Open Access

Draft genome sequence of the extremely halophilic archaeon Haladaptatus cibarius type strain D43T isolated from fermented seafood

  • Hae-Won Lee1, 2,
  • Dae-Won Kim3,
  • Mi-Hwa Lee4,
  • Byung-Yong Kim5,
  • Yong-Joon Cho5,
  • Kyung June Yim1,
  • Hye Seon Song1,
  • Jin-Kyu Rhee6,
  • Myung-Ji Seo7,
  • Hak-Jong Choi2,
  • Jong-Soon Choi1,
  • Dong-Gi Lee1,
  • Changmann Yoon1,
  • Young-Do Nam4Email author and
  • Seong Woon Roh1Email author
Contributed equally
Standards in Genomic Sciences201510:53

https://doi.org/10.1186/s40793-015-0051-8

Received: 10 September 2014

Accepted: 27 July 2015

Published: 13 August 2015

Abstract

An extremely halophilic archaeon, Haladaptatus cibarius D43T, was isolated from traditional Korean salt-rich fermented seafood. Strain D43T shows the highest 16S rRNA gene sequence similarity (98.7 %) with Haladaptatus litoreus RO1-28T, is Gram-negative staining, motile, and extremely halophilic. Despite potential industrial applications of extremely halophilic archaea, their genome characteristics remain obscure. Here, we describe the whole genome sequence and annotated features of strain D43T. The 3,926,724 bp genome includes 4,092 protein-coding and 57 RNA genes (including 6 rRNA and 49 tRNA genes) with an average G + C content of 57.76 %.

Keywords

Extremely halophilic archaea Haladaptatus cibarius Genome sequenceSalt-fermented seafoodGlycine betaineTrehalose

Introduction

The extremely halophilic archaea, called haloarchaea, possess the small retinal protein halorhodopsin [13] and currently consists of more than 47 genera that live in hypersaline environments [4, 5]. Three members of the genus Haladaptatus H. paucihalophilus [6], H. litoreus [7], and H. cibarius [8]—were isolated from a low-salt, sulfide-rich spring; marine solar saltern; and salt-fermented seafood, respectively. Haladaptatus comprises Gram-negative staining, non-motile haloarchaea that have polar lipids including phosphatidylglycerol, phosphatidylglycerol phosphate methyl ester, and phosphatidylglycerol sulfate [6]. The genomic analysis revealed that H. paucihalophilus survives in low salinity conditions because of trehalose synthesis with OtsAB pathway and trehalose glycosyl-transferring synthase pathway, and glycine betaine uptake [9]. However, other members in the genus Haladaptatus have not been analyzed at the genome level.

H. cibarius was isolated from the traditional Korean salt-fermented seafood, which is made with shellfish [8]. D43T (= DSM 19505T = JCM 15962T ) is a representative strain and designated as the type strain of the species. It can grow in 10%–30% (w/v) NaCl (optimum, 15%), with Mg2+ required for growth. In addition, cells are not lysed in distilled water. The genome sequences of this genus are expected to provide fundamental information for the halotolerant features and biotechnological applications of the haloarchaea. Here, we describe the first whole genome sequence of H. cibarius along with its annotated features, and summarize the taxonomic classification.

Organism information

Classification and features

The taxonomic position for H. cibarius D43T was identified with type strains obtained from the EzTaxon-e server [10]. The 16S rRNA sequences of D43T and closely related strains were aligned using the ClustalW multiple sequence alignment program [11] and were subsequently used for the phylogenetic analysis. Phylogenetic trees were constructed using the neighbor-joining [12], maximum-parsimony [13], and maximum likelihood [14] algorithms with bootstrap values of 1,000 using MEGA version 5 molecular evolutionary genetics analysis program [15]. Strain D43T clustered with type strains of Haladaptatus species (Fig. 1), exhibiting 16S rRNA gene sequence similarities of 98.7% and 95.1% between strain D43T (EF660747) and the type strain of H. litoreus and H. paucihalophilus , respectively. Classification and general features of H. cibarius D43T are shown in Table 1.
Fig. 1

