Open Access

Complete genome sequence of Desulfurococcus mucosus type strain (O7/1T)

  • Reinhard Wirth
  • , Olga Chertkov,
  • , Brittany Held,
  • , Alla Lapidus
  • , Matt Nolan
  • , Susan Lucas
  • , Nancy Hammon
  • , Shweta Deshpande
  • , Jan-Fang Cheng
  • , Roxane Tapia,
  • , Cliff Han,
  • , Lynne Goodwin,
  • , Sam Pitluck
  • , Konstantinos Liolios
  • , Pagani Ioanna
  • , Natalia Ivanova
  • , Konstantinos Mavromatis
  • , Natalia Mikhailova
  • , Amrita Pati
  • , Amy Chen
  • , Krishna Palaniappan
  • , Miriam Land,
  • , Loren Hauser,
  • , Yun-Juan Chang,
  • , Cynthia D. Jeffries,
  • , Yvonne Bilek
  • , Thomas Hader
  • , 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.1644004

Received: 29 April 2011

Published: 29 April 2011

Abstract

Desulfurococcus mucosus Zillig and Stetter 1983 is the type species of the genus Desulfurococcus, which belongs to the crenarchaeal family Desulfurococcaceae. The species is of interest because of its position in the tree of life, its ability for sulfur respiration, and several biotechnologically relevant thermostable and thermoactive extracellular enzymes. This is the third completed genome sequence of a member of the genus Desulfurococcus and already the 8th sequence from a member the family Desulfurococcaceae. The 1,314,639 bp long genome with its 1,371 protein-coding and 50 RNA genes is a part of the Genomic Encyclopedia of Bacteria and Archaea project.

Keywords:

hyperthermophileanaerobicorganotrophsulfur respirationspheroid-shapednon-motileextracellular enzymesDesulfurococcaceaeGEBA

Introduction

Strain O7/1T (= DSM 2162 = ATCC 35584 = JCM 9187) is the type strain of the species Desulfurococcus mucosus [1], which is the type species of its genus Desulfurococcus. The genus currently consists of five species with a validly published name [2]. For the genus name the Neo-Latin 'desulfo-' meaning 'desulfuricating, is used to characterize the dissimilatory sulfate-reducing feature of this spheroid-shaped 'coccus' [2]. The species epithet is derived from the Latin word 'mucosus' (slimy) [2]. Strain O7/1T was isolated from an acidic hot spring in Askja, Iceland and the name of the species was effectively published by Zillig et al. in 1982 [1]; valid publication of the name followed in 1983 [3]. The strain was an early target for phylogenetic studies of the domain Archaea (at that time termed ‘Archaebacteria’) via DNA-rRNA cross-hybridizations [4,5], as well as studies on the archaeal DNA-dependent RNA polymerase structure [6] and Archaea-specific quinones [7]. Subsequently, strain O7/1T was used for studies on thermostable extracellular enzymes such as proteinase [8] and pullulanase [9]. Here we present a summary classification and a set of features for D. mucosus strain O7/1T, together with a description of the complete genome sequencing and annotation.

Classification and features

The single genomic 16S rRNA sequence of strain O7/1T was compared using NCBI BLAST 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 [10] and the relative frequencies, weighted by BLAST scores, of taxa and keywords (reduced to their stem [11]) were determined. The five most frequent genera were Sulfolobus (27.8%), Aeropyrum (11.3%), Desulfurococcus (11.3%), Ignicoccus (6.5%) and Vulcanisaeta (6.2%) (100 hits in total). Regarding the five hits to sequences from other members of the genus, the average identity within HSPs was 96.7%, whereas the average coverage by HSPs was 97.4%. Among all other species, the one yielding the highest score was Desulfurococcus mobilis, which corresponded to an identity of 100.0% and an HSP coverage of 100.0%. The highest-scoring environmental sequence was AB462558 ('Microbial production and energy source hyperthermophilic prokaryotes geothermal hot spring pool clone DDP-A01'), which showed an identity of 95.8% and a HSP coverage of 98.2%. The five most frequent keywords within the labels of environmental samples which yielded hits were 'spring' (9.2%), 'microbi' (6.8%), 'hot' (6.2%), 'nation/park/yellowston' (5.4%) and 'popul' (4.8%) (150 hits in total), indicating a good fit to the original habitat of D. mucosus. Environmental samples which yielded hits of a higher score than the highest scoring species were not found.

Figure 1 shows the phylogenetic neighborhood of D. mucosus in a 16S rRNA based tree. A 16S rRNA reference sequence for D. mucosus has not been previously published.

Figure 1

Phylogenetic tree highlighting the position of D. mucosus relative to the other type strains within the family Desulfurococcaceae. The tree was inferred from 1,334 aligned characters [12,13] of the 16S rRNA gene sequence under the maximum likelihood criterion [14] 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 [15] if larger than 60%. Lineages with type strain genome sequencing projects registered in GOLD [16] are shown in blue, Staphylothermus hellenicus CP002051 and published genomes in bold [17-22].

