High-quality draft genome sequence of the Thermus amyloliquefaciens type strain YIM 77409(T) with an incomplete denitrification pathway.

Thermus amyloliquefaciens type strain YIM 77409(T) is a thermophilic, Gram-negative, non-motile and rod-shaped bacterium isolated from Niujie Hot Spring in Eryuan County, Yunnan Province, southwest China. In the present study we describe the features of strain YIM 77409(T) together with its genome sequence and annotation. The genome is 2,160,855 bp long and consists of 6 scaffolds with 67.4 % average GC content. A total of 2,313 genes were predicted, comprising 2,257 protein-coding and 56 RNA genes. The genome is predicted to encode a complete glycolysis, pentose phosphate pathway, and tricarboxylic acid cycle. Additionally, a large number of transporters and enzymes for heterotrophy highlight the broad heterotrophic lifestyle of this organism. A denitrification gene cluster included genes predicted to encode enzymes for the sequential reduction of nitrate to nitrous oxide, consistent with the incomplete denitrification phenotype of this strain.

Introduction

species have been isolated from both natural and man-made thermal environments such as hot springs, hot domestic water, deep mines, composting systems, and sewage sludge [1-5]. The genus has attracted considerable attention as a source of thermostable enzymes, which have important biotechnological applications [6], and as a model organism to study the mechanisms involved in bacterial adaptation to extreme environments [7]. Members of the genus were formerly considered to be strictly aerobic, based on the characteristics of the type species [2]. However, many studies have shown that strains also can grow as facultative anaerobes using nitrogen oxides, sulfur, or metals as terminal electron acceptors under oxygen-deprived conditions [8-10]. Cava et al. [11] demonstrated that different strains can grow anaerobically by reducing nitrate to nitrite or by reducing nitrite to a gaseous nitrogen product.

The nitrogen biogeochemical cycle has been investigated in a few geothermal systems [12], including Great Boiling Spring, a ~80 °C hot spring in the U.S. Great Basin [13-15]. Studies in GBS revealed a high flux of nitrous oxide, particularly in the ~80 °C source pool, suggesting the importance of incomplete denitrifiers in high-temperature environments. A subsequent cultivation and physiological study of heterotrophic denitrifiers suggested a significant role of and in denitrification in this hot spring [16]. A following study of the whole genomes of one strain from each species, JL-2 and JL-18, revealed that they have genes encoding the sequential reduction of nitrate to nitrous oxide but lack genes encoding the nitrous oxide reductase, and explains their incomplete denitrification phenotype [17].

strain YIM 77409 was isolated in the course of an investigation of the culturable thermophiles that inhabit geothermal springs in Yunnan Province, southwest China [18]. Strain YIM 77409 was cultured from a sediment sample collected from Niujie Hot Spring using the serial dilution technique on T5 agar. This organism was able to grow anaerobically using nitrate as a terminal electron acceptor, and may potentially impact the nitrogen biogeochemical cycle. Here we describe a summary classification and a set of the features of type strain YIM 77409, together with the genome sequence description and annotation. This work may help to better understand the physiological characters as well as the ecological role of this organism in hot spring ecosystems.

Organism information

Classification and features

A taxonomic study using a polyphasic approach placed strain YIM 77409 in the genus within the family of the phylum and resulted in the description of a novel species, , according to its ability to digest starch [18]. The highest 16S rRNA gene sequence pairwise similarities for strain YIM 77409 were found with the type strain of SE-1T (97.6 %), HN3-7T (96.6 %), YIM 77925 (96.5 %), and YIM 77924 (96.1 %) using EzTaxon-e [19]. The sequence similarities were less than 96.0 % with all other species. Phylogenetic analyses based on the 16S rRNA gene sequences show that YIM 77409 together with , , , and constitute a distinct monophyletic group within the genus (Fig. 1). The DNA-DNA hybridization value between strains YIM 77409 and SE-1T was 30.6 ± 1.6 % [18], which was lower than the threshold value (70 %) for the recognition of microbial species [20]. Similarly, the average nucleotide identity (ANI) score between the two strains based on genome-wide comparisons was 86.6  %, according to the algorithm proposed by Goris et al. [21], which is lower than the ANI threshold range (95–96 %) for species demarcation [22]. Those results indicate that strain YIM 77409 represents a distinct genospecies in the genus [18].

