Open Access

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

  • Senthil K. Murugapiran1,
  • Marcel Huntemann2,
  • Chia-Lin Wei2,
  • James Han2,
  • J. C. Detter3,
  • Cliff Han3,
  • Tracy H. Erkkila3,
  • Hazuki Teshima3,
  • Amy Chen2,
  • Nikos Kyrpides2,
  • Konstantinos Mavrommatis2,
  • Victor Markowitz2,
  • Ernest Szeto2,
  • Natalia Ivanova2,
  • Ioanna Pagani2,
  • Amrita Pati2,
  • Lynne Goodwin3,
  • Lin Peters2,
  • Sam Pitluck2,
  • Jenny Lam1,
  • Austin I. McDonald1,
  • Jeremy A. Dodsworth1,
  • Tanja Woyke2 and
  • Brian P. Hedlund1
Standards in Genomic Sciences20137:7030449

DOI: 10.4056/sigs.3667269

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

Thermus Thermus oshimai Thermus thermophilus thermophiles hot springs denitrification nitrous oxide Great 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 ̱C. A considerable volume of geomicrobiology research has been conducted in the GBS system, including coordinated cultivation-independent microbiology and geochemistry studies [14], 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 ̱C 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-4.1

Latitude

N40° 39.182′

TAS [1]

MIGS-4.2

Longitude

W119° 22.496′

 

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 [2022]

 

Species Thermus thermophilus

TAS [2527]

 

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-4.1

Latitude

N40° 39.182′

TAS [1]

MIGS-4.2

Longitude

W119° 22.506′

 

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).
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].

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

%age

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.

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.
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].

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.

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 [4143].

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 competence proteins 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

TtJL18_0836

Structural subunits

Theos_1236

>TtJL18_0835

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.

Abbreviations

NCBI: 

National Center for Biotechnology Information (Bethesda, MD, USA)

IMG: 

JGI Integrated Microbial Resource

Declarations

Acknowledgements

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.

Authors’ Affiliations

(1)
School of Life Sciences, University of Nevada Las Vegas
(2)
Department of Energy Joint Genome Institute
(3)
Los Alamos National Laboratory

