Open Access

Thermus oshimai JL-2 and T. thermophilus JL-18 genome analysis illuminates pathways for carbon, nitrogen, and sulfur cycling

  • Senthil K. Murugapiran
  • , Marcel Huntemann
  • , Chia-Lin Wei
  • , James Han
  • , J. C. Detter
  • , Cliff Han
  • , Tracy H. Erkkila
  • , Hazuki Teshima
  • , Amy Chen
  • , Nikos Kyrpides
  • , Konstantinos Mavrommatis
  • , Victor Markowitz
  • , Ernest Szeto
  • , Natalia Ivanova
  • , Ioanna Pagani
  • , Amrita Pati
  • , Lynne Goodwin
  • , Lin Peters
  • , Sam Pitluck
  • , Jenny Lam
  • , Austin I. McDonald
  • , Jeremy A. Dodsworth
  • , Tanja Woyke
  • and Brian P. Hedlund

DOI: 10.4056/sigs.3667269

Received: 25 February 2013

Accepted: 25 February 2013

Published: 25 February 2013

Abstract

The complete genomes of Thermus oshimai JL-2 and T. thermophilus JL-18 each consist of a circular chromosome, 2.07 Mb and 1.9 Mb, respectively, and two plasmids ranging from 0.27 Mb to 57.2 kb. Comparison of the T. thermophilus JL-18 chromosome with those from other strains of T. thermophilus revealed a high degree of synteny, whereas the megaplasmids from the same strains were highly plastic. The T. oshimai JL-2 chromosome and megaplasmids shared little or no synteny with other sequenced Thermus strains. Phylogenomic analyses using a concatenated set of conserved proteins confirmed the phylogenetic and taxonomic assignments based on 16S rRNA phylogenetics. Both chromosomes encode a complete glycolysis, tricarboxylic acid (TCA) cycle, and pentose phosphate pathway plus glucosidases, glycosidases, proteases, and peptidases, highlighting highly versatile heterotrophic capabilities. Megaplasmids of both strains contained a gene cluster encoding enzymes predicted to catalyze the sequential reduction of nitrate to nitrous oxide; however, the nitrous oxide reductase required for the terminal step in denitrification was absent, consistent with their incomplete denitrification phenotypes. A sox gene cluster was identified in both chromosomes, suggesting a mode of chemolithotrophy. In addition, nrf and psr gene clusters in T. oshmai JL-2 suggest respiratory nitrite ammonification and polysulfide reduction as possible modes of anaerobic respiration.

Keywords:

ThermusThermus oshimaiThermus thermophilusthermophileshot springsdenitrificationnitrous oxideGreat Basin

Introduction

The Great Boiling Spring (GBS) geothermal system is located in the northwestern Great Basin near the town of Gerlach, Nevada. Geothermal activity is driven by deep circulation of meteoric water, which rises along range-front faults at temperatures up to 96 ºC. A considerable volume of geomicrobiology research has been conducted in the GBS system, including coordinated cultivation-independent microbiology and geochemistry studies [1-4], habitat niche modeling [3], thermodynamic modeling [1,5], microbial cultivation and physiology [6,7], and integrated studies of the nitrogen biogeochemical cycle (N-cycle [5,6,8]). The latter group of studies is arguably the most detailed body of work on the N-cycle in any geothermal system. Those studies revealed a dissimilatory N-cycle based on oxidation and subsequent denitrification of ammonia supplied in the geothermal source water.

In high temperature sources such as GBS and Sandy’s Spring West (SSW), ammonia oxidation occurs at temperatures up to at least 82 ºC at rates comparable to those in nonthermal aquatic sediments [5]. Several lines of evidence, including deep 16S rRNA gene pyrosequencing datasets and quantitative PCR, suggest ammonia oxidation is carried out by a single species of ammonia-oxidizing archaea closely related to “Candidatus Nitrosocaldus yellowstonii”, which comprises a substantial proportion of the sediment microbial community in some parts of the springs [5,9]. Nitrite oxidation appears to be sluggish or non-existent in the high temperature source pools since nitrite accumulates in these systems and 16S rRNA gene sequences for nitrite-oxidizing bacteria have not been detected in clone library and pyrotag censuses [1,5]. Finally, the nitrite and nitrate that are produced are denitrified in the sediments to both nitrous oxide and dinitrogen; however, a high flux of nitrous oxide, particularly in the ~80 ºC source pool of GBS, suggested the importance of incomplete denitrifiers [6] and electron donor stimulation experiments suggested a key role for heterotrophic denitrifiers [5].

A subsequent cultivation study of heterotrophic denitrifiers in GBS and SSW resulted in the isolation of a large number of denitrifiers belonging to Thermus thermophilus and T. oshimai, including strains T. oshimai JL-2 and T. thermophilus JL-18 [6]. Strikingly, although Thermus strains were isolated using four different isolation strategies, nine different electron donor/acceptor combinations, and four different sampling dates, all isolates of these two species were able to convert nitrate-N stoichiometrically to nitrous oxide-N, but appeared unable to reduce nitrous oxide to dinitrogen. This physiology, combined with high nitrous oxide fluxes in situ suggested a significant role of T. oshimai and T. thermophilus in the unusual N-cycle in these hot springs. However, the genetic basis of this phenotype remained unknown. Here we present the complete genome sequences of T. oshimai JL-2 and T. thermophilus JL-18, compare them to genomes of other sequenced Thermus spp., and discuss them within the context of their potential impacts on biogeochemical cycling of carbon, nitrogen, sulfur, and iron.

Classification and features

The genus Thermus currently comprises 16 species and includes the well-known T. aquaticus and the genetically tractable T. thermophilus. The genome of T. oshimai JL-2 is the first finished genome to be reported from that species, while T. thermophilus JL-18 is the fourth genome to be sequenced from that species, the other being T. thermophilus HB27, HB8, and SG0.5JP17-16. Figure 1 shows the relationship of T. oshimai JL-2 and T. thermophilus JL-18 to other Thermus species, as determined by phylogenomic analysis of highly conserved genes, which supports the taxonomic identities previously determined by 16S rRNA gene phylogenetic analysis [6]. Table 1 shows general features of T. oshimai JL-2 and T. thermophilus JL-18.

Figure 1

Phylogenomic tree highlighting the position of Thermus oshimai JL-2 and Thermus thermophilus JL-18. Thirty-one bacterial phylogenetic markers were identified using Amphora [10]. Maximum-likelihood analysis was carried out with a concatenated alignment of all 31 proteins using RAxML Version 7.2.6 [11] and the tree was visualized using iTOL [12]. Red circles indicate bootstrap support >80% (100 replicates). Scale bar indicates 0.1 substitutions per position. The protein FASTA files for all the species are from NCBI, except for the following species, which are from IMG: Thermus igniterrae ATCC 700962 (Taxon OID: 2515935625), Thermus oshimai DSM 12092 (Taxon OID: 2515463139), Thermus oshimai JL-2 (Taxon OID: 2508706991), Thermus sp. RLM (Taxon OID: 2514335427).

Table 1(a)

Classification and general features of Thermus oshimai JL-2 according to the MIGS recommendations [13].

