Open Access

High quality draft genome sequence of an extremely halophilic archaeon Natrinema altunense strain AJ2T

Standards in Genomic Sciences201712:25

DOI: 10.1186/s40793-017-0237-3

Received: 21 May 2015

Accepted: 5 December 2016

Published: 1 March 2017

Abstract

Natrinema altunense strain AJ2T, a halophilic archaeal strain, was isolated from a high-altitude (3884 m) salt lake in Xinjiang, China. This strain requires at least 1.7 M NaCl to grow and can grow anaerobically in the presence of nitrate. To understand the genetics underlying its extreme phenotype, we de novo assembled the entire genome sequence of AJ2T (=CGMCC 1.3731T=JCM 12890T). We assembled 3,774,135 bp of a total of 4.4 Mb genome in only 20 contigs and noted its high GC content (64.6%). Subsequently we predicted the gene content and generated genome annotation to identify the relationship between the epigenetic characteristics and genomic features. The genome sequence contains 52 tRNA genes, 3 rRNA genes and 4,462 protein-coding genes, 3792 assigned as functional or hypothetical proteins in nr database. This Whole Genome Shotgun project was deposited in DDBJ/EMBL/GenBank under the accession JNCS00000000. We performed a Bayesian (Maximum-Likelihood) phylogenetic analysis using 16S rRNA sequence and obtained its relationship to other strains in the Natrinema and Haloterrigena genera. We also confirmed the ANI value between every two species of Natrinema and Haloterrigena genera. In conclusion, our analysis furthered our understanding of the extreme-environment adapted strain AJ2T by characterizing its genome structure, gene content and phylogenetic placement. Our detailed case study will contribute to our overall understanding of why Natrinema strains can survive in such a high-altitude salt lake.

Keywords

Halophilic archaea High-altitude Salt lake Rhodopsin Light-driven pumps

Introduction

When the genus Natrinema was first described in 1998, it contained two species, Natrinema pellirubrum and Natrinema pallidum [1]. The genus Natrinema belongs to family Halobacteriaceae , phylum Euryarchaeota . Five more species of this genus were isolated and characterized since then, including N. versiforme [2], N. altunense [3], N. gari [4], N. ejinorense [5] and N. salaciae [6]. For now, the genomic sequences of all but N. ejinorense and N. salaciae in the genus Natrinema are publicly available on Genomes Online Database [7] and/or NCBI Genbank. Our lab first identified the N. altunense strain AJ2T in 2005 in a salt lake [3]. Living cells in salt lake have made numerous adaptations to this special ecosystem, allowing them to flourish in a very harsh environment. To determine if the AJ2T genome contains genes for adaptation to a particular set of environmental restrictions and supply a version of genome assembly in the database, we sequenced its whole genome in 2011 and published the whole genome sequence in the WGS database in May, 2014 as the first reported whole genome sequence of its species.

Organism information

We isolated the strain AJ2T from a water sample collected from the edge of Ayakekum salt lake (37°37′ N, 89°29′ E) in Altun Mountain (Altyn-Tagh) National Nature Reserve in Xinjiang, China (Table 1). This salt lake is cold and exposed to strong ultraviolet radiation throughout the year due to its high altitude. It also has high salinity and lacks the common organic nutrients for microorganisms [3].
Table 1

Classification and general features of Natrinema altunense AJ2T [11]

MIGS ID

Property

Term

Evidence codea

 

Current classification

Domain Archaea

Phylum Euryarchaeota

Class Halobacteria

Order Halobacteriales

Family Halobacteriaceae

Genus Natrinema

Species Natrinema altunense

Type strain AJ2T=CGMCC 1.3731T=JCM 12890T

TAS [32]

TAS [33, 34]

TAS [33, 35]

TAS [35, 36]

TAS [37, 38]

TAS [1]

TAS [3]

 

Gram stain

-

TAS [3]

 

Cell shape

Rod

TAS [3]

 

Motility

Motile

TAS [3]

 

Sporulation

None

NAS [3]

 

Temperature range

Not reported

TAS [3]

 

Optimum temperature

Not reported

TAS [3]

 

pH range; Optimum

5.5–9.0; 6.5–7.5

IDA

 

Carbon source

Glucose, glycerol, maltose, glutamate, alanine, arginine, lysine, ornithine, acetate, fumarate, malate, propionate, pyruvate and succinate

TAS [3]

MIGS-6

Habitat

Salt lake

TAS [3]

MIGS-6.3

Salinity

Extremely halophilic. Growth requires 1.7 M NaCl (optimally 3.0–4.3 M) and grows in a wide range of 0.005–1.0 M MgCl2 (optimally 0.05–0.2 M).