References

  1. Costa KC, Navarro JB, Shock EL, Zhang CL, Soukup D, Hedlund BP. Microbiology and geochemistry of great boiling and mud hot springs in the United States Great Basin. Extremophiles 2009; 13:447–459. PubMed http://dx.doi.org/10.1007/s00792-009-0230-xView ArticlePubMedGoogle Scholar
  2. Huang Z, Hedlund BP, Wiegel J, Zhou J, Zhang CL. Molecular phylogeny of uncultivated Crenarchaeota in Great Basin hot springs of moderately elevated temperature. Geomicrobiol J 2007; 24:535–542. http://dx.doi.org/10.1080/01490450701572523View ArticleGoogle Scholar
  3. Miller-Coleman RL, Dodsworth JA, Ross CA, Shock EL, Williams AJ, Hartnett HE, McDonald AI, Havig JR, 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. PubMed http://dx.doi.org/10.1371/journal.pone.0035964PubMed CentralView ArticlePubMedGoogle Scholar
  4. Zhang CL, Ye Q, Huang Z, Li WJ, Chen J, Song Z, Zhao W, Bagwell C, Inskeep WP, Gao L, et al. Global occurrence and biogeography of putative archaeal amoA genes in terrestrial hot springs. Appl Environ Microbiol 2008; 74:6417–6426. PubMed http://dx.doi.org/10.1128/AEM.00843-08PubMed CentralView ArticlePubMedGoogle Scholar
  5. Dodsworth JA, Hungate BA, 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. PubMed http://dx.doi.org/10.1111/j.1462-2920.2011.02508.xView ArticlePubMedGoogle Scholar
  6. Hedlund BP, McDonald AI, Lam J, Dodsworth JA, Brown JR, 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. PubMed http://dx.doi.org/10.1111/j.1472-4669.2011.00295.xView ArticlePubMedGoogle Scholar
  7. Lefèvre CT, Abreu F, Schmidt ML, Lins U, Frankel RB, Hedlund BP, Bazylinski DA. Moderately thermophilic magnetotactic bacteria from hot springs in Nevada USA. Appl Environ Microbiol 2010; 76:3740–3743. PubMed http://dx.doi.org/10.1128/AEM.03018-09PubMed CentralView ArticlePubMedGoogle Scholar
  8. Dodsworth JA, Hungate B, de la Torre JR, Jiang H, Hedlund BP. Measuring nitrification, denitrification, and related biomarkers in continental geothermal ecosystems. Methods Enzymol 2011; 486:171–203. PubMed http://dx.doi.org/10.1016/B978-0-12-381294-0.00008-0View ArticlePubMedGoogle Scholar
  9. Cole JK, Peacock JP, Dodsworth JA, Williams AJ, Thompson DB, Dong H, Wu G, 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, Eisen JA. A simple, fast and accurate method of phylogenomic inference. Genome Biol 2008; 9:R151. PubMed http://dx.doi.org/10.1186/gb-2008-9-10-r151PubMed CentralView ArticlePubMedGoogle Scholar
  11. Stamatakis A. RAxML-VI-HPC: maximum likelihood-based phylogenetic analyses with thousands of taxa and mixed models. Bioinformatics 2006; 22:2688–2690. PubMed http://dx.doi.org/10.1093/bioinformatics/btl446View ArticlePubMedGoogle Scholar
  12. Letunic I, Bork P. Interactive Tree Of Life (iTOL): an online tool for phylogenetic tree display and annotation. Bioinformatics 2007; 23:127–128. PubMed http://dx.doi.org/10.1093/bioinformatics/btl529View ArticlePubMedGoogle Scholar
  13. Field D, Garrity G, Gray T, Morrison N, Selengut J, Sterk P, Tatusova T, Thomson N, Allen MJ, Angiuoli SV, et al. The minimum information about a genome sequence (MIGS) specification. Nat Biotechnol 2008; 26:541–547. PubMed http://dx.doi.org/10.1038/nbt1360PubMed CentralView ArticlePubMedGoogle Scholar
  14. Woese CR, Kandler O, Wheelis ML. Towards a natural system of organisms: proposal for the domains Archaea, Bacteria, and Eucarya. Proc Natl Acad Sci USA 1990; 87:4576–4579. PubMed http://dx.doi.org/10.1073/pnas.87.12.4576PubMed CentralView ArticlePubMedGoogle Scholar
  15. Weisburg WG, Giovannoni SJ, Woese CR. The Deinococcus-Thermus phylum and the effect of rRNA composition on phylogenetic tree construction. Syst Appl Microbiol 1989; 11:128–134. PubMed http://dx.doi.org/10.1016/S0723-2020(89)80051-7View ArticlePubMedGoogle Scholar
  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. PubMed http://dx.doi.org/10.1099/ijs.0.02358-0
  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.View ArticleGoogle Scholar
  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.Google Scholar