Fig. 1

Maximum-likelihood phylogenetic tree of the genus Thermus to highlight the position of Thermus amyloliquefaciens strain YIM 77409T. The tree was reconstructed based on 1374 aligned positions that remained after the application of the Lane mask to the 16S rRNA gene sequences using MEGA 5.0 [54]. Complete deletion of gaps and missing data and Kimura’s two-parameter model was applied. Bootstrap analysis was based on 1000 resamplings. Nodes supported in >75 % (black circles) or >50 % (grey circles) of bootstrap pseudoreplicates (1000 resamplings) for both maximum-likelihood and neighbor-joining methods are indicated. Bar, 0.02 changes per nucleotide. The number of genomes available for each species is included in parentheses (see Table 5) and the asterisk indicates that the genome of the type strain is available. The 16S rRNA gene sequences from Marinithermus hydrothermalis T1T/AB079382 and Rhabdothermus arcticus 2 M70-1T/HM856631 were used as outgroups

Table 5

Comparison of basic genome features of Thermus strains

Genome Name	Status	Genome Size (Mb)	GC Content (%)	Gene Count	No. of protein coding genes with function prediction	Percentage (%)	No. of protein coding genes with COGs	Percentage (%)	IMG Genome ID
T. amyloliquefaciens YIM 77409T	Draft	2.16	67.4	2313	1839	79.5	1558	67.4	2579778517
T. scotoductus SA-01	Finished	2.36	64.9	2514	1878	74.7	1704	67.8	649633105
T. scotoductus KI2	Draft	2.48	65.5	2643	2159	81.7	1808	68.4	2574179778
T. scotoductus DSM 8553T	Draft	2.07	64.8	2305	1816	78.8	1484	64.4	2518645614
T. antranikianii DSM 12462T	Draft	2.17	64.8	2321	1939	83.5	1654	71.3	2522572193
T. caliditerrae YIM 77777T	Draft	2.22	67.2	2327	1901	81.7	1646	70.7	2582581225
T. tengchongensis YIM 77401	Draft	2.56	66.4	2750	2158	78.5	1818	66.1	2574179781
T. arciformis CGMCC 1.6992T	Draft	2.44	68.7	2672	2052	76.8	1704	63.8	2617270932
T. thermophilus HB8T	Finished	2.12	69.5	2302	1498	65.1	1550	67.3	637000323
T. thermophilus JL-18	Finished	2.31	69.0	2508	1984	79.1	1717	68.5	2508501108
T. thermophilus SG0.5JP17-16	Finished	2.30	68.7	2488	2024	81.4	1700	68.3	2505679077
T. thermophilus HB27	Finished	2.13	69.4	2273	1517	66.7	1562	68.7	637000322
T. thermophilus ATCC 33923	Draft	2.15	69.4	2366	1928	81.5	1603	67.8	2554235155
T. islandicus DSM 21543T	Draft	2.26	68.4	2470	1965	79.6	1654	67.0	2524614852
T. oshimai JL-2	Finished	2.40	68.6	2548	2018	79.2	1735	68.1	2508501045
T. oshimai DSM 12092T	Draft	2.26	68.7	2409	1960	81.4	1700	70.6	2515154080
T. igniterrae ATCC 700962T	Draft	2.23	68.8	2379	1962	82.5	1661	69.8	2515154172
T. aquaticus Y51MC23	Draft	2.34	68.1	2595	1740	67.1	1530	59.0	645058872
T. brockianus	Draft	2.48	66.8	2789	2004	71.9	1709	61.3	2502171156
T. sp. CCB_US3_UF1	Finished	2.26	68.6	2333	1935	82.9	1655	70.9	2511231187
T. sp. RLM	Draft	2.04	68.3	2043	1636	80.1	1326	64.9	2513237279
T. sp. NMX2.A1	Draft	2.29	65.3	2522	1954	77.5	1666	66.1	2514885041

