Open Access

Complete genome sequence of Thermosediminibacter oceani type strain (JW/IW-1228PT)

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

DOI: 10.4056/sigs.1133078

Received: 28 September 2010

Published: 31 October 2010

Abstract

Thermosediminibacter oceani (Lee et al. 2006) is the type species of the genus Thermosediminibacter in the family Thermoanaerobacteraceae. The anaerobic, barophilic, chemoorganotrophic thermophile is characterized by straight to curved Gram-negative rods. The strain described in this study was isolated from a core sample of deep sea sediments of the Peruvian high productivity upwelling system. This is the first completed genome sequence of a member of the genus Thermosediminibacter and the seventh genome sequence in the family Thermoanaerobacteraceae. The 2,280,035 bp long genome with its 2,285 protein-coding and 63 RNA genes is a part of the Genomic Encyclopedia of Bacteria and Archaea project.

Keywords:

chemoorganotrophanaerobethermophilebarophileupwelling systemcore sampledeep sea sedimentThermoanaerobacteralesFirmicutesGEBA

Introduction

Strain JW/IW-1228PT (= DSM 16646 = ATCC BAA-1034) is the type strain of Thermosediminibacter oceani, which is the type species of the genus Thermosediminibacter [1], one out of nineteen genera in the family Thermoanaerobacteraceae [2-4]. The generic name derives from the Greek word ‘thermos’ meaning ‘hot’, the Latin word ‘sediment’ and the Latin word ‘bacter’ meaning ‘a rod or staff’, referring to its origin and growth temperature [1]. The species epithet is also derived from the Latin word ‘oceani’ meaning ‘of an ocean’, referring to its origin from the ocean [1]. Strain JW/IW-1228PT was described in 2005 by Lee as T. oceani [1] and validly published in 2006 [5]. Strain JW/IW-1228PT was isolated from a core sediment sample (core 201-1228E-1H-1) at 136-143 cm below the seafloor. The core sample was obtained from the outer shelf edge of the Peruvian high productivity upwelling system. The sea floor there was located at 252 m below the sea level with 12°C mud line temperature. Strain JW/IW-1228PT is of particular interest because it is able to ferment a significant number of polysaccharides [1]. Moreover, the strain JW/IW-1228PT is able to use thiosulfate, elemental sulfur and MnO2 as electron acceptors for growth. The only other species in the genus Thermosediminibacter is T. litoriperuensis, the type strain of which was isolated from the Peru Trench at 5,086 m below sea level with a mud-line temperature of 2°C [1]. Here we present a summary classification and a set of features for T. oceani JW/IW-1228PT, together with the description of the complete genomic sequencing and annotation.

Classification and features

The 16S rRNA gene sequence of JW/IW-1228PT is 98.4% identical to that of T. litoriperuensis JW/YJL-1230-2T, the type strain of the only other described species with a validly published name in the genus. The sequence similarities between strain JW/IW-1228PT and the type strains of the members of the genera Fervidicola and Caldanaerovirga are 94.4%, with the closest sequence match being that with F. ferrireducens and C. acetigignens [6]. Three significantly similar 16S rRNA gene sequences are known from uncultured clones of Thermovenabulum sp. from GenBank [7]: B5_otu10 (96%, DQ097675), B14_otu11 (95%, DQ097676) and B8_otu12 (95%, DQ097677), all from the Kongdian bed of the Dagang oil field (Hebei province, China) [7,8]. No phylotypes from environmental screening or genomic surveys could be linked to the species T. oceani or even the genus Thermosediminibacter, indicating a rather rare occurrence of these in the habitats screened so far (as of July 2010).

Figure 1 shows the phylogenetic neighborhood of T. oceani JW/IW-1228PT in a 16S rRNA based tree. The sequences of the three 16S rRNA gene copies in the genome differ from each other by up to one nucleotide and differ by only one nucleotide from the previously published sequence (AY703478).

Figure 1

Phylogenetic tree highlighting the position of T. oceani JW/IW-1228PT relative to the type strains of the other species within the family Thermoanaerobacteraceae. The trees were inferred from 1,316 aligned characters [9,10] of the 16S rRNA gene sequence under the maximum likelihood criterion [11] and rooted in accordance with the current taxonomy [12]. The branches are scaled in terms of the expected number of substitutions per site. Numbers above branches are support values from 850 bootstrap replicates [13] if larger than 60%. Lineages with type strain genome sequencing projects registered in GOLD [14] are shown in blue, published genomes in bold [32,33,CP001785,CP001145].