  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.Google Scholar
  20. Skerman VBD, McGowan V, Sneath PHA. Approved Lists of Bacterial Names. Int J Syst Bacteriol 1980; 30:225–420. http://dx.doi.org/10.1099/00207713-30-1-225View ArticleGoogle Scholar
  21. Brock TD, Freeze H. Thermus aquaticus gen. n. and sp. n., a nonsporulating extreme thermophile. J Bacteriol 1969; 98:289–297. PubMedPubMed CentralPubMedGoogle Scholar
  22. Nobre MF, Trüper HG, 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. http://dx.doi.org/10.1099/00207713-46-2-604View ArticleGoogle Scholar
  23. Williams RA, Smith KE, Welch SG, 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. PubMed http://dx.doi.org/10.1099/00207713-46-2-403View ArticlePubMedGoogle Scholar
  24. Ashburner M, Ball CA, Blake JA, Botstein D, Butler H, Cherry JM, Davis AP, Dolinski K, Dwight SS, Eppig JT, et al. Gene ontology: tool for the unification of biology. The Gene Ontology Consortium. Nat Genet 2000; 25:25–29. PubMed http://dx.doi.org/10.1038/75556PubMed CentralView ArticlePubMedGoogle Scholar
  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. http://dx.doi.org/10.1099/00207713-45-3-619
  26. Manaia CM, Hoste B, Gutierrez MC, Gillis M, Ventosa A, Kersters K, 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. http://dx.doi.org/10.1016/S0723-2020(11)80072-XView ArticleGoogle Scholar
  27. Oshima T, 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. http://dx.doi.org/10.1099/00207713-24-1-102View ArticleGoogle Scholar
  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.Google Scholar
  29. Pagani I, Liolios K, Jansson J, Chen IM, Smirnova T, Nosrat B, Markowitz VM, 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. PubMed http://dx.doi.org/10.1093/nar/gkr1100PubMed CentralView ArticlePubMedGoogle Scholar
  30. DOE Joint Genome Institute. http://my.jgi.doe.gov/general
  31. Bennett S. Solexa Ltd. Pharmacogenomics 2004; 5:433–438. PubMed http://dx.doi.org/10.1517/14622416.5.4.433View ArticlePubMedGoogle Scholar
  32. Ewing B, Green P. Base-calling of automated sequencer traces using Phred. II. Error probabilities. Genome Res 1998; 8:186–194. PubMedView ArticlePubMedGoogle Scholar
  33. Zerbino DR, Birney E. Velvet: algorithms for de novo short read assembly using de Bruijn graphs. Genome Res 2008; 18:821–829. PubMed http://dx.doi.org/10.1101/gr.074492.107PubMed CentralView ArticlePubMedGoogle Scholar
  34. Gordon D, Abajian C, Green P. Consed: a graphical tool for sequence finishing. Genome Res 1998; 8:195–202. PubMedView ArticlePubMedGoogle Scholar
  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, Hauser LJ. Prodigal: prokaryotic gene recognition and translation initiation site identification. BMC Bioinformatics 2010; 11:119. PubMed http://dx.doi.org/10.1186/1471-2105-11-119PubMed CentralView ArticlePubMedGoogle Scholar
  37. Pati A, Ivanova NN, Mikhailova N, Ovchinnikova G, Hooper SD, Lykidis A, Kyrpides NC. GenePRIMP: a gene prediction improvement pipeline for prokaryotic genomes. Nat Methods 2010; 7:455–457. PubMed http://dx.doi.org/10.1038/nmeth.1457View ArticlePubMedGoogle Scholar
  38. Henne A, Brüggemann H, Raasch C, Wiezer A, Hartsch T, Liesegang H, Johann A, Lienard T, Gohl O, Martinez-Arias R, et al. The genome sequence of the extreme thermophile Thermus thermophilus. Nat Biotechnol 2004; 22:547–553. PubMed http://dx.doi.org/10.1038/nbt956View ArticlePubMedGoogle Scholar
  39. Kurtz S, Phillippy A, Delcher AL, Smoot M, Shumway M, Antonescu C, Salzberg SL. Versatile and open software for comparing large genomes. Genome Biol 2004; 5:R12. PubMed http://dx.doi.org/10.1186/gb-2004-5-2-r12PubMed CentralView ArticlePubMedGoogle Scholar
  40. Yamada T, Letunic I, Okuda S, Kanehisa M, Bork P. iPath2.0: interactive pathway explorer. Nucleic Acids Res 2011; 39:W412–W415. PubMed http://dx.doi.org/10.1093/nar/gkr313PubMed CentralView ArticlePubMedGoogle Scholar