TAS [3]

MIGS-22

Oxygen requirement

Aerobic. But the isolate can grow anaerobically in the presence of nitrate.

TAS [3]

MIGS-15

Biotic relationship

Free-living

NAS

MIGS-14

Pathogenicity

Not reported

 

MIGS-4

Geographic location

Altun Mountain National Nature Reserve in Xinjiang, China

TAS [3]

MIGS-5

Sample collection

July, 2002

NAS

MIGS-4.1

MIGS-4.2

Latitude

Longitude

37.62° N

89.48° E

TAS [3]

TAS [3]

MIGS-4.4

Altitude

3884 m

TAS [3]

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 the Gene Ontology project [39]

Classification and features

N. altunense strain AJ2T is an extremely halophilic archaea growing at 1.7–4.3 M NaCl and 0.005–1.0 M MgCl2. Colonies in the agar plate have a vivid orange or red colour. Cells are rod-shaped, but can become pleomorphic under unfavourable conditions as reported in 2005 [3]. The 16S rRNA gene sequence analysis was submitted to the EzTaxon-e service [8] and revealed 95.77–98.50% sequence similarity to members of the genus Natrinema . Strain AJ2T exhibited the highest 16S rRNA gene sequence similarity with N. gari HIS40-3T (98.50%). Phylogenetic analysis based on 16S rRNA gene sequences showed that strain AJ2T clustered with most type strains of the genus Natrinema with a high bootstrap value (Fig. 1). The other three type strains, N. pellirubrum DSM 15624 T, N. salaciae MDB25T and N. ejinorense EJ-57T, were clustered with the genus Haloterrigena . In the 16S rRNA gene trees (Fig. 1) and rpoB’ (RNA polymerase subunit B′) gene trees [9], these three type strains of genus Natrinema showed unclear taxonomic positions [10]. The mixture phylogenetic relationship between these strains in the Natrinema and Haloterrigena genera were reported in 2003 [9]. This suggests that Haloterrigena maybe a later synonym (heterotypic) of genus Natrinema . The cell morphology and flagellum of N. altunense strain AJ2T were examined using transmission electron microscopy (JEM-1230, JEOL). The cells of strain AJ2T are straight and rods and have a diameter ranging 0.3–0.8 μm and length of 0.9–4.0 μm (Fig. 2). The cells are motile and their growth requires at least 1.7 M NaCl and 0.005–1 M MgCl2 (optimal 3.0–4.3 M NaCl and 0.05–0.2 M MgCl2). This strain is chemo-organotrophic and can anaerobically grow in the presence of nitrate. The strain had oxidase and catalase activity. The strain can reduce nitrate and nitrite and produce N2 gas. This strain can also hydrolyse gelatine and tweens 20, 40 and 80 as well as produce H2S from thiosulfate [3].
Fig. 1

Phylogenetic tree highlighting the position of the Natrinema altunense strain AJ2T relative to phylogenetically closely related type strains within the family Halobacteriaceae. These sequences were aligned on the SINA Online service [40] based on SILVA SSU/LSU databases. According to the best nucleotide substitution models found by the maximum-likelihood method in MEGA6 [41], the algorithm of the Jukes-Cantor model [42] was used to calculate the evolutionary distances in the neighbour-joining (NJ) method. Numbers at branch nodes refer to bootstrap values ≥ 50% (based on 1000 replicates). Halobacterium salinarum DSM 3754T (AJ496185) was used as an out-group. Bar, 0.01 substitutions per nucleotide position

Fig. 2

Transmission electron micrograph of cells of the strain AJ2T. Bar: 1 μm

Genome sequencing information

Genome project history

We selected N. altunense AJ2T for sequencing because its halophilic properties and high-altitude habitat may have caused interesting changes in its genome. Additionally, the five other members of genus Natrinema were sequenced and could be compared to our sequence (Table 2). This Whole Genome Shotgun project has been deposited in the DDBJ/EMBL/GenBank under the accession JNCS00000000. The version described in this paper is version JNCS00000000.1. Table 3 presents the project information and its association with MIGS version 2.0 compliance [11].
Table 2

The overall information of sequenced genomes about genus Natrinema

Species

Strain No.