Strain YIM 77409 is Gram-negative, facultatively anaerobic, non-motile, and rod shaped (Fig. 2). Cells are 0.4–0.6 μm wide and 1.5–4.5 μm long. Colonies grown on an R2A, T5, and agar plates for 2 days are yellow and circular. The strain degrades starch and is positive for nitrate reduction. The predominant menaquinone is MK-8. Major fatty acids (>10 %) are iso-C15:0 and iso-C17:0. The polar lipids consist of aminophospholipid, one unidentified phospholipid, and two unidentified glycolipids. Minimum Information about the Genome Sequence [23] of type strain YIM 77409 is provided in Table 1.

Fig. 2

Scanning electron microscopy image of Thermus amyloliquefaciens strain YIM 77409T grown in Thermus medium broth at 65 °C for 24 h

Table 1

Classification and general features of Thermus amyloliquefaciens strain YIM 77409T [23]

MIGS ID	Property	Term	Evidence codea
	Classification	Domain Bacteria	TAS [45]
		Phylum Deinococcus-Thermus	TAS [46]
		Class Deinococci	TAS [47, 48]
		Order Thermales	TAS [48, 49]
		Family Thermaceae	TAS [48, 50]
		Genus Thermus	TAS [2, 51, 52]
		Species Thermus amyloliquefaciens	TAS [18]
		Type strain: YIM 77409T	TAS [18]
	Gram stain	Negative	TAS [18]
	Cell shape	Rod	TAS [18]
	Motility	Non-motile	TAS [18]
	Sporulation	Nonsporulating	TAS [18]
	Temperature range	50–70 °C	TAS [18]
	Optimum temperature	60–65 °C	TAS [18]
	pH range; Optimum	6.0–8.0; 7.0	TAS [18]
	Carbon source	Glucose, sucrose, glycerol, maltose, raffinose, trehalose, rhamnose, inositol, xylitol, mannitol, sodium malate, mannose and L-arabinose	TAS [18]
MIGS-6	Habitat	Terrestrial hot springs	TAS [18]
MIGS-6.3	Salinity	Not reported
MIGS-22	Oxygen requirement	Facultatively anaerobic	TAS [18]
MIGS-15	Biotic relationship	Free-living	TAS [18]
MIGS-14	Pathogenicity	Non-pathogen	NAS
MIGS-4	Geographic location	Niujie hot spring in Eryuan County, Yunnan Province, southwest China	TAS [18]
MIGS-5	Sample collection	2010	NAS
MIGS-4.1	Latitude	N 26°15'01. 4"	NAS
MIGS-4.2	Longitude	E 99°59'22. 3"	NAS
MIGS-4.4	Altitude	2060 m	NAS

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 the Gene Ontology project [53]

a Evidence codes

Genome sequencing information

Genome project history

strain YIM 77409 was selected for whole genome sequencing based on its phylogenetic position, denitrifying phenotype, and also for its biotechnological potential. Comparison of the genome of this organism to that of other sequenced species may provide insights into the molecular basis of the denitrification process in this genus. The genome project for strain YIM 77409 was deposited in the Genomes OnLine Database [24] and the complete sequences were deposited in GenBank. Sequencing, finishing, and annotation were performed by the Department of Energy Joint Genome Institute (Walnut Creek, CA, USA) using state of the art sequencing technology [25]. A summary of the project information associated with MIGS version 2.0 compliance [23] is shown in Table 2.