  41. Gounder K, Brzuszkiewicz E, Liesegang H, Wollherr A, Daniel R, Gottschalk G, Reva O, Kumwenda B, Srivastava M, Bricio C. Berenguer. Sequence of the hyperplastic genome of the naturally competent Thermus scotoductus SA-01. BMC Genomics 2011; 12:577. PubMed http://dx.doi.org/10.1186/1471-2164-12-577PubMed CentralView ArticlePubMedGoogle Scholar
  42. Cava F, Laptenko O, Borukhov S, Chahlafi Z, Blas-Galindo E, Gómez-Puertas P, Berenguer J. Control of the respiratory metabolism of Thermus thermophilus by the nitrate respiration conjugative element NCE. Mol Microbiol 2007; 64:630–646. PubMed http://dx.doi.org/10.1111/j.1365-2958.2007.05687.xView ArticlePubMedGoogle Scholar
  43. Cava F, Zafra O, Magalon A, Blasco F, 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. PubMed http://dx.doi.org/10.1074/jbc.M404785200View ArticlePubMedGoogle Scholar
  44. Ramírez-Arcos S, Fernández-Herrero LA, Marín I, 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. PubMed http://dx.doi.org/10.1128/JB.182.8.2179-2183.2000View ArticleGoogle Scholar
  45. Brüggemann H, Chen C. Comparative genomics of Thermus thermophilus: Plasticity of the megaplasmid and its contribution to a thermophilic lifestyle. J Biotechnol 2006; 124:654–661. PubMed http://dx.doi.org/10.1016/j.jbiotec.2006.03.043View ArticlePubMedGoogle Scholar
  46. Moir JW, Wood NJ. Nitrate and nitrite transport in bacteria. Cell Mol Life Sci 2001; 58:215–224. PubMed http://dx.doi.org/10.1007/PL00000849View ArticlePubMedGoogle Scholar
  47. Wood NJ, Alizadeh T, Richardson DJ, Ferguson SJ, 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. PubMed http://dx.doi.org/10.1046/j.1365-2958.2002.02859.xView ArticlePubMedGoogle Scholar
  48. Jia W, Tovell N, Clegg S, Trimmer M, 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. PubMed http://dx.doi.org/10.1042/BJ20080746View ArticlePubMedGoogle Scholar
  49. Zumft WG. Cell biology and molecular basis of denitrification. Microbiol Mol Biol Rev 1997; 61:533–616. PubMedPubMed CentralPubMedGoogle Scholar
  50. Adman ET, Godden JW, 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. PubMed http://dx.doi.org/10.1074/jbc.270.46.27458View ArticlePubMedGoogle Scholar
  51. Edgar RC. MUSCLE: multiple sequence alignment with high accuracy and high throughput. Nucleic Acids Res 2004; 32:1792–1797. PubMed http://dx.doi.org/10.1093/nar/gkh340PubMed CentralView ArticlePubMedGoogle Scholar
  52. Fields BA, Guss JM, Freeman HC. Three-dimensional model for stellacyanin, a “blue” copper-protein. J Mol Biol 1991; 222:1053–1065. PubMed http://dx.doi.org/10.1016/0022-2836(91)90593-UView ArticlePubMedGoogle Scholar
  53. Hart PJ, Nersissian AM, Herrmann RG, Nalbandyan RM, Valentine JS, Eisenberg D. A missing link in cupredoxins: crystal structure of cucumber stellacyanin at 1.6 Å resolution. Protein Sci 1996; 5:2175–2183. PubMed http://dx.doi.org/10.1002/pro.5560051104PubMed CentralView ArticlePubMedGoogle Scholar
  54. Romero A, Hoitink CW, Nar H, Huber R, Messerschmidt A, Canters GW. X-ray analysis and spectroscopic characterization of M121Q azurin. A copper site model for stellacyanin. J Mol Biol 1993; 229:1007–1021. PubMed http://dx.doi.org/10.1006/jmbi.1993.1101View ArticlePubMedGoogle Scholar
  55. Hinchliffe P, Carroll J, Sazanov LA. Identification of a novel subunit of respiratory complex I from Thermus thermophilus. Biochemistry 2006; 45:4413–4420. PubMed http://dx.doi.org/10.1021/bi0600998View ArticlePubMedGoogle Scholar
  56. Simon J, Gross R, Einsle O, Kroneck PM, Kröger A, 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. PubMed http://dx.doi.org/10.1046/j.1365-2958.2000.01742.xView ArticlePubMedGoogle Scholar
  57. Mooser D, Maneg O, Corvey C, Steiner T, Malatesta F, Karas M, Soulimane T, Ludwig B. A four-subunit cytochrome bc1 complex complements the respiratory chain of Thermus thermophilus. Biochim Biophys Acta 2005; 1708:262–274. PubMed http://dx.doi.org/10.1016/j.bbabio.2005.03.008View ArticlePubMedGoogle Scholar
  58. Olendzenski L, Liu L, Zhaxybayeva O, Murphey R, Shin DG, Gogarten JP. Horizontal transfer of archaeal genes into the Deinococcaceae: detection by molecular and computer-based approaches. J Mol Evol 2000; 51:587–599. PubMedView ArticlePubMedGoogle Scholar