The cells of strain JW/IW-1228PT are straight to curved rods which occur singly, in pairs or in chains (Table 1 and Figure 2). They are between 0.2-0.7µm in diameter and 1.5-16 µm in length. In the late-exponential or stationary phase of growth the cells are swollen and subsequently form L-shaped autoplasts [1]. Strain JW/IW-1228PT is Gram-negative, although Thermosediminibacter belongs to the Gram-positive Bacillus-Clostridium subphylum [1]. The cells tend towards elongation and to form aggregates during growth. Motility has not been reported although flagella are observed on the cells (not visible in Figure 2), however, the cells are able to tumble [1], which might imply an impaired flagellar function. Strain JW/IW-1228PT is thermophilic and grows optimally at 68°C; the temperature range for growth is 52-76°C. The optimum pH25°C for growth is 7.5, with a range for growth at 6.3-9.3. The optimum salinity for growth is 1% (w/v), with a salinity range of 0-6% (w/v) [1]. Yeast extract is required for growth. The growth of strain JW/IW-1228PT is not observed on H2/CO2 (80:20, v/v) [1]. The strain produces α-glucosidase [22]. The carbon and energy sources used by JW/IW-1228PT include beef extract, casamino acids, cellobiose, fructose, galactose, glucose, inositol, lactate, maltose, mannose, pyruvate, raffinose, sorbitol, sucrose, trehalose, tryptone and xylose when 0.02% w/v of yeast extract is present in growth medium [1]. The fermentation product from glucose is acetate and occasionally trace amounts of propionate, isobutyrate and isovalerate. Acetate is a major product [1]. Strain JW/IW-1228PT does not utilize xylitol [22]. It is able to use thiosulfate, elemental sulfur and MnO2 as electron acceptors for growth. There is no indication that JW/IW-1228PT is able to grow chemolithoautotrophically; it does not reduce sulfate or Fe(III) [1].

Table 1

Classification and general features of T. oceani JW/IW-1228P T according to the MIGS recommendations [15].

MIGS ID

   Property

   Term

     Evidence code

   Current classification

   Domain Bacteria

     TAS [16]

   Phylum Firmicutes

     TAS [17,18]

   Class Clostridia

     TAS [2,19]

   Order Thermoanaerobacterales

     TAS [2,19,20]

   Family Thermoanaerobacteraceae

     TAS [2-4]

   Genus Thermosediminibacter

     TAS [1,5]

   Species Thermosediminibacter oceani

     TAS [1,5]

   Type strain JW/IW-1228P

     TAS [1,5]

   Gram stain

   negative

     TAS [1]

   Cell shape

   straight to curved rods, 0.2-0.7 ×1.5-16 µm.    cells tend to elongate and form   aggregates.

     TAS [1]

   Motility

   no motility, but tumbling (flagella observed)

     TAS [1]

   Sporulation

   not observed

     TAS [1]

   Temperature range

   52–76°C

     TAS [1]

   Optimum temperature

   68°C

     TAS [1]

   Salinity

   0-6% w/v NaCl (optimum at 1%)

     TAS [1]

MIGS-22

   Oxygen requirement

   anaerobic

     TAS [1]

   Carbon source

   carbohydrates

     TAS [1]

   Energy source

   chemoorganotroph

     TAS [1]

MIGS-6

   Habitat

   ocean subsurface sediments

     TAS [1]

MIGS-15

   Biotic relationship

   free-living

     NAS

MIGS-14

   Pathogenicity

   none

     NAS

   Biosafety level

   1

     TAS [21]

   Isolation

   core sample from deep sea sediment

     TAS [1]

MIGS-4

   Geographic location

   subseafloor, outer shelf edge of the    Peruvian high productivity upwelling    system, Peru

     TAS [1]

MIGS-5

   Sample collection time

   2002

     NAS

MIGS-4.1

   Latitude

   approx. S11° 11' 23"

     TAS [22]

MIGS-4.2

   Longitude

   approx. W79° 4' 33"

     TAS [22]

MIGS-4.3

   Depth

   136-143 cm below seafloor

     TAS [1]

MIGS-4.4

   Altitude

   252 m below sea level

     TAS [1]

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

Figure 2

Scanning electron micrograph of T. oceani JW/IW-1228P T

Chemotaxonomy

The peptidoglycan structure of strain JW/IW-1228PT is still unknown. The phospholipid fatty acid composition of strain JW/IW-1228PT consists of branched and straight chain saturated acids: iso-C15:0 (56.2%), iso-C17:0 (9.6%), C16:0 (7.5%), anteiso-C15:0 (6.7%), C16:1ω9c (5.6%), C15:0 (5.0%), C18:1 ω 9c (3.3%) and iso-C16:0 (1.9%) [1].

Genome sequencing and annotation

Genome project history

This organism was selected for sequencing on the basis of its phylogenetic position [24], and is part of the Genomic Encyclopedia of Bacteria and Archaea project [25]. The genome project is deposited in the Genome OnLine Database [14] 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

    Tree genomic libraries:    one Sanger 8 kb pMCL200 library,    one 454 pyrosequence standard library and    one Illumina standard library

MIGS-29

    Sequencing platforms

    ABI3730, Illumina GAii, 454 GS FLX    Titanium

MIGS-31.2

    Sequencing coverage

    5.3× Sanger; 34.3× Illumina, 25.4×    pyrosequence

MIGS-30

    Assemblers

    Newbler version 2.0.0-PostRelease-    07/15/2008, Velvet, phrap

MIGS-32

    Gene calling method

    Prodigal 1.4, GenePRIMP

    INSDC ID

    CP002131

    Genbank Date of Release

    August 5, 2010

    GOLD ID

    Gc01361

    NCBI project ID

    30983

    Database: IMG-GEBA

    2503242007

MIGS-13

    Source material identifier

    DSM 16646

    Project relevance

    Tree of Life, GEBA

Growth conditions and DNA isolation

T. oceani JW/IW-1228PT, DSM 16646, was grown anaerobically in DSMZ medium 664 (Thermotoga elfii medium) [26] at 68°C. DNA was isolated from 0.5-1 g of cell paste using Qiagen Genomic 500 DNA Kit (Qiagen, Hilden, Germany) following the standard protocol as recommended by the manufacturer, with modification st/LALMP for cell lysis as described in Wu et al. [25].