Build year

Contig num.

GC %

Proteins

Total length (bp)

N50 (bp)

GOLD ID

INSDC

Assembly ID

Level: Contig

Natrinema pellirubrum

DSM 15624T

1998

134

64.9

4176

4,264,455

83,437

Gi39311

AOIE00000000

GCA_000337635

Natrinema pallidum

DSM 3751T

1998

115

63.7

3844

3,915,814

88,603

Gi06160

AOII00000000.1

GCF_000337615.1

Natrinema versiforme

JCM 10478T

2000

72

64.0

4160

4,190,799

121,463

Gi0042913

AOID00000000.1

GCF_000337195.1

Natrinema altunense

AJ2 T

2005

20

64.6

4462

3,774,135

425,349

Gi0074394

JNCS00000000.1

GCA_000731985.1

Natrinema altunense

JCM 12890T

2005

52

64.5

3732

3,774,970

184,807

Gi06159

AOIK00000000.1

GCA_000337155.1

Natrinema gari

JCM 14663T

2008

88

63.7

4056

4,023,692

126,340

Gi0042887

AOIJ00000000.1

GCF_000337175.1

Natrinema sp.

J7-1

-

42

64.9

-

3,667,624

196,646

Gi17690

AJVG00000000.1

GCA_000493245.1

Level: Gapless Chromosome

Natrinema pellirubrum

DSM 15624T

1998

1 Chromosome

1 Plsm: pNATPE01

1 Plsm: pNATPE02

64.9

57.0

58.3

3688

266

245

4,354,100

-

Gi05999

Gc0016535

CP003372.1

CP003373.1

CP003374.1

GCA_000230735.3

Natrinema sp.

J7-2

-

1 Chromosome

1 Plsm: pJ7-I

64.3

58.6

4302

3,697,626 95,989

-

Gi18911

Gc02274

CP003412.1

CP003413.1

GCA_000281695.1

Level: Non-Sequenced

Natrinema ejinorense

JCM 13890T

2006

-

-

-

-

-

-

-

-

Natrinema salaciae

DSM 25055T

2012

-

-

-

-

-

-

-

-

The line highlighted with bold represent strain AJ2T

Table 3

Project information

MIGS ID

Property

Term

MIGS-31

Finishing quality

High-quality draft

MIGS-28

Libraries used

Paired-end 2000 bp library

MIGS-29

Sequencing platforms

GS FLX+ System

MIGS-31.2

Fold coverage

87× (2000 bp library)

MIGS-30

Assemblers

Newbler v. 2.5

MIGS-32

Gene calling method

Glimmer v3.02

Locus Tag

Genbank ID

ALTAJ2

JNCS00000000

Genbank Date of Release

July 21, 2014

GOLD ID

Gi0074394

BIOPROJECT

PRJNA248700

MIGS 13

Source Material Identifier

CGMCC 1.3731T=JCM 12890T

Project relevance

Ecosystem

Growth conditions and genomic DNA preparation

N. altunense strain AJ2T was aerobically cultivated at 37 °C for 3 days in modified CM medium, which contained the following (per liter distilled water): 7.5 g Casamino acid (Bacto), 10 g yeast extract (OXOID), 3 g trisodium citrate, 2 g KCl, 20 g MgSO4 · 7H2O and 200 g NaCl (pH 7.2). Genomic DNA was extracted according to the method described by Marmur & Doty [12]. The cells were suspended from 250 ml CM medium and washed once with 20% (w/v) NaCl solution. After extraction, the genomic DNA was dissolved in 1 ml of TE buffer. The quality and quantity of the genomic DNA was determined by 0.7% agarose gel electrophoresis with λ-Hind III digest and λ-EcoT14 I digest DNA marker (TaKaRa, Dalian, China) as well as by the DU800 spectrophotometer (Beckman Coulter, Inc.) with the nucleotide acid analysis method. The OD260/280 of genomic DNA was 1.92.