MIGS ID

     Property

     Term

    Evidence codea

     Current classification

     Domain Bacteria

    TAS [14]

     Phylum Deinococcus-Thermus

    TAS [15]

     Class Deinococci

    TAS [16,17]

     Order Thermales

    TAS [16,18]

     Family Thermaceae

    TAS [16,19]

     Genus Thermus

    TAS [20-22]

     Species Thermus oshimai

    TAS [23]

     Type strain JL-2

    TAS [6]

     Gram stain

     Negative

    TAS [13]

     Cell shape

     Rod

    TAS [6,23]

     Motility

     Non-motile

    NAS [13]

     Sporulation

     Nonsporulating

    TAS [13]

     Temperature range

     Not reported

     Optimum temperature

     70 °C

    TAS [13]

     Carbon source

     Several mono- and disaccharides; some organic acids and amino acids

    TAS [13]

     Energy source

     Chemoorganotroph

    TAS [6,23]

     Terminal electron acceptor

     O2, NO3-

    TAS [6,23]

MIGS-6

     Habitat

     Terrestrial hot springs

    TAS [6,23]

MIGS-6.3

     Salinity

     3.90 g/L total dissolved solids

    TAS [1]

MIGS-22

     Oxygen

     Facultative anaerobe (nitrate reduction)

    TAS [6,23]

MIGS-15

     Biotic relationship

     Free living

    TAS [6,23]

MIGS-14

     Pathogenicity

     Non-pathogenic

    NAS

MIGS-4

     Geographic location

     Sandy’s Spring West, Great Boiling Springs geothermal field, Nevada

    TAS [6]

MIGS-5

     Sample collection time

     October, 2008

    TAS [6]

MIGS-4.1MIGS-4.2

     Latitude     Longitude

     N40° 39.182’     W119° 22.496’

    TAS [1]

MIGS-4.3

     Depth

     Sediment/water interface (shallow)

    TAS [1]

MIGS-4.4

     Altitude

     1,203 m

    NAS

aEvidence codes - IDA: Inferred from Direct Assay; 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 Gene Ontology project [24].

Table 1(b)

Classification and general features of Thermus thermophilus JL-18 according to the MIGS recommendations [13].

MIGS ID

     Property

    Term

   Evidence codea

     Current classification

    Domain Bacteria

   TAS [14]

    Phylum Deinococcus-Thermus

   TAS [15]

    Class Deinococci

   TAS [16,17]

    Order Thermales

   TAS [16,18]

    Family Thermaceae

   TAS [16,19]

    Genus Thermus

   TAS [20-22]

    Species Thermus thermophilus

   TAS [25-27]

    Type strain JL-18

   TAS [28]

     Gram stain

    Negative

   TAS [28]

     Cell shape

    Rod

   TAS [6,28]

     Motility

    Non-motile

   TAS [28]

     Sporulation

    Nonsporulating

   TAS [28]

     Temperature range

    Not reported

     Optimum temperature

    70 °C

   TAS [28]

     Carbon source

    Several mono- and disaccharides; some organic acids and amino acids

   TAS [28]

     Energy source

    Chemoorganotroph

   TAS [28]

     Terminal electron acceptor

    O2, NO3-

   TAS [6]

MIGS-6

     Habitat

    Terrestrial hot springs

   TAS [6]

MIGS-6.3

     Salinity

    3.90 g/L total dissolved solids

   TAS [1]

MIGS-22

     Oxygen

    Facultative anaerobe (nitrate reduction)

   TAS [6,13]

MIGS-15

     Biotic relationship

    Free living

   TAS [6,13]

MIGS-14

     Pathogenicity

    Non-pathogenic

   NAS

MIGS-4

     Geographic location

    Sandy’s Spring West, Great Boiling Springs geothermal field, Nevada

   TAS [6]

MIGS-5

     Sample collection time

    12/2008

   TAS [6]

MIGS-4.1MIGS-4.2

     Latitude     Longitude

    N40° 39.182’    W119° 22.506’

   TAS [1]

MIGS-4.3

     Depth

    Sediment/water interface (shallow)

   TAS [1]

MIGS-4.4

     Altitude

    1,203 m

   NAS

aEvidence codes - IDA: Inferred from Direct Assay; 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 Gene Ontology project [24].

Genome sequencing information

Genome project history

T. oshimai JL-2 and T. thermophilus JL-18 were selected based on their important roles in denitrification and also for their biotechnological potential. The genome projects for both the organisms are deposited in the Genomes OnLine Database [29] and the complete sequences are deposited in GenBank. Sequencing, finishing, and annotation were performed by the DOE Joint Genome Institute (JGI). A summary of the project and information associated with MIGS version 2.0 compliance [13] are shown (T. oshimai JL-2; Table 2(a) and T. thermophilus JL-18; Table 2(b)).

Table 2(a)

Thermus oshimai JL-2 genome sequencing project information

MIGS ID

     Property

    Term

MIGS-31

     Finishing quality

    Finished

MIGS-28

     Libraries used

    454 standard and PE, Illumina

MIGS-29

     Sequencing platforms

    Illumina GAii, 454-GS-FLX-Titanium

MIGS-31.2

     Fold coverage

    38.3× (454), 2,228.9× (Illumina)

MIGS-30

     Assemblers

    Newbler v 2.3 (pre-release)

MIGS-32

     Gene calling method

    Prodigal 1.4, GenePRIMP

     Genome Date of Release

     Genbank ID

    CP003249.1 (chromosome)    CP003250.1 (Plasmid pTHEOS01)    CP003251.1 (Plasmid pTHEOS02)

     Genbank Date of Release

    November 5, 2012

     GOLD ID

    Gc02356

     Project relevance

    Biotechnological

Table 2(b)

Thermus thermophilus JL-18 genome sequencing project information

MIGS ID

     Property

     Term

MIGS-31

     Finishing quality

     Finished

MIGS-28

     Libraries used

     454 standard and PE, Illumina

MIGS-29

     Sequencing platforms

     Illumina GAii, 454-GS-FLX-Titanium

MIGS-31.2

     Fold coverage

     38.1× (454), 300× (Illumina)

MIGS-30

     Assemblers

     Newbler v 2.3 (pre-release)

MIGS-32

     Gene calling method

     Prodigal 1.4, GenePRIMP

     Genome Date of Release

     Oct 21, 2011

     Genbank ID

     CP003252.1 (chromosome)     CP003253.1 (plasmid pTTJL1801)     CP003254.1 (plasmid pTTJL1802)

     Genbank Date of Release

     April 9, 2012

     GOLD ID

     Gc02194

     Project relevance

     Biotechnological

Growth conditions and DNA isolation

Axenic cultures of T. oshimai JL-2 and T. thermophilus JL-18 were grown aerobically on Thermus medium as described [6] and DNA was isolated from 0.5-1.0 g of cells using the Joint Genome Institute's (JGI) cetyltrimethyl ammonium bromide protocol [30].

Genome sequencing and assembly

The draft genomes of Thermus oshimai JL-2 and Thermus thermophilus JL-18 were generated at the DOE Joint Genome Institute (JGI) using a combination of Illumina [31] and 454 technologies [32].

For T. oshimai JL-2, we constructed and sequenced an Illumina GAii shotgun library which generated 146,341,736 reads totaling 11,122 Mb, a 454 Titanium standard library which generated 181,476 reads and 1 paired end 454 library with an average insert size of 8 kb that generated 285,154 reads totaling 146.6 Mb of 454 data. For T. thermophilus JL-18, we constructed and sequenced an Illumina GAii shotgun library that generated 74,093,820 reads totaling 5,631.1 Mb, a 454 Titanium standard library that generated 212,217 reads and 1 paired end 454 library with an average insert size of 7 kb that generated 121,082 reads totaling 116.9 Mb of 454 data. All general aspects of library construction and sequencing performed at the JGI can be found at [30]. The initial draft assemblies of T. oshimai JL-2 and T. thermophilus JL-18 contained 39 contigs in 2 scaffolds and 75 contigs in 3 scaffolds, respectively.