Phylogenetic tree constructed using the neighbor-joining method based on 16S rRNA gene sequences, showing the taxonomic position of strain D43T in genus Haladaptatus. Bootstrap values (>70%) at nodes are shown as percentages calculated using the neighbor-joining/maximum parsimony/maximum likelihood probabilities based on 1000 replicates. Filled circles indicate identical branches generated using three algorithms. Methanosarcina semesiae MD1T was used as an outgroup. Bar, 0.05 substitutions per nucleotide position

Table 1

Classification and general features of Haladaptatus cibarius D43T [18]

MIGS ID

Property

Term

Evidence codea

 

Classification

Domain Archaea

TAS [24]

  

Phylum Euryarchaeota

TAS [25]

  

Class Halobacteria

TAS [26]

  

Order Halobacteriales

TAS [27, 28]

  

Family Halobacteriaceae

TAS [28, 29]

  

Genus Haladaptatus

TAS [6]

  

Species Haladaptatus cibarius

TAS [8]

  

Type strain D43T (DSM 19505, JCM 15962)

TAS [8]

 

Gram stain

Negative

TAS [8]

 

Cell shape

coccus or coccobacillus

TAS [8]

 

Motility

motile

TAS [8]

 

Sporulation

Not reported

TAS [8]

 

Temperature range

15–50 °C

TAS [8]

 

Optimum temperature

37 °C

TAS [8]

 

pH range; Optimum

6.0–8.0; 7.0

TAS [8]

 

Carbon source

Sucrose, D-fructose, D-glucose, lactose, formate, acetate

TAS [8]

MIGS-6

Habitat

Salt-fermented seafood

TAS [8]

MIGS-6.3

Salinity

35 % NaCl (w/v)

TAS [8]

MIGS-22

Oxygen requirement

Aerobic

TAS [8]

MIGS-15

Biotic relationship

Free-living

TAS [8]

MIGS-14

Pathogenicity

Not reported

 

MIGS-23.1

Isolation

Salt-fermented food

TAS [8]

MIGS-4

Geographic location

Republic of Korea

TAS [8]

MIGS-5

Sample collection time

Not reported

 

MIGS-4.1

Latitude

Not reported

 

MIGS-4.2

Longitude

Not reported

 

MIGS-4.3

Depth

Not reported

 

MIGS-4.4

Altitude

Not reported

 

a Evidence codes - TAS: traceable author statement (i.e., a direct report exists in the literature). These evidence codes are from the Gene Ontology project [30]

Strain D43T is a Gram-negative staining, coccus or coccobacillus, motile archaeon approximately 1.0 μm in diameter (Fig. 2). Catalase and oxidase tests yielded positive results, but reduction of nitrate to nitrite under aerobic conditions was negative. Cells contained the polar lipids phosphatidylglycerol, phosphatidylglycerol phosphate methyl ester, and two unidentified glycolipids. Strain D43T hydrolyzed gelatin and Tween 80, utilized formate and acetate as carbon sources, and produced acid from sucrose and d-glucose. The strain was sensitive to anisomycin, aphidicolin, chloramphenicol, and rifampicin, and was resistant to ampicillin, erythromycin, kanamycin, streptomycin, and polymycin B.
Fig. 2

Scanning electron micrographs of H. cibarius D43T obtained by SUPRA 55VP (Carl Zeiss, Jena, Germany). Scale bars represent 200 nm

Genome sequencing and annotation

Genome project history

The genome project and sequence of the H. cibarius D43T genome were deposited in the Genomes OnLine Database [16] (project ID: Gp0086819) and GenBank (accession number: JDTH00000000), respectively. The BioProject number was PRJNA236630. Sequencing and annotation were performed by Chun Lab Inc. (Seoul, Korea) and Integrated Microbial Genomes Expert Review (IMG-ER) [17].

Growth conditions and genomic DNA preparation

H. cibarius D43T grew optimally on halophilic medium [6] supplemented with 15% (w/v) NaCl and 20 mM Mg2+ adjusted to pH 7.0, producing colonies with a pink color after incubation at 37°C as previously described [8]. Genomic DNA was extracted and purified using a G-spin DNA extraction kit (iNtRON Biotechnology Inc., Sungnam, Korea), according to the manufacturer’s instructions.