Genome sequencing and assembly

The next-generation genome sequencing of N. altunense strain AJ2T and quality control was performed using pyrosequencing technology on a GS FLX+ system (454 Life Sciences, Roche). One library with an insert size 2,000 bp was constructed and a total of 380 Mb clean data was obtained after filtering the adapter, artificial or low quality sequence. In other words we sequenced for a genome-wide average coverage of 87. A total of 630,866 reads were used for assembly and produced 20 contigs using the Newbler v.2.5 (454 Life Sciences, Roche). The average contig size was 188,706 bp and the largest contig size was 837,556 bp with the N50 size of 425,349 bp.

Genome annotation

The tRNA genes of strain AJ2T were identified using tRNAscan-SE 1.21 [13] with an archaeal model, and its rRNA genes were found via RNAmmer 1.2 Server [14]. Other ORFs were predicted using Glimmer3 [15]. The predicted ORFs were translated and analysed using the BLASTp program (BLAST 2.2.26+) against the non-redundant, Swiss-Prot [16], Pfam [17] and COG [18] databases. Only results with an e-value smaller than 1 × e−5 were kept. For cross-validation purposes, we annotated the genome with a RAST server online [19]. KAAS [20] was used to assign the predicted amino acids into the KEGG Pathway [21] with the BBH method. Genes with transmembrane helices were predicted using TMHMM Server v.2.0 [22]. We attempted to predict signal peptides using SignalP 4.1 Server [23], but because there were not enough experimentally confirmed signal peptides in the Uni-Prot database [23], the online server failed to provide the archaeal group model. The circular map of the genome was obtained using a local CGView application [24] with adjusted parameters (−size medium -title ‘AJ2T’ -draw_divider_rings T -gene_decoration arc -linear circular). We uploaded the whole genome sequences in FASTA files and calculated the ANI value between every two genome sequences within the genus Natrinema and Haloterrigena on the EzGenome online server [25, 26]. Genome accession numbers for all five published Natrinema and Haloterrigena strains are listed as follows: N. altunense AJ2 (JNCS00000000); N. versiforme JCM 10478 (AOID00000000); N. pallidum DSM 3751 (AOII00000000); N. pellirubrum DSM 15624 (CP003372); N. gari JCM 14663 (AOIJ00000000); H. thermotolerans DSM 11522 (AOIR00000000); H. salina JCM 13891 (AOIS00000000); H. limicola JCM 13563 (AOIT00000000); H. turkmenica DSM 5511 (CP001860); and H. jeotgali A29 (JDTG00000000). Unless otherwise specified, we used default parameters for all software.

Genome properties

This high-quality draft genome sequence of N. altunense AJ2T revealed a genome size of 3,774,135 bp (all 20 contigs length, 64.56% GC content). We predicted 4517 genes; 4462 are protein-coding sequences. A total of 3792 protein-coding genes (83.95%) were assigned to a putative function or as hypothetical proteins. We also found 52 tRNA genes (removed 1 Pseudo tRNA) and 3 rRNA genes (one 23 S rRNA, one 16 S rRNA and one 5 S rRNA). We assigned 1929 protein-coding genes (42.71%) to Pfam domains and categorized 2255 (49.92%) protein-coding genes into COGs functional groups (Table 4 and Fig. 3). This genome has a gene content redundancy of 36.11%, and there are 1631 protein coding genes belonging to 540 paralog clusters. The genomic ANI values within the Natrinema and Haloterrigena genera are listed in Table 5. In the Richter & Rosselló-Móra report, the proposed ANI cut-off for the species boundary is at 95 ~ 96% [25]. According to our calculation data, the ANI values between any two species of Natrinema with published genome sequences were lower than 93.2% and this value was observed between strains AJ2T and Natrinema pallidum DSM 3751 T. We can also easily observe that N. pellirubrum show higher ANI values (>95%) with H. thermotolerans DSM 11522 T (95.4%) and H. jeotgali A29T (95.2%). These data are also identical to the phylogenetic distance in the 16S rRNA maximum-likelihood tree (Fig. 1). In the tree, the other two strains N. salaciae MDB25T and N. ejinorense EJ-57T, which are in the same clade as genus Haloterrigena , lack of genome information for considering their ANI values in this study.
Table 4