The 454 Titanium standard data and the 454 paired end data were assembled together with Newbler, version 2.3-PreRelease-6/30/2009. The Newbler consensus sequences were computationally shredded into 2 kb overlapping fake reads (shreds). Illumina sequencing data was assembled with VELVET, version 1.0.13 [33], and the consensus sequence were computationally shredded into 1.5 kb overlapping fake reads (shreds). We integrated the 454 Newbler consensus shreds, the Illumina VELVET consensus shreds and the read pairs in the 454 paired end library using parallel phrap, version SPS - 4.24 (High Performance Software, LLC). The software Consed [34] was used in the following finishing process. Illumina data was used to correct potential base errors and increase consensus quality using the software Polisher developed at JGI (Alla Lapidus, unpublished). Possible mis-assemblies were corrected using gapResolution (Cliff Han, unpublished), Dupfinisher [35] or sequencing cloned bridging PCR fragments with subcloning. Gaps between contigs were closed by editing in Consed, by PCR and by Bubble PCR (J-F Cheng, unpublished) primer walks. Additional reactions were necessary to close gaps and to raise the quality of the finished sequence (T. oshimai JL-2: 20 reactions; T. thermophilus JL-18: 45).

The total size of the genomes are 2,401,329 bp (T. oshimai JL-2) and 2,311,212 bp (T. thermophilus JL-18). The final assembly of T. oshimai JL-2 genome is based on 91.8 Mb of 454 draft data which provides an average 38.3× coverage of the genome and 5,349.4 Mb of Illumina draft data which provides an average 2,228.9× coverage of the genome. The final assembly of T. thermophilus JL-18 genome is based on 87.7 Mb of 454 draft data which provides an average 38.1× coverage of the genome and 690 Mb of Illumina draft data which provides an average 300× coverage of the genome. The data and metadata are made available at the JGI Integrated Microbial Resource website (IMG) [31].

Genome annotation

Initial identification of genes was done using Prodigal [36], a part of the DOE-JGI Annotation pipeline, followed by manual curation using GenePRIMP [37]. The predicted ORFs were translated into putative protein sequences and searched against databases including: NCBI nr, Uniprot, TIGR-Fam, Pfam, PRIAM, KEGG, COG, and Interpro. Additional annotations and curations were performed using the Integrated Microbial Genomes - Expert Review (IMG-ER) platform [33].

Genome properties

The T. oshimai JL-2 genome includes one circular chromosome of 2,072,393 bp (2205 predicted genes), a circular megaplasmid, pTHEOS01 (0.27 Mb, 268 predicted genes), and a smaller circular plasmid, pTHEOS02 (57.2 Kb, 75 predicted genes), for a total size of 2,401,329 bp. Of the total 2,548 predicted genes, 2,488 were protein-coding genes. A total of 2,015 (79%) protein-coding genes were assigned to a putative function with the remaining annotated as hypothetical proteins. The properties and the statistics of the genome are summarized in Table 3a, Table 3b, Table 3c and Figure 2).

Table 3(a)

Summary of Thermus oshimai JL-2 genome: one chromosome and two plasmids

Label

     Size (Mb)

    Topology

    INSDC identifier

    RefSeq ID

Chromosome

     2.072393

    Circular

    CP003249.1

    -

Plasmid pTHEOS01

     0.271713

    Circular

    CP003250.1

    -

Plasmid pTHEOS02

     0.057223

    Circular

    CP003251.1

    -

Table 3(b)

Nucleotide content and gene count levels of Thermus oshimai JL-2 genome

Attribute

    Value

    % of Totala

Genome size (bp)

    2,401,329

    100.00

DNA coding region (bp)

    2,251,025

    93.74

DNA G+C content (bp)

    1,646,250

    68.56

Total genesb

    2,548

    100.00

RNA genes

    60

    2.35

Protein-coding genes

    2,488

    97.65

Pseudogenes

    53

    2.08

Genes in paralog clusters

    1,099

    43.13

Genes with function prediction

    2,014

    79.04

Genes assigned to COGs

    2,003

    78.61

Genes assigned Pfam domains

    1,998

    78.41

Genes with signal peptides

    862

    33.83

Genes with transmembrane helices

    511

    20.05

CRISPR repeats

    5

aThe total is based on either the size of the genome in base pairs or the total number of protein coding genes in the annotated genome.

bPseudogenes may also be counted as protein coding or RNA genes, so is not additive under total gene count.

Table 3(c)

Number of Thermus oshimai JL-2 genes associated with the 25 general COG functional categories

Code

    Value

     %agea

      Description

J

    146

     6.67

      Translation

A

    4

     0.18

      RNA processing and modification

K

    114

     5.21

      Transcription

L

    117

     5.35

      Replication, recombination and repair

B

    2

     0.09

      Chromatin structure and dynamics

D

    35

     1.60

      Cell cycle control, mitosis and meiosis

Y

    0

     0

      Nuclear structure

V

    25

     1.14

      Defense mechanisms

T

    76

     3.47

      Signal transduction mechanisms

M

    90

     4.11

      Cell wall/membrane biogenesis

N

    23

     1.05

      Cell motility

Z

    1

     0.05

      Cytoskeleton

W

    0

     0

      Extracellular structures

U

    44

     2.01

      Intracellular trafficking and secretion

O

    85

     3.88

      Posttranslational modification, protein turnover, chaperones

C

    154

     7.04

      Energy production and conversion

G

    132

     6.03

      Carbohydrate transport and metabolism

E

    219

     10.01

      Amino acid transport and metabolism

F

    74

     3.38

      Nucleotide transport and metabolism

H

    126

     5.76

      Coenzyme transport and metabolism

I

    89

     4.07

      Lipid transport and metabolism

P

    99

     4.52

      Inorganic ion transport and metabolism

Q

    51

     2.33

      Secondary metabolites biosynthesis, transport and catabolism

R

    289

     13.21

      General function prediction only

S

    193

     8.82

      Function unknown

-

    545

     21.39

      Not in COGs

aThe total is based on the total number of protein coding genes in the annotated genome.

Figure 2

Map of T. oshimai JL-2 chromosome compared with other Thermus chromosomes. The outer four circles show the genes in forward and reverse strands and their corresponding COG categories. BLASTN hits (percentage identities) from T. thermophilus HB8 (1), T. thermophilus HB27 (2), and T. scotoductus SA-01 (3) chromosomes are shown in the inner three circles. Maps were created using CGView Comparison Tool [32].

The T. thermophilus JL-18 genome includes one circular chromosome of 1,902,595 bp (2,057 predicted genes), a circular megaplsmid, pTTJL1801 (0.26 Mb, 279 predicted genes), and a smaller circular plasmid, pTTJL1802 (0.14 Mb, 172 predicted genes), for a total size of 2,311,212 bp. Of the total 2,508 predicted genes, 2,452 were protein-coding genes. A total of 1,979 (79%) of protein-coding genes were assigned to a putative function with the remaining annotated as hypothetical proteins. The properties and the statistics of the genome are summarized in Table 4a, Table 4b, Table 4c and Figure 3.

Table 4a

Summary of Thermus thermophilus JL-18 genome: one chromosome and two plasmids

Label

    Size (Mb)

    Topology

    INSDC identifier

    RefSeq ID

Chromosome

    1.902595

    Circular

    CP003252.1

    NC_017587.1

Plasmid pTTJL1801

    0.265886

    Circular

    CP003253.1

    NC_017588.1

Plasmid pTTJL1802

    0.0142731

    Circular

    CP003254.1

    NC_017590.1

Table 4b

Nucleotide content and gene count levels of Thermus thermophilus JL-18 genome

Attribute

     Value

     % of totala

Genome size (bp)

     2,311,212

     100.00

DNA coding region (bp)

     2,172,588

     94.00

DNA G+C content (bp)

     1,594,227

     68.98

Total genesb

     2,508

     100.00

RNA genes

     56

     2.23

Protein-coding genes

     2,452

     97.77

Pseudogenes

     50

     1.99

Genes in paralog clusters

     1,069

     42.62

Genes with function prediction

     1,979

     78.91

Genes assigned to COGs

     1,992

     79.43

Genes assigned Pfam domains

     1,962

     78.23

Genes with signal peptides

     464

     18.5

Genes with transmembrane helices

     518

     20.65

CRISPR repeats

     3

aThe total is based on either the size of the genome in base pairs or the total number of protein coding genes in the annotated genome.

bPseudogenes may also be counted as protein coding or RNA genes, so is not additive under total gene count.

