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

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.

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 and , including strains JL-2 and JL-18 [6]. Strikingly, although 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 and 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 JL-2 and JL-18, compare them to genomes of other sequenced 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 currently comprises 16 species and includes the well-known and the genetically tractable . The genome of JL-2 is the first finished genome to be reported from that species, while JL-18 is the fourth genome to be sequenced from that species, the other being HB27, HB8, and SG0.5JP17-16. Figure 1 shows the relationship of JL-2 and JL-18 to other 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 JL-2 and JL-18.

Figure 1

Phylogenomic tree highlighting the position of JL-2 and 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: ATCC 700962 (Taxon OID: 2515935625), DSM 12092 (Taxon OID: 2515463139), JL-2 (Taxon OID: 2508706991), RLM (Taxon OID: 2514335427).

Table 1(a)

Classification and general features of 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].

Genome sequencing information

Genome project history

JL-2 and 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 ( JL-2; Table 2(a) and JL-18; Table 2(b)).

Table 2(a)

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)

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 JL-2 and JL-18 were grown aerobically on 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 JL-2 and JL-18 were generated at the DOE Joint Genome Institute (JGI) using a combination of Illumina [31] and 454 technologies [32].

For 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 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 JL-2 and 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 ( JL-2: 20 reactions; JL-18: 45).

The total size of the genomes are 2,401,329 bp ( JL-2) and 2,311,212 bp ( JL-18). The final assembly of 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 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 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 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 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 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 JL-2 chromosome compared with other chromosomes. The outer four circles show the genes in forward and reverse strands and their corresponding COG categories. BLASTN hits (percentage identities) from HB8 (1), HB27 (2), and SA-01 (3) chromosomes are shown in the inner three circles. Maps were created using CGView Comparison Tool [32].

The 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 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 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 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 JL-18 chromosome compared with other chromosomes. The outer four circles show the genes in forward and reverse strands and their corresponding COG categories. BLASTN hits (percentage identities) from HB8 (1), HB27 (2), and 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 JL-18 was compared with the chromosomes of 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. JL-2 chromosome and megaplasmid sequences were also compared with those of JL-18; however, little very synteny was apparent (Figure 4(b)).

Figure 4(a)

Dot plot comparison of 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 JL-2 and JL-18.

Profiles of metabolic networks and pathways

JL-2 and 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 HB27 [38] and SA-01 [41].

Figure 5

Metabolic pathways identified using iPATH2 [40]. Orange lines are common pathways that were identified in JL-2 and JL-18. Blue lines indicate pathways unique to JL-2 and red lines indicate pathways unique to 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 isolates, including JL-2 and JL-18 [6].

Figure 6 shows the organization of the nar operon and neighboring genes involved in denitrification in JL-2, JL-18, and SA-01. These gene clusters are located on the megaplasmids of JL-2 and JL-18, as in other strains [44,45]. They are located on the chromosome in 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 HB8 suggest NarK1 might also function in nitrite extrusion [39]. JL-2 and SA-01 also contain homologs of NarK2 (annotated as nep in 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 JL-2 and JL-18, consistent with the use of the Nar system in the .

Figure 6

Map showing the organization of nar operon and neighboring genes involved in denitrification located on the megaplasmids of JL-2 (pTHEOS01) and JL-18 (pTTJL1801) and the chromosome of SA-01. Fe: heme protein-containing nitrite reductase, Cu: copper-containing nitrite reductase. Numbers below the genes indicate the provisional ORF numbers in JL-2 (Theos_1057 - Theos_1036) and 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, JL-2 and 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 JL-2 and 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 strains as compared to strains reported previously, including strains JL-2 and JL-18 [6]. In those studies, nitrite accumulated in the medium at concentrations of <150 µM in strains, whereas it was rapidly produced to concentrations >200 µM but consumed rapidly to below method detection limits in 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 JL-2 and 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 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 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 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 JL-2, JL-18 and SA-01 (Figure 6).

Figure 7

. Amino acid sequences of known Cu-containing nitrite reductases from ( GI: 287907), (A. cycloclastes, GI: 157835402), ATCC 17025 ( 17025, GI: 146277634), KD131 ( KD131, GI: 221638756), (, GI: 393758960), (, GI: 422318032), (, GI: 30248928), Z2491 ( Z2491, GI: 218768658) and SA-01 ( SA-01, GI: 320450829) were aligned using Muscle v3.8.31 [51] along with JL-2 ( 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 JL-2 and SA-01.

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

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

Genes involved in iron reduction

SA-01 has been reported to be capable of dissimilatory Fe3+ reduction; however, the biochemical basis of iron reduction has not been elucidated in [41,59]. Sequences of proteins involved in iron reduction [60] in MR-1 (MtrA, MtrF, OmcA) and KN400 (OmcB, OmcE, OmcS, OmcT, OmcZ) were used as search queries into genomes using BLASTP. No hits were found in JL-2, JL-18, or SA-01. This suggests that the biochemical basis of iron reduction is distinct in compared to and and offers no predictive information on whether JL-2 and 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 JL-2 and JL-18 genomes. SoxCD is essential for chemotrophic growth of [61]. Taken together, this suggests that JL-2 and JL-18 may use thiosulfate as an electron donor and are similar to other sulfur-oxidizing strains including IT-7254 [62] and SA-01 [41]. Other genomes also harbor this gene cluster, suggesting thiosulfate oxidation may be widely distributed in [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 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 JL-2

In JL-2, three proteins showed high sequence identity to PsrA (88%; Theos_0751), PsrB (86%; Theos_0750), and PsrC (83%; Theos_0749) of HB27, which is likely involved in anaerobic respiration using polysulfide as a terminal electron acceptor. In 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 PsrABC proteins exhibit very low identity to PsrABC proteins that have been functionally characterized (PsrA: 33%, PsrB 46%, no clear BLASTP hits found in JL-2 for PsrC) [65]. In , formate dehydrogenase or hydrogenase and polysulfide reductase form the electron transport chain and mediate the reduction of polysulfide with formate or H2 [64]. In 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 , including SA-01 (TSC_c10040). Although the genes needed for polysulfide reduction are present, polysulfide reduction in 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]. 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 JL-2 and JL-18 both contain homologs of DNA transport genes (Table 5), suggesting that both JL-2 and JL-18 are naturally competent.

Table 5

Identification of competence proteins in JL-2 and 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 HB27 as queries. Table modified from [72].

Conclusions

We report the finished genomes of JL-2 and JL-18. JL-2 is the first complete genome to be reported for this species, while JL-18 is the fourth genome to be reported for . 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 isolates obtained from that system [6]. It is intriguing that 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 , although NAR1 appears to be capable of complete denitrification to N2 [73]. Another unusual feature of the JL-2 and 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.

JL-2 and 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 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 JL-2 and 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 JL-2 and JL-18 are valuable resources for both basic and applied research.

Table 1(b)

Classification and general features of 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].