Open Access

Permanent draft genome sequence of Dethiosulfovibrio peptidovorans type strain (SEBR 4207T)

  • Kurt LaButti
  • , Shanmugam Mayilraj,
  • , Alicia Clum
  • , Susan Lucas
  • , Tijana Glavina Del Rio
  • , Matt Nolan
  • , Hope Tice
  • , Jan-Fang Cheng
  • , Sam Pitluck
  • , Konstantinos Liolios
  • , Natalia Ivanova
  • , Konstantinos Mavromatis
  • , Natalia Mikhailova
  • , Amrita Pati
  • , Lynne Goodwin,
  • , Amy Chen
  • , Krishna Palaniappan
  • , Miriam Land,
  • , Loren Hauser,
  • , Yun-Juan Chang,
  • , Cynthia D. Jeffries,
  • , Manfred Rohde
  • , Stefan Spring
  • , Markus Göker
  • , Tanja Woyke
  • , James Bristow
  • , Jonathan A. Eisen,
  • , Victor Markowitz
  • , Philip Hugenholtz
  • , Nikos C. Kyrpides
  • , Hans-Peter Klenk
  • and Alla Lapidus
Corresponding author

DOI: 10.4056/sigs.1092865

Received: 20 August 2010

Published: 30 August 2010

Abstract

Dethiosulfovibrio peptidovorans Magot et al. 1997 is the type species of the genus Dethiosulfovibrio of the family Synergistaceae in the recently created phylum Synergistetes. The strictly anaerobic, vibriod, thiosulfate-reducing bacterium utilizes peptides and amino acids, but neither sugars nor fatty acids. It was isolated from an offshore oil well where it was been reported to be involved in pitting corrosion of mild steel. Initially, this bacterium was described as a distant relative of the genus Thermoanaerobacter, but was not assigned to a genus, it was subsequently placed into the novel phylum Synergistetes. A large number of repeats in the genome sequence prevented an economically justifiable closure of the last gaps. This is only the third published genome from a member of the phylum Synergistetes. The 2,576,359 bp long genome consists of three contigs with 2,458 protein-coding and 59 RNA genes and is part of the Genomic Encyclopedia of Bacteria and Archaea project.

Keywords:

anaerobicmotilevibrio-shapedthiosulfate-reducingH2S producingpeptide utilizationSynergistaceaeSynergistetesGEBA

Introduction

Strain SEBR 4207T (= DSM 11002 = JCM 15826) is the type strain of the species Dethiosulfovibrio peptidovorans (‘curved rod-shaped [vibrio] bacterium that reduces thiosulfate devouring peptides’), which represents the type species of the genus Dethiosulfovibrio [1]. D. peptidovorans strain SEBR 4207T was isolated in 1989 from an offshore oil well in the Congo (Brazzaville) and initially described by Magot et al. in 1997 [1]. The strain provided the first experimental evidence for the involvement of microbial thiosulfate reduction in the corrosion of steel (pitting corrosion). Strain SEBR 4207T utilizes only peptides and amino acids, but no sugar or fatty acids. For the first few years neither the strain nor the genus Dethiosulfovibrio could be assigned to an established higher taxon, except that the distant relationship to the genus Thermanaerovibrio was reported [1]. The taxonomic situation of the species was only recently further enlightened, when Jumas-Bilak et al. [2] combined several genera with anaerobic, rod-shaped, amino acid degrading, Gram-negative bacteria into the novel phylum Synergistetes [2]. The phylum Synergistetes contains organisms isolated from humans, animals, terrestrial and oceanic habitats: Thermanaerovibrio, Dethiosulfovibrio, Aminiphilus, Aminobacterium, Aminomonas, Anaerobaculum, Jonquetella, Synergistes and Thermovirga. Given the novelty of the phylum it is not surprising that many of the type strains from these genera are already subject to genome sequencing projects. Here we present a summary classification and a set of features for D. peptidovorans strain SEBR 4207T, together with the description of the genomic sequencing and annotation.

Classification and features

The 16S rRNA genes of the four other type strains in the genus Dethiosulfovibrio share between 94.2% (D. salsuginis [3]) and 99.2% (D. marinus [4]) sequence identity with strain SEBR 4207T, whereas the other type strains from the family Synergistaceae share 83.6 to 86.6% sequence identity [5]. There are no other cultivated strains that closely related. Uncultured clones with high sequence similarity to strain SEBR 4207T were identified in a copper-polluted sediment in Chile (clones LC6 and LC23, FJ024724 and FJ024721, 99.1%). Metagenomic surveys and environmental samples based on 16S rRNA gene sequences provide no indication for organisms with sequence similarity values above 88% to D. peptidovorans SEBR 4207T, indicating that members of this species are not abundant in habitats screened thus far. The majority of these 16S rRNA gene sequences with similarity between 88% and 93% originate from marine metagenomes (status July 2010).