Table 2

Project information

MIGS ID	Property	Term
MIGS 31	Finishing quality	Permanent Draft
MIGS-28	Libraries used	PacBio 10 kb
MIGS 29	Sequencing platforms	PacBio RS
MIGS 31.2	Fold coverage	384.9X PacBio
MIGS 30	Assemblers	HGAP version 2.1.1
MIGS 32	Gene calling method	Prodigal 2.5; GenePRIMP
	Locus Tag	BS74
	Genbank ID	JQMV00000000
	GenBank Date of Release	August 28, 2014
	Database: IMG	2579778517
	GOLD ID	Gp0050852
	BIOPROJECT	PRJNA234787
MIGS 13	Source Material Identifier	YIM 77409T
	Project relevance	Biotechnological

Growth conditions and genomic DNA preparation

type strain YIM 77409 was grown aerobically in medium at 65 °C for 2 days [18] and DNA was isolated from 0.5–1.0 g of cell pellet using the Joint Genome Institute CTAB bacterial genomic DNA isolation protocol [26].

Genome sequencing and assembly

The draft genome of type strain YIM 77409 was generated at the DOE JGI using Pacific Biosciences sequencing technology [27]. A PacBio SMRTbell™ library was constructed and sequenced on the PacBio RS platform using three SMRT cells, which generated 264,235 filtered subreads totaling 751.5 Mbp with an N50 contig length of 2,065,958 bp. All general aspects of library construction and sequencing can be found at the JGI website. All raw reads were assembled using HGAP version 2.1.1 [28]. The final draft assembly produced 6 contigs in 6 scaffolds, totaling 2.16 Mbp in size. The input read coverage was 384.9 × .

Genome annotation

Genes were identified using Prodigal [29] as part of the JGI microbial annotation pipeline [30], followed by a round of manual curation using the JGI GenePRIMP pipeline [31]. The predicted coding sequences were translated and used to search against the Integrated Microbial Genomes non-redundant database, UniProt, TIGRfam, Pfam, PRIAM, KEGG, COG, and InterPro databases. These data sources were combined to assert a product description for each predicted protein. The rRNA genes are predicted using hmmsearch tool from the package HMMER 3.0 [32] and a set of in-house curated HMMs derived from an alignment of full-length rRNA genes selected from IMG isolate genomes; tRNA genes were found using tRNAscan-SE 1.3.1 [33]; other non-coding RNAs and regulatory RNA features were found by searching the genome for the corresponding Rfam profiles using INFERNAL 1.0.2 package [34]. Additional gene prediction analysis and manual functional annotation was performed using the Integrated Microbial Genomes Expert Review platform developed by the JGI [35]. The analysis of the genome presented here and the annotations are for the version available through IMG (2579778517).

Genome properties

The YIM 77409 high quality draft genome is 2,160,855 bp long with a 67.4 % G + C content. The genomes comprise 2,257 protein-coding genes and 56 RNA genes. The coding regions accounted for 94 % of the whole genome and 1,839 genes were assigned to a putative function with the remaining annotated as hypothetical proteins. A total of 1,558 genes (67.4 %) were assigned to COGs. The properties and the statistics of the genome are presented in Table 3. The distribution of genes into COG functional categories is presented in Table 4.

Table 3

Genome statistics

Attribute	Value	% of Totala
Genome size (bp)	2,160,855	100.0
DNA coding (bp)	2,031,100	94.0
DNA G + C (bp)	1,457,281	67.4
DNA scaffolds	6	100.0
Total genes	2,313	100.0
Protein coding genes	2,257	97.6
RNA genes	56	2.4
Pseudo genesb	74	3.2
Genes in internal clusters	1,932	83.5
Genes with function prediction	1,839	79.5
Genes assigned to COGs	1,558	67.4
Genes with Pfam domains	1,842	79.6
Genes with signal peptides	110	4.8
Genes with transmembrane helices	439	19.0
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 4