Genome sequencing and assembly

Genomic sequences of H. cibarius D43T were generated from a total of 9,237,360 quality-filtered reads (710.3-fold coverage) by combining 5,074,634 reads (374.9-fold coverage) obtained from Mi-Seq 300 bp paired-end library (Illumina, San Diego, CA, USA), 4,112,798 reads (292.1-fold coverage) obtained from an Ion Torrent Personal Genome Machine 318v2 chip (Life Technologies, Carlsbad, CA, USA), and 49,928 reads (43.3-fold coverage) obtained from PacBio RS 10 kb library (Pacific Biosciences, Menlo Park, USA). Illumina and PGM data were assembled de novo with CLC Genomics Workbench 6.5.1 (CLC bio, Boston, MA, USA) and PacBio data were assembled using the HGAP2 algorithm in SMRT Analysis 2.1 (Pacific Biosciences). Resultant contigs were assembled with CodonCode Aligner 3.7 (CodonCode Corporation, Centerville, MA, USA). Sequences were assembled to 13 scaffolds with an N50 contig size of 985,075 bp; the genome sequencing project information and its associated MIGS version 2.0 compliance levels [18] are shown in Table 2.
Table 2

Project information

MIGS ID

Property

Term

MIGS-31

Finishing quality

Improved high-quality draft

MIGS-28

Libraries used

Illumina PE, Ion PGM, and PacBio libraries

MIGS-29

Sequencing platforms

Illumina Mi-seq, Ion PGM, and PacBio RS systems

MIGS-31.2

Fold coverage

374.92 × Illumina; 292.08 × Ion PGM; 43.25 × PacBio

MIGS-30

Assemblers

CLC Genomics Workbench 6.5.1, SMRT Analysis 2.1

MIGS-32

Gene calling method

IMG-ER

 

Locus Tag

HL45

 

GenBank ID

JDTH0000000

 

GenBank Date of Release

June 20, 2014

 

GOLD ID

Gi0069860

 

BIOPROJECT

PRJNA236630

MIGS-13

Source material identifier

D43T

 

Project relevance

Environmental and biotechnological

Genome annotation

The open reading frames of the assembled genome were predicted and annotated using IMG-ER [17], NCBI COG [19], Pfam [20], and EzTaxon-e [10] databases. The rRNA and tRNA genes were identified using RNAmmer 1.2 [21] and tRNA scan-SE 1.23 [22], respectively.

Genome properties

The draft genome sequence for H. cibarius D43T contained 3,926,724 bp, with 13 scaffolds. The G + C content was 57.76 % (Fig. 3 and Table 3), and 4,092 protein-coding genes were predicted along with 57 RNA genes, including six rRNA (two 5S, three 16S, and one 23S rRNA), 49 tRNA, and two additional RNA genes. There were 2,676 protein-coding genes with predicted functions: 773 were enzymes, 98 encoded signal peptides, and 1,049 encoded transmembrane proteins. The distribution of genes in the COG functional categories is shown in Table 4. A large number of genes were associated with the COG functional categories of cell wall biogenesis (79, 3.3 %); transcription (100, 4.1 %); and transport and metabolism of amino acids (299, 12.3 %), carbohydrates (121, 5.0 %), and lipids (80, 3.3 %). Further analysis with dbCAN [23], a database for annotation of carbohydrate-active enzymes, showed that the genome contains genes encoding various enzymes for the breakdown and biosynthesis of carbohydrates such as chitinase (GH18), chitosanase (GH5), pullulanase (GH13), trehalose synthase (GT4 and 20), cellulose synthase (GT2), and alginate lyase (PL6).
Fig. 3

Graphical map of the H. cibarius D43T pseudochromosome. From outside to center: RNA genes (red, tRNA and blue, rRNA) and genes on the antisense and sense strands (colored according to COG categories). Inner circle shows the GC skew, with yellow and blue indicating positive and negative values, respectively. GC content is indicated in red and green. The genome map was visualized using CLgenomics 1.06 (Chun Lab Inc.)