Table 4c

Number of Thermus thermophilus JL-18 genes associated with the 25 general COG functional categories

Code

    Value

    %agea

     Description

J

    148

    6.79

     Translation

A

    1

    0.05

     RNA processing and modification

K

    104

    4.77

     Transcription

L

    130

    5.97

     Replication, recombination and repair

B

    2

    0.09

     Chromatin structure and dynamics

D

    33

    1.51

     Cell cycle control, mitosis and meiosis

Y

    0

    0

     Nuclear structure

V

    25

    1.15

     Defense mechanisms

T

    67

    3.07

     Signal transduction mechanisms

M

    87

    3.99

     Cell wall/membrane biogenesis

N

    30

    1.38

     Cell motility

Z

    1

    0.05

     Cytoskeleton

W

    0

    0

     Extracellular structures

U

    57

    2.62

     Intracellular trafficking and secretion

O

    82

    3.76

     Posttranslational modification, protein turnover, chaperones

C

    149

    6.84

     Energy production and conversion

G

    125

    5.74

     Carbohydrate transport and metabolism

E

    216

    9.91

     Amino acid transport and metabolism

F

    64

    2.94

     Nucleotide transport and metabolism

H

    119

    5.46

     Coenzyme transport and metabolism

I

    94

    4.31

     Lipid transport and metabolism

P

    96

    4.41

     Inorganic ion transport and metabolism

Q

    57

    2.62

     Secondary metabolites biosynthesis, transport and catabolism

R

    291

    13.35

     General function prediction only

S

    201

    9.22

     Function unknown

-

    516

    20.57

     Not in COGs

aThe total is based on the total number of protein coding genes in the annotated genome.

Figure 3

Map of T. thermophilus JL-18 chromosome compared with other Thermus chromosomes. The outer four circles show the genes in forward and reverse strands and their corresponding COG categories. BLASTN hits (percentage identities) from T. thermophilus HB8 (1), T. thermophilus HB27 (2), and T. scotoductus SA-01 (3) chromosomes are shown in the inner three circles. Maps were created using CGView Comparison Tool [32].

Comparison with other sequenced genomes

The chromosome of T. thermophilus JL-18 was compared with the chromosomes of T. thermophilus strains HB8 and HB27 [38] using nucmer [39]. The megaplasmid pTTJL1801 was also compared with the megaplasmid sequences of HB8 and HB27. Dot plot results from this analysis (Figure 4(a)) demonstrate a high degree of synteny between the chromosomes of JL-18, HB8, and HB27; however, little synteny exists between the megaplasmids. T. oshimai JL-2 chromosome and megaplasmid sequences were also compared with those of T. thermophilus JL-18; however, little very synteny was apparent (Figure 4(b)).

Figure 4(a)

Dot plot comparison of T. thermophilus JL-18 chromosome and megaplasmid DNA sequence with those of the strains HB8 and HB27.

Figure 4(b)

Dot plot comparing the chromosome and megaplasmid DNA sequence of T. oshimai JL-2 and T. thermophilus JL-18.

Profiles of metabolic networks and pathways

T. oshimai JL-2 and T. thermophilus JL-18 genomes encode genes for complete glycolysis, tricarboxylic acid (TCA) cycle, and pentose phosphate pathway (Figure 5). The genomes also encode glucosidases, glycosidases, proteases, and peptidases, highlighting the ability of these species to use various carbohydrate and peptide substrates. Thus, central carbon metabolic pathways are very similar to those of T. thermophilus HB27 [38] and T. scotoductus SA-01 [41].

Figure 5

Metabolic pathways identified using iPATH2 [40]. Orange lines are common pathways that were identified in T. oshimai JL-2 and T. thermophilus JL-18. Blue lines indicate pathways unique to T. oshimai JL-2 and red lines indicate pathways unique to T. thermophilus JL-18.

Genes involved in denitrification

Denitrification involves the conversion of nitrate to dinitrogen through the intermediates nitrite, nitric oxide, and nitrous oxide and is mediated by nar, nir, nor, and nos genes [4]. Incomplete denitrification phenotypes terminating in the production of nitrous oxide have recently been reported for a large number of Thermus isolates, including T. oshimai JL-2 and T. thermophilus JL-18 [6].

Figure 6 shows the organization of the nar operon and neighboring genes involved in denitrification in T. oshimai JL-2, T. thermophilus JL-18, and T. scotoductus SA-01. These gene clusters are located on the megaplasmids of T. oshimai JL-2 and T. thermophilus JL-18, as in other T. thermophilus strains [44,45]. They are located on the chromosome in T. scotoductus SA-01 [41]. The nar operons show a high degree of synteny and all include genes encoding the membrane-bound nitrate reductase (NarGHI), the associated periplasmic cytochrome NarC, and the dedicated chaperone NarJ. All three strains contained homologs of NarK1, which is a member of the major facilitator superfamily that likely functions as a nitrate/proton symporter [46,47]. However, some experiments in T. thermophilus HB8 suggest NarK1 might also function in nitrite extrusion [39]. T. oshimai JL-2 and T. scotoductus SA-01 also contain homologs of NarK2 (annotated as nep in T. scotoductus SA-01 [41]), which likely encodes a nitrate/nitrite antiporter [44,48]. No significant BLASTP hits for periplasmic nitrate reductase subunits NapB and NapC were found in T. oshimai JL-2 and T. thermophilus JL-18, consistent with the use of the Nar system in the Thermales.

Figure 6

Map showing the organization of nar operon and neighboring genes involved in denitrification located on the megaplasmids of T. oshimai JL-2 (pTHEOS01) and T. thermophilusJL-18 (pTTJL1801) and the chromosome of T. scotoductus SA-01. Fe: heme protein-containing nitrite reductase, Cu: copper-containing nitrite reductase. Numbers below the genes indicate the provisional ORF numbers in T. oshimai JL-2 (Theos_1057 - Theos_1036) and T. thermophilus JL-18 (TtJL18_2297 to TtJL18_2327), the locations in the megaplasmid are indicated below. nar: nitrate reductase; nir: nitrite reductase; nos: nitric oxidereductase; dnr: denitrification regulator [41-43].

All three strains contain a dnrST operon adjacent to, but divergently transcribed from, the narGHJIK operon. dnrST encodes transcriptional activators responsible for upregulation of the nitrate respiration pathway in the absence of O2 and the presence of nitrogen oxides or oxyanions [42] (Figure 6).

Both the species contain a putative nirK, which encodes the NO-forming, Cu-containing nitrite reductase. In addition, T. oshimai JL-2 and T. scotoductus SA-01 both harbor nirS [41], which encodes the isofunctional tetraheme cytochrome cd1-containing nitrite reductase. Previous studies have suggested that bacteria use either NirK or NirS, but not both, for the reduction of nitrite [49]. The unique presence of NirK and NirS in T. oshimai JL-2 and T. scotoductus SA-01 likely enhances their denitrification abilities since isoenzymes are typically kinetically distinct and/or regulated differently. This idea is consistent with the distinct denitrification phenotypes of T. oshimai strains as compared to T. thermophilus strains reported previously, including strains T. oshimai JL-2 and T. thermophilus JL-18 [6]. In those studies, nitrite accumulated in the medium at concentrations of <150 µM in T. thermophilus strains, whereas it was rapidly produced to concentrations >200 µM but consumed rapidly to below method detection limits in T. oshimai strains.