Number of genes associated with general COG functional categories

Code

Value

% age

Description

J

163

5.98

Translation, ribosomal structure and biogenesis

A

1

0.04

RNA processing and modification

K

155

5.69

Transcription

L

135

4.95

Replication, recombination and repair

B

3

0.11

Chromatin structure and dynamics

D

26

0.95

Cell cycle control, Cell division, chromosome partitioning

V

39

1.43

Defense mechanisms

T

120

4.40

Signal transduction mechanisms

M

97

3.56

Cell wall/membrane biogenesis

N

19

0.70

Cell motility

U

26

0.95

Intracellular trafficking and secretion

O

123

4.51

Posttranslational modification, protein turnover, chaperones

C

188

6.90

Energy production and conversion

G

98

3.60

Carbohydrate transport and metabolism

E

225

8.26

Amino acid transport and metabolism

F

74

2.72

Nucleotide transport and metabolism

H

147

5.39

Coenzyme transport and metabolism

I

112

4.11

Lipid transport and metabolism

P

183

6.72

Inorganic ion transport and metabolism

Q

48

1.76

Secondary metabolites biosynthesis, transport and catabolism

R

471

17.28

General function prediction only

S

272

9.98

Function unknown

-

2073

46.46

Not in COGs

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

Fig. 3

Graphical circular map of the genome of N. altunense AJ2T. Labelling from outside to the center: circle 1, CDSs on the forward strand (coloured by COG categories); circle 2, CDSs on the reverse strand (coloured by COG categories); circle 3, RNA genes (tRNAs red and rRNAs blue); circle 4, G + C content (peaks out/inside the circle indicate values higher or lower than the average G + C content 64.65%, respectively); circle 5, GC skew (calculated as (G-C)/(G + C) using a window size of 10000 and step of 100, green/purple peaks out/inside the circle indicates values higher or lower than average GC skew value (−0.0047), respectively); and circle 6, Genome size (Mbp)

Table 5

ANI values between genome pairs within genus Natrinema and Haloterrigena

N. altunense

N. versiforme

N. pallidum

N. pellirubrum

N. gari

H. thermotolerans

H. salina

H. limicola

H. turkmenica

H. jeotgali

N. altunense

-

83.50%

93.22%

82.89%

92.75%

82.82%

79.84%

80.99%

79.94%

82.89%

N. versiforme

83.09%

-

82.96%

82.40%

82.92%

82.31%

80.41%

80.90%

80.20%

82.37%

N. pallidum

93.21%

82.75%

-

82.75%

91.65%

82.74%

79.78%

80.93%

79.70%

82.79%

N. pellirubrum

83.00%

82.36%

82.98%

-

82.69%

95.39%

80.00%

80.83%

80.20%

95.16%

N. gari

92.82%

82.59%

91.89%

82.39%

-

82.48%

79.63%

80.69%

79.75%

82.65%

H. thermotolerans

82.65%

82.29%

82.35%

95.49%

82.33%

-

80.03%

80.64%

80.30%

97.36%

H. salina

79.45%

79.87%

79.38%

79.91%

79.13%

79.91%

-

78.77%

90.60%

79.96%

H. limicola

80.98%

81.15%

80.89%

81.12%

80.81%

80.88%

79.43%

-

79.49%

81.34%

H. turkmenica

79.69%

80.25%

79.42%

80.22%

79.41%

80.12%

91.13%

79.14%

-

80.29%

H. jeotgali

82.56%

82.42%

82.79%

95.14%

82.43%

97.41%

80.11%

80.81%

80.35%

-

The calculated genomic sequence used: N. altunense AJ2 (JNCS00000000); N. versiforme JCM 10478 (AOID00000000); N. pallidum DSM 3751 (AOII00000000); N. pellirubrum DSM 15624 (CP003372); N. gari JCM 14663 (AOIJ00000000); H. thermotolerans DSM 11522 (AOIR00000000); H. salina JCM 13891 (AOIS00000000); H. limicola JCM 13563 (AOIT00000000); H. turkmenica DSM 5511 (CP001860); H. jeotgali A29 (JDTG00000000)