  59. Kieft TL, Fredrickson JK, Onstott TC, Gorby YA, Kostandarithes HM, Bailey TJ, Kennedy DW, Li SW, Plymale AE, Spadoni CM, Gray MS. Dissimilatory reduction of Fe(III) and other electron acceptors by a Thermus isolate. Appl Environ Microbiol 1999; 65:1214–1221. PubMedPubMed CentralPubMedGoogle Scholar
  60. Richter K, Schicklberger M, Gescher J. Dissimilatory reduction of extracellular electron acceptors in anaerobic respiration. Appl Environ Microbiol 2012; 78:913–921. PubMed http://dx.doi.org/10.1128/AEM.06803-11PubMed CentralView ArticlePubMedGoogle Scholar
  61. Friedrich CG, Bardischewsky F, Rother D, Quentmeier A, Fischer J. Prokaryotic sulfur oxidation. Curr Opin Microbiol 2005; 8:253–259. PubMed http://dx.doi.org/10.1016/j.mib.2005.04.005View ArticlePubMedGoogle Scholar
  62. Skirnisdottir S, Hreggvidsson GO, Holst O, Kristjansson JK. Isolation and characterization of a mixotrophic sulfur-oxidizing Thermus scotoductus. Extremophiles 2001; 5:45–51. PubMed http://dx.doi.org/10.1007/s007920000172View ArticlePubMedGoogle Scholar
  63. Friedrich CG, Rother D, Bardischewsky F, Quentmeier A, Fischer J. Oxidation of reduced inorganic sulfur compounds by bacteria: emergence of a common mechanism? Appl Environ Microbiol 2001; 67:2873–2882. PubMed http://dx.doi.org/10.1128/AEM.67.7.2873-2882.2001PubMed CentralView ArticlePubMedGoogle Scholar
  64. Jormakka M, Yokoyama K, Yano T, Tamakoshi M, Akimoto S, Shimamura T, Curmi P, Iwata S. Molecular mechanism of energy conservation in polysulfide respiration. Nat Struct Mol Biol 2008; 15:730–737. PubMed http://dx.doi.org/10.1038/nsmb.1434PubMed CentralView ArticlePubMedGoogle Scholar
  65. Krafft T, Gross R, 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. PubMed http://dx.doi.org/10.1111/j.1432-1033.1995.0601h.xView ArticlePubMedGoogle Scholar
  66. Glaser P, Danchin A, Kunst F, Zuber P, Nakano MM. Indentification and isolation of a gene required for nitrate assimilation and anaerobic growth of Bacillus subtilis. J Bacteriol 1995; 177:1112–1115. PubMedPubMed CentralPubMedGoogle Scholar
  67. Aravind L, Tatusov RL, Wolf YI, Walker DR, Koonin EV. Evidence for massive gene exchange between archaeal and bacterial hyperthermophiles. Trends Genet 1998; 14:442–444. PubMed http://dx.doi.org/10.1016/S0168-9525(98)01553-4View ArticlePubMedGoogle Scholar
  68. Dodsworth JA, Li L, Wei S, Hedlund BP, Leigh JA, de Figueiredo P. Interdomain conjugal transfer of DNA from bacteria to archaea. Appl Environ Microbiol 2010; 76:5644–5647. PubMed http://dx.doi.org/10.1128/AEM.00967-10PubMed CentralView ArticlePubMedGoogle Scholar
  69. Schwarzenlander C, Averhoff B. Characterization of DNA transport in the thermophilic bacterium Thermus thermophilus HB27. FEBS J 2006; 273:4210–4218. PubMed http://dx.doi.org/10.1111/j.1742-4658.2006.05416.xView ArticlePubMedGoogle Scholar
  70. Schwarzenlander C, Haase W, 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. PubMed http://dx.doi.org/10.1111/j.1462-2920.2008.01801.xView ArticlePubMedGoogle Scholar
  71. Markowitz VM, Mavromatis K, Ivanova NN, Chen IM, Chu K, Kyrpides NC. IMG ER: a system for microbial genome annotation expert review and curation. Bioinformatics 2009; 25:2271–2278. PubMed http://dx.doi.org/10.1093/bioinformatics/btp393View ArticlePubMedGoogle Scholar
  72. Averhoff B. Shuffling genes around in hot environments: the unique DNA transporter of Thermus thermophilus. FEMS Microbiol Rev 2009; 33:611–626. PubMed http://dx.doi.org/10.1111/j.1574-6976.2008.00160.xView ArticlePubMedGoogle Scholar
  73. Cava F, Zafra O, da Costa MS, 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. PubMed http://dx.doi.org/10.1111/j.1462-2920.2007.01472.xView ArticlePubMedGoogle Scholar
  74. Dodsworth JA, Hungate BA, 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. PubMed http://dx.doi.org/10.1111/j.1462-2920.2011.02508.xView ArticlePubMedGoogle Scholar

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