Table 3

Genome statistics

Attribute

Value

% of Total

Genome size (bp)

3,926,724

100.00

DNA coding (bp)

3,378,684

86.04

DNA G + C (bp)

2,267,915

57.76

DNA scaffolds

13

100.00

Total genes

4,149

100.00

Protein-coding genes

4,092

98.63

RNA genes

57

1.37

Genes in internal clusters

3,135

75.56

Genes with function prediction

2,676

64.50

Genes assigned to COGs

2,188

52.74

Genes assigned Pfam domains

2,699

65.05

Genes with signal peptides

98

2.36

Genes with transmembrane helices

1049

25.28

CRISPR repeats

4

 
Table 4

Number of genes associated with general COG functional categories

Code

Value

% age

Description

J

164

6.76

Translation, ribosomal structure, and biogenesis

A

1

0.04

RNA processing and modification

K

100

4.12

Transcription

L

102

4.20

Replication, recombination, and repair

B

3

0.12

Chromatin structure and dynamics

D

20

0.82

Cell cycle control, cell division, chromosome partitioning

Y

0

0.00

Nuclear structure

V

37

1.53

Defense mechanisms

T

55

2.27

Signal transduction mechanisms

M

79

3.26

Cell wall/membrane biogenesis

N

28

1.15

Cell motility

Z

0

0.00

Cytoskeleton

W

0

0.00

Extracellular structures

U

28

1.15

Intracellular trafficking and secretion, and vesicular transport

O

88

3.63

Post-translational modification, protein turnover, chaperones

C

162

6.68

Energy production and conversion

G

121

4.99

Carbohydrate transport and metabolism

E

299

12.32

Amino acid transport and metabolism

F

76

3.13

Nucleotide transport and metabolism

H

109

4.49

Coenzyme transport and metabolism

I

80

3.30

Lipid transport and metabolism

P

173

7.13

Inorganic ion transport and metabolism

Q

46

1.90

Secondary metabolism biosynthesis, transport, and catabolism

R

392

16.16

General function prediction only

S

263

10.84

Function unknown

-

1961

47.26

Not in COGs

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

Insights from the genome sequence

The genome analysis of H. cibarius D43T revealed genes involved in glycine betaine synthesis—including betaine aldehyde dehydrogenase, glycine betaine demethylase, and choline-glycine betaine transporter gene—that allow H. cibarius to maintain osmotic balance in hypersaline environments. In addtion, trehalose-related genes of trehalose-6-phosphate synthase, trehalose-6-phosphatase, trehalose-6-phosphate synthase and trehalose-6-phosphate hydrolase, and trehalose-utilization protein genes were analyzed in the genome sequences of H. cibarius D43T. The genes related with trehalose synthesis in the genome show the possibility of trehalose production that is important in food industry.

Conclusions

The draft genome sequences of the extremely halophilic archaeon isolated from the salt-fermented seafood were analyzed. Genes related with glycine betaine and trehalose for the survival in extreme environments were identified. The extremely halophilic archaeon could be a valuable resource for biotechnological applications because hypersaline conditions minimize the risk of contamination by other microorganisms. Further characterization of halophilic enzymes of the haloarchaea based on the genomic analyses can provide more detailed information on enzyme structures and potential industrial applications.

Notes

Abbreviations

PGM: 

Personal genome machine

IMG-ER: 

Integrated microbial genomes expert review

ORF: 

Open reading frame

Declarations

Acknowledgements

This research was supported by the Basic Science Research Program through the National Research Foundation of Korea (2012R1A1A2040922) and a Korea Basic Science Institute NAP grant (T34780).

Open Access This 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)
Biological Disaster Analysis Group, Korea Basic Science Institute
(2)
World Institute of Kimchi
(3)
Systems Biology Team, Center for Immunity and Pathology, Korea National Institute of Health
(4)
Research Group of Gut Microbiome, Korea Food Research Institute
(5)
ChunLab Inc., Seoul National University
(6)
Department of Food Science and Engineering, Ewha Womans University
(7)
Division of Bioengineering, Incheon National University

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Copyright

© Lee et al. 2015