The non-motile cells of strain O7/1T are spheroid with diameters of 0.3 to 2.0 µm [1] (Figure 2), sometimes up to 10 µm [23], surrounded by a slimy mucoid layer, which covers the envelope and consists of neutral sugars and a small fraction of amino sugars [24] (Figure 2). In growing cultures, cells of strain O7/1T were often found in pairs [2] (Table 1). Cells of strain O7/1T can be differentiated from those of D. mobilis, the closest relative of D. mucosus, which are mobile by monopolar polytrichous flagella and devoid of the mucous polymer surrounding the D. mucosus cells [1,23]. Strain O7/1T can utilize yeast extract and casein or its tryptic digests, but not casamino acids as the sole carbon source, by sulfur respiration with the production of H2S and CO2, or by fermentation [1]. Growing cultures synthesize a strong smelling uncharacterized product [1]. Cultures require little or no NaCl in growth media [1,23]. The temperature range for growth of strain O7/1T is 76 to 93ºC, with an optimum at 85ºC [1,23]. At the optimal growth temperature, the generation time of strain O7/1T was about four hours [1]. The pH range is 4.5 to 7.0, with an optimum at 6.0 [1,23]. Sugars, starch, glycogen, alcohols and intermediary metabolites are also not utilized [1]. Strain O7/1T lacks an intron in the 23S RNA gene, which has been described for its close relative D. mobilis [35].

Figure 2

Scanning electron micrograph of D. mucosus strain O7/1T

Table 1

Classification and general features of D. mucosus 07/1T according to the MIGS recommendations [25].

MIGS ID

    Property

     Term

  Evidence code

    Current classification

     Domain Archaea

  TAS [26]

     Phylum Crenarchaeota

  TAS [27,28]

     Class Thermoprotei

  TAS [27,29]

     Order Desulfurococcales

  TAS [27,30]

     Family Desulfurococcaceae

  TAS [2,3,31]

     Genus Desulfurococcus

  TAS [1,3,32]

     Species Desulfurococcus mucosus

  TAS [1,3]

     Type strain O7/1

  TAS [1]

    Gram stain

     negative

  TAS [1]

    Cell shape

     spheroid, often in pairs

  TAS [1]

    Motility

     non-motile

  TAS [1]

    Sporulation

     none

  NAS

    Temperature range

     76°C-93°C

  TAS [23]

    Optimum temperature

     85°C

  TAS [1,23]

    Salinity

     around 0

  TAS [23]

MIGS-22

    Oxygen requirement

     strictly anaerobic

  TAS [1]

    Carbon source

     yeast extract, casein or its tryptic digest

  TAS [1]

    Energy metabolism

     organotroph

  TAS [1]

MIGS-6

    Habitat

     fresh water, sulfur spring

  TAS [1]

MIGS-15

    Biotic relationship

     free living

  TAS [1]

MIGS-14

    Pathogenicity

     none

  NAS

    Biosafety level

     1

  TAS [33]

    Isolation

     acidic hot spring

  TAS [1]

MIGS-4

    Geographic location

     Askja, Iceland

  TAS [1]

MIGS-5

    Sample collection time

     1981 or before

  TAS [1]

MIGS-4.1

    Latitude

     65.05

  NAS

MIGS-4.2

    Longitude

     -16.8

  NAS

MIGS-4.3

    Depth

     not reported

  NAS

MIGS-4.4

    Altitude

     approx. 1,053 m

  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 [34]. 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

According to Zillig et al. 1982 [1], the cell envelope of the strain O7/1T is flexible and probably composed of two layers of which at least the outer one appears to consist of subunits perpendicular to the surface [1]. Scarce information is available regarding the lipid composition of D. mucosus. The lipids in the strain O7/1T are composed of phytanol and C40 polyisoprenoid dialcohols [1]. The polar lipid profile of the closely related D. mobilis has been studied and the structure of its three complex lipids has been elucidated in detail [36].

Genome sequencing and annotation

Genome project history

This organism was selected for sequencing on the basis of its phylogenetic position [37], and is part of the Genomic Encyclopedia of Bacteria and Archaea project [38]. The genome project is deposited in the Genomes On Line Database [16] 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: one 454 pyrosequence standard library,    one 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

    75.7 × Illumina; 44.8 × pyrosequence

MIGS-30

     Assemblers

    Newbler version 2.5-internal-10Apr08-1-threads,    Velvet, phrap

MIGS-32

     Gene calling method

    Prodigal 1.4, GenePRIMP

     INSDC ID

    CP002363

     Genbank Date of Release

    January 20, 2011

     GOLD ID

    Gc02914

     NCBI project ID

    48641

     Database: IMG-GEBA

    2503538025

MIGS-13

     Source material identifier

    DSM 2162

     Project relevance

    Tree of Life, GEBA

Growth conditions and DNA isolation

D. mucosus strain 07/1T, DSM 2162, was grown anaerobically in DSMZ medium 184 (Desulfurococcus medium) [39] at 85°C. DNA was isolated from 0.5-1 g of cell paste using Qiagen Genomic 500 DNA kit (Qiagen 10262) following the standard protocol as recommended by the manufacturer, with no modification. DNA is available through the DNA Bank Network [40].