NirK functions as a homo-trimer [50] and contains type 1 (blue) and type 2 (non-blue) copper-binding residues [49]. Comparison of the NirK from T. oshimai JL-2 and T. scotoductus SA-01 with previously studied NirK amino acid sequences revealed that six of the seven copper-binding residues are conserved, except for a single methionine (M) to glutamine (Q) substitution in both Thermus proteins (Figure 7; indicated by an asterisk (*)). Glutamine, not methionine, is the copper-binding ligand in the case of stellacyanin, a blue (type 1) copper-containing protein [52,53]. A M121Q recombinant protein of Alcaligenes denitrificans azurin showed similar electron paramagnetic resonance (EPR), but exhibited a 100-fold lower redox activity when compared to wild-type azurin [54]. Therefore, although the methionine is replaced with a glutamine in the T. oshimai JL-2 NirK, it is possible that this glutamine residue can function as a copper-binding ligand similar to stellacyanin and azurin. The large and small subunits of nitric oxide reductase (NorB and NorC) are predicted to be co-transcribed along with nitrite reductases in T. oshimai JL-2, T. thermophilus JL-18 and T. scotoductus SA-01 (Figure 6).

Figure 7

Thermus oshimai JL-2 gene Theos_1053 encodes a Copper-containing nitrite reductase. Amino acid sequences of known Cu-containing nitrite reductases from Pseudomonas aureofaciens (P. aureofaciens, GI: 287907), Achromobacter cycloclastes (A. cycloclastes, GI: 157835402), Rhodobacter sphaeroides ATCC 17025 (R. sphaeroides 17025, GI: 146277634), Rhodobacter sphaeroides KD131 (R. sphaeroides KD131, GI: 221638756), Alcaligenes faecalis (A. faecalis, GI: 393758960), Alcaligenes xylosoxidans (A. xylosoxidans, GI: 422318032), Nitrosomonas europaea (N. europaea, GI: 30248928), Neisseria meningitidis Z2491 (N. meningitidis Z2491, GI: 218768658) and Thermus scotoductus SA-01 (T. scotoductus SA-01, GI: 320450829) were aligned using Muscle v3.8.31 [51] along with Thermus oshimai JL-2 (T. oshimai JL-2, GI: 410732282) Theos_1053. Putative copper-binding residues are indicated with downward arrows according to their classes: 1: type 1 (blue) Cu; 2: type 2 (nonblue) Cu [49]. Numbers on left and right of the alignments refer to positions in the alignment. Asterisk (*) indicates the M→Q substitution in T. oshimai JL-2 and T. scotoductus SA-01.

Genes encoding the 15 subunit NADH-quinone oxidoreductase [55] were identified in both genomes (Theos_0703 to 0716, 1811 in T. oshimai JL-2; TTJL18_1786 to 1799, 1580 T. thermophilus JL-18). nrcDEFN, a four gene operon encoding a novel NADH dehydrogenase, is adjacent to the nar operon in the megaplasmid of T. thermophilus HB8 and has been previously implicated in nitrate reduction [43]. In T. thermophilus JL-18, the operon is present (Figure 6), although (TTJL18_2313) is truncated (NarE in HB8: 232 AA, in JL-18: 78 AA). In T. oshimai JL-2, only nrcN is present. Theos_0161 and Theos_0162, orthologs of Wolinella succinogenes NrfA and NrfH [56], respectively, were identified in T. oshimai JL-2 suggesting that T. oshimai JL-2 may be capable of respiratory nitrite ammonification, although this phenotype has not yet been observed in Thermus [6].

Other possible electron transport components include a ba3-type heme-copper oxidase (Theos_1499, 1498, 1497, T. oshimai JL-2; TTJL18_0925, 0926, 0927 T. thermophilus JL-18) and bc1 complex encoded by the FbcCDFB operon [57]. (Theos_0106 to 0109, T. oshimai JL-2; TTJL18_2018 to 2021 T. thermophilus JL-18). In addition, both T. oshimai JL-2 and T. thermophilus JL-18 harbor genes for archaeal-type V0-V1 (vacuolar) type ATPases, which appears to have been acquired from Archaea prior to the divergence of the modern Thermales [58].

Genes involved in iron reduction

T. scotoductus SA-01 has been reported to be capable of dissimilatory Fe3+ reduction; however, the biochemical basis of iron reduction has not been elucidated in Thermus [41,59]. Sequences of proteins involved in iron reduction [60] in Shewanella oneidensis MR-1 (MtrA, MtrF, OmcA) and Geobacter sulfurreducens KN400 (OmcB, OmcE, OmcS, OmcT, OmcZ) were used as search queries into Thermus genomes using BLASTP. No hits were found in T. oshimai JL-2, T. thermophilus JL-18, or T. scotoductus SA-01. This suggests that the biochemical basis of iron reduction is distinct in Thermus compared to Shewanella and Geobacter, and offers no predictive information on whether T. oshimai JL-2 and T. thermophilus JL-18 may be able to respire iron.

Genes involved in sulfur oxidation

A complete sox cluster comprising of 15 genes, including soxCD, is present in T. oshimai JL-2 and T. thermophilus JL-18 genomes. SoxCD is essential for chemotrophic growth of P. pantotrophus [61]. Taken together, this suggests that T. oshimai JL-2 and T. thermophilus JL-18 may use thiosulfate as an electron donor and are similar to other sulfur-oxidizing Thermus strains including T. scotoductus IT-7254 [62] and T. scotoductus SA-01 [41]. Other T. thermophilus genomes also harbor this gene cluster, suggesting thiosulfate oxidation may be widely distributed in Thermus [38].

A variety of chemotrophs and anoxygenic phototrophs can oxidize hydrogen sulfide, organic sulfur compounds, sulfite, and thiosulfate as electron donors for respiration [63]. Reconstituted proteins of SoxXA, SoxYZ, SoxB and SoxCD together, but not alone, mediate the oxidation of thiosulfate, sulfite, sulfur, and hydrogen sulfide in Paratrophus pantotrophus [61]. The absence of free intermediates of sulfur oxidation and the occurrence of sulfite oxidation without SoxCD in P. pantotrophus excludes SoxCD as a sulfite dehydrogenase and provides evidence to its role as a sulfur dehydrogenase with protein-bound sulfur atom [61].

Polysulfide reductase in T. oshimai JL-2

In T. oshimai JL-2, three proteins showed high sequence identity to PsrA (88%; Theos_0751), PsrB (86%; Theos_0750), and PsrC (83%; Theos_0749) of T. thermophilus HB27, which is likely involved in anaerobic respiration using polysulfide as a terminal electron acceptor. In T. thermophilus HB27, PsrA is the putative catalytic subunit containing two molybdopterin guanine dinucleotide co-factors and a cubane-type [4Fe-4S] cluster. Electron transfer is likely mediated by PsrB, which also contains a [4Fe-4S] cluster, while PsrC is a putative transmembrane protein that contains the electron carrier menaquinone-7 (MK-7). PSR functions as a hexamer (composed of 2 subunits each of A, B and C) and catalyzes the reactions: MKH2→MK + 2H+ + 2e- in the membrane, and Sn2-+ 2e- + 2H+ + Sn-12- + H2S in the periplasm [64]. However, the Thermus PsrABC proteins exhibit very low identity to Wolinella succinogenes PsrABC proteins that have been functionally characterized (PsrA: 33%, PsrB 46%, no clear BLASTP hits found in T. oshimai JL-2 for W. succinogenes PsrC) [65]. In Wolinella succinogenes, formate dehydrogenase or hydrogenase and polysulfide reductase form the electron transport chain and mediate the reduction of polysulfide with formate or H2 [64]. In T. oshimai JL-2, Theos_1377 encodes a putative formate dehydrogenase alpha subunit. Another gene, Theos_1111, encodes a putative formate dehydrogenase family accessory protein (FdhD), which is required for regulation of the formate dehydrogenase catalytic subunit [66] and is conserved in many members of the Thermaceae, including T. scotoductus SA-01 (TSC_c10040). Although the genes needed for polysulfide reduction are present, polysulfide reduction in T. oshimai JL-2 has not been tested.