Number of genes associated with general COG functional categories

Code	Value	%age	Description
J	179	10.4	Translation, ribosomal structure and biogenesis
A	4	0.2	RNA processing and modification
K	76	4.4	Transcription
L	63	3.7	Replication, recombination and repair
B	2	0.1	Chromatin structure and dynamics
D	22	1.3	Cell cycle control, Cell division, chromosome partitioning
V	35	2.0	Defense mechanisms
T	66	3.8	Signal transduction mechanisms
M	77	4.5	Cell wall/membrane biogenesis
N	16	0.9	Cell motility
U	14	0.8	Intracellular trafficking and secretion
O	90	5.2	Posttranslational modification, protein turnover, chaperones
C	131	7.6	Energy production and conversion
G	105	6.1	Carbohydrate transport and metabolism
E	183	10.6	Amino acid transport and metabolism
F	75	4.4	Nucleotide transport and metabolism
H	121	7.0	Coenzyme transport and metabolism
I	91	5.3	Lipid transport and metabolism
P	82	4.8	Inorganic ion transport and metabolism
Q	40	2.3	Secondary metabolites biosynthesis, transport and catabolism
R	170	9.9	General function prediction only
S	68	4.0	Function unknown
-	755	32.6	Not in COGs

The total is based on the total number of protein coding genes in the genome

Insights from the genome sequence

Comparisons with other spp. genomes

Twenty-two genomes from 12 different species have been sequenced, including type strain YIM 77409, and 7 of them have finished genome sequences. The phylogenetic coverage of these genomes is shown in Fig. 1 and their basic properties are summarized in Table 5. The genomes range in size from 2.04 Mb ( sp. RLM) to 2.56 Mb (YIM 77401); GC contents vary from 64.8 % (DSM 8553) to 69.5 % ( HB8T), predicted gene number range from 2,043 (T. sp. RLM) to 2,789 (). The genome size (2.16 Mb) and GC contents (67.4 %) of strain YIM 77409 are around the average value, but the gene number of this strain is lower than the average, possibly indicating gene loss through genomic streamlining in this species. In addition, the percentage of protein-coding genes with functional prediction (79.5 %) is higher than the average, whereas the percentage of protein-coding genes with COGs (67.4 %) is similar to the average of the genus .

Profiles of metabolic network and pathway

The YIM 77409 genome encodes genes for complete glycolysis, gluconeogenesis, tricarboxylic acid cycle, pyruvate dehydrogenase, and pentose phosphate pathway. Twenty ABC transporters were identified in the YIM 77409 genome, including amino acid, oligopeptide/dipeptide, N-acetyl-D-glucosamine, maltose, nucleoside, sugar, phosphonate, phosphate, thiamin, cation, and ammonium transporters as well as other permeases. The genome also encodes glucosidases, glycosidases, proteases, and peptidases. The finding of three genes probably coding for esterase (BS74_RS04020, BS74_RS04625, BS74_RS10315) and one gene probably coding for amylopullulanase (BS74_RS00620) are consistent with the observed lipase and amylase activities observed in strain YIM 77409. A number of genes assigned to a classical electron transport chain have been identified in the strain YIM 77409 genome. Respiratory complex I NADH quinone oxidoreductases consists of NADH quinone oxidoreductase chains A-N (BS74_RS03070-BS74_RS03135), NADH quinone oxidoreductase subunit 15 (BS74_RS02790), and two quinone oxidoreductases (BS74_RS00610, BS74_RS06600). Complex II consists of succinate dehydrogenase (cytochrome b556 subunit SdhC (BS74_RS07950), SdhA (BS74_RS07940), SdhB (BS74_RS07935), and SdhD (BS74_RS07945). A four-subunit cytochrome bc1 complex found in was also identified in strain YIM 77409 (BS74_RS10415-BS74_RS10430) [36, 37]. The terminal cytochrome oxidase is encoded by four cytochrome c oxidase genes ctaC1 (BS74_RS00820), caaA (BS74_RS00825), ctaD2 (BS74_RS04775), and ctaC2 (BS74_RS04780). Other cytochrome c oxidase genes observed in SA-01, ctaH, ctaE1, ctaE2, ctaD1, and coxM (TSC_C00960-TSC_C01000), were not found in the YIM 77409 genome.