Figure 1 shows the phylogenetic neighborhood of D. peptidovorans SEBR 4207T in a 16S rRNA based tree. The five copies of the 16S rRNA gene differ by up to one nucleotide from each other and by eight nucleotides from the previously published sequence generated from DSM 11002 (DPU52817).

Figure 1

Phylogenetic tree highlighting the position of D. peptidovorans SEBR 4207T relative to the other type strains within the phylum Synergistetes. The tree was inferred from 1,328 aligned characters [6,7] of the 16S rRNA gene sequence under the maximum likelihood criterion [8] and rooted in accordance with the current taxonomy [9]. 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 greater than 60%. Lineages with type strain genome sequencing projects registered in GOLD [10] are shown in blue, published genomes in bold [11,12].

Cells of D. peptidovorans SEBR 4207T stain Gram-negative [1]. Cells are vibriod with pointed or round ends and lateral flagella (Figure 2, flagella not visible) and a size of 3-5 by 1 µm [1] (Table 1). Spores were not detected [1]. Optimal growth rate was observed at 42°C, pH 7.0 in 3% NaCl [1]. D. peptidovorans is capable of utilizing peptides and amino acids as a sole carbon and energy source and can ferment serine and histidine. In the presence of thiosulfate, strain SEBR 4207T is capable of utilizing alanine, arginine, asparagines, glutamate, isoleucine, leucine, methionine and valine as an electron acceptor. The strain is capable of producing acetate, isobutyrate, isovalerate, 2-methylbutyrate, CO2 and H2 from peptides. The strain uses elemental sulfur and thiosulfate but not sulfate as electron acceptor. H2S is produced with a decrease in H2. Cells do not have cytochrome or desulfoviridin [1]. When yeast extract was added as sole carbon and energy source together with trypticase, thiosulfate was used as sole electron acceptor. Strain SEBR 4207T was not able to utilize gelatine, casein, arabinose, fructose, galactose, glucose, lactose, maltose, mannose, rhamnose, ribose, sucrose, sorbose, trehalose, xylose, acetate, propionate, butyrate, citrate and lactate.

Figure 2

Scanning electron micrograph of D. peptidovorans SEBR 4207T

Table 1

Classification and general features of D. peptidovorans SEBR 4207T according to the MIGS recommendations [13].

MIGS ID

  Property

  Term

   Evidence code

  Current classification

  Domain Bacteria

   TAS [14]

  Phylum Synergistetes

   TAS [2]

  Class Synergistia

   TAS [2]

  Order Synergistales

   TAS [2]

  Family Synergistaceae

   TAS [2]

  Genus Dethiosulfovibrio

   TAS [1]

  Species Dethiosulfovibrio peptidovorans

   TAS [1]

  Type strain SEBR 4207

   TAS [1]

  Gram stain

  negative

   TAS [1]

  Cell shape

  curved rods (vibrioid)

   TAS [1]

  Motility

  motile via lateral flagella

   TAS [1]

  Sporulation

  non-sporulating

   TAS [1]

  Temperature range

  mesophile, 20-45°C

   TAS [1]

  Optimum temperature

  42°C

   TAS [1]

  Salinity

  slightly halophilic, optimum 3% NaCl

   TAS [1]

MIGS-22

  Oxygen requirement

  anaerobic

   TAS [1]

  Carbon source

  peptides and amino acids

   TAS [1]

  Energy source

  peptides and amino acids

   TAS [1]

MIGS-6

  Habitat

  marine, oil wells

   TAS [1]

MIGS-15

  Biotic relationship

  free living

   NAS

MIGS-14

  Pathogenicity

  non pathogenic

   NAS

  Biosafety level

  1

   TAS [15]

  Isolation

  from corroding off-shore oil wells

   TAS [1]

MIGS-4

  Geographic location

  Emeraude oil field, Congo (Brazzaville)

   TAS [1]

MIGS-5

  Sample collection time

  before 1989

   TAS [1]

MIGS-4.1MIGS-4.2

  Latitude  Longitude

  -5.05  11.78

   NAS

MIGS-4.3

  Depth

  not reported

MIGS-4.4

  Altitude

  about sea level

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

Chemotaxonomy

None of the classical chemotaxonomic features (peptidoglycan structure, cell wall sugars, cellular fatty acid profile, menaquinones, or polar lipids) are known for D. peptidovorans SEBR 4207T or any of the other members of the genus Dethiosulfovibrio.