Genes involved in DNA uptake

A significant number of genes in hyperthermophilic bacteria are of archaeal origin, and appear to have been acquired through inter-domain gene transfer [67], which is mediated by both transformation and conjugation systems [68]. T. thermophilus HB27 is naturally competent to both linear and circular DNA, and DNA transport mechanisms in this species have been well studied [69,70]. The genome of T. oshimai JL-2 and T. thermophilus JL-18 both contain homologs of DNA transport genes (Table 5), suggesting that both T. oshimai JL-2 and T. thermophilus JL-18 are naturally competent.

Table 5

Identification of competence proteins in T. oshimai JL-2 and T. thermophilus JL-18 by IMG/ER [71].

Known competenceproteins in HB27

    T. oshimai JL-2

    T. thermophilus JL-18

     Potential Function

ComEC

    Theos_2202

    TtJL18_2054

     DNA transport through the IM

ComEA

    Theos_2201

    TtJL18_2053

     DNA binding

DprA

    Theos_0224

    TtJL18_1834

     Transport of ssDNA to RecA

PilA1

    Theos_1235,    Theos_1236

    TtJL18_0836,    TtJL18_0835

     Structural subunits

PilA2

    Theos_1237

    TtJL18_0834

     Structural subunits

PilA3

    Theos_1238

    TtJL18_0833

     Structural subunits

PilA4

    Theos_1240

    TtJL18_0837

     Structural subunits

PilD

    Theos_1920

    TtJL18_0122

     Export and maturation of prepilins

PilF

    Theos_1970

    TtJL18_0018

     Retraction of pili proteins and DNA translocation

PilC

    Theos_0570

    TtJL18_1257

     Linkage of periplasmic and cytoplasmic proteins

PilQ

    Theos_0435

    TtJL18_0665

     Directing DNA transporter through OM

ComZ

    Theos_1239

    TtJL18_0832

     IM protein, function unknown

PilM

    Theos_0439

    TtJL18_0669

     ATPase, function unknown

PilN

    Theos_0438

    TtJL18_0668

     IM protein, function unknown

PilO

    Theos_0437

    TtJL18_0667

     IM protein, function unknown

PilW

    Theos_0436

    TtJL18_0666

     OM protein, stabilization of PilQ

BLASTP analysis using sequences of known competence proteins from T. thermophilus HB27 as queries. Table modified from [72].

Conclusions

We report the finished genomes of T. oshimai JL-2 and T. thermophilus JL-18. T. oshimai JL-2 is the first complete genome to be reported for this species, while T. thermophilus JL-18 is the fourth genome to be reported for T. thermophilus. Analysis of the genomes revealed that they encode enzymes for the reduction of nitrate to nitrous oxide, which is consistent with the high flux of nitrous oxide reported in GBS [6], and explains the truncated denitrification phenotype reported for many Thermus isolates obtained from that system [6]. It is intriguing that Thermus scotoductus SA-01 also has genes encoding the sequential reduction of nitrate to nitrous oxide but lacks genes encoding the nitrous oxide reductase. The high degree of synteny in the respiratory gene cluster combined with the conserved absence of the nitrous oxide reductase suggests incomplete denitrification might be a previously unrecognized but conserved feature of denitrification pathways in the genus Thermus, although T. thermophilus NAR1 appears to be capable of complete denitrification to N2 [73]. Another unusual feature of the T. oshimai JL-2 and T. scotoductus SA-01 denitrification systems is the apparent presence of the NO-forming, Cu-containing nitrite reductase, NirK, and the isofunctional tetraheme cytochrome cd1-containing nitrite reductase, NirS.

T. oshimai JL-2 and T. thermophilus JL-18 also may be capable of sulfur oxidation since they both encode a complete, chromosomal sox cluster. However, experiments with GBS sediments failed to demonstrate a stimulation of denitrification when thiosulfate was added in excess [74], suggesting thiosulfate oxidation may not be coupled to denitrification in these organisms. The presence of psrA, psrB and psrC genes encoding polysulfide reducatase in T. oshimai JL-2 suggests the ability to reduce polysulfide. The function of these putative pathways could be tested with pure cultures in the laboratory.

The presence of complete macromolecular machinery for natural competence and the presence of megaplasmids harboring genes for nitrate/nitrite reduction and thermophily points out that T. oshimai JL-2 and T. thermophilus JL-18 could have acquired innumerable genes through intra- and inter-domain gene transfer, and suggests considerable plasticity in denitrification pathways. Considering the importance of these organisms in the nitrogen biogeochemical cycle, and their potential as sources of enzymes for biotechnology applications, the complete genome sequences of T. oshimai JL-2 and T. thermophilus JL-18 are valuable resources for both basic and applied research.

Declarations

Acknowledgments

The work conducted by the US Department of Energy Joint Genome Institute is supported by the Office of Science of the US Department of Energy under Contract No. DE-AC02-05CH11231. Additional support was supported by NSF Grant Numbers MCB-0546865 and EPS-9977809. We are also grateful for support from Greg Fullmer through the UNLV Foundation.