Genome sequencing and assembly

The genome was sequenced using a combination of Sanger, Illumina and 454 sequencing platforms. All general aspects of library construction and sequencing can be found at the JGI website (Web Site). Pyrosequencing reads were assembled using the Newbler assembler version 2.0.0-PostRelease-07/15/2008 (Roche). The initial Newbler assembly consisted of 83 contigs in 32 scaffolds which was converted into a phrap assembly by making fake reads from the consensus. Illumina GAii sequencing data was assembled with Velvet [27] and the consensus sequences were shredded into 1.5 kb overlapped fake reads and assembled together with the 454 data. Draft assemblies were based on 166.4 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 (Web Site) 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 Dupfinisher [28], 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 625 additional reactions and two shatter libraries were necessary to close gaps and to raise the quality of the finished sequence. Illumina data was used to correct potential base errors and increase consensus quality using a software Polisher developed at JGI [29]. The error rate of the completed genome sequence is less than 1 in 100,000. Together, the combination of the Sanger, Illumina and 454 sequencing platforms provided 65.0 ×coverage of the genome.

Genome annotation

Genes were identified using Prodigal [30] as part of the Oak Ridge National Laboratory genome annotation pipeline, followed by a round of manual curation using the JGI GenePRIMP pipeline [31]. 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 Integrated Microbial Genomes - Expert Review (IMG-ER) platform [32].

Genome properties

The genome consists of a 2,280,035 bp long chromosome with a 468% GC content (Table 3 and Figure 3). Of the 2,348 genes predicted, 2,285 were protein-coding genes, and 63 RNAs; eighty eight pseudogenes were also identified. The majority of the protein-coding genes (73.3%) 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)

  2,280,035

     100.00%

DNA coding region (bp)

  1,991,971

     87.37%

DNA G+C content (bp)

  1,067,515

     46.82%

Number of replicons

  1

Extrachromosomal elements

  0

Total genes

  2,348

     100.00%

RNA genes

  63

     2.68%

rRNA operons

  3

Protein-coding genes

  2,285

     97.32%

Pseudo genes

  88

     3.75%

Genes with function prediction

  1,722

     73.34%

Genes in paralog clusters

  366

     15.59%

Genes assigned to COGs

  1,751

     74.57%

Genes assigned Pfam domains

  1,925

     81.98%

Genes with signal peptides

  280

     11.93%

Genes with transmembrane helices

  563

     23.98%

CRISPR repeats

  5

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

    140

   7.3

    Translation, ribosomal structure and biogenesis

A

    0

   0.0

    RNA processing and modification

K

    122

   6.4

    Transcription

L

    191

   10.0

    Replication, recombination and repair

B

    1

   0.1

    Chromatin structure and dynamics

D

    35

   1.8

    Cell cycle control, cell division, chromosome partitioning

Y

    0

   0.0

    Nuclear structure

V

    28

   1.5

    Defense mechanisms

T

    96

   5.0

    Signal transduction mechanisms

M

    107

   5.6

    Cell wall/membrane/envelope biogenesis

N

    58

   3.0

    Cell motility

Z

    0

   0.0

    Cytoskeleton

W

    0

   0.0

    Extracellular structures

U

    49

   2.6

    Intracellular trafficking and secretion, and vesicular transport

O

    58

   3.0

    Posttranslational modification, protein turnover, chaperones

C

    142

   7.4

    Energy production and conversion

G

    117

   6.1

    Carbohydrate transport and metabolism

E

    147

   7.7

    Amino acid transport and metabolism

F

    51

   2.7

    Nucleotide transport and metabolism

H

    94

   4.9

    Coenzyme transport and metabolism

I

    33

   1.7

    Lipid transport and metabolism

P

    85

   4.5

    Inorganic ion transport and metabolism

Q

    25

   1.3

    Secondary metabolites biosynthesis, transport and catabolism

R

    171

   9.0

    General function prediction only

S

    158

   8.3

    Function unknown

-

    597

   25.4

    Not in COGs

Declarations

Acknowledgements

We would like to gratefully acknowledge the help of Maren Schröder for growing T. oceani cultures and Susanne Schneider for DNA extraction and quality analysis (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, as well as German Research Foundation (DFG) INST 599/1-2 and SI 1352/1-2 and Thailand Research Fund Royal Golden Jubilee Ph.D. Program No. PHD/0019/2548 for MY.


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