Insights from the genome sequence

We compared all sequenced strains in the genus Natrinema with strain AJ2T according to the contig numbers, G + C content, predicted protein numbers, total length and N50, which are listed below (Table 6). The other relevant genomic features were listed in Table 7. According to the chemotaxonomic information and characteristic features of strain AJ2T that was mentioned before, the strain contains a flagellin domain protein in its genomic features to support cell motility. It also has DNA repair systems for protecting the stability of its genome from potential damage caused by UV radiation. Additionally, the energy converting system and light-driven pumps are introduced below.
Table 6

Genome statistics

Attribute

Value

% of total

Genome Size (bp)

3,774,135

-

DNA coding (bp)

3,316,088

87.86

DNA G + C (bp)

2,436,432

64.56

DNA scaffolds

20

-

Total genes

4517

-

Protein-coding genes

4462

98.78

RNA genes

55

1.22

Pseudo genes

-

-

Genes in internal clusters

540

11.95

Genes with function prediction

2215

49.04

Genes assigned to COGs

2255

49.92

Genes with Pfam domains

1929

42.71

Genes with signal peptides

-

-

Genes with transmembrane helices

879

19.46

CRISPR repeats

-

-

Table 7

The relevance characteristics with genomic features annotation

Relevant characteristics

ID

Contig

Position

Strand

Annotation

Tween degradation

AJ2_rast_231

1

198927:200015

+

esterase/lipase

AJ2_rast_522

1

476323:477450

putative esterase

Thiosulfate degradation

AJ2_rast_3344

11

30688:31554

thiosulfate sulfurtransferase2C rhodanese (EC 2.8.1.1)

AJ2_rast_3346

11

31834:32646

+

thiosulfate sulfurtransferase (EC:2.8.1.1)

H2O2 degradation

AJ2_rast_1204

2

332019:334157

+

catalase (EC 1.11.1.6)/Peroxidase (EC 1.11.1.7)

AJ2_rast_3782

16

4816:5718

catalase (EC:1.11.1.6)

Nitrous oxide reductase

AJ2_rast_1974

4

104337:105296

nitrous oxide reductase maturation transmembrane protein NosY

AJ2_rast_2203

4

324166:325008

+

nitrous oxide reductase maturation transmembrane protein NosY

AJ2_rast_3199

10

18205:19152

nitrous oxide reductase maturation transmembrane protein NosY

AJ2_rast_3201

10

20059:21438

nitrous oxide reductase maturation protein NosD

AJ2_rast_3203

10

22285:24204

nitrous-oxide reductase (EC 1.7.99.6)

Motility

AJ2_rast_1043

2

171217:173058

+

flagella-related protein FlaI

AJ2_rast_1170

2

296018:296341

+

chemotaxis regulator CheY

AJ2_rast_1825

3

392764:394281

+

conserved flagella cluster protein

AJ2_rast_2104

4

230767:231792

signal peptidase2C type IV - prepilin/preflagellin

DNA repair

AJ2_rast_1703

3

284669:285400

+

DNA repair and recombination protein RadB

AJ2_rast_2261

4

382800:384209

single-stranded-DNA-specific exonuclease RecJ (EC 3.1.-.-)

AJ2_rast_2296

4

413891:414922

+

DNA repair and recombination protein RadA

AJ2_rast_2880

8

16958:18862

+

RecJ like exonuclease

Light-driven pumps

The strict living environment and lack of nutritious carbon/nitrogen sources cause diversification of metabolic pathway strain AJ2T and similar halophilic archaea, as well as for haloarchaea, with more resources. Strain AJ2T might use sunlight to produce ATP. We predicted the existence of two light-energy-converting system genes in the AJ2T genome, namely bop and hop. The two encode homologous proteins bacteriorhodopsin and halorhodopsin, respectively. Bacteriorhodopsin and halorhodopsin share 36% of the amino acid residues in the transmembrane part and 19% in the surface connecting loops [27].

Bacteriorhodopsin is an integral membrane protein, called purple membrane, located in the archaea cell membrane, and it acts as a light-driven proton pump. It is mainly found in the Halobacteriaceae family [28, 29]. It captures and uses light energy to move protons out of the cell membrane, resulting in a proton electrochemical gradient. Subsequently, the gradient is converted into chemical energy through ATP synthesis or is used to fuel flagellar motility and other energy requiring processes [30]. We obtained the complete bop gene (AY279548, JQ406920, and AFB77278) in the strain AJ2T by the LPA method. We then successfully expressed the AJ2T bacteriorhodopsin protein in E.coli BL21 with recombinant pET28a plasmid. This result indicates that the prediction of the bop gene is correct. Halorhodopsin is a light-activated chloride pump that is also found in archaea. It utilizes light to transfer the chloride ions into the cytoplasm and increase the electrochemical potential of the proton gradient [31]. This gene is extremely important for salty environment tolerance and, by reporting the existence of a hop gene in the N. altunense strain AJ2T, we shed light on the potential mechanism of its adaptation to high salinity.