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 [41]. Pyrosequencing reads were assembled using the Newbler assembler version 2.5-internal-10Apr08-1-threads (Roche). The initial Newbler assembly consisting of three contigs in one scaffold was converted into a phrap assembly [42] by making fake reads from the consensus, to collect the read pairs in the 454 paired end library. Illumina GAii sequencing data (99.5 Mb) were assembled with Velvet [43] 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 546.5 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 [42] 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 [41], Dupfinisher [44], or sequencing cloned bridging PCR fragments with subcloning or transposon bombing (Epicentre Biotechnologies, Madison, WI). Gaps between contigs were closed by editing in Consed, by PCR and by Bubble PCR primer walks (J.-F.Chang, unpublished). A total of 12 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 [45]. 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 120.5 × coverage of the genome. The final assembly contained 264,988 pyrosequence and 1,310,055 Illumina reads.

Genome annotation

Genes were identified using Prodigal [46] as part of the Oak Ridge National Laboratory genome annotation pipeline, followed by a round of manual curation using the JGI GenePRIMP pipeline [47]. The predicted CDSs were translated and used to search the National Center for Biotechnology Information (NCBI) nonredundant database, UniProt, TIGR-Fam, Pfam, PRIAM, KEGG, COG, and InterPro databases. Additional gene prediction analysis and functional annotation were performed within the Integrated Microbial Genomes - Expert Review (IMG-ER) platform [48].

Genome properties

The genome consists of a 1,314,639 bp long chromosome with a G+C content of 53.1% (Table 3 and Figure 3). Of the 1,421 genes predicted, 1,371 were protein-coding genes, and 50 RNAs; 26 pseudogenes were also identified. The majority of the protein-coding genes (65.5%) 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)

    1,314,639

     100.00%

DNA coding region (bp)

    1,186,810

     90.28%

DNA G+C content (bp)

    698,621

     53.14%

Number of replicons

    1

Extrachromosomal elements

    0

Total genes

    1,421

     100.00%

RNA genes

    50

     3.52%

rRNA operons

    1

Protein-coding genes

    1,371

     96.48%

Pseudo genes

    26

     1.83%

Genes with function prediction

    931

     65.52%

Genes in paralog clusters

    103

     7.25%

Genes assigned to COGs

    1,001

     70.44%

Genes assigned Pfam domains

    1,010

     71.08%

Genes with signal peptides

    146

     10.27%

Genes with transmembrane helices

    296

     20.83%

CRISPR repeats

    3

Figure 3

Graphical circular map of 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

   148

    13.9

    Translation, ribosomal structure and biogenesis

A

   2

    0.2

    RNA processing and modification

K

   50

    4.7

    Transcription

L

   62

    5.8

    Replication, recombination and repair

B

   1

    0.1

    Chromatin structure and dynamics

D

   7

    0.7

    Cell cycle control, cell division, chromosome partitioning

Y

   0

    0.0

    Nuclear structure

V

   10

    0.9

    Defense mechanisms

T

   14

    1.3

    Signal transduction mechanisms

M

   37

    3.5

    Cell wall/membrane/envelope biogenesis

N

   4

    0.4

    Cell motility

Z

   0

    0.0

    Cytoskeleton

W

   0

    0.0

    Extracellular structures

U

   10

    0.9

    Intracellular trafficking, secretion, and vesicular transport

O

   45

    4.2

    Posttranslational modification, protein turnover, chaperones

C

   97

    9.1

    Energy production and conversion

G

   52

    4.9

    Carbohydrate transport and metabolism

E

   77

    7.2

    Amino acid transport and metabolism

F

   39

    3.7

    Nucleotide transport and metabolism

H

   45

    4.2

    Coenzyme transport and metabolism

I

   14

    1.3

    Lipid transport and metabolism

P

   81

    7.6

    Inorganic ion transport and metabolism

Q

   3

    0.3

    Secondary metabolites biosynthesis, transport and catabolism

R

   170

    16.0

    General function prediction only

S

   96

    9.0

    Function unknown

-

   420

    29.6

    Not in COGs

Declarations

Acknowledgements

We would like to gratefully acknowledge the help of Olivier D. Ngatchou-Djao (HZI) in preparing the manuscript. 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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