High quality draft genome sequence of an extremely halophilic archaeon Natrinema altunense strain AJ2T
© The Author(s). 2016
Received: 21 May 2015
Accepted: 5 December 2016
Published: 1 March 2017
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.
KeywordsHalophilic archaea High-altitude Salt lake Rhodopsin Light-driven pumps
When the genus Natrinema was first described in 1998, it contained two species, Natrinema pellirubrum and Natrinema pallidum . The genus Natrinema belongs to family Halobacteriaceae , phylum Euryarchaeota . Five more species of this genus were isolated and characterized since then, including N. versiforme , N. altunense , N. gari , N. ejinorense  and N. salaciae . For now, the genomic sequences of all but N. ejinorense and N. salaciae in the genus Natrinema are publicly available on Genomes Online Database  and/or NCBI Genbank. Our lab first identified the N. altunense strain AJ2T in 2005 in a salt lake . 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.
Classification and general features of Natrinema altunense AJ2T 
Species Natrinema altunense
Type strain AJ2T=CGMCC 1.3731T=JCM 12890T
pH range; Optimum
Glucose, glycerol, maltose, glutamate, alanine, arginine, lysine, ornithine, acetate, fumarate, malate, propionate, pyruvate and succinate
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).
Aerobic. But the isolate can grow anaerobically in the presence of nitrate.
Altun Mountain National Nature Reserve in Xinjiang, China
Classification and features
Genome sequencing information
Genome project history
The overall information of sequenced genomes about genus Natrinema
Total length (bp)
Level: Gapless Chromosome
1 Plsm: pNATPE01
1 Plsm: pNATPE02
1 Plsm: pJ7-I
Paired-end 2000 bp library
GS FLX+ System
87× (2000 bp library)
Newbler v. 2.5
Gene calling method
Genbank Date of Release
July 21, 2014
Source Material Identifier
CGMCC 1.3731T=JCM 12890T
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 . 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.
The tRNA genes of strain AJ2T were identified using tRNAscan-SE 1.21  with an archaeal model, and its rRNA genes were found via RNAmmer 1.2 Server . Other ORFs were predicted using Glimmer3 . The predicted ORFs were translated and analysed using the BLASTp program (BLAST 2.2.26+) against the non-redundant, Swiss-Prot , Pfam  and COG  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 . KAAS  was used to assign the predicted amino acids into the KEGG Pathway  with the BBH method. Genes with transmembrane helices were predicted using TMHMM Server v.2.0 . We attempted to predict signal peptides using SignalP 4.1 Server , but because there were not enough experimentally confirmed signal peptides in the Uni-Prot database , the online server failed to provide the archaeal group model. The circular map of the genome was obtained using a local CGView application  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.
Number of genes associated with general COG functional categories
Translation, ribosomal structure and biogenesis
RNA processing and modification
Replication, recombination and repair
Chromatin structure and dynamics
Cell cycle control, Cell division, chromosome partitioning
Signal transduction mechanisms
Cell wall/membrane biogenesis
Intracellular trafficking and secretion
Posttranslational modification, protein turnover, chaperones
Energy production and conversion
Carbohydrate transport and metabolism
Amino acid transport and metabolism
Nucleotide transport and metabolism
Coenzyme transport and metabolism
Lipid transport and metabolism
Inorganic ion transport and metabolism
Secondary metabolites biosynthesis, transport and catabolism
General function prediction only
Not in COGs
ANI values between genome pairs within genus Natrinema and Haloterrigena
Insights from the genome sequence
% of total
Genome Size (bp)
DNA coding (bp)
DNA G + C (bp)
Genes in internal clusters
Genes with function prediction
Genes assigned to COGs
Genes with Pfam domains
Genes with signal peptides
Genes with transmembrane helices
The relevance characteristics with genomic features annotation
thiosulfate sulfurtransferase2C rhodanese (EC 126.96.36.199)
thiosulfate sulfurtransferase (EC:188.8.131.52)
catalase (EC 184.108.40.206)/Peroxidase (EC 220.127.116.11)
Nitrous oxide reductase
nitrous oxide reductase maturation transmembrane protein NosY
nitrous oxide reductase maturation transmembrane protein NosY
nitrous oxide reductase maturation transmembrane protein NosY
nitrous oxide reductase maturation protein NosD
nitrous-oxide reductase (EC 18.104.22.168)
flagella-related protein FlaI
chemotaxis regulator CheY
conserved flagella cluster protein
signal peptidase2C type IV - prepilin/preflagellin
DNA repair and recombination protein RadB
single-stranded-DNA-specific exonuclease RecJ (EC 3.1.-.-)
DNA repair and recombination protein RadA
RecJ like exonuclease
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 .
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 . 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 . 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 and halorhodopsin in the genomes of genus Natrinema
N. altunense a
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.
Average Nucleotide Identity
Bi-directional Best Hit
KEGG Automatic Annotation Server
Ligation-mediated PCR Amplification
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).
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.
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.
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