Bacteriorhodopsin, halorhodopsin and several related bacterio-opsin activator HTH domain proteins were also found in the other sequenced type strains N. pellirubrum , N. pallidum , N. gari and strain Natrinema sp. J7-2 (listed in Table 8). As the haloarchaea species of the genus Natrinema typically live in similar environment, this type of bacteriorhodopsin/halorhodopsin-based phototrophy can help them adapt to extremely hypersaline and oligotrophic niches.
Table 8

Bacteriorhodopsin and halorhodopsin in the genomes of genus Natrinema

Species

Strain

Bacteriorhodopsin

Halorhodopsin

Size/aa

Accession No.

Size/aa

Accession No.

N. pellirubrum

DSM 15624T

223

WP_006180343

281

WP_006179856

N. pallidum

DSM 3751T

223

WP_006186147

282

WP_006185564

N. altunense a

AJ2T

223

AFB77278

285

KY435894

N. gari

JCM 14663T

223

WP_008455435

282

WP_008453746

Natrinema sp.

J7-2

223

YP_006542121

278

YP_006540994

aThis data line represents the closest output obtained using BLASTp program against the nr database. These two genes are on contig 1 (position:629096–629767, forward strand) and contig 3 (position:389528–390385, forward strand) of the genome of strain AJ2T, respectively

Conclusions

The genome of strain AJ2T did not have the longest length in the sequenced strains of Natrinema , but it had most predicted proteins. Meanwhile, the assembled result in the strain AJ2T had the lowest contig numbers and largest N50 length. This indicated the larger size of the library (2000 bp library) and the longer read length (up to 1000 bp with an average read length 603 bp) may significantly improve the assembling quality.

Our genomic analysis of strain AJ2T shed light on its ability to survive in the Ayakekum salt lake of Altun Mountain National Nature Reserve in Xinjiang, China. This lake is regarded as a relatively extreme environment with low nutrient levels, a cool temperature, strong sunlight and high-altitude. We found evidence for an alternative energy converting system to gain a supplementary energy source. The energy converting system, bacteriorhodopsin, halorhodopsin and HTH domain proteins, were also found in comparison it to all other sequenced strains in the genus Natrinema and they mostly share this energy-producing pathway.

More intensive study and data-mining need to be considered in genomes of the genus Natrinema or another halophilic archaeon. Then, we might find some reasons for these ancient archaeon to have so much vitality and prosperity in extreme environment on planet Earth.

Abbreviations

ANI: 

Average Nucleotide Identity

BBH: 

Bi-directional Best Hit

KAAS: 

KEGG Automatic Annotation Server

LPA: 

Ligation-mediated PCR Amplification

Plsm: 

Plasmid

Declarations

Acknowledgements

We would like to thank Qi-Lan Wang for help with coding custom scripts and performing gene annotations. We also thank Chenling Antelope for language editing and her advice about the phylogenetic analysis. This work was supported by the National Natural Science Foundation of China (Project No. 31170001, No. 31470005, No. 41276173), Zhejiang Provincial Natural Science Foundation of China (No. LQ13D060002) and Scientific Research Fund of the Second Institute of Oceanography, SOA (No. JT1305).

Authors’ contributions

Hong Cheng designed and performed experiments, analysed the data and wrote the paper; Ying-Yi Huo performed experiments and edited the paper; Jing Hu collected and analysed genome data; and Xue-Wei Xu and Min Wu conceived of the experiments and wrote the paper. All authors read and approved of the final manuscript.

Competing interests

The authors declare that they have no competing interests.

Open AccessThis article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.

Authors’ Affiliations

(1)
Laboratory of Marine Ecosystem and Biogeochemistry, Second Institute of Oceanography, State Oceanic Administration
(2)
College of Life Sciences, Zhejiang University

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Copyright

© The Author(s). 2016