This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

References

  1. Costa KC, Navarro JB, Shock EL, Zhang CL, Soukup D and Hedlund BP. Microbiology and geochemistry of great boiling and mud hot springs in the United States Great Basin. Extremophiles. 2009; 13:447-459 View ArticlePubMed
  2. Huang Z, Hedlund BP, Wiegel J, Zhou J and Zhang CL. Molecular phylogeny of uncultivated Crenarchaeota in Great Basin hot springs of moderately elevated temperature. Geomicrobiol J. 2007; 24:535-542 View Article
  3. Miller-Coleman RL, Dodsworth JA, Ross CA, Shock EL, Williams AJ, Hartnett HE, McDonald AI, Havig JR and Hedlund BP. Korarchaeota diversity, biogeography, and abundance in Yellowstone and Great Basin hot springs and ecological niche modeling based on machine learning. PLoS ONE. 2012; 7:e35964 View ArticlePubMed
  4. Zhang CL, Ye Q, Huang Z, Li WJ, Chen J, Song Z, Zhao W, Bagwell C, Inskeep WP and Gao L. Global occurrence and biogeography of putative archaeal amoA genes in terrestrial hot springs. Appl Environ Microbiol. 2008; 74:6417-6426 View ArticlePubMed
  5. Dodsworth JA, Hungate BA and Hedlund BP. Ammonia oxidation, denitrification and dissimilatory nitrate reduction to ammonium in two US Great Basin hot springs with abundant ammonia-oxidizing archaea. Environ Microbiol. 2011; 13:2371-2386 View ArticlePubMed
  6. Hedlund BP, McDonald AI, Lam J, Dodsworth JA, Brown JR and Hungate BA. Potential role of Thermus thermophilus and T. oshimai in high rates of nitrous oxide (N2O) production in ~80 °C hot springs in the US Great Basin. Geobiology. 2011; 9:471-480 View ArticlePubMed
  7. Lefèvre CT, Abreu F, Schmidt ML, Lins U, Frankel RB, Hedlund BP and Bazylinski DA. Moderately thermophilic magnetotactic bacteria from hot springs in Nevada USA. Appl Environ Microbiol. 2010; 76:3740-3743 View ArticlePubMed
  8. Dodsworth JA, Hungate B, de la Torre JR, Jiang H and Hedlund BP. Measuring nitrification, denitrification, and related biomarkers in continental geothermal ecosystems. Methods Enzymol. 2011; 486:171-203 View ArticlePubMed
  9. Cole JK, Peacock JP, Dodsworth JA, Williams AJ, Thompson DB, Dong H, Wu G and Hedlund BP. Sediment Microbial Communities in Great Boiling Spring are Controlled by Temperature and Distinct from Water Communities. [In press]. ISME J. 2013
  10. Wu M and Eisen JA. A simple, fast and accurate method of phylogenomic inference. Genome Biol. 2008; 9:R151 View ArticlePubMed
  11. Stamatakis A. RAxML-VI-HPC: maximum likelihood-based phylogenetic analyses with thousands of taxa and mixed models. Bioinformatics. 2006; 22:2688-2690 View ArticlePubMed
  12. Letunic I and Bork P. Interactive Tree Of Life (iTOL): an online tool for phylogenetic tree display and annotation. Bioinformatics. 2007; 23:127-128 View ArticlePubMed
  13. 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
  14. 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
  15. Weisburg WG, Giovannoni SJ and Woese CR. The Deinococcus-Thermus phylum and the effect of rRNA composition on phylogenetic tree construction. Syst Appl Microbiol. 1989; 11:128-134 View ArticlePubMed
  16. Validation List no. 85. Validation of publication of new names and new combinations previously effectively published outside the IJSEM. Int J Syst Evol Microbiol. 2002; 52:685-690 View ArticlePubMed
  17. Garrity GM, Holt JG. Class I. Deinococci class. nov. In: Garrity GM, Boone DR, Castenholz RW (eds), Bergey's Manual of Systematic Bacteriology, Second Edition, Volume 1, Springer, New York, 2001, p. 395.
  18. Rainey FA, da Costa MS. Order II. Thermales ord. nov. In: Garrity GM, Boone DR, Castenholz RW (eds), Bergey's Manual of Systematic Bacteriology, Second Edition, Volume 1, Springer, New York, 2001, p. 403.
  19. da Costa MS, Rainey FA. Family I. Thermaceae fam. nov. In: Garrity GM, Boone DR, Castenholz RW (eds), Bergey's Manual of Systematic Bacteriology, Second Edition, Volume 1, Springer, New York, 2001, p. 403-404.
  20. Skerman VBD, McGowan V and Sneath PHA. Approved Lists of Bacterial Names. Int J Syst Bacteriol. 1980; 30:225-420 View Article
  21. Brock TD and Freeze H. Thermus aquaticus gen. n. and sp. n., a nonsporulating extreme thermophile. J Bacteriol. 1969; 98:289-297PubMed
  22. Nobre MF, Trüper HG and da Costa MS. Transfer of Thermus ruber (Loginova et al. 1984), Thermus silvanus (Tenreiro et al. 1995), and Thermus chliarophilus (Tenreiro et al. 1995) to Meiothermus gen. nov. as Meiothermus ruber comb. nov., Meiothermus silvanus comb. nov., and Meiothermus chliarophilus comb. nov., respectively, and emendation of the genus Thermus. Int J Syst Bacteriol. 1996; 46:604-606 View Article
  23. Williams RA, Smith KE, Welch SG and Micallef J. Thermus oshimai sp. nov., isolated from hot springs in Portugal, Iceland, and the Azores, and comment on the concept of a limited geographical distribution of Thermus species. Int J Syst Bacteriol. 1996; 46:403-408 View ArticlePubMed
  24. 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. The Gene Ontology Consortium. Nat Genet. 2000; 25:25-29 View ArticlePubMed
  25. Validation List no. 54. Validation of the publication of new names and new combinations previously effectively published outside the IJSB. Int J Syst Bacteriol. 1995; 45:619-620 View Article
  26. Manaia CM, Hoste B, Gutierrez MC, Gillis M, Ventosa A, Kersters K and da Costa MS. Halotolerant Thermus strains from marine and terrestrial hot springs belong to Thermus thermophilus, ex Oshima and Imahori, 1974 nom. rev. emend. Syst Appl Microbiol. 1994; 17:526-532 View Article
  27. Oshima T and Imahori K. Description of Thermus thermophilus (Yoshida and Oshima) comb. nov. a nonsporulating thermophilic bacterium from a Japanese thermal spa. Int J Syst Bacteriol. 1974; 24:102-112 View Article
  28. da Costa MS, Nobre MF, Rainey FA. Genus I. Thermus brock and freeze 1969, 295AL, emend. Nobre, Trüper, and da Costa 1996b, 605, p.404-414. In Boone, D., Castenholz, R., and Garrity, G. (ed.), Bergey's Manual of Systematic Bacteriology, 2nd ed. Springer-Verlag, New York, N.Y; 2001.
  29. 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
  30. . Web Site
  31. Bennett S. Solexa Ltd. Pharmacogenomics. 2004; 5:433-438 View ArticlePubMed
  32. Ewing B and Green P. Base-calling of automated sequencer traces using Phred. II. Error probabilities. Genome Res. 1998; 8:186-194PubMed
  33. 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
  34. Gordon D, Abajian C and Green P. Consed: a graphical tool for sequence finishing. Genome Res. 1998; 8:195-202PubMed
  35. Han C, Chain P. 2006. Finishing repeat regions automatically with Dupfinisher. In Proceeding of the 2006 international conference on bioinformatics & computational biology. Hamid R. Arabnia & Homayoun Valafar (Eds), CSREA Press 2006:141-146.
  36. 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
  37. 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
  38. Henne A, Brüggemann H, Raasch C, Wiezer A, Hartsch T, Liesegang H, Johann A, Lienard T, Gohl O and Martinez-Arias R. The genome sequence of the extreme thermophile Thermus thermophilus. Nat Biotechnol. 2004; 22:547-553 View ArticlePubMed
  39. Kurtz S, Phillippy A, Delcher AL, Smoot M, Shumway M, Antonescu C and Salzberg SL. Versatile and open software for comparing large genomes. Genome Biol. 2004; 5:R12 View ArticlePubMed