Genome sequencing and annotation

Genome project history

This organism was selected for sequencing on the basis of its phylogenetic position [17], and is part of the Genomic Encyclopedia of Bacteria and Archaea project [18]. The genome project is deposited in the Genome OnLine Database [10] 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

    Permanent draft

MIGS-28

   Libraries used

    One 8 kb pMCL200 Sanger library,    one 454 pyrosequence standard library    and one Solexa library

MIGS-29

   Sequencing platforms

    ABI3730, 454 Titanium, Illumina GAii

MIGS-31.2

   Sequencing coverage

    8.0 x Sanger; 55.0 x pyrosequence

MIGS-30

   Assemblers

    Newbler version 1.1.02.15, Arachne

MIGS-32

   Gene calling method

    Prodigal 1.4, GenePRIMP

   INSDC ID

    ABTR00000000

   Genbank Date of Release

    May 1, 2009

   GOLD ID

    Gc01332

   NCBI project ID

    20741

   Database: IMG-GEBA

    2501533205

MIGS-13

   Source material identifier

    DSM 11002

   Project relevance

    Tree of Life, GEBA

Growth conditions and DNA isolation

D. peptidovorans SEBR 4207T, DSM 11002, was grown anaerobically in DSMZ medium 786 (Dethiosulfovibrio peptidovorans Medium) [19] at 42°C. DNA was isolated from 0.5-1 g of cell paste using Qiagen Genomic 500 DNA Kit (Qiagen, Hilden, Germany) following the protocol as recommended by the manufacturer, with modification st/FT for cell lysis as described in Wu et al. [18].

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 the JGI website (Web Site). Pyrosequencing reads were assembled using the Newbler assembler version 1.1.02.15 (Roche). Large Newbler contigs were broken into 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 adjust inflated q-scores. A hybrid 454/Sanger assembly was made using Arachne assembler. Possible mis-assemblies were corrected and gaps between contgis were closed by primer walks off Sanger clones and bridging PCR fragments and by editing in Consed. A total of 392 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 [20] ). The error rate of the final genome sequence is less than 1 in 100,000. Together, the combination of the Sanger and 454 sequencing platforms provided 63.0× coverage of the genome. The final assembly contains 35,314 Sanger reads and 626,193 pyrosequencing reads.

Genome annotation

Genes were identified using Prodigal [21] as part of the Oak Ridge National Laboratory genome annotation pipeline, followed by a round of manual curation using the JGI GenePRIMP pipeline [22]. 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 [23].

Genome properties

The genome is 2,576,359 bp long and assembled in one large contig and two small contigs (7,415 bp and 1,508 bp) with a 54.0% G+C content (Table 3 and Figure 3). Of the 2,517 genes predicted, 2,458 were protein-coding genes, and 59 RNAs; No pseudogenes were identified. The majority of the protein-coding genes (75.0%) 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)

  2,576,359

   100.00%

DNA coding region (bp)

  2,391,158

   92.81%

DNA G+C content (bp)

  1,401,945

   54.42%

Number of repolicons

  3

Extrachromosomal elements

  2

Total genes

  2,517

   100.00%

RNA genes

  59

   1.40%

rRNA operons

  5

Protein-coding genes

  2,458

   97.27%

Pseudo genes

  0

   0.00%

Genes with function prediction

  1,888

   75.01%

Genes in paralog clusters

  438

   17.41%

Genes assigned to COGs

  1,952

   77.55%

Genes assigned Pfam domains

  2,007

   79.74%

Genes with signal peptides

  420

   16.69%

Genes with transmembrane helices

  619

   24.59%

CRISPR repeats

  2

Figure 3

Graphical circular map of the genome (without the two small 1.5 and 7.4 kbp 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 4

Number of genes associated with the general COG functional categories

Code

    value

    %age

   Description

J

    149

    6.7

   Translation, ribosomal structure and biogenesis

A

    0

    0.0

   RNA processing and modification

K

    129

    5.9

   Transcription

L

    115

    5.3

   Replication, recombination and repair

B

    0

    0.0

   Chromatin structure and dynamics

D

    28

    1.3

   Cell cycle control, mitosis and meiosis

Y

    0

    0.0

   Nuclear structure

V

    32

    1.5

   Defense mechanisms

T

    133

    6.1

   Signal transduction mechanisms

M

    119

    5.5

   Cell wall/membrane biogenesis

N

    75

    3.5

   Cell motility

Z

    0

    0.0

   Cytoskeleton

W

    0

    0.0

   Extracellular structures

U

    46

    2.1

   Intracellular trafficking and secretion, and vesicular transport

O

    70

    3.2

   Posttranslational modification, protein turnover, chaperones

C

    142

    6.5

   Energy production and conversion

G

    113

    5.2

   Carbohydrate transport and metabolism

E

    252

    11.6

   Amino acid transport and metabolism

F

    65

    3.0

   Nucleotide transport and metabolism

H

    99

    4.6

   Coenzyme transport and metabolism

I

    44

    2.0

   Lipid transport and metabolism

P

    125

    5.8

   Inorganic ion transport and metabolism

Q

    31

    1.4

   Secondary metabolites biosynthesis, transport and catabolism

R

    243

    11.2

   General function prediction only

S

    161

    7.4

   Function unknown

-

    565

    22.5

   Not in COGs

Declarations

Acknowledgements

We would like to gratefully acknowledge the help of Esther Schüler for growing D. peptidovorans 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'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, 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. The Indian Council of Scientific and Industrial Research provided a Raman Research Fellowship to Shanmugam Mayilraj.


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