Genes involved in denitrification

Denitrification is a respiratory process to reduce nitrate or nitrite stepwise to nitrogen gas (NO3− → NO2− → NO → N2O → N2), and plays a major role in converting bioavailable nitrogen to recalcitrant dinitrogen gas [38]. Denitrification normally occurs under oxygen-limiting conditions, and is catalyzed by four types of nitrogen oxide reductases in sequence: nitrate reductase (Nar or Nap), nitrite reductase (Nir), nitric oxide reductase (Nor), and nitrous oxide reductase (Nos) [39, 40]. Previous studies have demonstrated that some species have incomplete denitrification phenotypes terminating with the production of nitrite or nitrous oxide [16, 41]. This incomplete denitrification is partly encoded by a conjugative element (nitrate conjugative element, NCE) that can be transferred among strains [42]. The NCE is composed of two main operons, nar and nrc, and the transcription factors DnrS and DnrT, which are required for their expression under anaerobic conditions when nitrate is present [43, 44]. The periplasmic nitrate reductase subunits NapB and NapC were not found in the genome of YIM 77409, consistent with the use of the Nar system in the . Figure 3 shows the organization of the nar operon and neighboring genes involved in denitrification in YIM 77409, YIM 77401, and SA-01. They are located on the chromosome in strains YIM 77409 and YIM 77401, as in SA-01. However, these gene clusters are located on megaplasmids in and strains [17]. The nar operons show a high degree of synteny and consist of narCGHJIKT encoding the associated periplasmic cytochrome NarC, the membrane-bound nitrate reductase (NarGHI), the dedicated chaperone NarJ, the nitrate/proton symporter (NarK1), which might also function in nitrite extrusion in HB8T, and the nitrate/nitrite antiporter (NarK2). Regulatory protein A and a denitrification regulator gene operon dnrST are adjacent to the nar operons. Strain YIM 77409 contains a putative nirS, which encodes the isofunctional tetraheme cytochrome cd1-containing nitrite reductase. The nirK, encoding a Cu-containing nitrite reductase in SA-01, is absent in strain YIM 77409 and YIM 77401. Genes encoding conserved hypothetical proteins, coenzyme PQQ synthesis protein (PqqE), and nitric oxide reductase subunit b (NorB) and c (NorC) were also presented in the YIM 77409 genome. Genes encoding the periplasmic multicopper enzyme nitrous oxide reductase (Nos), which catalyzes the last step of the denitrification (N2O → N2), were not observed in the YIM 77409 genome or in any spp. genomes. Physiological experiments with nitrate as the sole terminal electron acceptor also confirm that strain YIM 77409 can convert nitrate to nitrous oxide under anaerobic conditions, but not to nitrogen gas.

Fig. 3

Molecular organization of identified nar operon and neighboring genes involved in denitrification located on the chromosome of T. amyloliquefaciens YIM 77409T, T. tengchongensis YIM 77401, and 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. amyloliquefaciens YIM 77409T and T. tengchongensis YIM 77401, the locations in the chromosome are indicated below. nar: nitrate reductase gene; nir: nitrite reductase gene; nor: nitric oxide reductase gene; dnr: denitrification regulator gene [43, 55, 56]. This figure is modified from Murugapiran et al. [17]

Conclusions

The genus is the archetypal thermophilic bacterium and has been isolated from both natural and man-made thermal environments. Members of this genus are of significance as a source of thermophilic enzymes of great biotechnological interest and as an excellent laboratory models to study the molecular basis of thermal stability. Here, we report the annotation of a high quality draft genome sequence of YIM 77409. Analysis of the genome revealed that strain YIM 77409 encodes enzymes involved in complete glycolysis, pentose phosphate pathway, tricarboxylic acid cycle, pyruvate dehydrogenase, and pentose phosphate pathway. The genome sequence of strain YIM 77409 provides insights to better understand the molecular mechanisms of the incomplete denitrification phenotype and the ecological roles that species play in nitrogen cycling. Combined analysis of this genome and other genomes also provides important insights into the evolution and ecology of this group and the role it may play in the high-temperature nitrogen biogeochemical cycle.