  40. Yamada T, Letunic I, Okuda S, Kanehisa M and Bork P. iPath2.0: interactive pathway explorer. Nucleic Acids Res. 2011; 39:W412-W415 View ArticlePubMed
  41. Gounder K, Brzuszkiewicz E, Liesegang H, Wollherr A, Daniel R, Gottschalk G, Reva O, Kumwenda B, Srivastava M and Bricio C. Berenguer. Sequence of the hyperplastic genome of the naturally competent Thermus scotoductus SA-01. BMC Genomics. 2011; 12:577 View ArticlePubMed
  42. Cava F, Laptenko O, Borukhov S, Chahlafi Z, Blas-Galindo E, Gómez-Puertas P and Berenguer J. Control of the respiratory metabolism of Thermus thermophilus by the nitrate respiration conjugative element NCE. Mol Microbiol. 2007; 64:630-646 View ArticlePubMed
  43. Cava F, Zafra O, Magalon A, Blasco F and Berenguer J. A new type of NADH dehydrogenase specific for nitrate respiration in the extreme thermophile Thermus thermophilus. J Biol Chem. 2004; 279:45369-45378 View ArticlePubMed
  44. Ramírez-Arcos S, Fernández-Herrero LA, Marín I and Berenguer J. Two nitrate/nitrite transporters are encoded within the mobilizable plasmid for nitrate respiration of Thermus thermophilus HB8. J Bacteriol. 2000; 182:2179-2183 View ArticlePubMed
  45. Brüggemann H and Chen C. Comparative genomics of Thermus thermophilus: Plasticity of the megaplasmid and its contribution to a thermophilic lifestyle. J Biotechnol. 2006; 124:654-661 View ArticlePubMed
  46. Moir JW and Wood NJ. Nitrate and nitrite transport in bacteria. Cell Mol Life Sci. 2001; 58:215-224 View ArticlePubMed
  47. Wood NJ, Alizadeh T, Richardson DJ, Ferguson SJ and Moir JW. Two domains of a dual-function NarK protein are required for nitrate uptake, the first step of denitrification in Paracoccus pantotrophus. Mol Microbiol. 2002; 44:157-170 View ArticlePubMed
  48. Jia W, Tovell N, Clegg S, Trimmer M and Cole J. A single channel for nitrate uptake, nitrite export and nitrite uptake by Escherichia coli NarU and a role for NirC in nitrite export and uptake. Biochem J. 2009; 417:297-304 View ArticlePubMed
  49. Zumft WG. Cell biology and molecular basis of denitrification. Microbiol Mol Biol Rev. 1997; 61:533-616PubMed
  50. Adman ET, Godden JW and Turley S. The structure of copper-nitrite reductase from Achromobacter cycloclastes at five pH values, with NO2- bound and with type II copper depleted. J Biol Chem. 1995; 270:27458-27474 View ArticlePubMed
  51. Edgar RC. MUSCLE: multiple sequence alignment with high accuracy and high throughput. Nucleic Acids Res. 2004; 32:1792-1797 View ArticlePubMed
  52. Fields BA, Guss JM and Freeman HC. Three-dimensional model for stellacyanin, a "blue" copper-protein. J Mol Biol. 1991; 222:1053-1065 View ArticlePubMed
  53. Hart PJ, Nersissian AM, Herrmann RG, Nalbandyan RM, Valentine JS and Eisenberg D. A missing link in cupredoxins: crystal structure of cucumber stellacyanin at 1.6 Å resolution. Protein Sci. 1996; 5:2175-2183 View ArticlePubMed
  54. Romero A, Hoitink CW, Nar H, Huber R, Messerschmidt A and Canters GW. X-ray analysis and spectroscopic characterization of M121Q azurin. A copper site model for stellacyanin. J Mol Biol. 1993; 229:1007-1021 View ArticlePubMed
  55. Hinchliffe P, Carroll J and Sazanov LA. Identification of a novel subunit of respiratory complex I from Thermus thermophilus. Biochemistry. 2006; 45:4413-4420 View ArticlePubMed
  56. Simon J, Gross R, Einsle O, Kroneck PM, Kröger A and Klimmek O. A NapC/NirT-type cytochrome c (NrfH) is the mediator between the quinone pool and the cytochrome c nitrite reductase of Wolinella succinogenes. Mol Microbiol. 2000; 35:686-696 View ArticlePubMed
  57. Mooser D, Maneg O, Corvey C, Steiner T, Malatesta F, Karas M, Soulimane T and Ludwig B. A four-subunit cytochrome bc1 complex complements the respiratory chain of Thermus thermophilus. Biochim Biophys Acta. 2005; 1708:262-274 View ArticlePubMed
  58. Olendzenski L, Liu L, Zhaxybayeva O, Murphey R, Shin DG and Gogarten JP. Horizontal transfer of archaeal genes into the Deinococcaceae: detection by molecular and computer-based approaches. J Mol Evol. 2000; 51:587-599PubMed
  59. Kieft TL, Fredrickson JK, Onstott TC, Gorby YA, Kostandarithes HM, Bailey TJ, Kennedy DW, Li SW, Plymale AE, Spadoni CM and Gray MS. Dissimilatory reduction of Fe(III) and other electron acceptors by a Thermus isolate. Appl Environ Microbiol. 1999; 65:1214-1221PubMed
  60. Richter K, Schicklberger M and Gescher J. Dissimilatory reduction of extracellular electron acceptors in anaerobic respiration. Appl Environ Microbiol. 2012; 78:913-921 View ArticlePubMed
  61. Friedrich CG, Bardischewsky F, Rother D, Quentmeier A and Fischer J. Prokaryotic sulfur oxidation. Curr Opin Microbiol. 2005; 8:253-259 View ArticlePubMed
  62. Skirnisdottir S, Hreggvidsson GO, Holst O and Kristjansson JK. Isolation and characterization of a mixotrophic sulfur-oxidizing Thermus scotoductus. Extremophiles. 2001; 5:45-51 View ArticlePubMed
  63. Friedrich CG, Rother D, Bardischewsky F, Quentmeier A and Fischer J. Oxidation of reduced inorganic sulfur compounds by bacteria: emergence of a common mechanism? Appl Environ Microbiol. 2001; 67:2873-2882 View ArticlePubMed
  64. Jormakka M, Yokoyama K, Yano T, Tamakoshi M, Akimoto S, Shimamura T, Curmi P and Iwata S. Molecular mechanism of energy conservation in polysulfide respiration. Nat Struct Mol Biol. 2008; 15:730-737 View ArticlePubMed
  65. Krafft T, Gross R and Kröger A. The function of Wolinella succinogenes psr genes in electron transport with polysulphide as the terminal electron acceptor. Eur J Biochem. 1995; 230:601-606 View ArticlePubMed
  66. Glaser P, Danchin A, Kunst F, Zuber P and Nakano MM. Indentification and isolation of a gene required for nitrate assimilation and anaerobic growth of Bacillus subtilis. J Bacteriol. 1995; 177:1112-1115PubMed
  67. Aravind L, Tatusov RL, Wolf YI, Walker DR and Koonin EV. Evidence for massive gene exchange between archaeal and bacterial hyperthermophiles. Trends Genet. 1998; 14:442-444 View ArticlePubMed
  68. Dodsworth JA, Li L, Wei S, Hedlund BP, Leigh JA and de Figueiredo P. Interdomain conjugal transfer of DNA from bacteria to archaea. Appl Environ Microbiol. 2010; 76:5644-5647 View ArticlePubMed
  69. Schwarzenlander C and Averhoff B. Characterization of DNA transport in the thermophilic bacterium Thermus thermophilus HB27. FEBS J. 2006; 273:4210-4218 View ArticlePubMed
  70. Schwarzenlander C, Haase W and Averhoff B. The role of single subunits of the DNA transport machinery of Thermus thermophilus HB27 in DNA binding and transport. Environ Microbiol. 2009; 11:801-808 View ArticlePubMed
  71. Markowitz VM, Mavromatis K, Ivanova NN, Chen IM, Chu K and Kyrpides NC. IMG ER: a system for microbial genome annotation expert review and curation. Bioinformatics. 2009; 25:2271-2278 View ArticlePubMed
  72. Averhoff B. Shuffling genes around in hot environments: the unique DNA transporter of Thermus thermophilus. FEMS Microbiol Rev. 2009; 33:611-626 View ArticlePubMed
  73. Cava F, Zafra O, da Costa MS and Berenguer J. The role of the nitrate respiration element of Thermus thermophilus in the control and activity of the denitrification apparatus. Environ Microbiol. 2008; 10:522-533 View ArticlePubMed
  74. Dodsworth JA, Hungate BA and Hedlund BP. Ammonia oxidation, denitrification and dissimilatory nitrate reduction to ammonium in two US Great Basin hot springs with abundant ammonia-oxidizing archaea. Environ Microbiol. 2011; 13:2371-2386 View ArticlePubMed