984Google Scholar
  4. Shah HN, Collins MD. Genus Bacteroides: a chemotaxonomical perspective. J Appl Bacteriol 1983; 55:403–416. PubMedView ArticlePubMedGoogle Scholar
  5. Paster BJ, Ludwig W, Weisburg WG, Stackebrandt E, Hespell RB, Hahn CM, Reichenbach H, Stetter KO, Woese CR. A phylogenetic grouping of the Bacteroides, Cytophagas, and certain Flavobacteria. Syst Appl Microbiol 1985; 6:34–42.View ArticleGoogle Scholar
  6. Rivière D, Desvignes V, Pelletier E, Chaussonnerie S, Guermazi S, Weissenbach J, Li T, Camacho P, Sghir A. Towards the definition of a core of microorganisms involved in anaerobic digestion of sludge. ISME J 2009; 3:700–714. PubMed doi:10.1038/ismej.2009.2View ArticlePubMedGoogle Scholar
  7. Lehman RM, Lundgren JG, Petzke LM. Bacterial communities associated with the digestive tract of the predatory ground beetle, Poecilus chalcites, and their modifications by laboratory rearing ans antibiotic treatment. Microb Ecol 2009; 57:349–358. PubMed doi:10.1007/s00248-008-9415-6View ArticlePubMedGoogle Scholar
  8. Chun J, Lee JH, Jung Y, Kim M, Kim S, Kim BK, Lim YW. EzTaxon: a web-based tool for the identification of prokaryotes based on 16S ribosomal RNA gene sequences. Int J Syst Evol Microbiol 2007; 57:2259–2261. PubMed doi:10.1099/ijs.0.64915-0View ArticlePubMedGoogle Scholar
  9. Castresana J. Selection of conserved blocks from multiple alignments for their use in phylogenetic analysis. Mol Biol Evol 2000; 17:540–552. PubMedView ArticlePubMedGoogle Scholar
  10. Lee C, Grasso C, Sharlow MF. Multiple sequence alignment using partial order graphs. Bioinformatics 2002; 18:452–464. PubMed doi:10.1093/bioinformatics/18.3.452View ArticlePubMedGoogle Scholar
  11. Stamatakis A, Hoover P, Rougemont J. A rapid bootstrap algorithm for the RAxML web servers. Syst Biol 2008; 57:758–771. PubMed doi:10.1080/10635150802429642View ArticlePubMedGoogle Scholar
  12. Liolios K, Chen IM, Mavromatis K, Tavernarakis N, Hugenholtz P, Markowitz VM, Kyrpides NC. The Genomes On Line Database (GOLD) in 2009: status of genomic and metagenomic projects and their associated metadata. Nucleic Acids Res 2010; 38:D346–D354. PubMed doi:10.1093/nar/gkp848PubMed CentralView ArticlePubMedGoogle Scholar
  13. Ivanova N, Gronow S, Lapidus A, Copeland A, Glavina Del Rio T, Nolan M, Lucas S, Cheng F, Tice H, Cheng JF, et al. Leptotrichia buccalis type strain (C-1013-bT). Stand Genomic Sci 2009; 1:126–132. doi:10.4056/sigs.1854PubMed CentralView ArticlePubMedGoogle Scholar
  14. Nolan M, Gronow S, Lapidus A, Ivanova N, Copeland A, Lucas S, Glavina Del Rio T, Chen F, Tice H, Pitluck S, et al. Streptobacillus moniliformis type strain (9901T). Stand Genomic Sci 2009; 1:300–397. doi:10.4056/sigs.48727PubMed CentralPubMedGoogle Scholar
  15. 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/nbt1360PubMed CentralView ArticlePubMedGoogle Scholar
  16. 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.87.12.4576PubMed CentralView ArticlePubMedGoogle Scholar
  17. Garrity GM, Holt JG. Taxonomic Outline of the Archaea and Bacteria. In: Garrity GM, Boone DR, Castenholz RW (eds), Bergey’s Manual of Systematic Bacteriology, Second Edition, Volume 1, Springer, New York, 2001, p. 155–166.Google Scholar
  18. Ludwig W, Euzeby J, Whitman WG. Draft taxonomic outline of the Bacteroidetes, Planctomycetes, Chlamydiae, Spirochaetes, Fibrobacteres, Fusobacteria, Acidobacteria, Verrucomicrobia, Dictyoglomi, and Gemmatimonadetes. http://www.bergeys.org/outlines/Bergeys_Vol_4_Outline.pdf. Taxonomic Outline 2008.
  19. Skerman VBD, McGowan V, Sneath PHA. Approved Lists of Bacterial Names. Int J Syst Bacteriol 1980; 30:225–420. doi:10.1099/00207713-30-1-225View ArticleGoogle Scholar
  20. CDC’s Office of Health and Safety. http://www.cdc.gov/od/ohs/biosfty/bmbl5/bmbl5toc.htm
  21. 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:10.1038/75556PubMed CentralView ArticlePubMedGoogle Scholar
  22. 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/nature08656PubMed CentralView ArticlePubMedGoogle Scholar
  23. Growth media used at ATCC: http://www.atcc.org/Attachments/2718.pdf
  24. Sims D, Brettin T, Detter J, 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.761PubMed CentralView ArticlePubMedGoogle Scholar
  25. Hyatt D, Chen GL, Locascio PF, Land ML, Larimer FW, Hauser LJ. Prodigal: Prokaryotic Dynamic Programming Genefinding Algorithm. BMC Bioinformatics 2010; 11:119. PubMed doi:10.1186/1471-2105-11-119PubMed CentralView ArticlePubMedGoogle Scholar
  26. 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 (In press).Google Scholar
  27. 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/btp393View ArticlePubMedGoogle Scholar

Copyright

© The Author(s) 2010